Bacteriophage Ecology Group Reference Abstracts (2000)
	Dedicated to the ecology and evolutionary biology of the parasites of unicellular organisms (UOPs)
	© Stephen T. Abedon
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© Phage et al.	last updated on Wednesday, December 26, 2001

1. The murky origin of Snow White and her T-even dwarfs. Abedon, S.T. (2000). Genetics 155:481-486. The T-even bacteriophages—T2, T4, and T6—represent facile experimental systems that are both relatively complex and meticulously well defined. They played essential roles in the birth and early nurturing of the field of molecular genetics, and could serve similarly as model organisms for ecology. Identification of the source habitat from which these phages were isolated would be satisfying from an ecological as well as historical perspective. Here I infer, mostly from published materials, the habitats from which these three phages were isolated, plus I delve into the history of their host, Eschcerichia coli B. [TOP OF PAGE]

2. The evolution of pathogen-host interactions mediated by bacteriophages. Ai, Y.-C., Meng, F., Zeng, Y. (2000). Acta Microbiologica Sinica 40:657-661. [TOP OF PAGE]

3. Phage resistance in Lactococcus lactis subsp. lactis strains isolated from traditional fermented milk products in Turkey. Akcelik, M., Sanlibaba, P., Tükel, C. (2000). International Journal of Food Science & Technology 35:473-481. Lactococcus lactis subsp. lactis strains isolated from traditional fermented milk products in Turkey were used to determine their phage resistance against three different lactic phages. The following modes of action were examined: phage adsorption inhibition in five strains, abortive infection (heat sensitive phage resistance) in three strains, restriction/modification in four strains and blocking of phage DNA injection in one strain. The genetic nature of the phage resistance systems in these strains was determined by comparison of phage proliferation parameters, e.g. adsorption (%), EOP, burst size, latent period and production of major capsid protein, between wild-type strains and their plasmid-cured derivatives. [TOP OF PAGE]

4. Serotype-converting bacteriophages and O-antigen modification in Shigella flexneri. Allison, G.E., Verma, N.K. (2000). Trends in Microbiology 8:17-23. O-antigen modification (serotype conversion) in Shigella flexneri, which is an important virulence determinant, is conferred by temperate bacteriophages. Several serotype-converting phages have been isolated and preliminary characterization has identified the genes involved in O-antigen modification, and has also provided insight into the molecular biology of these phages. [TOP OF PAGE]

5. Inactivation of MS-2 phage and poliovirus in groundwater. Alvarez, M.E., Aguilar, M., Fountain, A., Gonzalez, N., Rascon, O., Saenz, D. (2000). Canadian Journal of Microbiology 46:159-165. Since temperature affects the inactivation rate of viruses in natural water systems, the aim of this study was to determine if a temperature shift could influence the structural integrity of model viruses. When crude lysates of MS-2 phage were seeded into groundwater microcosms and incubated at 27 degrees C, complete virus inactivation took place in eight days. The temperature was then shifted to 4 degrees C. Three days after the temperature shift, a two-log increase in virus titer (reactivation) occurred. However, when purified MS-2 lysates were added to groundwater microcosms, no reactivation was obtained. No reactivation of poliovirus took place when similar microcosm experiments were done. The sedimentation coefficients of MS-2 shifted from 80S to 58S, 48S, 37S, 32S, and 18S as inactivation proceeded in groundwater and distilled water controls. Similarly, the sedimentation coefficients of polioviruses changed from 156S to 142S, 135S, 117S, 105S, 95S, and 80 S as inactivation took place. There was no correlation between % virus inactivation and % decrease in virions with intact sedimentation coefficients, as reported earlier for poliovirus inactivated by chlorine. The results presented support our hypothesis that virus inactivation proceeds gradually, involving the rearrangement and (or) loss of capsomere components that may eventually lead to the ejection of nucleic acids. The intermediate particles generated as inactivation proceeds may be in a reversibly inactivated state, and may revert back to a fully infectious state when chemical components stabilize the virus particle. [TOP OF PAGE]

6. Going through phages. Anonymous (2000). Science 290:227 [TOP OF PAGE]

7. SNDV, a novel virus of the extremely thermophilic and acidophilic archaeon Sulfolobus. Arnold, H.P., Ziese, U., Zillig, W. (2000). Virology 272:409-416. We describe a novel virus, SNDV (Sulfolobus neozealandicus droplet-shaped virus), of the crenarchaeotal archaeon Sulfolobus, which was found in a carrier state in a Sulfolobus strain isolated from a field sample from New Zealand. SNDV particles are droplet-shaped and densely covered by thin tail fibers at their pointed ends. The virion consists of a core and a coat. The latter has the appearance of a beehive and has a surface that is either helically ribbed or a stack of hoops. The genome is cccDNA of 20 kb, which is modified by dam-like methylation. It is cleaved by only a few type II restriction enzymes e.g., DpnI but not MboI, demonstrating an N(6)-methylation of the adenine residue in GATC sequences. The DNA-modifying system differentiates between virus and host. We postulate a virus-encoded methylase that is active on hemimethylated DNA. The host range of SNDV is confined to few Sulfolobus strains from New Zealand. The virus persists in an unstable carrier state rather than as a prophage. Due to its uniqueness we propose to assign it to a novel virus family termed Guttaviridae. Copyright 2000 Academic Press. [TOP OF PAGE]

8. Seasonal population dynamics and interactions of competing bacteriophages and their host in the rhizosphere. Ashelford, K.E., Norris, S.J., Fry, J.C., Bailey, M.J., Day, M.J. (2000). Appl. Environ. Microbiol. 66:4193-4199. We describe two prolonged bacteriophage blooms within sugar beet rhizospheres ensuing from an artificial increase in numbers of an indigenous soil bacterium, Further, we provide evidence of in situ competition between these phages, This is the first in situ demonstration of such microbial interactions in soil, To achieve this, sugar beet seeds were inoculated with Serratia liquefaciens CP6RS or its lysogen, CP6RS-ly-Phi 1. These were sown, along with uninoculated seeds, in 36 field plots arranged in a randomized Latin square. The plots were then sampled regularly over 194 days, and the plants were assayed for the released bacteria and any infectious phages, Both the lysogen and nonlysogen forms of CP6RS survived equally well in situ, contradicting earlier work suggesting lysogens have a competitive disadvantage in nature. A Podoviridae phage, identified as Phi CP6-4, flourished on the nonlysogen-inoculated plants in contrast to those plants inoculated with the lysogen. Conversely, the Siphoviridae phage Phi CP6-1 (used to construct the released lysogen) was isolated abundantly from the lysogen-treated plants but almost never on the nonlysogen-inoculated plants. The uninoculated plants also harbored some Phi CP6-1 phage up to day 137, yet hardly any Phi CP6-4 phages were found, and this was consistent with previous years. We sinew that the different temporal and spatial distributions of these two physiologically distinct phages can be explained by application of optimal foraging theory to phage ecology. This is the first time that such in situ evidence has been provided in support of this theoretical model. [TOP OF PAGE]

9. Experimental design optimization of filamentous phage transfection into mammalian cells by cationic lipids. Aujame, L., Seguin, D., Droy, C., Hessler, C. (2000). BioTechniques 28:1202-1213. A previous study showed that filamentous phage could be efficiently transfected into mammalian cells in the presence of the cationic lipid Transfectam. In the present study, we used an experimental plan based on a uniform network (Doehlert) matrix to estimate optimal transfection conditions in two different cell lines, CHO and Cos-7. Using the cationic lipid RPR120535b as a model, we show that optimal conditions can be determined much more readily than with standard response curves. Under optimal conditions as analyzed by FACS, up to 60% of Cos-7 and 50% of CHO cells can be transfected. Furthermore, a comparison of different lipids (Transfectam, RPR120535b, TC1-12 and GAP-DLRIE/DOPE) suggests that lipids with multiple amine groups are more efficient for the transfection of filamentous phage. [TOP OF PAGE]

10. The presence of viruses and bacteria along the Adriatic Coast. Aulicino, F.A., Ammazzalorso, P., Ercolessi, M., Banini, L., Silverii, G., Orsini, P., Mastrantonio, A., Bellucci, C., Carere, M. (2000). Igiene Moderna 113:99-116. A study was carried out on seawater samples, collected from the Adriatic sea near the coast of Pesaro, to determine the presence of enteric viruses and Escherichia coli bacteriophages besides the common indicators of fecal pollution and of trophic conditions of the marine environment (Pseudomonas, Vibrio, algae). During 1994-95, seawater samples were tested in 8 stations located in seaside resorts; in 1994 samples of sediment were also analyzed. Generally the results showed a good situation from the microbiological and eutrophic point of view. Only 2 stations showed fecal pollution. Enteroviruses were not detected while Reovirus was isolated from samples of the two most contaminated stations and from a not polluted area. [TOP OF PAGE]

11. Microbiological quality of the Catania coastal sea water. Aulicino, F.A., Mauro, L., Marranzano, M., Biondi, M., Ursino, A., Carere, M. (2000). Annali di Igiene 12:533-541. This study was carried out from 1997 to 1998 along a selected coastal area near Catania to ascertain bacteriological and virological quality of marine waters. 44 seawater samples, collected from 4 stations, were assayed for the presence of total and fecal coliforms, fecal streptococci, coliphages, Salmonellae and enteric viruses. Two stations localized at canal outfalls showed high levels of fecal pollution. The other stations were of good microbiological quality and showed a limited number of samples exceeding the standards laid down as guide values for bathing waters by Italian normative during the bathing period. Salmonellae were isolated in 8 out of 44 sea water samples (18%). Their presence was ascertained mainly in samples of the two polluted stations. Enteroviruses were not isolated. Enteric viruses such as Reoviruses were isolated from all stations, in 12 out of 44 samples (27%). The presence of these viruses was ascertained only during autumnal and winter seasons. The results of this study showed that, notwithstanding some stations showed high levels of bacteriological indicators of fecal pollution and presence of Salmonellae, enteroviruses growing on cell cultures were not isolated. Reoviruses confirmed their high diffusion in marine waters. [TOP OF PAGE]

12. Application of wild starter cultures for flavour development in pilot plant cheese making. Ayad, E.H.E., Verheul, A., Wouters, J.T.M., Smit, G. (2000). International Dairy Journal 10:169-179. A number of wild lactococci of dairy and non-dairy origin which have the ability to produce unusual new flavours in model systems were studied with regard to various characteristics important for cheese making. All strains were found to be non-lysogenic and resistant to phages affecting strains present in commercial starters. Since the overall acidifying activity of many potentially interesting strains is rather low, they were used in combination with commercial starters. Defined-strain starter cultures (DSS) were prepared, composed of a combination of wild strains together with industrial strains, and tested in real cheese making (Gouda-type) experiments. The population dynamics of DSS were studied to understand the behaviour of the selected wild strains in the cheese environment. Wild strains showed various interactions with industrial strains in a defined-strain starter culture. Some wild strains, which were able to grow well together with industrial strains could be used relatively easily for practical applications. Other strains appeared to inhibit the growth of the industrial strains, due to the production of bacteriocins. In many cases the bacteriocin appeared to be nisin. Sensory evaluation revealed that the selected wild strains also produced typical flavours in a real cheese environment which corroborated the results obtained in model systems. GC/MS data confirmed the results of sensory evaluations. [TOP OF PAGE]

13. A proposal for the reclassification of Bdellovibrio stolpii and Bdellovibrio starrii into a new genus, Bacteriovorax gen. nov. as Bacteriovorax stolpii comb. nov. and Bacteriovorax starrii comb. nov., respectively. Baer, M.L., Ravel, J., Chun, J., Hill, R.T., Williams, H.N. (2000). Int J Syst Evol Microbiol 50 Pt 1:219-224. Bdellovibrios are unique bacteria with the ability to prey upon a wide variety of susceptible Gram-negative bacteria. Micro-organisms exhibiting this trait have been included in the genus Bdellovibrio despite their isolation from diverse habitats and relatively unstudied taxonomic relatedness. In this study, 16S rDNA sequences were compared from known terrestrial Bdellovibrio species, Bdellovibrio bacteriovorus 100T, Bdellovibrio stolpii Uki2T and Bdellovibrio starrii A3.12T in order to study their phylogenetic relationship. The two sequences from B. stolpii Uki2T and B. starrii A3.12T were 90.0% similar to each other but exhibited only 81.7% and 81.2% similarity, respectively to B. bacteriovorus 100T. Phylogenetic analysis indicated that B. bacteriovorus 100T clustered in a separate clade from B. starrii A3.12T and B. stolpii Uki2T, demonstrating only a distant relationship between B. bacteriovorus 100T and the other two recognized type species. DNA-DNA hybridization experiments also demonstrated <4% hybridization between these three species. On the basis of the results obtained from the phylogenetic analysis and DNA-DNA hybridization studies, it is proposed that B. stolpii Uki2T and B. starrii A3.12T should be transferred to a new genus, Bacteriovorax gen. nov. as Bacteriovorax stolpii comb. nov. and Bacteriovorax starrii comb. nov., respectively. It is also proposed that the type species for the new genus Bacteriovorax should be Bacteriovorax stolpii comb. nov. [TOP OF PAGE]

14. Light scattering by viral suspensions. Balch, W.M., Vaughn, J., Novotny, J., Drapeau, D.T., Vaillancourt, R., Lapierre, J., Ashe, A. (2000). Limnol. Oceanogr. 45:492-498. Viruses represent one of the most abundant, ocean-borne particle types and have significant potential for affecting optical backscattering. Experiments addressing the light-scattering properties of viruses have heretofore not been conducted. Here we report the results of laboratory experiments in which the volume-scattering functions of several bacterial viruses (bacteriophages) were measured at varying concentrations with a laser light-scattering photometer using a He-Ne and/or Argon ion laser (632.8 and 514.0 nm, respectively). Four bacterial viruses of varying size were examined, including the coliphages MS-2 (capsid size 25-30 nm) and T-4 (capsid size apprx100 nm), and marine phages isolated from Saco Bay, Maine (designation Y-1, capsid size 50-80 nm) and Boothbay Harbor, Maine (designation C-2, capsid size apprx110 nm). Volume-scattering functions (VSFs) were fitted with the Beardsley-Zaneveld function and then integrated in the backward direction to calculate backscattering cross section. This was compared to the virus geometric cross section as determined by transmission electron microscopy and flow-field fractionation. Typical backscattering efficiencies varied from 20 X 10^-6 to 1,000 X 10^-6. Data on particle size and backscattering efficiencies were incorporated into Mie scattering calculations to estimate refractive index of viruses. The median relative refractive index of the four viruses was apprx1.06. Results presented here suggest that viruses, while highly abundant in the sea, are not a major source of backscattering. [TOP OF PAGE]

15. Evolution of life's fringes. Balter, M. (2000). Science 289:1866-1867. Fresh evidence that viruses have existed for billions of years has scientists wondering what role these stripped-down microbes played in evolution. [TOP OF PAGE]

16. A bacteriophage-like particle from Bartonella bacilliformis. Barbian, K.D., Minnick, M.F. (2000). Microbiology 146 ( Pt 3):599-609. Bartonella bacilliformis and Bartonella henselae, the respective agents of Oroya fever and cat-scratch disease in humans, are known to produce bacteriophage-like particles (BLPs) that package 14 kbp segments of the host chromosome. Data from this study suggest that other Bartonella species including Bartonella quintana, Bartonella doshiae and Bartonella grahamii also contain similar BLPs, as evidenced by the presence of a 14 kbp extrachromosomal DNA element in their genomes, whereas Bartonella elizabethae and Bartonella clarridgeiae do not. A purification scheme utilizing chloroform, DNase I and centrifugation was devised to isolate BLPs from B. bacilliformis. Intact BLPs were observed by transmission electron microscopy and were round to icosahedral in shape and approximately 80 nm in diameter. RFLP and Southern blot analysis of BLP DNA from B. bacilliformis suggest that packaging, while non-selective, is less than the near-random packaging previously reported for the B. henselae phage. Data also suggest that the linear, double-stranded BLP DNA molecules have blunt ends with noncovalently closed termini. Packaging of the BLP DNA molecules into a protein coat appears to be closely related to nucleic acid synthesis, as unpackaged phage DNA is not detectable within the host cell. SDS-PAGE analysis of purified BLPs from B. bacilliformis showed three major proteins with apparent molecular masses of 32, 34 and 36 kDa; values that closely correspond to proteins found in B. henselae BLPs. Western blot analysis performed with patient convalescent serum showed that BLP proteins are slightly immunogenic in humans. To determine if BLPs contribute to horizontal gene transfer, mutants of B. bacilliformis were generated by allelic exchange with an internal fragment of the 16S-23S rDNA intergenic spacer region and a suicide vector construct, termed pKB1. BLPs from one of the resultant strains were able to package the mutagenized region containing the kanamycin-resistance cassette; however, numerous approaches and attempts at intraspecies transduction using these BLPs were unsuccessful. [TOP OF PAGE]

17. A shared strategy for virulence. Barinaga, M. (2000). Science 272:1261-1263. [TOP OF PAGE]

18. Diversity in the arrangement of the CTX prophages in classical strains of Vibrio cholerae O1. Basu, A., Mukhopadhyay, A.K., Garg, P., Chakraborty, S., Ramamurthy, T., Yamasaki, S., Takeda, Y., Nair, G.B. (2000). FEMS Microbiol. Let. 182:35-40. This study reports the results of a molecular analysis of the CTX prophages in classical biotype strains of Vibrio cholerae O1 of clinical origin isolated between 1970 and 1979 in India. All strains were sensitive to group IV classical phage and polymyxin B but resistant to group 5 El Tor phage. These phenotypic traits are consistent to that exhibited by the classical biotype. PCR studies reconfirmed their biotype assignment and showed the presence of intact CTX prophages and the presence of the recently described toxin linked cryptic plasmid. Restriction fragment length polymorphism of rRNA genes and pulsed-field gel electrophoresis showed clonal diversity among the strains. The most notable observation was the finding that one strain (GP13) has three CTX prophages while another (GP147) has four CTX prophages. This is the first time heterogeneity is reported in the arrangement of the CTX prophages among classical strains of V. cholerae O1. [TOP OF PAGE]

19. 'Deja vu all over again': the similar structures of bacteriophage PRD1 and adenovirus. Belnap, D.M., Steven, A.C. (2000). Trends in Microbiology 8:91-93. [TOP OF PAGE]

20. The complete cDNA sequence of a type II Trichomonas vaginalis virus. Bessarab, I.N., Liu, H.W., Ip, C.F., Tai, J.H. (2000). Virology 267:350-359. Trichomonas vaginalis viruses (TVV), which may regulate P270 gene expression in the protozoan pathogen T. vaginalis, are a group of divergent double-stranded (ds) RNA viruses. In the present study, the complete 4674-bp cDNA sequence of a 4.6-kb ds RNA from a newly identified TVV2-1 isolate was determined. The sequence of the plus-strand mRNA contains four open reading frames, which encode overlapping cap and pol genes in the reading frame 2 and reading frame 1, respectively, and two putative serine-threonine-rich basic proteins VP3 and VP4 in the third reading frame. An 85-kDa capsid protein and a 160-kDa CAP-POL fusion protein were identified in crude viruses by Western blotting experiments using antisera raised against gene-specific oligopeptides. In conjunction with the presence of a potential ribosomal slippery heptanucleotide G GGC CCC within the overlap of the cap and pol genes, these observations suggest that the pol gene of TVV2-1 is translated via a -1 ribosomal frameshifting event during translation of the cap gene. Our results also provide insight into the conservation among divergent dsRNA species from TVV and suggest that the genome of TVV2-1 may encode two extra genes in addition to the cap and pol genes. Copyright 2000 Academic Press. [TOP OF PAGE]

21. A comparison of methods for counting viruses in aquatic systems. Bettarel, Y., Sime-Ngando, T., Amblard, C., Laveran, H. (2000). Appl. Environ. Microbiol. 66:2283-2289. In this study, we compared different methods-including transmission electron microscopy-and various nucleic acid labeling methods in which we used the fluorochromes 4',6'-diamidino-2-phenylindole (DAPI), 4-[3-methyl-2,3-dihydro-(benzo-1, 3-oxazole)-2-methylmethyledene]-1-(3'-trimethyl ammoniumpropyl)-quinilinium diioide (YOPRO-1), and SYBR Green I, which can be detected by epifluorescence microscopy (EM), for counting viruses in samples obtained from freshwater ecosystems whose trophic status varied and from a culture of T7 phages. From a quantitative and qualitative viewpoint, our results showed that the greatest efficiency for all ecosystems was obtained when we used the EM counting protocol in which YOPRO-1 was the label, as this fluorochrome exhibited strong and very stable fluorescence. A modification of the original protocol in which YOPRO-1 was used is recommended, because this modification makes the protocol faster and allows it to be used for routine analysis of fixed samples. Because SYBR Green I fades very quickly, the use of this fluorochrome is not recommended for systems in which the viral content is very high (>10⁸ particles/ml), such as treated domestic sewage effluents. Experiments in which we used DNase and RNase revealed that the number of viruses determined by EM was slightly overestimated (by approximately 15%) because of interference caused by the presence of free nucleic acids. [TOP OF PAGE]

22. Thermal and chemical inactivation of indigenous Streptococcus thermophilus bacteriophages isolated from Argentinian dairy plants. Binetti, A.G., Reinheimer, J.A. (2000). Journal of Food Protection 63:509-515. Thermal and chemical resistance of five autochthonal bacteriophages of Streptococcus thermophilus, isolated from Cuartirolo cheese wheys and yogurt, was investigated. Times to obtain 99% inactivation of phages (T99) at 63 degrees C and 72 degrees C in three suspension media (enriched tryptic soy broth, reconstituted commercial nonfat skim milk, and tris magnesium gelatin buffer) were determined. The thermal resistance was dependent on the phages studied but not detectable counts (<10 PFU/ml) were only achieved by heating at 90 degrees C during 5 min. The data obtained for the three assayed media did not permit verifying significant differences among them. Sodium hypochlorite (100 ppm) provided a fast inactivation of bacteriophage particles (<10 PFU/ml after 5 min). Ethanol, at concentrations of 75% and 100%, was also effective for phage destruction. Isopropanol was slightly less effective than ethanol at the same concentrations. Peracetic acid (0.15%) was also a very effective agent for phage inactivation. The results showed that these autochthonal bacteriophages were not completely inactivated neither by normal pasteurization treatments nor by some biocides commonly used in disinfection, except sodium hypochlorite and peracetic acid. The practical implications of these findings have pointed out the necessity of recognizing the importance of establishing adequate conditions to assure effective thermal and chemical treatments in dairy plants and laboratory environments. [TOP OF PAGE]

23. Characterization of mesophilic mixed starter cultures used for the manufacture of aged cheddar cheese. Bissonnette, F., Labrie, S., Deveau, H., Lamoureux, M., Moineau, S. (2000). Journal of Dairy Science 83:620-627. Seventy-one different Lactococcus lactis subsp. cremoris strains were isolated from seven mesophilic mixed starters used in the manufacture of aged Cheddar cheese in Canada. Based on plasmid profiles and growth in milk (with or without glucose, Casamino Acids or both), two mixed starters were highly heterogeneous, containing at least 18 to 24 distinct L. lactis strains. Three mixed starters were comprised of seven to nine strains, whereas two starters were relatively homogeneous, containing two or three strains. Many strains with similar plasmid profiles behaved differently during growth in milk, indicating variability in the phenotypes. Only 20% of the strains could grow in plain milk, whereas 30% could not grow in milk supplemented with glucose and Casamino Acids. Twenty-five lactococcal bacteriophages were also isolated from whey samples with single strains as hosts. Eighteen phages belonged to the 936 species and seven to the c2 species. Thirteen strains were insensitive to all 25 phages. Almost all sensitive strains were phage species-specific. The 936-like phages had a broader host range. [TOP OF PAGE]

24. Low-frequency transduction of imipenem resistance and high-frequency transduction of ceftazidime and aztreonam resistance by the bacteriophage AP-151 isolated from a Pseudomonas aeruginosa strain. Blahova, J., Kralikova, K., Krcmery, V., Sr., Jezek, P. (2000). JOURNAL OF CHEMOTHERAPY 12:482-486. Bacteriophage AP-151, isolated from a multidrug resistant Pseudomonas aeruginosa strain, was found to transduce antibiotic resistance determinants to recipient strains of P. aeruginosa. Resistance to cefotaxime, ceftazidime, aztreonam, imipenem and meropenem was transduced as a block, at different frequencies, to two P. aeruginosa strains. Resistance was two logarithms higher (in the range 10^-5) for cefotaxime, ceftazidime or aztreonam than for imipenem in recipient strain PAO-1670. The frequency of transduced imipenem resistance was also lower in recipient strain ML-1008. This phenomenon reflects the difference in the lytic activity of AP-151 in both strains, as the titer of the AP-151 phage in the PAO strain was found to be restricted to 10^-4-10^-5 in contrast to the titer of the same phage in the ML strain which was 10^-10. The limited lytic activity in the PAO recipient strain was correlated with higher transducing activity. It can be concluded that some wild-type bacteriophages of P. aeruginosa might have highly individual relations between lytic and transducting activity in various potential recipient nosocomial strains of P. aeruginosa. The nature of resistance to ceftazidime and imipenem was studied using clavulanate and EDTA as inhibitors of individual class of beta-lactamases, indicating the presence of extended-spectrum beta-lactamase and a metallo-beta-lactamase in this isolate. [TOP OF PAGE]

25. The relative importance of competition and predation varies with productivity in a model community. Bohannan, B.J.M., Lenski, R.E. (2000). Am. Nat. 156:329-340. Recent theory predicts that productivity can influence the relative importance of predation and competition in determining patterns in abundance, diversity, and community structure. In low-productivity systems, competition is predicted to be the major influence on community patterns, while at high productivity, the major influence is predicted to be predation. We directly tested this theory using a laboratory model community. Our model community consisted of the bacteriophage T2 (a virus that feeds on Escherichia coli) and two populations of E. coli, in glucose-limited chemostats. One E. coli population consisted of individuals that were sensitive to predation by T2 ("vulnerable" E. coli), and the other population consisted of individuals that were partially resistant to predation by T2 ("less vulnerable" E. coli). We manipulated productivity in this experiment by running replicate chemostats with different input concentrations of glucose. Our observations were consistent with theoretical predictions. We observed the decline of the more vulnerable prey population at higher productivity but not at lower productivity, and the decline of the less vulnerable prey population at lower productivity but not at higher productivity. However, the rate of decline in some replicates was slower than predicted, and extinctions were not observed during the experiments, contrary to theoretical predictions. We present some testable hypotheses that might explain the slow rate of decline observed. [TOP OF PAGE]

26. Linking genetic change to community evolution: insights from studies of bacteria and bacteriophage. Bohannan, B.J.M., Lenski, R.E. (2000). Ecological Letters 3:362-377. A major goal of community ecology is to link biological processes at lower scales with community patterns. Microbial communities are especially powerful model systems for making these links. In this article, we review recent studies of laboratory communities of bacteria and bacteriophage (viruses that infect bacteria). We focus on the ecology and evolution of bacteriophage-resistance as a case study demonstrating the relationship between specific genes, individual interactions, population dynamics, community structure, and evolutionary change. In laboratory communities of bacteria and bacteriophage, bacteria rapidly evolve resistance to bacteriophage infection. Different resistance mutations produce distinct resistance phenotypes, differing, for example, in whether resistance is partial or complete, in the magnitude of the physiological cost associated with resistance, and in whether the mutation can be countered by a host-range mutation in the bacteriophage. These differences determine whether a mutant can invade, the effect its invasion has on the population dynamics of sensitive bacteria and phage, and the resulting structure of the community. All of these effects, in turn, govern the community's response to environmental change and its subsequent evolution. [TOP OF PAGE]

27. Integrated management of bacterial leaf spot of mungbean with bacteriophages of Xav and chemicals. Borah, P.K., Jindal, J.K., Verma, J.P. (2000). Journal of Mycology and Plant Pathology 30:19-21. The population of Xanthomonas axonopodis pv vignaeradiatae (Xav) was completely eliminated from mungbean seeds by lytic action of bacteriophage (XMP-1) and streptomycin when the seeds were treated with Xav, phages and streptomycin at a ratio/concentration of 1: 60 + 300 mug ml-1. These results confirmed the synergistic action between phage (XMP-1) and streptomycin, as their combination could eradicate Xav from mungbean seeds at a much lower concentration as compared to when used singly. Moreover, the seed treatment with phage lysate + streptomycin 300 mug ml-1 was also found most effective in checking seedling infection of mungbean by Xav. The seedling infection was 4 per cent as compared to 68 per cent in control. The percentage of seed germination was also increased to 86 per cent in comparison to 75 per cent in control. [TOP OF PAGE]

28. Pressure cycling technology: a novel approach to virus inactivation in plasma. Bradley, D.W., Hess, R.A., Tao, F., Sciaba-Lentz, L., Remaley, A.T., Laugharn, J.J., Manak, M. (2000). Transfusion 40:193-200. BACKGROUND: Hydrostatic-pressure virus inactivation is a novel approach to the inactivation of pathogens in plasma and blood-derived components, that retains the therapeutic properties of these products. STUDY DESIGN AND METHODS: A custom-built apparatus was used to pressurize human plasma samples spiked with lambda phage. Phage titer and plasma protein activities were monitored after pressure treatment. RESULTS: Pressure-mediated inactivation of lambda phage was found to be an effective means for virus inactivation, particularly when performed at near-zero (0 degrees C) temperatures, rather than at temperatures above 20 degrees C and below -40 degrees C. The efficiency of inactivation was improved by an increase in applied pressure and repeated cycling from atmospheric to high pressure. In contrast, activities of plasma proteins alkaline phosphatase and total amylase did not vary with temperature and remained within 29 percent and 6 percent, respectively, of starting values after the same pressure treatments. By combining cycling, near-zero temperatures, and high pressure, phage titers in serum were reduced approximately 6 log after 10 to 20 minutes of treatment. Activities of plasma proteins IgG, IgM, and factor X were at 104 percent, 89 percent, and 80 percent, respectively, of starting values after 20 minutes of the same temperature and pressure treatment. CONCLUSION: High-pressure procedures may be useful for the inactivation of viruses in blood and other protein-containing components. [TOP OF PAGE]

29. Viruses of fungi and protozoans: Is everyone sick? Bruenn, J.A. (2000). pp. 297-317. In In Hurst, C.J. (ed.), Viral Ecology. Academic Press, San Diego. [no abstract?[. [TOP OF PAGE]

30. Flow cytometric detection of viruses. Brussaard, C.P.D., Marie, D., Bratbak, G. (2000). Journal of Virological Methods 85:175-182. Representatives from several different virus families (Baculoviridae, Herpesviridae, Myoviridae, Phycodnaviridae, Picornaviridae, Podoviridae, Retroviridae, and Siphoviridae) were stained using a variety of highly fluorescent nucleic acid specific dyes (SYBR Green I, SYBR Green II, OliGreen, PicoGreen) and examined using a standard flow cytometer equipped with a standard 15 mW argon-ion laser. The highest green fluorescence intensities were obtained using SYBR Green I. DNA viruses with genome sizes between 48.5 and 300 kb could easily be detected. The fluorescence signals of the small genome-sized RNA viruses (7.4–14.5 kb) were found at the limit of detection. No significant linear relationship could be found between genome size and the green fluorescence intensity of the SYBR Green I stained virus preparations. To our knowledge, this is the first report of detecting and discriminating between a wide range of different viruses directly using flow cytometry. This rapid and precise assay represents a new and promising tool in the field of virology. [TOP OF PAGE]

31. Big-benefit mutations in a bacteriophage inhibited with heat. Bull, J.J., Badgett, M.R., Wichman, H.A. (2000). Molecular Biology and Evolution 17:942-950. High temperature inhibits the growth of the wild-type bacteriophage phiX174. Three different point mutations were identified that each recovered growth at high temperature. Two affected the major capsid protein (residues F188 and F242), and one affected the internal scaffolding protein (B114). One of the major capsid mutations (F242) is located in a beta strand that contacts B114 in the procapsid during viral maturation, whereas the other capsid mutation (F188) is involved in subunit interactions at the threefold axis of symmetry. Selective coefficients of these mutations ranged from 13.9 to 3.8 in the inhibitory, hot environment, but all mutations reduced fitness at normal temperature. The selective effect of one of the mutations (F242) was evaluated at high temperature in four different genetic backgrounds and exhibited epistasis of diminishing returns: as log fitness of the background genotype increased from -0.1 to 4.1, the fitness boost provided by the F242 mutation decreased from 3.9 to 0. 8. These results support a model in which viral fitness is bounded by an upper limit and the benefit of a mutation is scaled according to the remaining opportunity for fitness improvement in the genome. [TOP OF PAGE]

32. Evolvability of an RNA virus is determined by its mutational neighbourhood. Burch, C.L., Chao, L. (2000). Nature 406:625-628. The ubiquity of mechanisms that generate genetic variation has spurred arguments that evolvability, the ability to generate adaptive variation, has itself evolved in response to natural selection. The high mutation rate of RNA viruses is postulated to be an adaptation for evolvability, but the paradox is that whereas some RNA viruses evolve at high rates, others are highly stable. Here we show that evolvability in the RNA bacteriophage phi6 is also determined by the accessibility of advantageous genotypes within the mutational neighbourhood (the set of mutants one or a few mutational steps away). We found that two phi6 populations that were derived from a single ancestral phage repeatedly evolved at different rates and toward different fitness maxima. Fitness measurements of individual phages showed that the fitness distribution of mutants differed between the two populations. Whereas population A, which evolved toward a higher maximum, had a distribution that contained many advantageous mutants, population B, which evolved toward a lower maximum, had a distribution that contained only deleterious mutants. We interpret these distributions to measure the fitness effects of genotypes that are mutationally available to the two populations. Thus, the evolvability of phi6 is constrained by the distribution of its mutational neighbours, despite the fact that this phage has the characteristic high mutation rate of RNA viruses. [TOP OF PAGE]

33. Selective accumulation may account for shellfish-associated viral illness. Burkhardt, W., Calci, K.R. (2000). Appl. Environ. Microbiol. 66:1375-1378. From 1991 through 1998, 1,266 cases of shellfish-related illnesses were attributed to Norwalk-like viruses. Seventy-eight percent of these illnesses occurred following consumption of oysters harvested from the Gulf Coast during the months of November through January. This study investigated the ability of eastern oysters (Crassostrea virginica) to accumulate indicator microorganisms (i.e., fecal coliforms, Escherichia coli, Clostridium perfringens, and F(+) coliphage) from estuarine water. One-week trials over a 1-year period were used to determine if these indicator organisms could provide insight into the seasonal occurrence of these gastrointestinal illnesses. The results demonstrate that oysters preferentially accumulated F(+) coliphage, an enteric viral surrogate, to their greatest levels from late November through January, with a concentration factor of up to 99-fold. However, similar increases in accumulation of the other indicator microorganisms were not observed. These findings suggest that the seasonal occurrence of shellfish-related illnesses by enteric viruses is, in part, the result of seasonal physiological changes undergone by the oysters that affect their ability to accumulate viral particles from estuarine waters. [TOP OF PAGE]

34. Inactivation of indicator microorganisms in estuarine waters. Burkhardt, W., III, Calci, K.R., Watkins, W.D., Rippey, S.R., Chirtel, S.J. (2000). Water Res. 34:2207-2214. In the United States, shellfish growing areas are classified, in part, using standards based on the densities of either the total or fecal coliform groups in surface waters. However, the standards currently employed may not reliably index the presence of certain enteric pathogens, particularly enteric viruses responsible for human illnesses, even though both the pathogens and indicators derive from the same fecal contamination. To some extent, this may be due to differences in the survival of these pathogens in the environment relative to that of the bacterial indicators. This investigation was conducted to assess the effects of temperature, salinity, dissolved oxygen, geographic location, season, and solar radiation on the survival of selected indicator microorganisms in estuarine waters. The indicators examined included fecal coliforms, Escherichia coli, Clostridium perfringens, and male-specific bacteriophage (MSB), a potential indicator of enteric viruses. In situ experiments were performed in estuarine waters of Alabama and Rhode Island. Among the parameters examined, sunlight and/or temperature most significantly affected indicator decay rates. In general, the effects from exposure to sunlight accounted for up to 83, 84, and 99% of the density reductions of MSB, C. perfringens and fecal coliforms, respectively. Thus, the effects from sunlight were greatest on fecal coliforms and much less pronounced on MSB and C. perfringens. For fecal coliforms, the effect of sunlight was more pronounced during the winter than the summer. In the absence of sunlight, the rate of MSB decline was strongly negatively correlated with estuarine water temperatures and dissolved oxygen. Overall, fecal coliform decay rates were dissimilar to those found for MSB. From this, it would appear that fecal coliforms may not be reliable indicators of viruses in estuarine waters. [TOP OF PAGE]

35. Mathematical analysis of growth and interaction dynamics of streptomycetes and a bacteriophage in soil. Burroughs, N.J., Marsh, P., Wellington, E.M.H. (2000). Appl. Environ. Microbiol. 66:3868-3877. We observed the infection cycle of the temperate actinophage KC301 in relation to the growth of its host Streptomyces lividans TK24 in sterile soil microcosms. Despite a large increase in phage population following germination of host spores, there was no observable impact on host population numbers as measured by direct plate counts. The only change in the host population following infection was the establishment of a small subpopulation of KC301 lysogens. The interaction of S. lividans and KC301 in soil was analyzed with a population-dynamic mathematical model to determine the underlying mechanisms of this low susceptibility to phage attack relative to aquatic environments. This analysis suggests that the soil environment is a highly significant component of the phage-host interaction, an idea consistent with earlier observations on the importance of the environment in determining host growth and phage-host dynamics. Our results demonstrate that the accepted phage-host interaction and host life cycle, as determined from agar plate studies and liquid culture, is sufficient for quantitative agreement with observations in soil, using soil-determined rates. There are four significant effects of the soil environment: (i) newly germinated spores are more susceptible to phage lysis than are hyphae of developed mycelia, (ii) substrate mycelia in mature colonies adsorb about 98% of the total phage protecting susceptible young hyphae from infection, (iii) the burst size of KC301 is large in soil (> 150, 90% confidence) relative to that observed in liquid culture (120, standard error of the mean (SEM), 6), and (iv) there is no measurable impact on the host in terms of reduced growth by the phage. We hypothesize that spatial heterogeneity is the principal cause of these effects and is the primary determinant in bacterial escape of phage lysis in soil. [TOP OF PAGE]

36. Large-plaque mutants of Sindbis virus show reduced binding to heparan sulfate, heightened viremia, and slower clearance from the circulation. Byrnes, A.P., Griess, G.A. (2000). J. Virol. 74:644-651. Laboratory strains of Sindbis virus must bind to the negatively charged glycosaminoglycan heparan sulfate in order to efficiently infect cultured cells. During infection of mice, however, we have frequently observed the development of large-plaque viral mutants with a reduced ability to bind to heparan sulfate. Sequencing of these mutants revealed changes of positively charged amino acids in putative heparin-binding domains of the E2 glycoprotein. Recombinant viruses were constructed with these changes as single amino acid substitutions in a strain Toto 1101 background. All exhibited decreased binding to heparan sulfate and had larger plaques than Toto 1101. When injected subcutaneously into neonatal mice, large-plaque viruses produced higher-titer viremia and often caused higher mortality. Because circulating heparin-binding proteins are known to be rapidly sequestered by tissue heparan sulfate, we measured the kinetics of viral clearance following intravenous injection. Much of the parental small-plaque Toto 1101 strain of Sindbis virus was cleared from the circulation by the liver within minutes, in contrast to recombinant large-plaque viruses, which had longer circulating half-lives. These findings indicate that a decreased ability to bind to heparan sulfate allows more efficient viral production in vivo, which may in turn lead to increased mortality. Because Sindbis virus is only one of a growing number of viruses from many families which have been shown to bind to heparan sulfate, these results may be generally applicable to the pathogenesis of such viruses. [TOP OF PAGE]

37. Effect of five dietary antimutagens on the genotoxicity of six mutagens in the microscreen prophage-induction assay. Cabrera, G. (2000). Environmental and Molecular Mutagenesis 36:206-220. Dietary antimutagens have been studied extensively in the last two decades, using mainly bacterial and mammalian cells. These studies have shown that certain dietary antimutagens, acting individually or as mixtures, are useful in counteracting the effects of certain mutagens and/or carcinogens to which humans are commonly exposed. However, there are some inconsistencies among publications using different bioassays. The general purpose of the research presented here was to conduct a comparative study of the antigenotoxic activity of five dietary antimutagens against six mutagens, using three rather different short-term tests: the Microscreen prophage-induction assay, the Tradescantia micronucleus test, and the Salmonella/mammalian microsome test. In this study I report the results with the Microscreen prophage-induction assay. The antimutagens selected were chlorophyllin, beta-carotene, and vitamins A, C, and E. The mutagens selected were 2-aminoanthracene, benzo[a]pyrene, 2-nitrofluorene, toxaphene, dichlorvos, and nitrofen. The results show that chlorophyllin and beta-carotene inhibited the genotoxicity of all six mutagens; vitamin E inhibited all except dichlorvos; and vitamins C and A inhibited 2-aminoanthracene, benzo[a]pyrene, 2-nitrofluorene, and nitrofen. [TOP OF PAGE]

38. Lateral gene transfer in prokaryotes. Campbell, A.M. (2000). Theor. Pop. Biol. 57:71-77. Evolutionists have traditionally depicted organismal descent on a tree, where all lineages branch from a com- mon ancestor. Such a tree phylogeny implies that all genetic traits within a lineage are derived from its founder. Lateral (horizontal) gene transfer negates this exclusive relationship between ancestor and descendants by the introduction into a lineage of genes originating elsewhere. [TOP OF PAGE]

39. Helicobacter pylori-antigen-binding fragments expressed on the filamentous M13 phage prevent bacterial growth. Cao, J., Sun, Y., Berglindh, T., Mellgard, B., Li, Z., Mardh, B., Mardh, S. (2000). Biochim. Biophys. Acta 1474:107-113. Colonization of the human stomach by Helicobacter pylori is associated with the development of gastritis, duodenal ulcer, mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric cancer. H. pylori-antigen-binding single-chain variable fragments (ScFv) were derived from murine hybridomas producing monoclonal antibodies and expressed as a g3p-fusion protein on a filamentous M13 phage. The recombinant ScFv-phage reacted specifically with a 30-kDa monomeric protein of a H. pylori surface antigen preparation and by means of immunofluorescence microscopy the phage was shown to bind to both the spiral and coccoid forms of the bacterium. In vitro, the recombinant phage exhibited a bacteriocidal effect and inhibited specifically the growth of all the six strains of H. pylori tested. When H. pylori was pretreated with the phage 10 min before oral inoculation of mice, the colonization of the mouse stomachs by the bacterium was significantly reduced (P<0.01). The results suggest that genetic engineering may be used to generate therapy-effective phages. [TOP OF PAGE]

40. Transport and retention of bacteriophages in two types of willow-cropped lysimeters. Carlander, A., Aronsson, P., Allestam, G., Stenstrom, T.A., Perttu, K. (2000). Journal of Environmental Science and Health Part A Toxic-Hazardous Substances & Environmental Engineering A35:1477-1492. Irrigation and fertilization of short-rotation willow coppice with wastewater is a new way of reusing wastewater in Sweden. To evaluate the possible impact of viruses on groundwater quality, the transport and retention of the bacteriophage Salmonella Typhimurium type 28B were studied in two types of willow-cropped field lysimeters containing clay or sand soil. Phages were applied to the soil surface and moderate irrigation was done daily under field-like conditions. In the clay, soil rapid transport of bacteriophages was recorded with breakthrough at 1.2-m depth after 2-24 hours indicating macropore flow through the soil. Phage transport through the sand oil varied considerably, but was in general much slower and the phage retention much higher compared with the clay soil. The willow plants were not found to facilitate phage leaching. Instead, the results indicate the presence of phage retaining processes in the rhizosphere. [TOP OF PAGE]

41. Development and evaluation of a phage typing scheme for Vibrio cholerae O139. Chakrabarti, A.K., Ghosh, A.N., Nair, G.B., Niyogi, S.K., Bhattacharya, S.K., Sarkar, B.L. (2000). Journal of Clinical Microbiology 38:44-49. The scenario of cholera that existed previously changed in 1992 and 1993 with the emergence of toxigenic Vibrio cholerae O139 in India. The genesis of the new serogroup formed the impetus to search for O139 phages in and around the country. A total of five newly isolated phages lytic to V. cholerae O139 strains were used for the development of this phage typing scheme. These phages differed from each other and also differed from the existing O1 phages in their lytic patterns, morphologies, restriction endonuclease digestion profiles, and immunological criteria. With this scheme, 500 V. cholerae O139 strains were evaluated for their phage types, and almost all strains were found to be typeable. The strains clustered into 10 different phage types, of which type 1 (38.2%) was the dominant type, followed by type 2 (22.4%) and type 3 (18%). Additionally, a comparative study of phage types in 1993 and 1994 versus those from 1996 to 1998 for O139 strains showed a higher percentage of phage type 1 (40.5%), followed by type 3 (18.8%) during the period between 1993 and 1994, whereas phage type 2 (32. 1%) was the next major type during the period from 1996 to 1998. This scheme comprising five newly isolated phages would be another useful tool in the study of the epidemiology of cholera caused by V. cholerae O139. [TOP OF PAGE]

42. Genome organization and the evolution of the virulence gene locus in Listeria species. Chakraborty, T., Hain, T., Domann, E. (2000). IJMM International Journal of Medical Microbiology 290:167-174. The chromosomal region of Listeria monocytogenes harboring the gene cluster prfA-plcA-hly-mpl-actA-plcB (virulence gene cluster; vgc) harbors virulence genes critical for the survival of the bacteria following infection. Previous studies have implicated it as an ancestral pathogenicity island, derivatives of which are present in the species L. ivanovii and L. seeligeri, but absent in non-pathogenic species such as L. innocua. We cloned the corresponding region from L. innocua and L. welshimeri and compared its sequences to those from L. monocytogenes, L. ivanovii and L. seeligeri. The analysis allowed exact determination of delineation and size of the vgc and suggests that these genes may have been acquired by bacteriophage transduction. Thus, here we present an alternative view of the evolution of Listeria spp. and suggest that L. monocytogenes may be the primordial species of this genus. [TOP OF PAGE]

43. Emmental cheese: A complex microbial ecosystem. Consequences on selection and use of starters. Chamba, J.F. (2000). Sciences des Aliments 20:37-54. Two fermentations usually take place in emmental cheese, lactic acid fermentation, followed by propionic acid fermentation. Unfortunately, an undesirable fermentation sometimes occurs during cheese ripening: the butyric acid fermentation. Numerous microorganisms cooperate and interact in these fermentations: lactococci, thermophilic streptococci and lactobacilli, heterofermentative lactobacilli and pediococci, propionic acid bacteria. Thus, the cheese is a microbial ecosystem which, in addition to bacteriophages, has other parasites. The properties and the role of each species, the effects of various strains (and the interactions between them) on curd acidification, proteolysis during ripening, eye formation and sensorial characteristics of emmental cheese is demonstrated using examples. Such properties should be taken into account during the selection of lactic and propionic acid bacteria for use as starters. [TOP OF PAGE]

44. Evolution of virulence in parasites: making hard and soft choices. Chao, L., Hanley, K.A., Burch, C.L., Dahlberg, C., Turner, P.E. (2000). Q. Rev. Biol. 75:261-275. [TOP OF PAGE]

45. A high yielding mutant of mycobacteriophage L1 and its application as a diagnostic tool. Chatterjee, S., Mitra, M., Das, G.S. (2000). FEMS Microbiol. Let. 188:47-53. L1 is a lysogenic phage of mycobacteria, which along with L5 and D29 constitute a closely linked family of homoimmune mycobacteriophages. These phages can be potentially used for genetic engineering of mycobacteria and diagnosis of mycobacterial infection. The effectiveness of such phage based systems depends on the efficiency with which they infect and grow within target cells. While working with phage L1c1ts which is a temperature sensitive mutant of phage L1, we observed that high yielding phage stocks were generated by repeated passage through the host, Mycobacterium smegmatis. A plaque purified mutant L1-P2, obtained from one such high yielding stock, when analyzed further was found to infect host cells with increased efficiency. The DNA obtained from L1-P2 was examined by restriction digestion, and it was observed that spontaneous loss of DNA fragment from the right arm, which encodes early regulatory factors, had occurred. It has been further demonstrated that the high yielding property of the mutant phage could be utilized to increase the sensitivity of mycobacteriophage-based detection systems. [TOP OF PAGE]

46. Forces dictating colloidal interactions between viruses and soil. Chattopadhyay, D., Puls, R.W. (2000). Chemosphere 41:1279-1286. The fate and transport of viruses in soil and aquatic environments were studied with respect to the different forces involved in the process of sorption of these viruses on soil particles. In accordance with the classical DLVO theory, we have calculated the repulsive electrostatic forces and the attractive van der Waals forces. Bacteriophages have been used as model sorbates, while different clays have been used as model sorbents. The equations used for the determination of the change in free energy for the process (?G) takes into consideration the roughness of the sorbent surfaces. Results indicate that attractive van der Waals forces predominate the process of sorption of the selected bacteriophages on clays. [TOP OF PAGE]

47. Isolation of a virulent bacteriophage from a Propionibacterium species in the sheep rumen. Cheong, J.P.E., Brooker, J.D. (2000). Australian Journal of Agricultural Research 51:119-123. Propionibacterium is a facultative anaerobe associated with the rumen epithelium, the presence of which may influence the anaerobic environment through oxygen scavenging, as well as providing a source of propionate. Factors such as bacteriophages that influence Propionibacterium populations may therefore be important regulators of rumen function. This study describes the isolation and identification of a ruminal Propionibacterium bacteriophage. Sheep rumen fluid was screened for Propionibacterium species and 3 isolates were identified and characterised. One isolate, PA1, was used as an indicator strain to screen for the presence of Propionibacterium-specific virulent bacteriophages. A virulent bacteriophage, PB2, was isolated from clear plaques on a lawn of PA1 cells and was shown by transmission electron microscopy to be a siphovirus-like particle comprising an icosahedral head 50 nm in diameter and a tail 140 nm in length. The bacteriophage was visibly attached to and within PA1 cells, and was shown to infect all 3 ruminal isolates of Propionibacterium and 4 of 6 clinical isolates of P. acnes. Restriction mapping of bacteriophage PB2 demonstrated a 30.8 kb genome. [TOP OF PAGE]

48. Transfer of conjugative plasmids and bacteriophage lambda occurs in the presence of antibiotics that prevent de novo gene expression. Cooper, T.F., Heinemann, J.A. (2000). Plasmid ???:???-??? [TOP OF PAGE]

49. Evolutionary Reversals During Viral Adaptation to Alternating Hosts. Crill, W.D., Wichman, H.A., Bull, J.J. (2000). Genetics 154:27-37. Experimental adaptation of the bacteriophage fX174 to a Salmonella host depressed its ability to grow on the traditional Escherichia host, whereas adaptation to Escherichia did not appreciably affect growth on Salmonella. Continued host switching consistently exhibited this pattern. Growth inhibition on Escherichia resulted from two to three substitutions in the major capsid gene. When these phages were forced to grow again on Escherichia, fitness recovery occurred predominantly by reversions at these same sites, rather than by second-site compensatory changes, the more frequently observed mechanism in most microbial systems. The affected residues lie on the virion surface and they alter attachment efficiency, yet they occur in a region distinct from a putative binding region previously identified from X-ray crystallography. These residues not only experienced high rates of evolution in our experiments, but also exhibited high levels of radical amino acid variation among fX174 and its known relatives, consistent with a history of adaptation involving these sites. [TOP OF PAGE]

50. Determination of enteroviruses, hepatitis A virus, bacteriophages and Escherichia coli in Adriatic Sea mussels. Croci, L., De, M.D., Scalfaro, C., Fiore, A., Divizia, M., Donia, D., Cosentino, A.M., Moretti, P., Costantini, G. (2000). Journal of Applied Microbiology 88:293-298. The aim of the present study was to evaluate the incidence of enteric viruses in mussels and to verify the possibility of using phages as indirect indicators of mussel viral contamination. Mussels (36 samples) collected from three different areas of the Adriatic Sea were analysed to determine the following parameters: Escherichia coli, somatic coliphage (T6 phage), F-Plus (MS2 phage), B40-8 (phage of Bacteroides fragilis), enteroviruses and hepatitis A virus. Most of the results of the bacteriological analysis (most probable number (MPN) ml-1) were in accordance with the bacteriological limits established by European law, with the exception of seven samples. The bacteriophage analyses were always negative for F-Plus and B40-8, with the exception of a few samples, whereas the somatic coliphages were generally between 0 and 20 MPN g-1, with the exception of two samples (110 MPN g-1). The virological analysis showed five samples positive for the presence of enteroviruses and 13 for the presence of hepatitis A virus (in three samples both viruses were present). Most of these samples presented acceptable bacteriological parameters and the bacteriophages were absent or their value was generally very low. The results show that the detection of E. coli and phages does not seem to be a good indicator of viral contamination. [TOP OF PAGE]

51. Development of a genetically modified bacteriophage for use in tracing sources of pollution. Daniell, T.J., Davy, M.L., Smith, R.J. (2000). Journal of Applied Microbiology 88:860-869. Bacteriophage are frequently used as biotracers to identify the source of water pollutants. Genetic manipulation of bacteriophage M13mp18 has been used to enhance this technique by creating a library in which each recombinant bacteriophage genome contains a unique identification sequence. Techniques that identify a recombinant bacteriophage by the presence of the identification sequence, including polymerase chain reaction, restriction site polymorphism and plaque hybridization, have been developed. Recombinant bacteriophage can be used to test a large number of suspected sources simultaneously. The identification sequence also eliminates confusion with natural bacteriophage present in water samples. The performance of the modified bacteriophage and the techniques were assessed in simulated field trials on a restricted site carried out under a consent for environmental release of a genetically modified organism. The techniques were also field tested at sites in northwest England using wild-type M13 bacteriophage. [TOP OF PAGE]

52. CTX prophages in classical biotype Vibrio cholerae: functional phage genes but dysfunctional phage genomes. Davis, B.M., Moyer, K.E., Boyd, E.F., Waldor, M.K. (2000). J. Bacteriol. 182:6992-6998. CTXphi is a filamentous, lysogenic bacteriophage whose genome encodes cholera toxin, the primary virulence factor produced by Vibrio cholerae. CTX prophages in O1 El Tor and O139 strains of V. cholerae are found within arrays of genetically related elements integrated at a single locus within the V. cholerae large chromosome. The prophages of O1 El Tor and O139 strains generally yield infectious CTXphi. In contrast, O1 classical strains of V. cholerae do not produce CTXphi, although they produce cholera toxin and they contain CTX prophages integrated at two sites. We have identified the second site of CTX prophage integration in O1 classical strains and characterized the classical prophage arrays genetically and functionally. The genes of classical prophages encode functional forms of all of the proteins needed for production of CTXphi. Classical CTX prophages are present either as solitary prophages or as arrays of two truncated, fused prophages. RS1, a genetic element that is closely related to CTXphi and is often interspersed with CTX prophages in El Tor strains, was not detected in classical V. cholerae. Our model for CTXphi production predicts that the CTX prophage arrangements in classical strains will not yield extrachromosomal CTX DNA and thus will not yield virions, and our experimental results confirm this prediction. Thus, failure of O1 classical strains of V. cholerae to produce CTXphi is due to overall deficiencies in the structures of the arrays of classical prophages, rather than to mutations affecting individual CTX prophage genes. [TOP OF PAGE]

53. Convergence of the secretory pathways for cholera toxin and the filamentous phage, CTXphi. Davis, B.M., Lawson, E.H., Sandkvist, M., Ali, A., Sozhamannan, S., Waldor, M.K. (2000). Science 288:333-335. Virulence of Vibrio cholerae depends on secretion of cholera toxin (CT), which is encoded within the genome of a filamentous phage, CTXphi. Release of CT is mediated by the extracellular protein secretion (eps) type II secretion system. Here, the outer membrane component of this system, EpsD, was shown to be required for secretion of the phage as well. Thus, EpsD plays a role both in pathogenicity and in horizontal transfer of a key virulence gene. Genomic analysis suggests that additional filamentous phages also exploit chromosome-encoded outer membrane channels. [TOP OF PAGE]

54. Effect of deleterious mutation-accumulation on the fitness of RNA bacteriophage MS2. de la Pena, M., Elena, S.F., Moya, A. (2000). Evolution 54:686-691. RNA viruses show the highest mutation rate in nautre. It has been extensively demonstrated that, in the absence of purifying selection, RNA viruses accumulate deleterious mutations at a high rate. However, the parameters describing this accumulation are, in general, poorly understood. The present study reports evidences for fitness declines by the accumulation of deleterious mutations in the bacteriophage MS2. We estimated the rate of fitness decline to be as high as 16% per bottleneck transfer. In addition, our results agree with an additive model of fitness effects. [TOP OF PAGE]

55. An introduction to the evolutionary ecology of viruses. DeFilippis, V.R., Villarreal, L.P. (2000). pp. 125-208. In In Hurst, C.J. (ed.), Viral Ecology. Academic Press, San Diego. [no abstract?]. [TOP OF PAGE]

56. Comparative genomics of the late gene cluster from Lactobacillus phages. Desiere, F., Pridmore, R.D., Brossow, H. (2000). Virology 275:294-305. Three prophage sequences were identified in the Lactobacillus johnsoni strain NCC533. Prophage Lj965 predicted a gene map very similar to those of pac-site Streptococcus thermophilus phages over its DNA packaging and head and tail morphogenesis modules. Sequence similarity linked the putative DNA packaging and head morphogenesis genes at the protein level. Prophage Lj965/S. thermophilus phage Sfi 11/Lactococcus lactis phage TP901-1 on one band and Lactobacillus delbrueckii phage LL-H/Lactbacillus plantarum phage phig 1e/Listeria monocytogenes phage A118 on the other hand defined two sublines of structural gene clusters in pac-site Siphoviridae from low-GC Gram-positive bacteria. Bacillus subtilis phage SPP1 linked both sublines. The putative major head and tail proteins from Lj965 shared weak sequence similarity with phages from Gram-negative bacteria. A clearly independent line of structural genes in Siphoviridae from low-GC Grampositive bacteria is defined by temperate cos-site phages including Lactobacillus gasserl phage adh, which also shared sequence similarity with phage D3 infecting a Gram-negative bacterium. A phylogenetic tree analysis demonstrated that the ClpP-like protein identified in four cos-site Siphoviridae from Lactobacillus, Lactococcus, Streptococcus, and Pseudomonas showed graded sequence relationships The tree suggested that the ClpP-like proteins from the phages were not acquired by horizontal gene transfer from their corresponding bacterial hosts. [TOP OF PAGE]

57. Present and potential applications of genetic engineering in agronomy and agro-food. Desmazeaud, M. (2000). Comptes Rendus de l'Academie d'Agriculture de France 86:97-102. In agronomy, genetically engineered Rhizobium can improve plant growth. In the case of food productions, a bakery yeast was modified for increasing maltose utilization. Brewery and wine yeasts were modified for better properties than wild strains about alcohol or pH or sulfite resistance. New flavor profiles are obtained also: Kluyveromyces lactis can produce chymosin A used in cheese-making; lactic acid bacteria (mainly Lactococcus lactis) are modified for a controlled action during cheese ripening; genetic engineering can produce bacteriophage resistant strains. [TOP OF PAGE]

58. Pathogenicity islands and phage conversion: Evolutionary aspects of bacterial pathogenesis. Dobrindt, U., Reidl, J. (2000). IJMM International Journal of Medical Microbiology 290:519-527. Horizontal gene transfer plays a key role in the generation of novel bacterial pathogens. Besides plasmids and bacteriophages, large genomic regions termed pathogenicity islands (PAIs) can be transferred horizontally. All three mechanisms for DNA exchange or transfer may be important for the evolution of bacterial pathogens. [TOP OF PAGE]

59. [Water quality control of 2 swimming pools in Rome. Evaluation of the bacteriophage parameter]. Donia, D., Divizia, M., della, S.M., Pana, A. (2000). Annali di Igiene 12:35-39. [TOP OF PAGE]

60. Evaluation of F-specific RNA bacteriophage as a candidate human enteric virus indicator for bivalve molluscan shellfish. Dore, W.J., Henshilwood, K., Lees, D.N. (2000). Appl. Environ. Microbiol. 66:1280-1285. Escherichia coli is a widely utilized indicator of the sanitary quality of bivalve molluscan shellfish sold for human consumption. However, it is now well documented that shellfish that meet the E. coli standards for human consumption may contain human enteric viruses that cause gastroenteritis and hepatitis. In this study we investigated using F-specific RNA bacteriophage (FRNA bacteriophage) to indicate the likely presence of such viruses in shellfish sold for consumption. FRNA bacteriophage and E. coli levels were determined over a 2-year period for oysters (Crassostrea gigas) harvested from four commercial sites chosen to represent various degrees of sewage pollution. Three sites were classified as category B sites under the relevant European Community (EC) Directive (91/492), which required purification (depuration) of oysters from these sites before sale. One site was classified as a category A site, and oysters from this site could be sold directly without further processing. Samples were tested at the point of sale following commercial processing and packaging. All of the shellfish complied with the mandatory EC E. coli standard (less than 230 per 100 g of shellfish flesh), and the levels of contamination for more than 90% of the shellfish were at or below the level of sensitivity of the assay (20 E. coli MPN per 100 g), which indicated good quality based on this criterion. In contrast, FRNA bacteriophage were frequently detected at levels that exceeded 1,000 PFU per 100 g. High levels of FRNA bacteriophage contamination were strongly associated with harvest area fecal pollution and with shellfish-associated disease outbreaks. Interestingly, FRNA bacteriophage contamination exhibited a marked seasonal trend that was consistent with the trend of oyster-associated gastroenteritis in the United Kingdom. The correlation between FRNA bacteriophage contamination and health risk was investigated further by using a reverse transcription-PCR assay for Norwalk-like virus (NLV). NLV contamination of oysters was detected only at the most polluted site and also exhibited a seasonal trend that was consistent with the trend of FRNA bacteriophage contamination and with the incidence of disease. The results of this study suggest that FRNA bacteriophage could be used as viral indicators for market-ready oysters. [TOP OF PAGE]

61. Genomic relatedness of Staphylococcus aureus phages of the International Typing Set and detection of serogroup A, B, and F prophages in lysogenic strains. Doskar, J., Pallova, P., Pant, c., Rosypal, S., R, z., Pant, c., Kailerova, J., Kleparnik, K., Mala, Z., Bocek, P. (2000). Canadian Journal of Microbiology 46:1066-1076. On the basis of HindIII-restriction digest analysis of genomic DNAs, the S. aureus bacteriophages of the International Typing Set were divided into five clusters designated as A, F, Ba, Bb, and Bc. The clusters A and F include all the phages of serogroups A and F and correspond to species 3A and 77 proposed by Ackermann and DuBow (1987). On the other hand, the phages of serogroup B were divided into three clusters designated as Ba, Bb, and Bc that differ significantly each from the other in their restriction patterns. The clusters Ba and Bb may represent two separate species, while the cluster Bc may include more than one phage species. For each of the phage serogroups A, B, and F, common HindIII-restriction fragments of phage 3A (1700 bp), of 53 (4060 bp), and of 77 (8300 bp) were used for the preparation of probes specific to the phages of serogroups A, B, and F. These probes were very effective, making it possible to detect up to three different prophages in a given lysogenic strain at the same time. Restriction enzyme maps of phages 3A, 53, and 77, each representing a different serogroup, were constructed. The restriction maps of phage 3A and that of phage 77 are linear, whereas that of phage 53 is circular and exhibits a circular permutation. DNAs of the phages of serogroups A and F have cohesive ends. On each restriction map, the sites corresponding to specific probes are indicated. The size of intact genomic DNA of all phages estimated by PFGE varies within the range of 41.5-46.2 kb. [TOP OF PAGE]

62. Genetic analysis of chromosomal regions of Lactococcus lactis acquired by recombinant lytic phages. Durmaz, E., Klaenhammer, T.R. (2000). Appl. Environ. Microbiol. 66:895-903. Recombinant phages are generated when Lactococcus lactis subsp. lactis harboring plasmids encoding the abortive type (Abi) of phage resistance mechanisms is infected with small isometric phages belonging to the P335 species. These phage variants are likely to be an important source of virulent new phages that appear in dairy fermentations. They are distinguished from their progenitors by resistance to Abi defenses and by altered genome organization, including regions of L. lactis chromosomal DNA. The objective of this study was to characterize four recombinant variants that arose from infection of L. lactis NCK203 (Abi(+)) with phage phi31. HindIII restriction maps of the variants (phi31.1, phi31.2, phi31.7, and phi31.8) were generated, and these maps revealed the regions containing recombinant DNA. The recombinant region of phage phi31.1, the variant that occurred most frequently, was sequenced and revealed 7.8 kb of new DNA compared with the parent phage, phi31. This region contained numerous instances of homology with various lactococcal temperate phages, as well as homologues of the lambda recombination protein BET and Escherichia coli Holliday junction resolvase Rus, factors which may contribute to efficient recombination processes. A sequence analysis and phenotypic tests revealed a new origin of replication in the phi31.1 DNA, which replaced the phi31 origin. Three separate HindIII fragments, accounting for most of the recombinant region of phi31.1, were separately cloned into gram-positive suicide vector pTRK333 and transformed into NCK203. Chromosomal insertions of each plasmid prevented the appearance of different combinations of recombinant phages. The chromosomal insertions did not affect an inducible prophage present in NCK203. Our results demonstrated that recombinant phages can acquire DNA cassettes from different regions of the chromosome in order to overcome Abi defenses. Disruption of these regions by insertion can alter the types and diversity of new phages that appear during phage-host interactions. [TOP OF PAGE]

63. Bacteriophages of spirochetes. Eggers, C.H., Casjens, S., Hayes, S.F., Garon, C.F., Damman, C.J., Oliver, D.B., Samuels, D.S. (2000). J Mol Microbiol Biotechnol 2:365-373. Historically, a number of bacteriophage-like particles have been observed in association with members of the bacterial order Spirochetales, the spirochetes. In the last decade, several spirochete bacteriophages have been isolated and characterized at the molecular level. We have recently characterized a bacteriophage of the Lyme disease agent, Borrelia burgdorferi, which we have designated phiBB-1. Here we review the history of the association between the spirochetes and their bacteriophages, with a particular emphasis on phiBB-1 and its prophage, the 32-kb circular plasmid family of B. burgdorferi. [TOP OF PAGE]

64. Characterization of a Phage Resistance Plasmid, pLKS, of Silage-Making Lactobacillus plantarum NGRI0101. Eguchi, T., Doi, K., Nishiyama, K., Ohmomo, S., Ogata, S. (2000). Biosci. Biotech. Biochem. 64:751-756. [TOP OF PAGE]

65. The two faces of mutation: Extinction and adaptation in RNA viruses. Elena, S.F., Miralles, R., Cuevas, J.M., Turner, P.E., Moya, A. (2000). IUBMB Life 49:5-9. From a population standpoint, two main features characterize the replication of RNA viruses and viruses that use RNA as a replicative intermediate: high genetic variability, and enormous fluctuations in population size. Their genetic variability mainly reflects a lack of the proof-reading and post-replicative error correction mechanisms that operate during cellular DNA replication, but recombination and segment exchange can also play an important role, Viral population size can change tremendously as a consequence of transmission between hosts or between different tissues within an infected host. A new infection can be initiated with very few particles that subsequently expand many trillion-fold. Repeated bottleneck events can lead to drastic fitness losses or even to viral extinction, whereas continuously large population sizes result in fitness gains and adaptation. Here we review experimental evidence for the effects of mutation, selection, and genetic drift on the adaptation and extinction of RNA viruses. [TOP OF PAGE]

66. Toward antiviral strategies that resist viral escape. Endy, D., Yin, J. (2000). Antimicrobial Agents and Chemotherapy 44:1097-1099. We studied the effect on viral growth of drugs targeting different virus functions using a computer simulation for the intracellular growth of bacteriophage T7. We found that drugs targeting components of negative-feedback loops gain effectiveness against mutant viruses that attenuate the drug-target interaction. The greater inhibition of such mutants than of the wild type suggests a drug design strategy that would hinder the development of drug resistance. [TOP OF PAGE]

67. Computation, prediction, and experimental tests of fitness for bacteriophage T7 mutants with permuted genomes. Endy, D., You, L., Yin, J., Molineux, I.J. (2000). Proc. Natl. Acad. Sci. USA 97:5375-5380. We created a simulation based on experimental data from bacteriophage T7 that computes the developmental cycle of the wild-type phage and also of mutants that have an altered genome order. We used the simulation to compute the fitness of more than 10⁵ mutants. We tested these computations by constructing and experimentally characterizing T7 mutants in which we repositioned gene 1, coding for T7 RNA polymerase. Computed protein synthesis rates for ectopic gene 1 strains were in moderate agreement with observed rates. Computed phage-doubling rates were close to observations for two of four strains, but significantly overestimated those of the other two. Computations indicate that the genome organization of wild-type T7 is nearly optimal for growth: only 2.8% of random genome permutations were computed to grow faster, the highest 31% faster, than wild type. Specific discrepancies between computations and observations suggest that a better understanding of the translation efficiency of individual mRNAs and the functions of qualitatively "nonessential" genes will be needed to improve the T7 simulation. In silico representations of biological systems can serve to assess and advance our understanding of the underlying biology. Iteration between computation, prediction, and observation should increase the rate at which biological hypotheses are formulated and tested. [TOP OF PAGE]

68. Sunlight-Induced Propagation of the Lysogenic Phage Encoding Cholera Toxin. Faruque, S.M., Rahman, A.M.M., Waldor, M.K., Sack, D.A. (2000). Infect. Immun. 68:4795-4801. [TOP OF PAGE]

69. Control of bacterial spot on tomato in the greenhouse and field with h-mutant bacteriophages. Flaherty, J.E., Jones, J.B., Harbaugh, B.K., Somodi, G.C., Jackson, L.E. (2000). Hortscience 35:882-884. A mixture of host-range mutant (h-mutant) bacteriophages specific for tomato race 1 (T1) and race 3 (T3) of the bacterial spot pathogen, Xanthomonas campestris pv. vesicatoria (Doidge) Dye was evaluated for biological control of bacterial spot on 'Sunbeam' tomato (Lycopersicon esculentum Mill.) transplants and field-grown plants for two seasons (Fall 1997 and Fall 1998). Foliar applications of bacteriophages were compared with similar applications of water (control) and of copper/mancozeb bactericides, the commonly used chemical control strategy for tomato seedling and field production. In 1997, the incidence of bacterial spot on greenhouse-grown seedlings was reduced from 40.5% (control) to 5.5% or 0.9% for bactericide- or bacteriophage-treated plants, respectively. In 1998, the incidence of bacterial spot was 17.4% on control plants vs. 5.5% and 2.7% for bactericide- and bacteriophage-treated plants, respectively, although these differences were not statistically significant at P ltoreq 0.05. Applications of bacteriophages to field-grown tomatoes decreased disease severity as measured by the area under the disease progress curve (AUDPC) by 17.5% (1997) and 16.8% (1998) compared with untreated control plants. Preharvest plant vigor ratings, taken twice during each field season, were higher in the bacteriophage-treated plants than in either bactericide-treated plants or nontreated controls except for the early vigor rating in 1998. Use of bacteriophages increased total weight of extra-large fruit 14.9% (1997) and 24.2% (1998) relative to that of nontreated control plants, and 37.8% (1997) and 23.9% (1998) relative to that of plants treated with the chemical bactericides. Chemical names used: manganese, zinc, carboxy-ethylene his dithiocarbamate (mancozeb). [TOP OF PAGE]

70. Elimination of enteroviruses, other enteric viruses, F-specific coliphages, somatic coliphages and E. coli in four sewage treatment plants of southern Germany. Fleischer, J., Schlafmann, K., Otchwemah, R., Botzenhart, K. (2000). Journal of Water Supply Research and Technology - AQUA 49:127-138. The reduction processes at four advanced sewage treatment plants in Baden-Wuerttemberg were evaluated with regard to virus elimination and the elimination of indicator organisms from wastewater. The results of virus elimination were compared with the reduction of somatic and male specific bacteriophages and of E. coli. In total, 222 water samples were examined. The results obtained for the different treatment plants show reduction rates from 80.0% to 99.9% for enteroviruses, enumerated as PFU l^-1 on BGM cell line, and reduction rates from 59.4% to 99.9% for other enteric viruses, enumerated as MPN l-1 on MA-104 cell line. Identification of the isolated enteroviruses yielded 88.3% for Coxsackie virus B (1-5), 18.3% were positive for Polio (1-3) and 8.3% for Echo virus (1+11). The reduction rates of somatic bacteriophages ranged from 76.4% to 99.90%, for male specific bacteriophages from 87.5% to 99.9% and for E. coli from 75.0% to 99.9% respectively. Two of the plants use standard chemical precipitation and the other two employ combinations of chemical and biological elimination techniques to reduce the concentrations of phosphorus and nitrogen. A correlation between the amount of precipitators and the elimination rates of the tested microorganisms could not be demonstrated, perhaps due to the fact that the treatment conditions could not be modified by the investigators. It is concluded that the tested treatment plants using combinations of chemical and biological techniques for P and N removal show equal or higher elimination rates than conventional treatment processes using chemical elimination techniques. [TOP OF PAGE]

71. Evaluation of CD4+ T cell function In vivo in HIV-infected patients as measured by bacteriophage phiX174 immunization. Fogelman, I., Davey, V., Ochs, H.D., Elashoff, M., Feinberg, M.B., Mican, J., Siegel, J.P., Sneller, M., Lane (2000). J. Infect. Dis. 182:435-441. Bacteriophage phiX174 immunization was used to measure CD4(+) T cell function in vivo in human immunodeficiency virus (HIV)-infected patients across all disease stages. Function was evaluated by measuring the ability of T cells to provide help to B cells in antibody production, amplification, and isotype switching. A total of 33 patients and 10 controls received 3 bacteriophage phiX174 immunizations 6 weeks apart. The patients' responses regarding bacteriophage-specific total antibody titers and IgG titers were quantitatively and qualitatively inferior to the controls' responses. Overall, 7 of 33 patients had normal T cell function. Baseline CD4 counts provided the strongest correlation with total antibody and IgG titers. HIV RNA had a weaker association with responses but had some predictive power among patients with a CD4 count >200 cells/microL. Bacteriophage phiX174 immunization seems to be a useful tool for measuring immune function in vivo, which suggests that most HIV-infected patients may have abnormal CD4(+) T cell function despite adequate antiretroviral treatment. [TOP OF PAGE]

72. Widespread distribution of a group I intron and its three deletion derivatives in the lysin gene of Streptococcus thermophilus bacteriophages. Foley, S., Bruttin, A., Brussow, H. (2000). J. Virol. 74:611-618. Of 62 Streptococcus thermophilus bacteriophages isolated from various ecological settings, half contain a lysin gene interrupted by a group IA2 intron. Phage mRNA splicing was demonstrated. Five phages possess a variant form of the intron resulting from three distinct deletion events located in the intron-harbored open reading frame (orf 253). The predicted orf 253 gene sequence showed a significantly lower GC content than the surrounding intron and lysin gene sequences, and the predicted protein shared a motif with endonucleases found in phages from both gram-positive and gram-negative bacteria. A comparison of the phage lysin genes revealed a clear division between intron-containing and intron-free alleles, leading to the establishment of a 14-bp consensus sequence associated with intron possession. The conserved intron was not found elsewhere in the phage or S. thermophilus bacterial genomes. Folding of the intron RNA revealed secondary structure elements shared with other phage introns: first, a 38-bp insertion between regions P3 and P4 that can be folded into two stem-loop structures (shared with introns from Bacillus phage SPO1 and relatives); second, a conserved P7.2 region (shared with all phage introns); third, the location of the stop codon from orf 253 in the P8 stem (shared with coliphage T4 and Bacillus phage SPO1 introns); fourth, orf 253, which has sequence similarity with the H-N-H motif of putative endonuclease genes found in introns from Lactococcus, Lactobacillus, and Bacillus phages. [TOP OF PAGE]

73. Occurrence and distribution of microbiological indicators in groundwater and stream water. Francy, D.S., Helsel, D.R., Nally, R.A. (2000). Water Environment Research 72:152-161. A total of 136 stream water and 143 groundwater samples collected in five important hydrologic systems of the United States were analyzed for microbiological indicators to test monitoring concepts in a nationally consistent program. Total coliforms were found in 99%, Escherichia coli in 97%, and Clostridium perfringens in 73% of stream water samples analyzed for each bacterium. Total coliforms were found in 20%, E. coli in less than 1%, and C. perfringens in none of the groundwater samples analyzed for each bacterium. Although coliphage analyses were performed on many of the samples, contamination in the laboratory and problems discerning discrete plaques precluded quantification. Land use was found to have the most significant effect on concentrations of bacterial indicators in stream water. Presence of septic systems on the property near the sampling site and well depth were found to be related to detection of coliforms in groundwater, although these relationships were not statistically significant. A greater diversity of sites, more detailed information about some factors, and a larger dataset may provide further insight to factors that affect microbiological indicators. [TOP OF PAGE]

74. Titration of infective and noninfective Ff filamentous bacteriophages using a monoclonal antibody against g3p. Frisch, C., Knappik, A., Choidas, M., Tesar, M. (2000). BioTechniques 29:26-8, 30. [TOP OF PAGE]

75. Effect of host bacteria genotype on spontaneous reversions of Bacillus subtilis bacteriophage PHI29 sus17 nonsense codon. Fucik, V., Beran, J., Krasny, L., Jonak, J. (2000). FEMS Microbiol. Let. 183:143-146. Gene 17 of Bacillus subtilis bacteriophage PHI29 is an early gene playing a role in DNA replication. Its mutant sus17(112) carries the TAA nonsense triplet at the fifth codon of the gene. We isolated and sequenced 73 spontaneous revertants producing normal-size plaques on bacteria without an informational suppressor gene. In all revertants, the TAA triplet was changed by a one-base substitution and the sequences CAA, AAA, TTA, TAC and TAT were recovered at its place. The spectrum of these mutations was markedly influenced by the genotype of the bacteria in which the revertants arose. In agreement with the results described in Escherichia coli, the ratio of transversions to transitions (CAA being the only transition acceptable) was higher in strains harboring the functional allele recA+ than in those with recA4. Our results support the idea that also in the Gram-positive B. subtilis, the spectra of spontaneous mutations are specifically modified by an SOS function. It is assumed that the single-stranded DNA chains generated in the course of phage DNA replication might act as an inducing factor. [TOP OF PAGE]

76. Horizontal gene transfer in bacterial and archaeal complete genomes. Garcia-Vallve, S., Romeu, A., Palau, J. (2000). Genome Research 10:1719-1725. There is growing evidence that horizontal gene transfer is a potent evolutionary force in prokaryotes, although exactly how potent is not known. We have developed a statistical procedure for predicting whether genes of a complete genome have been acquired by horizontal gene transfer. It is based on the analysis of G+C contents, codon usage, amino acid usage, and gene position. When we applied this procedure to 17 bacterial complete genomes and seven archaeal ones, we found that the percentage of horizontally transferred genes varied from 1.5% to 14.5%. Archaea and nonpathogenic bacteria had the highest percentages and pathogenic bacteria, except for Mycoplasma genitalium, had the lowest. As reported in the literature, we found that informational genes were less likely to be transferred than operational genes. Most of the horizontally transferred genes were only present in one or two lineages. Some of these transferred genes include genes that form part of prophages, pathogenecity islands, transposases, integrases, recombinases, genes present only in one of the two Helicobacter pylori strains, and regions of genes functionally related. All of these findings support the important role of horizontal gene transfer in the molecular evolution of microorganisms and speciation. [TOP OF PAGE]

77. Sensitivity of microorganisms to different wavelengths of UV light: Implications on modeling of medium pressure UV systems. Giese, N., Darby, J. (2000). Water Res. 34:4007-4013. The responses of three species of coliform bacteria (Citrobacter diversus, Citrobacter freundii and Klebsiella pneumoniae) and the bacteriophage (virus) variant phiX-174 to three wavelengths of UV light (254, 280 and 301 nm) were measured. The values of germicidal efficiency at 280 nm determined for each of the microorganisms were not significantly different. At 301 nm, the values of germicidal efficiency were significantly different, but all values were too small (< 0.06) to warrant consideration in the modeling of the germicidal intensity delivered by a medium pressure UV system. The data from this study provide some evidence that the values of germicidal efficiency determined for one species of bacteria or virus may be used to represent the relative responses of all bacteria and viruses to medium pressure UV irradiation. [TOP OF PAGE]

78. Phage-displayed peptides as biosensor reagents. Goldman, E.R., Pazirandeh, M.P., Mauro, J.M., King, K.D., Frey, J.C., Anderson, G.P. (2000). Journal of Molecular Recognition 13:382-387. This study investigated the potential to utilize phage-displayed peptides as reagents in sensor applications. A library of random 12-mers displayed on phage was panned against staphylococcal enterotoxin B (SEB), a causative agent of food poisoning. Nine SEB binding phage clones were isolated, all of which share the consensus sequence Trp His Lys at their amino terminus. Binding of several of these phage was shown to be inhibited when they were assayed in a competitive enzyme-linked immunosorbent assay (ELISA) format with synthesized peptide corresponding to the peptide-encoding region of one of the clones. Whole phage were labeled with the dye Cy5, and incorporated into fluoroimmunoassays. Labeled phage were able to detect SEB down to a concentration of 1.4 ng/well in a fluorescence-based immunoassay. When incorporated into an automated fluorescence-based sensing assay, Cy5-labeled phage bound to probes coated with SEB generated a robust signal of about 10,000 pA, vs a signal of 1000 pA using a control fiber coated with streptavidin. These results demonstrate the potential for development of phage-based sensor reagents. [TOP OF PAGE]

79. Bacterial indicator occurrence and the use of an F+ specific RNA coliphage assay to identify fecal sources in Homosassa Springs, Florida. Griffin, D.W., Stokes, R., Rose, J.B., Paul, J.H., III (2000). Microb. Ecol. 39:56-64. A microbiological water quality study of Homosassa Springs State Wildlife Park (HSSWP) and surrounding areas was undertaken. Samples were collected in November of 1997 (seven sites) and again in November of 1998 (nine sites). Fecal bacterial concentrations (total and fecal coliforms, Clostridium perfringens, and enterococci) were measured as relative indicators of fecal contamination. F⁺-specific coliphage genotyping was performed to determine the source of fecal contamination at the study sites. Bacterial levels were considerably higher at most sites in the 1997 sampling compared to the 1998 sampling, probably because of the greater rainfall that year. In November of 1997, 2 of the 7 sites were in violation of all indicator standards and guidance levels. In November of 1998, 1 of 9 sites was in violation of all indicator standard and guidance levels. The highest concentrations of all fecal indicators were found at a station downstream of the animal holding pens in HSSWP. The lowest levels of indicators were found at the Homosassa Main Spring vent. Levels of fecal indicators downstream of HSSWP (near the point of confluence with the river) were equivalent to those found in the Southeastern Fork and areas upstream of the park influences. F⁺ specific RNA coliphage analysis indicated that fecal contamination at all sites that tested positive was from animal sources (mammals and birds). These results suggest that animal (indigenous and those in HSSWP) and not human sources influenced microbial water quality in the area of Homosassa River covered by this study. [TOP OF PAGE]

80. Characterization of natural isolates of Lactobacillus strains to be used as starter cultures in dairy fermentation. Hebert, E.M., Raya, R.R., Tailliez, P., de, G.G. (2000). Int. J. Food Microbiol. 59:19-27. The technological relevant characteristics of five homofermentative lactobacilli strains, isolated from natural fermented hard cheeses, were studied. Isolates CRL 581 and CRL 654, from Argentinian artesanal hard cheeses, and isolates CRL 1177, CRL 1178, and CRL 1179, from Italian Grana cheeses, were identified as Lactobacillus delbrueckii subsp. lactis and Lactobacillus helveticus, respectively, by physiological and biochemical tests, SDS-PAGE of whole-cell proteins and sequencing of the variable (V1) region of the 16S ribosomal DNA. All strains showed high levels of beta-galactosidase activity. However, proteolytic activity varied widely among isolates. Strains CRL 581, CRL 654, and CRL 1177 hydrolyzed alpha- and beta-caseins and were able to coagulate reconstituted skim milk in less than 16 h at 42 degrees C. According to the substrate specificity, these proteinases have a caseinolytic activity comparable to that of the P(III)-type of lactococcal proteinases. No strains produced inhibitor substances (bacteriocin) and all were insensitive to attack by 14 L. helveticus- and L. delbrueckii subsp. lactis-specific bacteriophages. [TOP OF PAGE]

81. The origins and ongoing evolution of viruses. Hendrix, R.W., Lawrence, J.G., Hatfull, G.F., Casjens, S. (2000). Trends in Microbiology 8:504-508. Genome analyses of double strand DNA tailed bacteriophages argue that they evolve by recombinational reassortment of genes and by the acquisition of novel genes as simple genetic elements termed morons. These processes suggest a model for early virus evolution, wherein viruses can be regarded less as having derived from cells and more as being partners in their mutual co-evolution. [TOP OF PAGE]

82. Intramuscular immunization with genetically inactivated (ghosts) Actinobacillus pleuropneumoniae serotype 9 protects pigs against homologous aerosol challenge and prevents carrier state. Hensel, A., Huter, V., Katinger, A., Raza, P., Strnistschie, C., Roesler, U., Brand, E., Lubitz, W. (2000). Vaccine 18:2945-2955. Bacterial ghosts are empty cell envelopes achieved by the expression of a cloned bacteriophage lysis gene and, unlike classical bacterins, suffer no denaturing steps during their production. These properties may lead to a superior presentation of surface antigens to the immune system. Currently available porcine Actinobacillus pleuropneumoniae vaccines afford only minimal protection by decreasing mortality but not morbidity. Pigs which survive infection can still be carriers of the pathogen, so a herd once infected remains infected. Carrier pigs harbour A. pleuropneumoniae in their nasal cavities, in their tonsils, or within lung lesions. A dose-defined nose-only aerosol infection model for pigs was used to study the immunogenic and protective potential of systemic immunization with ghosts made from A. pleuropneumoniae serotype 9 reference strain CVI 13261 against an homologous aerogenous challenge. Pigs were vaccinated twice intramuscularly with a dose of 5x10(9) CFU ghosts (GVPs) or formalin-inactivated A. pleuropneumoniae bacterins (BVPs). After 2 weeks vaccinated pigs and non-vaccinated placebo controls (PCs) were challenged with a dose of 10(9) CFU by aerosol. The protective efficacy of immunization was evaluated by clinical, bacteriological, serological and post-mortem examinations. Bronchoalveolar lavage in pigs was performed during the experiment to obtain lavage samples (BALF) for assessment of local antibodies. Isotype-specific antibody responses in serum and BALF were determined by ELISAs based on whole-cell antigen. Immunization with ghosts did not cause clinical side-effects. After aerosol challenge PCs developed fever and pleuropneumonia. GVPs or BVPs were found to be fully protected against clinical disease or lung lesions in both vaccination groups, whereas colonization of the respiratory tract with A. pleuropneumoniae was only prevented in GVPs. Specific immunoglobins against A. pleuropneumoniae were not detectable in BALF after immunization. A significant systemic increase of IgM, IgA, IgG(Fc'), or IgG(H+L) antibodies reactive with A. pleuropneumoniae was measured in GVPs and BVPs when compared to the non-exposed controls. BVPs reached higher titers of IgG(Fc') and IgG(H+L) than GVPs. However, prevention of carrier state in GVPs coincided with a significant increase of serum IgA when compared to BVPs. These results suggest that immunization with ghosts, that bias antibody populations specific to non-denaturated surface antigens, may be more efficacious in protecting pigs against colonization and infection than bacterins. [TOP OF PAGE]

83. Characterization of PHI8, a bacteriophage containing three double-stranded RNA genomic segments and distantly related to PHI6. Hoogstraten, D., Qiao, X., Sun, Y., Hu, A., Onodera, S., Mindich, L. (2000). Virology 272:218-224. The three double-stranded RNA genomic segments of bacteriophage PHI8 were copied as cDNA, and their nucleotide sequences were determined. Although the organization of the genome is similar to that of PHI6, there is no similarity in either the nucleotide sequences or the amino acid sequences, with the exception of the motifs characteristic of viral RNA polymerases that are found in the presumptive polymerase sequence. Several features of the viral proteins differ markedly from those of PHI6. Although both phages are covered by a lipid-containing membrane, the protein compositions are very different. The most striking difference is that protein P8, which constitutes a shell around the procapsid in PHI6, is part of the membrane in PHI8. The host attachment protein consists of two peptides rather than one and the phage attaches directly to the lipopolysaccharide of the host rather than to a type IV pilus. The host range of PHI8 includes rough strains of Salmonella typhimurium and of pseudomonads. [TOP OF PAGE]

84. Phage infection of the obligate intracellular bacterium, Chlamydia psittaci strain Guinea Pig Inclusion Conjunctivitis. Hsia, R.C., Ohayon, H., Gounon, P., Dautry-Varsat, A., Bavoil, P.M. (2000). Microbes and Infection 2:761-772. The infectious cycle of phiCPG1, a bacteriophage that infects the obligate intracellular pathogen, Chlamydia psittaci strain Guinea Pig Inclusion Conjunctivitis, was observed using transmission electron microscopy of phage-hyperinfected, Chlamydia-infected HeLa cells. Phage attachment to extracellular, metabolically dormant, infectious elementary bodies and cointernalisation are demonstrated. Following entry, phage infection takes place as soon as elementary bodies differentiate into metabolically active reticulate bodies. Phage-infected bacteria follow an altered developmental path whereby cell division is inhibited, producing abnormally large reticulate bodies, termed maxi-reticulate bodies, which do not mature to elementary bodies. These forms eventually lyse late in the chlamydial developmental cycle, releasing abundant phage progeny in the inclusion and, upon lysis of the inclusion membrane, into the cytosol of the host cell. Structural integrity of the hyperinfected HeLa cell is markedly compromised at late stages. Released phage particles attach avidly to the outer leaflet of the outer membranes of lysed and unlysed Chlamydiae at different stages of development, suggesting the presence of specific phage receptors in the outer membrane uniformly during the chlamydial developmental cycle. A mechanism for phage infection is proposed, whereby phage gains access to replicating chlamydiae by attaching to the infectious elementary body, subsequently subverting the chlamydial developmental cycle to its own replicative needs. The implications of phage infection in the context of chlamydial infection and disease are discussed. [TOP OF PAGE]

85. Microvirus of Chlamydia psittaci strain Guinea pig inclusion conjunctivitis: Isolation and molecular characterization. Hsia, R.C., Ting, L.M., Bavoil, P.M. (2000). Microbiology (Reading) 146:1651-1660. The authors report the isolation and molecular characterization of a bacteriophage, phiCPG1, which infects Chlamydia psittaci strain Guinea pig Inclusion Conjunctivitis. Purified virion preparations contained isometric particles of 25 nm diameter, superficially similar to spike-less members of the phiX174 family of bacteriophages. The single-stranded circular DNA genome of phiCPG1 included five large ORFs, which were similar to ORFs in the genome of a previously described Chlamydia bacteriophage (Chp1) that infects avian C. psittaci. Three of the ORFs encoded polypeptides that were similar to those in a phage infecting the mollicute Spiroplasma melliferum, a pathogen of honeybees. Lesser sequence similarities were seen between two ORF products and the major capsid protein of the phiX174 coliphage family and proteins mediating rolling circle replication initiation in phages, phagemids and plasmids. Phage phiCPG1 is the second member of the genus Chlamydiamicrovirus, the first to infect a member of a Chlamydia species infecting mammals. Similarity searches of the nucleotide sequence further revealed a highly conserved (75% identity) 375 base sequence integrated into the genome of the human pathogen Chlamydia pneumoniae. This genomic segment encodes a truncated 113 residue polypeptide, the sequence of which is 72% identical to the amino-terminal end of the putative replication initiation protein of phiCPG1. This finding suggests that C. pneumoniae has been infected by a phage related to phiCPG1 and that infection resulted in integration of some of the phage genome into the C. pneumoniae genome. [TOP OF PAGE]

86. Control of the eel (Anguilla japonica) pathogens, Aeromonas hydrophila and Edwardsiella tarda, by bacteriophages. Hsu, C.H., Lo, C.Y., Liu, J.K., Lin, C.S. (2000). Journal of the Fisheries Society of Taiwan 27:21-31. Aeromonas hydrophila and Edwardsiella tarda are the two major pathogens of the eel, Anguilla japonica. The prevalent method to control the diseases is antibiotics. Long term and large scale application of the drugs results in resistance which makes disease control difficult. In the nature, bacteriophages are an important factor in controling bacterial population. The purpose of this research is to study the capability of the phages to control the pathogens in pond water. Several bacteriophages of A. hydrophila and E. tarda were isolated from the water samples of southern Taiwan. In pure culture, the phages could reduce the host 3 orders of magnitude in 2 hr when the multiplicity of infection (moi) was above 11.5 at 25ºC. In the pond water with added A. hydrophila to 6 X 10⁵ / ml, the number dropped 250 folds at phage moi of 0.23 in 8 hr with accompanying phage multiplication to the level of 106 PFU/ml in the water. Most (85%) of the surviving hosts were still vulnerable to the phage. The resistant strains (15%) appeared to be lysogens since the culture broth of the strains could form phage plaques on A. hydrophila. In the case of E. tarda, the bacteria subsided rapidly even in the absence of phage in 48 hr in the pond water. [TOP OF PAGE]

87. Characterization of Streptococcus thermophilus strains that undergo lysis under unfavourable environmental conditions. Husson-Kao, C., Mengaud, J., Gripon, J.C., Benbadis, L., Chapot-Chartier, M.P. (2000). Int. J. Food Microbiol. 55:209-213. The autolysis of starter lactic acid bacteria appears as a promising way to enhance the flavour of fermented dairy products. The present work was aimed at investigating the autolysis phenomenon in Streptococcus thermophilus, a thermophilic lactic acid bacteria involved in the starters used for the production of yoghurts, Italian and Swiss-type cheeses. Out of 146 strains screened for their aptitude to spontaneously lyse at the end of growth in M17 medium containing lactose in limited concentration, six strains, among which is the type strain CNRZ 1358, were found to be highly autolytic. These autolytic strains are characterized by a typical bell-shaped growth curve. Lysis of the type strain, which was studied as the model, was triggered under unfavourable environmental conditions, such as lactose depletion and NaCl or organic solvents addition. The lysogenic character of this strain was evidenced. Taken together, our results indicate that the autolytic phenotype in S. thermophilus is linked to the lysogenic character but does not result from the massive prophage induction under stressing conditions. [TOP OF PAGE]

88. The Streptococcus thermophilus autolytic phenotype results from a leaky prophage. Husson-Kao, C., Mengaud, J., Cesselin, B., van, S.D., Benbadis, L., Chapot-Chartier, M.P. (2000). Appl. Environ. Microbiol. 66:558-565. Streptococcus thermophilus autolytic strains are characterized by a typical bell-shaped growth curve when grown under appropriate conditions. The cellular mechanisms involved in the triggering of lysis and the bacteriolytic activities of these strains were investigated in this study. Lactose depletion and organic solvents (ethanol, methanol, and chloroform) were shown to trigger a premature and immediate lysis of M17 exponentially growing cells. These factors and compounds are suspected to act by altering the cell envelope properties, causing either the permeabilization (organic solvents) or the depolarization (lactose depletion) of the cytoplasmic membrane. The autolytic character was shown to be associated with lysogeny. Phage particles, most of which were defective, were observed in the culture supernatants after both mitomycin C-induced and spontaneous lysis. By renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a bacteriolytic activity was detected at 31 kDa exclusively in the autolytic strains. This enzyme was detected during both growth and spontaneous lysis with the same intensity. We have shown that it was prophage encoded and homologous to the endolysin Lyt51 of the streptococcal temperate bacteriophage phi01205 (M. Sheehan, E. Stanley, G. F. Fitzgerald, and D. van Sinderen, Appl. Environ. Microbiol. 65:569-577, 1999). It appears from our results that the autolytic properties are conferred to the S. thermophilus strains by a leaky prophage but do not result from massive prophage induction. More specifically, we propose that phagic genes are constitutively expressed in almost all the cells at a low and nonlethal level and that lysis is controlled and achieved by the prophage-encoded lysis proteins. [TOP OF PAGE]

89. Protocol for the manufacture of miniature washed-curd cheeses under controlled microbiological conditions. Hynes, E., Ogier, J.C., Delacroix-Buchet, A. (2000). International Dairy Journal 10:733-737. A protocol for the preparation of miniature washed-curd cheeses under controlled bacteriological conditions was designed and tested for reproducibility. The process was adapted from "Saint-Paulin" technology, and involves inoculation and renneting in autoclaved bottles, and cutting, stirring, curd washing and removal of whey by centrifugation. Pressing was simulated by low-speed centrifugation. All operations were performed using sterile techniques and autoclaved equipment. Forty miniature cheeses (approximately 40 g) were produced over 10 working days, and ripened for 28 days. Gross composition (dry matter, salt-in-moisture and pH) of the one-day-old cheeses did not differ significantly between cheesemaking days, and average values were 45.16, 2.46 and 5.15%, respectively. Adventitious Lactobacillus population remained less than 200 CFU g^-1 all during ripening, and phages were absent. Nitrogen soluble at pH 4.4 and in phosphotungstic acid attained 21 and 3% of total nitrogen, respectively, in 28-day-old cheeses. The proposed model was shown to be suitable for the preparation of miniature cheese specimens for use in microbiological studies of cheese manufacture and ripening. [TOP OF PAGE]

90. Inducible stx2 phages are lysogenized in the enteroaggregative and other phenotypic Escherichia coli O86:HNM isolated from patients. Iyoda, S., Tamura, K., Itoh, K., Izumiya, H., Ueno, N., Nagata, K., Togo, M., Terajima, J., Watanabe, H. (2000). FEMS Microbiol. Let. 191:7-10. We characterized two Shiga toxin-producing Escherichia coli (STEC) O86:HNM isolates from a patient with hemolytic uremic syndrome (HUS) or bloody diarrhea. Both of them did not possess the eaeA gene. However, the isolate from a HUS patient carried genetic markers of enteroaggregative E. coli (EAEC) and showed aggregative adherence pattern to HEp-2 cells. The other isolate from bloody diarrhea, which was negative with EAEC markers, was diffusely adhered to HEp-2 cells. The stx2 gene in both E. coli O86:HNM strains was encoded in each infectious phage, which was partially homologous to that of strain EDL933, a STEC O157:H7. These results will help to explain the genotypic divergences of STEC. [TOP OF PAGE]

91. Ultraviolet radiation effects on bacterioplankton and viruses in marine ecosystems. Jeffrey, W.H., Kase, J.P., Wilhelm, S.W. (2000). pp. 206-236. In In De Mora, S.J. and et al. (eds.), Effects Of UV Radiation On Marine Ecosystems. Cambridge University Press, Cambridge. [TOP OF PAGE]

92. Virus removal and transport in saturated and unsaturated sand columns. Jin, Y., Chu, Y., Li, Y. (2000). Journal of Contaminant Hydrology 43:111-128. The purpose of this research was to determine the role that unsaturated flow conditions play in virus sorption and inactivation during transport through sand columns. Column flow experiments were conducted in Ottawa sand under both saturated and unsaturated flow conditions using two bacteriophages, MS2 and phiX174. Input solution containing bromide (Br-) tracer and the viruses was applied to the column as a step function and samples were collected at the effluent end using a fraction collector. The convection-dispersion equation, partially calibrated with the transport parameters measured from the Br- signal, was used to evaluate the sorption and inactivation characteristics of the viruses. We found that, while removal of both MS2 and phiX174 increased significantly under unsaturated flow conditions, the mechanisms responsible for removing the two viruses seemed to be different. The results from elution experiments using beef extract solution revealed that the increased removal of phiX174 in the Ottawa sand under unsaturated conditions appeared to be caused by increased sorption whereas the increased removal of MS2 was due to inactivation. The difference in virus removal and transport behavior between saturated and unsaturated conditions was likely caused by additional sorption at the solid surfaces and the presence of the air-water interface (AWI) in the unsaturated system. [TOP OF PAGE]

93. The microbial genetics of antibiotic cycling. John, J.F.J., Rice, L.B. (2000). INFECTION CONTROL AND HOSPITAL EPIDEMIOLOGY 21:S22-S31 Cycling of currently available antibiotics to reduce resistance is an attractive concept. For cycling strategies to be successful, their implementation must have a demonstrable impact on the prevalence of resistance determinants already dispersed throughout the hospital and associated healthcare facilities. While antibiotic use in hospitals clearly constitutes a stimulus for the emergence of resistance, it is by no means the only important factor. The incorporation of resistance determinants into potentially stable genetic structures, including bacteriophages, plasmids, transposons, and the more newly discovered movable elements termed integrons and gene cassettes, forces some degree of skepticism about the potential for such strategies in institutions where resistance determinants are already prevalent. In particular, the expanding role of integrons may pose an ultimate threat to formulary manipulations such as cycling. Despite these concerns, the crisis posed by antimicrobial resistance warrants investigation of any strategy with the potential for reducing the prevalence of resistance. Over the next decade, new studies with carefully designed outcomes should determine the utility of antibiotic cycling as one control measure for nosocomial resistance. [TOP OF PAGE]

94. Structures of virus and virus-like particles. Johnson, J.E., Chiu, W. (2000). Current Opinion in Structural Biology 10:229-235. Virus structures continue to be the basis for mechanistic virology and serve as a paradigm for solutions to problems concerning macromolecular assembly and function in general. The use of X-ray crystallography, electron cryomicroscopy and computational and biochemical methods has provided not only details of the structural folds of individual viral components, but also insights into the structural basis of assembly, nucleic acid packaging, particle dynamics and interactions with cellular molecules. [TOP OF PAGE]

95. Bacteriophage infections in the industrial acetone butanol (AB) fermentation process. Jones, D.T., Shirley, M., Wu, X., Keis, S. (2000). J Mol Microbiol Biotechnol 2:21-26. The reported incidence and effects of bacteriophage infections occurring in the industrial acetone butanol (AB) fermentation processes operated in the USA, Japan, and Puerto Rico during the earlier part of the twentieth century is reviewed. The growth characteristics and solvent-producing ability of a lysogenic strain of Clostridium madisonii isolated from a phage infection in Puerto Rico was determined in molasses fermentation medium. The host strain harbours a large lysogenic phage belonging to the Siphoviridae and the growth rate of the lysogenic strain was found to be slower than the non-lysogenic parent strain and exhibited reduced solvent production. The history of phage infections that occurred in the South African AB process is documented along with the various remedial actions that were taken to restore production. A more detailed account of the last phage infection that occurred in 1980 involving a small pseudo-lysogenic phage belonging to the Podoviridae is given. This phage infected Clostridium beijerinckii P260 and a number of closely related industrial strains. Factory-scale fermentations contaminated by this phage were compared with equivalent laboratory-scale control fermentations. The effect of the phage infection in the full-scale and laboratory-scale fermentations were monitored. Results obtained in laboratory-based studies included an assessment of the effect of the multiplicity of infection and the timing of phage infection. The general effects and symptoms of phage infections in the industrial AB fermentation are reviewed including gross changes in the fermentation and changes in cell morphology. Common techniques used for the diagnosis of phage infections and approaches for controlling phage contamination in the AB fermentation are discussed. Prevention strategies included good factory hygiene, sterilisation, decontamination and disinfection, and the use of resistant strains immunised against specific phages. [TOP OF PAGE]

96. Isolation of coliphages specific to enterotoxigenic E. coli (ETEC). Jothikumar, N., Reddy, C.G., Sundari, R.B., Saigopal, D.V. (2000). Journal of Environmental Monitoring 2:372-374. Bacteriophages specific to Enterotoxigenic E. coli (ETEC) are reported for the first time. Out of 15 isolated phages only 10 were specific to strains of ETEC. All ten phages of dsDNA could be grouped into three different genotypes based on their RAPD patterns observed and it is likellly that they belong to only 3 different strains. The three phages yielded clear plaques on 10 strains of ETEC within 4-6 h at 37 degrees C. [TOP OF PAGE]

97. Genomic sequences of bacteriophages HK97 and HK022: Pervasive genetic mosaicism in the lambdoid bacteriophages. Juhala, R.J., Ford, M.E., Duda, R.L., Youlton, A., Hatfull, G.F., Hendrix, R.W. (2000). J. Mol. Biol. 299:27-51. We report the complete genome DNA sequences of HK97 (39,732 bp) and HK022 (40,751 bp), double-stranded DNA bacteriophages of Escherichia coli and members of the lambdoid or lambda-like group of phages. We provide a comparative analysis of these sequences with each other and with two previously determined lambdoid family genome sequences, those of E. coli phage lambda and Salmonella typhimurium phage P22. The comparisons confirm that these phages are genetic mosaics, with mosaic segments separated by sharp transitions in the sequence. The mosaicism provides clear evidence that horizontal exchange of genetic material is a major component of evolution for these viruses. The data suggest a model for evolution in which diversity is generated by a combination of illegitimate and homologous recombination and mutational drift, and selection for function produces a population in which most of the surviving mosaic boundaries are located at gene boundaries or, in some cases, at protein domain boundaries within genes. Comparisons of these genomes highlight a number of differences that allow plausible inferences of specific evolutionary scenarios for some parts of the genome. The comparative analysis also allows some inferences about function of genes or other genetic elements. We give examples for the generalized recombination genes of HK97, HK022 and P22, and for a putative headtail adaptor protein of HK97 and HK022. We also use the comparative approach to identify a new class of genetic elements, the morons, which consist of a protein-coding region flanked by a putative sigma70 promoter and a putative factor-independent transcription terminator, all located between two genes that may be adjacent in a different phage. We argue that morons are autonomous genetic modules that are expressed from the repressed prophage. Sequence composition of the morons implies that they have entered the phages' genomes by horizontal transfer in relatively recent evolutionary time. [TOP OF PAGE]

98. Influence of infected cell growth state on bacteriophage reactivation levels. Kadavy, D.R., Shaffer, J.J., Lott, S.E., Wolf, T.A., Bolton, C.E., Gallimore, W.H., Martin, E.L., Nickerson, K.W., Kokjohn, T.A. (2000). Appl. Environ. Microbiol. 66:5206-5212. Reactivation of UV-C-inactivated Pseudomonas aeruginosa bacteriophages D3C3, F116, G101, and UNL-1 was quantified in host cells infected during the exponential phase, during the stationary phase, and after starvation (1 day, 1 and 5 weeks) under conditions designed to detect dark repair and photoreactivation. Our experiments revealed that while the photoreactivation capacity of stationary-phase or starved cells remained about the same as that of exponential-phase cells, in some cases their capacity to support dark repair of UV-inactivated bacteriophages increased over 10-fold. This enhanced reactivation capacity was correlated with the ca. 30-fold-greater UV-C resistance of P. aeruginosa host cells that were in the stationary phase or exposed to starvation conditions prior to irradiation. The dark repair capacity of P. aeruginosa cells that were infected while they were starved for prolonged periods depended on the bacteriophage examined. For bacteriophage D3C3 this dark repair capacity declined with prolonged starvation, while for bacteriophage G101 the dark repair capacity continued to increase when cells were starved for 24 h or 1 week prior to infection. For G101, the reactivation potentials were 16-, 18-, 10-, and 3-fold at starvation intervals of 1 day, 1 week, 5 weeks, and 1. 5 years, respectively. Exclusive use of exponential-phase cells to quantify bacteriophage reactivation should detect only a fraction of the true phage reactivation potential. [TOP OF PAGE]

99. Photocatalytic inactivation of Lactobacillus PL-1 phages by a thin film of titania. Kakita, Y., Obuchi, E., Nakano, K., Murata, K., Kuroiwa, A., Miake, F., Watanabe, K. (2000). Biocontrol Science 5:73-79. A thin film of titanium dioxide (TiO₂, anatase crystalline form) coated on a glass plate inactivated Lactobacillus casei PL-1 phages suspended in a buffer solution, when the reaction mixture was illuminated with a black-light lamp (maximum wave length, 365 nm) and shaken gently. When the reaction mixture was not illuminated, the TiO₂ film exhibited no phage-inactivating activity. TiO₂ was not photoexcited by white (fluorescent)-light lamp. The degree of phage inactivation was directly proportional to the surface area of the TiO₂ film. The phage inactivation approximately followed first-order reaction kinetics. The phage inactivation by photoexcited TiO₂ film was inhibited by superoxide dismutase and D-mannitol, and accelerated by hydrogen peroxide, indicating that the phage inactivation is due to the active oxygen species generated on the surface of TiO2 film under the black-light illumination. Electron microscopic observation of the negatively-stained preparation revealed that about 43% of the phages treated with photoexcited TiO₂ film were converted into ghost-particles with empty heads. [TOP OF PAGE]

100. Mating in Bacillus thuringiensis can induce plasmid integrative prophage J7W-1. Kanda, K., Takada, Y., Kawasaki, F., Kato, F., Murata, A. (2000). Acta Virol. 44:189-193. Bacillus thuringiensis serovar israelensis, a bacterium which possesses plasmid transfer ability after mating, has been lysogenized by plasmid integrative phage J7W-1. The induction of phage in this J7W-1 lysogen was observed after mating with phage-insensitive strains, such as B. thuringiensis serovar thuringiensis, B. cereus and B. subtilis, as well as the phage-sensitive strain serovar israelensis. The phage induction was not observed after mating with B. thuringiensis strains AF101, serovar dendrolimus and serovar indiana. Because these strains are naturally associated with J7W-1 or its related phage, the data strongly suggest a constitutive expression of the repressor encoded by the prophage in these strains. However, the phage induction was observed in B. thuringiensis serovar aizawai, although it contained the J7W-1 DNA homologous region(s). [TOP OF PAGE]

101. Temperature influences induction of a J7W-1-related phage in Bacillus thuringiensis serovar indiana. Kanda, K., Kayashima, T., Kato, F., Murata, A. (2000). Acta Virol. 44:183-187. Induction of a plasmid-integrative J7W-1-related phage in Bacillus thuringiensis serovar indiana by ethidium bromide was influenced by the temperature at which the host cells were cultured. Under optimal growth conditions, the maximum titer of the phage produced by the serovar indiana reached 1.2 X 10⁶ PFU/ml at 37ºC while at 27ºC it was lower by an order of magnitude (1.3 X 10⁵ PFU/ml). The temperature-sensitive period was estimated to occur early during the phage induction. However, the temperature effect observed with the serovar indiana did not occur with the serovar israelensis. In the latter case, the phage induction was the same at 37ºC or 27ºC. Thus we assume that the temperature sensitive phage induction observed with the serovar indiana as host was not a phenomenon caused by the phage genome but rather by product(s) encoded by certain host gene(s). [TOP OF PAGE]

102. [A method for detection of coliphages in the drinking water]. Kashkarova, G.P., Dorodnikov, A.I. (2000). Gigiena i Sanitariia 66-68. [TOP OF PAGE]

103. Rapid titration of multiple samples of filamentous bacteriophage (M13) on nitrocellulose filters. Koch, J., Breitling, F., Duebel, S. (2000). BioTechniques 29:1196-1202. [TOP OF PAGE]

104. [Effect of chitosan derivatives on the reproduction of Coliphages T2 and T7]. Kochkina, Z.M., Chirkov, S.N. (2000). Mikrobiologiia 69:257-260. The effect of chitosan derivatives with different degrees of polymerization and deamination, as well as of chitosan 6-O-sulfate and chitosan N-succinate-6-O-sulfate, on the reproduction of coliphages T2 and T7 in Escherichia coli and on the growth of this bacterium was studied. Chitosan derivatives decreased the yield of coliphages and exhibited bactericidal activity. The efficiency of inhibition of viral infection and the bactericidal activity of chitosan were found to be dependent on the degree of its polymerization. At the same time, there was no correlation between the degree of chitosan deamination and the extent of inhibition of viral infection. Anionic chitosan derivatives virtually did not possess antiviral or bactericidal activity. It is assumed that chitosan blocks some stages of phage reproduction. The decrease in the phage-producing ability of E. coli may also be due to the bactericidal effect of chitosan. [TOP OF PAGE]

105. [Effect of chitosan derivatives on the development of phage infection in cultured Bacillus thuringiensis]. Kochkina, Z.M., Chirkov, S.N. (2000). Mikrobiologiia 69:266-269. The influence of chitosan fragments with different degrees of polymerization and some chemical chitosan derivatives on the infection of Bacillus thuringiensis by phage 1-97A was studied. It was shown that chitosan inhibits phage infection and inactivates phage particles. The extent of inhibition of phage infection inversely depended on the degree of polymerization of chitosan fragments. On the contrary, the extent of inactivation of phage virulence was proportional to the degree of polymerization. Chitosan derivatives did not inhibit the growth of bacilli. Deaminated chitosan derivatives at a concentration of 100 mg/ml efficiently inhibited phage reproduction, exhibiting no correlation between the degree of deamination and antiviral activity. The anionic derivative chitosan sulfate and N-succinate-6-O-sulfate did not inactivate phage, did not influence bacterial growth, and did not inhibit the process of viral infection. [TOP OF PAGE]

106. Inactivation of coliphages by chitosan derivatives. Kochkina, Z.M., Surgucheva, N.A., Chirkov, S.N. (2000). Mikrobiologiya 69:261-265. The effect of chitosan fragments with different degrees of polymerization and the chemical derivatives of chitosan differing in the number of amino groups and total molecule charge on phages T2, T4, and T7 was studied. The interaction of chitosan with bacteriophage particles inactivated them to the extent dependent on the chemical properties of chitosan and its concentration. Phage T2 was found to be most susceptible to inactivation by chitosan. The polycationic nature of chitosan plays an important role in the inactivation of phages. It is assumed that the abnonnal rearrangement of the basal plate of phages, the loss of long tail fibers, and, probably, modification of the receptor-recognizing phage proteins may be responsible for the inactivation of coliphages by chitosan. [TOP OF PAGE]

107. [Coliphages inactivation using chitosan derivatives]. Kochkina, Z.M., Surgucheva, N.A., Chirkov, S.N. (2000). Mikrobiologiia 69:261-265. The effect of chitosan fragments with different degrees of polymerization and the chemical derivatives of chitosan differing in the number of amino groups and total molecule charge on phages T2, T4, and T7 was studied. The interaction of chitosan with bacteriophage particles inactivated them to the extent dependent on the chemical properties of chitosan and its concentration. Phage T2 was found to be most susceptible to inactivation by chitosan. The polycationic nature of chitosan plays an important role in the inactivation of phages. It is assumed that the abnormal rearrangement of the basal plate of phages, the loss of long tail fibers, and probably, modification of the receptor-recognizing phage proteins may be responsible for the inactivation of coliphages by chitosan. [TOP OF PAGE]

108. Antibacterials that are used as growth promoters in animal husbandry can affect the release of Shiga-toxin-2-converting bacteriophages and Shiga toxin 2 from Escherichia coli strains. Kohler, B., Karch, H., Schmidt, H. (2000). Microbiology 146:1085-1090. Antibiotics are commonly used as growth promoters in animal husbandry worldwide. This practice has been linked to the emergence of particular antibiotic-resistant bacteria, and is now controversial. In this study, the ability of growth-promoting antibiotics to induce Shiga toxin (Stx)-converting bacteriophages from Stx-producing Escherichia coli (STEC) strains was investigated. Subinhibitory concentrations of the antibacterial growth promoters olaquindox, carbadox, tylosin and monensin were used for induction experiments. The amount of mature Stx-converting phage particles released from induced and non-induced cultures was determined, and the production of Stx was simultaneously measured by ELISA. Whereas the quinoxaline-1,4-dioxide-type antibiotics olaquindox and carbadox enhanced the release of Stx-converting phage particles from STEC cells, tylosin and monensin decreased phage induction. The production of Stx increased or decreased simultaneously with the amount of free phages. The results of this study show that particular antibacterial growth promoters can induce Stx phages. In vivo induction of Stx phages from lysogenic STEC may increase the amount of free phages in the intestine and therefore may contribute to the spread of STEC and development of new STEC pathotypes. [TOP OF PAGE]

109. Impact of acid on survival of Vibrio vulnificus and Vibrio vulnificus phage. Koo, J., Depaola, A., Marshall, D.L. (2000). Journal of Food Protection 63:1049-1052. Three strains of Vibrio vulnificus and V. vulnificus phages were tested for acid sensitivity at 21 degrees C. V. vulnificus strain 304 was more resistant to pH 4.0 than strains CVD-1 and A-9, whereas acid sensitivities of V. vulnificus strains at pH 3.0 and 2.0 were similar. V. vulnificus phage strain 110A-7 was more resistant to pH 4.0 than strain 153A-7, whereas acid sensitivities of phage strains at pH 3.5 and 3.0 were similar. Numbers of V. vulnificus and its phage were close to the limit of detection after 100 s at pH 2.0 and after 24 min at pH 3.0. Acid D-values at 21 degrees C decreased as pH decreased for both V. vulnificus and phages. D-values of phage strains at pH 3.5 were 10-fold greater than those of host strain at pH 4.0. D-values of phage strains were slightly greater than those of host strain at pH 3.0. These results suggest that V. vulnificus and its phage were very sensitive to pH of less than 3.0, although V. vulnificus phages were more resistant to acid than their host. [TOP OF PAGE]

110. Effect of simulated gastric fluid and bile on survival of Vibrio vulnificus and Vibrio vulnificus phage. Koo, J., Depaola, A., Marshall, D.L. (2000). Journal of Food Protection 63:1665-1669. Bacteria and phages may be exposed to acid conditions in the stomach and to bile in the intestine. Survival of three strains of Vibrio vulnificus and three strains of its phages was examined at 37ºC after exposure to simulated gastric fluid at pH 3 to 4 or to 0, 1, and 2% bile in broth or buffer. Mean D-values (decimal reduction times) at pH 4 and 3 were 3.3 and 1.3 min for V. vulnificus and 97.8 and 0.7 min for its phages. No V. vulnificus survivors were found at pH 2.0. There were few survival differences among strains of V. vulnificus or its phages. Numbers of V. vulnificus increased 1 log in tryptic soy broth containing 1 or 2% bile after 3 h. Numbers of V. vulnificus and its phages remained constant in phosphate-buffered saline regardless of bile concentrations up to 3 h. Those V. vulnificus bacteria and phages that survive stomach acidity may proliferate in the small intestine, since they are resistant to bile. [TOP OF PAGE]

111. Sequence of the genome of the temperate, serotype-converting, Pseudomonas aeruginosa bacteriophage D3. Kropinski, A.M. (2000). J. Bacteriol. 182:6066-6074. Temperate bacteriophage D3, a member of the virus family Siphoviridae, is responsible for serotype conversion in its host, Pseudomonas aeruginosa. The complete sequence of the double-stranded DNA genome has been determined. The 56,426 bp contains 90 putative open reading frames (ORFs) and four genes specifying tRNAs. The latter are specific for methionine (AUG), glycine (GGA), asparagine (AAC), and threonine (ACA). The tRNAs may function in the translation of certain highly expressed proteins from this relatively AT-rich genome. D3 proteins which exhibited a high degree of sequence similarity to previously characterized phage proteins included the portal, major head, tail, and tail tape measure proteins, endolysin, integrase, helicase, and NinG. The layout of genes was reminiscent of lambdoid phages, with the exception of the placement of the endolysin gene, which parenthetically also lacked a cognate holin. The greatest sequence similarity was found in the morphogenesis genes to coliphages HK022 and HK97. Among the ORFs was discovered the gene encoding the fucosamine O-acetylase, which is in part responsible for the serotype conversion events. [TOP OF PAGE]

112. Morphology of bacteriophages of E. Hammarström's set for typing Shigella sonnei. Krzywy, T., Kucharewicz-Krukowska, A., Slopek, S. (2000). Arch. Immunol. Ther. Exp. (Warsz) 20:73-83. [TOP OF PAGE]

113. Vibrio cholerae O139 bacteriophages. Kudryakova, T.A., Makedonova, L.D., Kachkia, G.V., Sayamov, S.R. (2000). Zhurnal Mikrobiologii Epidemiologii i Immunobiologii 28-30. Cholera bacteriophages have been isolated from 27 lesogenic cultures of V. cholerae O139. As shown the pages under study belog to two morphological groups A1 and F1 and serological types II and XII. The use of prophage typing and the sensitivity test to specific phage made it possible to differetiate V. cholerae strains, serogroup O139. [TOP OF PAGE]

114. Target genes for virulence assessment of Escherichia coli isolates from water, food and the environment. Kuhnert, P., Boerlin, P., Frey, J. (2000). FEMS Microbiol. Rev. 24:107-117. The widespread species Escherichia coli includes a broad variety of different types, ranging from highly pathogenic strains causing worldwide outbreaks of severe disease to avirulent isolates which are part of the normal intestinal flora or which are well characterized and safe laboratory strains. The pathogenicity of a given E. coli strain is mainly determined by specific virulence factors which include adhesins, invasins, toxins and capsule. They are often organized in large genetic blocks either on the chromosome ('pathogenicity islands'), on large plasmids or on phages and can be transmitted horizontally between strains. In this review we summarize the current knowledge of the virulence attributes which determine the pathogenic potential of E. coli strains and the methodology available to assess the virulence of E. coli isolates. We also focus on a recently developed procedure based on a broad-range detection system for E. coli-specific virulence genes that makes it possible to determine the potential pathogenicity and its nature in E. coli strains from various sources. This makes it possible to determine the pathotype of E. coli strains in medical diagnostics, to assess the virulence and health risks of E. coli contaminating water, food and the environment and to study potential reservoirs of virulence genes which might contribute to the emergence of new forms of pathogenic E. coli. [TOP OF PAGE]

115. High-frequency interconversion of turbid and clear plaque strains of bacteriophage f1 and associated host cell death. Kuo, M.Y., Yang, M.K., Chen, W.P., Kuo, T.T. (2000). Canadian Journal of Microbiology 46:841-847. Under normal cultivation conditions, a mixture of turbid and clear plaques is often apparent in cultures of bacterial cells infected with filamentous bacteriophages. Beginning with a culture of wild-type filamentous phage f1, which itself produces turbid plaques, a clear plaque strain (c1) was isolated. From c1, the turbid plaque strain t1 was isolated; from t1, the clear plaque strain c2 was isolated; and from c2, the turbid plaque strain t2 was isolated. Each of these strains was generated with a frequency of approximately 1 X 10-4. Although filamentous phages have been thought not to induce host cell death, both turbid and clear plaque strains of f1 killed host bacteria. Plating of bacterial cells 1 h after infection revealed that colonies produced by cells infected with either wild-type f1 or strain c2 were smaller than those derived from uninfected cells, and that colony formation by infected cells was reduced by 15% and 38%, respectively. The time course of bacterial growth revealed that, at 4 h after infection, the number of CFU per milliliter of culture of cells infected with wild-type f1 or with strain c2 was reduced by 27% and 95%, respectively, compared with that for uninfected cells. Microculture analysis also revealed that the percentages of nondividing cells in f1 or c2 infected were 19% and 52%, respectively, 4 h after infection with wild-type f1 or with strain c2; no such cells were detected in cultures of uninfected cells. Negative staining and electron microscopy showed that 20% and 61% of cells infected with wild-type f1 or with strain c2 were dead 4 h postinfection. Finally, although the rates of DNA synthesis were similar for infected and uninfected cells, the rates of RNA and protein synthesis were markedly reduced in infected cells. [TOP OF PAGE]

116. Multiplex PCR for detection and identification of lactococcal bacteriophages. Labrie, S., Moineau, S. (2000). Appl. Environ. Microbiol. 66:987-994. Three genetically distinct groups of Lactococcus lactis phages are encountered in dairy plants worldwide, namely, the 936, c2, and P335 species. The multiplex PCR method was adapted to detect, in a single reaction, the presence of these species in whey samples or in phage lysates. Three sets of primers, one for each species, were designed based on conserved regions of their genomes. The c2-specific primers were constructed using the major capsid protein gene (mcp) as the target. The mcp sequences for three phages (eb1, Q38, and Q44) were determined and compared with the two available in the databases, those for phages c2 and bIL67. An 86.4% identity was found over the five mcp genes. The gene of the only major structural protein (msp) was selected as a target for the detection of 936-related phages. The msp sequences for three phages (p2, Q7, and Q11) were also established and matched with the available data on phages sk1, bIL170, and F4-1. The comparison of the six msp genes revealed an 82. 2% identity. A high genomic diversity was observed among structural proteins of the P335-like phages suggesting that the classification of lactococcal phages within this species should be revised. Nevertheless, we have identified a common genomic region in 10 P335-like phages isolated from six countries. This region corresponded to orfF17-orf18 of phage r1t and orf20-orf21 of Tuc2009 and was sequenced for three additional P335 phages (Q30, P270, and ul40). An identity of 93.4% within a 739-bp region of the five phages was found. The detection limit of the multiplex PCR method in whey was 10(4) to 10(7) PFU/ml and was 10(3) to 10(5) PFU/ml with an additional phage concentration step. The method can also be used to detect phage DNA in whey powders and may also detect prophage or defective phage in the bacterial genome. [TOP OF PAGE]

117. Genetic analysis of a bacterial genetic exchange element: the gene transfer agent of Rhodobacter capsulatus. Lang, A.S., Beatty, J.T. (2000). Proc. Natl. Acad. Sci. USA 97:859-864. An unusual system of genetic exchange exists in the purple nonsulfur bacterium Rhodobacter capsulatus. DNA transmission is mediated by a small bacteriophage-like particle called the gene transfer agent (GTA) that transfers random 4.5-kb segments of the producing cell's genome to recipient cells, where allelic replacement occurs. This paper presents the results of gene cloning, analysis, and mutagenesis experiments that show that GTA resembles a defective prophage related to bacteriophages from diverse genera of bacteria, which has been adopted by R. capsulatus for genetic exchange. A pair of cellular proteins, CckA and CtrA, appear to constitute part of a sensor kinase/response regulator signaling pathway that is required for expression of GTA structural genes. This signaling pathway controls growth-phase-dependent regulation of GTA gene messages, yielding maximal gene expression in the stationary phase. We suggest that GTA is an ancient prophage remnant that has evolved in concert with the bacterial genome, resulting in a genetic exchange process controlled by the bacterial cell. [TOP OF PAGE]

118. [Variants of Yersinia pestis resistant to a diagnostic bacteriophage and the problems related to them]. Lebedeva, S.A. (2000). Zhurnal Mikrobiologii, Epidemiologii i Immunobiologii 99-104. The data of literature on the pleiotropic variability of the resistance of Y. pestis mutants to diagnostic phage are presented. The conditions of reversion to the initial phenotype are characterized. The mechanisms of the appearance of such variability of Y. pestis, as well as problems arising in connection with this variability and linked with the pathogenic activity of Y. pestis, low effectiveness of the diagnostic methods used in the inspection of the natural foci of plaque, the reservation of microbes in nature during the periods between epidemics, are discussed. [TOP OF PAGE]

119. Bacteriophages as indicators of enteric viruses and public health risk in groundwaters. Leclerc, H., Edberg, S., Pierzo, V., Delattre, J.M. (2000). Journal of Applied Microbiology 88:5-21. Low concentrations of all types of bacteriophages in groundwater limit their power to predict the presence of enteric viruses. There is little concordance in the literature regarding phage detection methods, thus making comparisons extremely difficult. Different authors have used different hosts, phage concentration methods, and end-point determinations. Also, markedly different volumes of sample have been employed, varying from 1 litre to 400 l. Bacteriophage concentration methods are not reproducible. There has been marked variability among groups in the natural substrates used (for example, beef extract), the type of adsorbing filter used, centrifugation instruments and conditions, and the delivery of the concentrate to the host cells. There is no consensus on the best bacterial host strain. Currently, several are employed with each showing differential sensitivities and specificities. In particular, host stability must be considered. Host stability has two components: the ability of the host to continue to be receptive to the bacteriophage after continued sub-culture, and the lack of lysogenic or temperate bacteriophage in the host cell line which may be randomly and unpredictably activated. There is a lack of consistent recovery of bacteriophages from individual faecal specimens. In particular, only approximately 3% of individual humans carry the FRNA phages. While there is some evidence to indicate that the phages multiply in sewage, it is not clear how they do so since the host pili should not be produced at lower temperatures. These ecological factors need to be understood. Of all the phages thus far studied, Bacteroides fragilis HSP40 has the highest recovery rate from individual people. However, Bacteroides, being an anaerobe, is a difficult host for routine laboratory analysis. Methods for the enumeration of F(+)-specific phages and Bacteroides phages are complex, time-consuming, costly and not reproducible. Conversely, somatic coliphage methods are simpler and results can be available in 4-6 h. The occurrence of phages and viruses in groundwater depends on physicochemical characteristics that control their fate and transport in the groundwater/aquifer environment. There are very little actual data taken from the field that allow an understanding of the ecology and life span of phages in their natural environment. Moreover, the ability of phages to serve as a source of food for other microbes needs to be understood. There has been a lack of association of bacteriophage recovery with gastroenteritis outbreaks due to enteric viruses. There is only a small epidemiological database concerning the occurrence of enteric viruses in groundwater. [TOP OF PAGE]

120. Forced retroevolution of an RNA bacteriophage. Licis, N., Balklava, Z., Van, D.J. (2000). Virology 271:298-306. The operator hairpin ahead of the replicase gene in RNA bacteriophage MS2 contains overlapping signals for binding the coat protein and ribosomes. Coat protein binding inhibits further translation of the gene and forms the first step in capsid formation. The hairpin sequence was partially randomized to assess the importance of this structure element for the bacteriophage and to monitor alternative solutions that would evolve on the passaging of mutant phages. The evolutionary reconstruction of the operator failed in the majority of mutants. Instead, a poor imitation developed containing only some of the recognition signals for the coat protein. Three mutants were of particular interest in that they contained double nonsense codons in the lysis reading frame that runs through the operator hairpin. The simultaneous reversion of two stop codons into sense codons has a very low probability of occurring. Therefore the phage solved the problem by deleting the nonsense signals and, in fact, the complete operator, except for the initiation codon of the replicase gene. Several revertants were isolated with activities ranging from 1% to 20% of wild type. The operator, long thought to be a critical regulator, now appears to be a dispensable element. In addition, the results indicate how RNA viruses can be forced to step back to an attenuated form. [TOP OF PAGE]

121. Molecular characterization of a bacteriophage (Chp2) from Chlamydia psittaci. Liu, B.L., Everson, J.S., Fane, B., Giannikopoulou, P., Vretou, E., Lambden, P.R., Clarke, I.N. (2000). J. Virol. 74:3464-3469. Comparisons of the proteome of abortifacient Chlamydia psittaci isolates from sheep by two-dimensional gel electrophoresis identified a novel abundant protein with a molecular mass of 61.4 kDa and an isoelectric point of 6.41. C-terminal sequence analysis of this protein yielded a short peptide sequence that had an identical match to the viral coat protein (VP1) of the avian chlamydiaphage Chp1. Electron microscope studies revealed the presence of a 25-nm-diameter bacteriophage (Chp2) with no apparent spike structures. Thin sections of chlamydia-infected cells showed that Chp2 particles were located to membranous structures surrounding reticulate bodies (RBs), suggesting that Chp2 is cytopathic for ovine C. psittaci RBs. Chp2 double-stranded circular replicative-form DNA was purified and used as a template for DNA sequence analysis. The Chp2 genome is 4,567 bp and encodes up to eight open reading frames (ORFs); it is similar in overall organization to the Chp1 genome. Seven of the ORFs (1 to 5, 7, and 8) have sequence homologies with Chp1. However, ORF 6 has a different spatial location and no cognate partner within the Chp1 genome. Chlamydiaphages have three viral structural proteins, VP1, VP2, and VP3, encoded by ORFs 1 to 3, respectively. Amino acid residues in the phiX174 procapsid known to mediate interactions between the viral coat protein and internal scaffolding proteins are conserved in the Chp2 VP1 and VP3 proteins. We suggest that VP3 performs a scaffolding-like function but has evolved into a structural protein. [TOP OF PAGE]

122. Complete nucleotide sequence, molecular analysis and genome structure of Listeria monocytogenes bacteriophage A118: implications for phage evolution. Loessner, M.J., Inman, R.B., Lauer, P., Calendar, R. (2000). Molecular Microbiology 35:324-340? [TOP OF PAGE]

123. Rapid in vivo evolution of a beta-lactamase using phagemids. Long-McGie, J., Liu, A.D., Schellenberger, V. (2000). Biotechnology and Bioengineering 68:121-125. RNA viruses are capable of undergoing extremely rapid evolution due to their high rates of reproduction, small genome size, and a high frequency of spontaneous mutagenesis. Here we demonstrate that a virus-like, evolutionary state can be created by propagating a phagemid population in a hypermutator strain of Escherichia coli in the presence of a helper phage. This enables one to subject individual phagemid-encoded genes to rapid in vivo evolution. We applied this approach to TEM-1 beta-lactamase which confers resistance to 0.05 mg/L of the antibiotic cefotaxime. After 3 weeks of in vivo evolution we were able to isolate a double mutant, E104K/G238S, of the enzyme which confers a 500-fold increased level of resistance to cefotaxime compared to the starting enzyme. In two independent experiments we obtained a triple mutant, E104K/G238S/T263M, which confers a 1000-fold increase in resistance compared to the wild type enzyme. The same three mutations have been previously observed in TEM-4 beta-lactamase which was discovered in a highly cefotaxime-resistant clinical isolate. The probability of randomly obtaining a beta-lactamase carrying three identical point mutations is less than 10(-10). This indicates that phagemid evolution can rapidly reproduce evolution occurring in nature. [TOP OF PAGE]

124. Broad-range bacteriophage resistance in Streptococcus thermophilus by insertional mutagenesis. Lucchini, S., Sidoti, J., Brussow, H. (2000). Virology 275:267-277. Streptococcus thermophilus is a lactic acid bacterium used in industrial milk fermentation. To obtain phage-resistant starters, S. thermophilus strain Sfi1 was submitted to mutagenesis with the thermolabile insertional vector pG+host9:ISS1 followed by a challenge with the lytic S. thermophilus phage Sfi19. Vector insertions into four distinct sites led to a phage-resistance phenotype. Three mutants were characterized further. They were protected against the homologous challenging phage and 14 heterologous phages. All three mutants adsorbed phages. No intracellular phage DNA synthesis was observed in mutants R7 and R71, while mutant R24 showed a delayed and diminished phage DNA synthesis compared to the parental Sfi1 strain. In mutant R7 a short deletion occurred next to the insertion site which removed the upstream sequences and the 15 initial codons from orf 394, encoding a likely transmembrane protein. Analogy with other phage systems suggests an involvement of this protein in the phage DNA injection process. In mutant R24 the vector was inserted into orf 269 predicting an oxido-reductase. When the vector sequence was removed via homologous recombination across the duplicated insertion elements, mutant R24 returned to the phage susceptibility of the parental strain. This observation suggested that inactivation of orf 269 was not crucial for the resistance phenotype. A gene encoding a likely restriction subunit of a type 1 restriction-modification system was located directly downstream of the insertion site in mutant R24. hsdM and hsdS gene encoding the modification and specificity subunits of a type 1 R-M system and biological evidence for an active R-M system were detected in strain Sfi1, suggesting involvement of a type 1 R-M system in the resistance phenotype of R24. [TOP OF PAGE]

125. Influence of salts on virus adsorption to microporous filters. Lukasik, J., Scott, T.M., Andryshak, D., Farrah, S.R. (2000). Appl. Environ. Microbiol. 66:2914-2920. We investigated the direct and indirect effects of mono-, di-, and trivalent salts (NaCl, MgCl(2), and AlCl(3)) on the adsorption of several viruses (MS2, PRD-1, phiX174, and poliovirus 1) to microporous filters at different pH values. The filters studied included Millipore HA (nitrocellulose), Filterite (fiberglass), Whatman (cellulose), and 1MDS (charged-modified fiber) filters. Each of these filters except the Whatman cellulose filters has been used in virus removal and recovery procedures. The direct effects of added salts were considered to be the effects associated with the presence of the soluble salts. The indirect effects of the added salts were considered to be (i) changes in the pH values of solutions and (ii) the formation of insoluble precipitates that could adsorb viruses and be removed by filtration. When direct effects alone were considered, the salts used in this study promoted virus adsorption, interfered with virus adsorption, or had little or no effect on virus adsorption, depending on the filter, the virus, and the salt. Although we were able to confirm previous reports that the addition of aluminum chloride to water enhances virus adsorption to microporous filters, we found that the enhanced adsorption was associated with indirect effects rather than direct effects. The increase in viral adsorption observed when aluminum chloride was added to water was related to the decrease in the pH of the water. Similar results could be obtained by adding HCl. The increased adsorption of viruses in water at pH 7 following addition of aluminum chloride was probably due to flocculation of aluminum, since removal of flocs by filtration greatly reduced the enhancement observed. The only direct effect of aluminum chloride on virus adsorption that we observed was interference with adsorption to microporous filters. Under conditions under which hydrophobic interactions were minimal, aluminum chloride interfered with virus adsorption to Millipore, Filterite, and 1MDS filters. In most cases, less than 10% of the viruses adsorbed to filters in the presence of a multivalent salt and a compound that interfered with hydrophobic interactions (0.1% Tween 80 or 4 M urea). [TOP OF PAGE]

126. The life cycles of the temperate lactococcal bacteriophage phiLC3 monitored by a quantitative PCR method. Lunde, M., Blatny, J.M., Kaper, F., Nes, I.F., Lillehaug, D. (2000). FEMS Microbiol. Let. 192:119-124. We present here a new and general approach for monitoring the life cycles of temperate bacteriophages which establish lysogeny by inserting their genomes site-specifically into the bacterial host chromosome. The method is based on quantitative amplification of specific DNA sites involved in various cut-and-join events during the life cycles of the phages (i.e. the cos, attP, attB, attL and attR sites) with the use of sequence-specific primers. By comparing the amounts of these specific DNA sites at different intervals, we were able to follow the development of the lytic and lysogenic life cycles of the temperate lactococcal bacteriophage phiLC3 after infection of its bacterial host Lactococcus lactis ssp. cremoris IMN-C18. [TOP OF PAGE]

127. Biological significance of lysogenic conversion. Masuda, S. (2000). Jikeikai Medical Journal 47:129-130. [TOP OF PAGE]

128. Distribution and evolution of bacteriophage WO in Wolbachia, the endosymbiont causing sexual alterations in arthropods. Masui, S., Kamoda, S., Sasaki, T., Ishikawa, H. (2000). J. Mol. Evol. 51:491-497. Wolbachia are obligatory intracellular and maternally inherited bacteria, known to infect many species of arthropod. In this study, we discovered a bacteriophage-like genetic element in Wolbachia, which was tentatively named bacteriophage WO. The phylogenetic tree based on phage WO genes of several Wolbachia strains was not congruent with that based on chromosomal genes of the same strains, suggesting that phage WO was active and horizontally transmitted among various Wolbachia strains. All the strains of Wolbachia used in this study were infected with phage WO. Although the phage genome contained genes of diverse origins, the average G+C content and codon usage of these genes were quite similar to those of a chromosomal gene of Wolbachia. These results raised the possibility that phage WO has been associated with Wolbachia for a very long time, conferring some benefit to its hosts. The evolution and possible roles of phage WO in various reproductive alterations of insects caused by Wolbachia are discussed. [TOP OF PAGE]

129. Characterization of a novel Vibrio parahaemolyticus phage, KVP241, and its relatives frequently isolated from seawater. Matsuzaki, S., Inoue, T., Tanaka, S., Koga, T., Kuroda, M., Kimura, S., Imai, S. (2000). Microbiology and Immunology 44:953-956. A vibriophage, KVP241, and six of its relatives were isolated independently from seawater using Vibrio parahaemolyticus as the host. All of the phages had the same morphology (a hexagonal head and a tail with a contractile sheath) and the same host range (specific for some V. parahaemolyticus strains). DNA-DNA hybridization experiments elucidated that their genomes are highly homologous to each other. Analyses of amino acid sequences of putative major capsid proteins indicated that KVP241 may be weakly related to T4-type phages having a more elongated head. [TOP OF PAGE]

130. Bacterial growth rate and marine virus–host dynamics. Middelboe, M. (2000). Microb. Ecol. 40:114-124. The dynamics of a marine virus–host system were investigated at different steady state growth rates in chemostat cultures and the data were analyzed using a simple model. The virus–host interactions showed strong dependence on host cell growth rate. The duration of the infection cycle and the virus burst size were found to depend on bacterial growth rate, and the rate of cell lysis and virus production were positively correlated with steady state growth rate in the cultures (r ² > 0.96, p < 0.05). At bacterial growth rates of 0.02 to 0.10 h^-1 in the chemostats the virus burst size increased from 12 ± 4 to 56 ± 4, and the latent period decreased from 2.0 to 1.7 h. Resistant clones of the host strain were present in the cultures from the beginning of the experiment and replaced the sensitive host cells following viral lysis in the cultures. Regrowth of resistant cells correlated significantly (r ² = 1.000, p < 0.02) with the lysis rate of sensitive cells, indicating that release of viral lysates stimulated growth of the non-infected, resistant cells. The constructed model was suitable for simulating the observed dynamics of the sensitive host cells, viruses and resistant clones in the cultures. The model was therefore used in an attempt to predict the dynamics of this virus–host interaction in a natural marine environment during a certain set of growth conditions. The simulation indicated that a steady state relationship between the specific viruses and sensitive and resistant bacterial clones may occur at densities that are reasonable to assume for natural environments. The study demonstrates that basic characterization and modeling of specific virus–host interactions may improve our understanding of the behavior of bacteria and viruses in natural systems. [TOP OF PAGE]

131. Diminishing returns of population size in the rate of RNA virus adaptation. Miralles, R., Moya, A., Elena, S.F. (2000). J. Virol. 74:3566-3571. Whenever an asexual viral population evolves by adapting to new environmental conditions, beneficial mutations, the ultimate cause of adaptation, are randomly produced and then fixed in the population. The larger the population size and the higher the mutation rate, the more beneficial mutations can be produced per unit time, With the usually high mutation rate of RNA viruses and in a large enough population, several beneficial mutations could arise at the same time but in different genetic backgrounds, and if the virus is asexual, they will never be brought together through recombination, Thus, the best of these genotypes must outcompete each other on their way to fixation, This competition among beneficial mutations has the effect of slowing the overall rate of adaptation. This phenomenon is known as clonal interference. Clonal interference predicts a speed limit for adaptation as the population size increases. In the present report, by varying the size of evolving vesicular stomatitis virus populations, we found evidence clearly demonstrating this speed limit and thus indicating that clonal interference might be an important factor modulating the rate of adaptation to an in vitro cell system. Several evolutionary and epidemiological implications of the clonal interference model applied to RNA viruses are discussed. [TOP OF PAGE]

132. Properties of natural interspecific hybrids of transposable phages of Pseudomonas aeruginosa: specific characteristics of phage PL24 transposition. Mit'kina, L.N., Krylov, V.N. (2000). Genetika 36:1330-1339. Properties of natural hybrid transposable phages (TP) of Pseudomonas aeruginosa, including phage PL24 and lysogens for this phage, were studied. PL24 possesses the properties of TP from two previously described groups, B3 and D3112. Its genome, unlike the genome of D3112, contains many sites susceptible to the SalGI restriction endonuclease and possesses no more than 100 nucleotides of bacterial origin located at the left genome end. However, unlike B3, phage PL24 failed to induce auxotrophic mutants upon integration in the bacterial genome. This phage differed from both B3 and D3112 in sensitivity to chloroform treatment. A more detailed examination of a group containing 25 randomly isolated lysogens for phage PL24 revealed previously unknown processes occurring at early stages of bacterial lysogenization. There are at least two different modes of cell lysogenization with phage PL24. In the first case, the emerging lysogens contained a single prophage genome located (in each lysogen) at individual sites. In the second case, polylysogenic bacteria appeared, and, after primary integration of a phage genome, replicative transposition occurred at new sites (often accompanied by the appearance of prophage clusters at these sites). The choice of the mode of lysogenization can be determined both by differences in the physiological state of bacteria and by specific features of phage PL24, which possibly affect the time of repressor accumulation to the concentration sufficient for blocking phage growth or the stability of the lysogenic state. [TOP OF PAGE]

133. Conversion of Vibrio eltor MAK757 to classical biotype: role of phage PS166. Mitra, S.N., Mukhopadhyay, R., Ghosh, A.N., Ghosh, R.K. (2000). Virology 273:36-43. Temperate phage PS166 infection of Vibrio eltor MAK757 resulted in complete changes in all biotype-specific determinants. About 10% of the PS166 lysogens of MAK757 lost their eltor-specific determinants, namely, the ability to produce soluble hemolysin, cell-associated hemagglutinin for chicken erythrocytes, and resistance to polymyxin B, as well as resistance to Mukherjee's group IV phage and sensitivity to eltor phage e4. These lysogens were found to have acquired the properties of classical strains, most significantly becoming sensitive to group IV phage but resistant to eltor-specific e4. The remainder of these lysogens, however, retained their parental biotype and serotype but acquired auxotrophy for glycine and histidine. The differential behavior of the two types of lysogen was due to the integration of the phage PS166 genome at different locations in the host chromosome. A 800-bp BglII fragment was found to contain the attP site. Phage PS166 has a polyhedral head (95 nm in diameter) and a contractile tail (98 nm in length). The phage chromosome is a linear double-stranded DNA of 110 kb and a G + C content of 58.7%. Copyright 2000 Academic Press. [TOP OF PAGE]

134. Multiple independent horizontal transfers of informational genes from bacteria to plasmids and phages: implications for the origin of bacterial replication machinery. Moreira, D. (2000). Molecular Microbiology 35:1-5. In contrast to the universality of other central genetic mechanisms, the replication machinery of Bacteria is clearly different from those of Archaea and Eukaryotes. A large number of bacterial genes involved in DNA replication can also be found in plasmids and phages. Based on this, it has been recently proposed that the ancestral bacterial genes were displaced by non-orthologous replication genes from plasmids and phages, which would explain the profound difference between Bacteria and the other domains of life. The alternative hypothesis is that these DNA replication genes have been frequently transferred from bacterial hosts to the genomes of their plasmids and phages. The phylogenetic analysis of the bacterial DNA replication proteins most abundant in databases (replicative helicase DnaB, single-strand binding protein Ssb and topoisomerase TopB) presented here supports the latter hypothesis. Each protein tree shows that sequences from plasmids and phages branch close to their bacterial-specific hosts, suggesting multiple independent horizontal transfers. Therefore, there is no evidence so far for non-orthologous gene displacement of these genes. [TOP OF PAGE]

135. Evolution of microbial pathogens. Morschhaeuser, J., Koehler, G., Ziebuhr, W., Blum-Oehler, G., Dobrindt, U., Hacker, J. (2000). Philosophical Transactions of the Royal Society of London B Biological Sciences 355:695-704. Various genetic mechanisms including point mutations, genetic rearrangements and lateral gene transfer processes contribute to the evolution of microbes. Long-term processes leading to the development of new species or subspecies are termed macroevolution, and short-term developments, which occur during days or weeks, are considered as microevolution. Both processes, macro- and microevolution need horizontal gene transfer, which is particularly important for the development of pathogenic microorganisms. Plasmids, bacteriophages and so-called pathogenicity islands (PAIs) play a crucial role in the evolution of pathogens. During microevolution, genome variability of pathogenic microbes leads to new phenotypes, which play an important role in the acute development of an infectious disease. Infections due to Staphylococcus epidermidis, Candida albicans and Escherichia coli will be described with special emphasis on processes of microevolution. In contrast, the development of PAIs is a process involved in macroevolution. PAIs are especially important in processes leading to new pathotypes or even species. In this review, particular attention will be given to the fact that the evolution of pathogenic microbes can be considered as a specific example for microbial evolution in general. [TOP OF PAGE]

136. Characterization of the DNA replication module of bacteriophage A2 and use of its origin of replication as a defense against infection during milk fermentation by Lactobacillus casei. Moscoso, M., Suarez, J.E. (2000). Virology 273:101-111. Adjacent to the lysis/lysogeny cassette of the A2 phage genome lies a stretch of over 8 kb, which contains a series of genes probably involved in DNA replication. Fifteen open reading frames (orfs) were identified, 13 of which are encoded on the main coding strand and only two on the complementary strand. Database searches and comparative analyses allowed the identification of an open reading frame (orf455) that shows similarity with DNA helicases and contains a variant zinc-finger motif known from the phage T7 helicase/primase. Orf770 showed similarity to putative plasmid and phage DNA primases. Downstream of orf770 is a noncoding 258-bp region rich in direct and inverted repeats, which specifically binds to proteins whose synthesis is induced during phage infection. When present in a plasmid, this region can direct a partial bacteriophage resistance phenotype due to interference with phage DNA replication, both under laboratory conditions and during milk fermentation. It is deduced that this stretch contains the origin of replication of phage A2. [TOP OF PAGE]

137. The evolution of RNA viruses: A population genetics view. Moya, A., Elena, S.F., Bracho, A., Miralles, R., Barrio, E. (2000). Proc. Natl. Acad. Sci. USA 97:6967-6973. RNA viruses are excellent experimental models for studying evolution under the theoretical framework of population genetics. For a proper justification of this thesis we have introduced some properties of RNA viruses that are relevant for studying evolution. On the other hand, population genetics is a reductionistic theory of evolution. It does not consider or make simplistic assumptions on the transformation laws within and between genotypic and phenotypic spaces. However, such laws are minimized in the case of RNA viruses because the phenotypic space maps onto the genotypic space in a much more linear way than on higher DNA-based organisms. Under experimental conditions, we have tested the role of deleterious and beneficial mutations in the degree of adaptation of vesicular stomatitis virus (VsV). a nonsegmented virus of negative strand. We also have studied how effective population size, initial genetic variability in populations, and environmental heterogeneity shapes the impact of mutations in the evolution of vesicular stomatitis virus. Finally, in an integrative attempt, we discuss pros and cons of the quasispecies theory compared with classic population genetics models for haploid organisms to explain the evolution of RNA viruses. [TOP OF PAGE]

138. Viral contamination of shellfish: Evaluation of methods and analysis of bacteriophages and human viruses. Muniain-Mujika, I., Girones, R., Lucena, F. (2000). Journal of Virological Methods 89:109-118. Viral outbreaks attributed to the consumption of contaminated shellfish have been clearly demonstrated. Thirty-five samples of mussels collected from areas with two different levels of faecal pollution were analysed for somatic coliphages, F-RNA phages and bacteriophages infecting Bacteroides fragilis HSP40 and RYC2056 following standardised protocols, and for enterovirus, human adenovirus and hepatitis A virus by nucleic acid amplification (Nested-PCR and RT-PCR). Four methods for viral recovery from shellfish have been compared. The first method is based on the borate buffer at pH 9.5 as eluent, the second is based on glycine buffer at pH 10 as eluent, a third method is based on glycine buffer at pH 7.5 and changes in conductivity and the fourth method on nutritive broth with Tween 80 as eluent. The results obtained were analysed statistically and the method based in glycine buffer at pH 10 seems to be the most efficient and useful for the recovery of phages and human viruses. The results also show a different pattern in the proportions between the viral parameters when the source of the faecal pollution is close to or distant from the shellfish growing area. [TOP OF PAGE]

139. Characterization of a shiga toxin 2e-converting bacteriophage from an Escherichia coli strain of human origin. Muniesa, M., Recktenwald, J., Bielaszewska, M., Karch, H., Schmidt, H. (2000). Infect. Immun. 68:4850-4855. An infectious Shiga toxin (Stx) 2e-converting bacteriophage (phiP27) was isolated from Stx2e-producing Escherichia coli ONT:H(-) isolate 2771/97 originating from a patient with diarrhea. The phage could be transduced to E. coli laboratory strain DH5alpha, and we could show that lysogens were able to produce biologically active toxin in a recA-dependent manner. By DNA sequence analysis of a 6,388-bp HindIII restriction fragment of phiP27, we demonstrated that the stx(2e) gene was located directly downstream of ileZ and argO tRNA genes. Although no analogue of an antiterminator Q encoding gene was present on this fragment, a lysis cassette comprising two holin genes which are related to the holin genes of Pseudomonas aeruginosa phage phiCTX and a gene homologous to the endolysin gene gp19 of phage PS3 were detected. The results of our study demonstrated for the first time that Stx2e can be encoded in the genome of an infectious bacteriophage. [TOP OF PAGE]

140. Occurrence of phages infecting Escherichia coli O157:H7 carrying the Stx 2 gene in sewage from different countries. Muniesa, M., Jofre, J. (2000). FEMS Microbiol. Let. 183:197-200. Shiga toxin-converting bacteriophages are involved in the pathogenicity of some enteric bacteria, such as Escherichia coli O157:H7. Recent studies have demonstrated a relatively high presence of Shiga toxin 2 phages in sewage from Spain, but no data on sewage from other areas were available. In order to evaluate the presence of such phages in sewage from diverse geographical origins, 33 sewage samples, including samples from eight different European countries as well as from New Zealand and South Africa were analysed. Using an experimental approach based on the detection of Stx 2 gene by a phage enrichment culture followed by PCR, bacteriophages infecting E. coli O157:H7 carrying the Shiga toxin 2 gene were detected in 15 of the samples studied. Results presented here show that the presence of phages carrying the Stx 2 gene is common in sewage from developed countries. [TOP OF PAGE]

141. Molecular characterization and allelic distribution of the phage-mediated hyaluronidase genes hylP and hylP2 among Group A streptococci from western Norway. Mylvaganam, H., Bjorvatn, B., Hofstad, T., Osland, A. (2000). Microbial Pathogenesis 29:145-153. Forty-two isolates of group A streptococcus from patients with invasive and non-invasive diseases in western Norway, belonging to the emm sequence types emml, emm3, emm6, emm22, emm28, emm75 and emm78 were screened by PCR for the phage-mediated hyaluronidase genes hylP and hylP2. The amplified genes were characterized by nucleotide sequencing and/or by PCR-RFLP, with the objective of looking for possible associations between alleles of these two genes and invasiveness. The hylP was amplified from all isolates and two main alleles were found: hylP-emm3 in all emm3 isolates and hylP-emm6A in all emm6 isolates, the latter possibly generated by an intergenic recombination between hylP and hylP2. The isolates of the other sequence types had either of these two alleles, or both. Only 27 isolates gave amplicons of the appropriate size with the primers targeting hylP2. Sequencing of these amplicons showed two main types: one was similar to the published hylP2 and the other (hylP-emm6B) was probably a variant of hylP. PCR-RFLP revealed the presence of both hylP-emm6B and hylP2 in at least six of the emm6 isolates. The alleles of both hylP and hylP2 seemed to have emm sequence type preferences. No association between invasiveness and specific phage-mediated hyaluronidase genes/alleles or the production of extracellular hyaluronidase was observed. [TOP OF PAGE]

142. The R-type pyocin of Pseudomonas aeruginosa is related to P2 phage, and the F-type is related to lambda phage. Nakayama, K., Takashima, K., Ishihara, H., Shinomiya, T., Kageyama, M., Kanaya, S., Ohnishi, M., Murata, T., Mori, H., Hayashi, T. (2000). Molecular Microbiology 38:213-231. Pseudomonas aeruginosa produces three types of bacteriocins: R-, F- and S-type pyocins. The S-type pyocin is a colicin-like protein, whereas the R-type pyocin resembles a contractile but non-flexible tail structure of bacteriophage, and the F-type a flexible but non-contractile one. As genetically related phages exist for each type, these pyocins have been thought to be variations of defective phage. In the present study, the nucleotide sequence of R2 pyocin genes, along with those for F2 pyocin, which are located downstream of the R2 gene cluster on the chromosome of P. aeruginosa PAO1, was analysed in order to elucidate the relationship between the pyocins and bacteriophages. The results clearly demonstrated that the R-type pyocin is derived from a common ancestral origin with P2 phage and the F-type from lambda phage. This notion was supported by identification of a lysis gene cassette similar to those for bacteriophages. The gene organization of the R2 and F2 pyocin gene cluster, however, suggested that both pyocins are not simple defective phages, but are phage tails that have been evolutionarily specialized as bacteriocins. A systematic polymerase chain reaction (PCR) analysis of P. aeruginosa strains that produce various subtypes of R and F pyocins revealed that the genes for every subtype are located between trpE and trpG in the same or very similar gene organization as for R2 and F2 pyocins, but with alterations in genes that determine the receptor specificity. [TOP OF PAGE]

143. Microbiological survey of shellfish. Nanni, H., Bronzetti, L., Fabio, G., Pupillo, M., Quaglio, P. (2000). Igiene Moderna 114:113-127. Raw and partially cooked molluscan shellfish appear to be a common vehicle for trasmission of foodborne bacterial and viral infections. Coliphages have been proposed as indicators of viral contamination owing to their biological characteristics which are similar to those of enteroviruses. The presence of standard indicators of faecal pollution (faecal coliforms, Escherichia coli, Salmonella), Vibrio parahaemolyticus and coliphages was tested in 264 shellfish samples (87 Mytilus galloprovincialis, 177 Tapes philippinarum) harvested in sea water. The search for coliphages was carried out with "the plaque count agar" method. V. parahaemolyticus was never detected. Two strains belonging to Salmonella (S. typhimurium and S. napoli) were isolated from clams; two strains of S. tennessee were isolated from mussels. We found high levels of coliphages during winter and autumn but much lower levels during dry weather. Clam samples appeared the most contaminated ones. Furthemore, we did not observe any relationship between the presence of enterobacteria commonly considered as faecal contamination and coliphages. [TOP OF PAGE]

144. A Filamentous Phage Associated with Recent Pandemic Vibrio parahaemolyticus O3:K6 Strains. Nasu, H., Iida, T., Sugahara, T., Yamaichi, Y., Park, K.-S., Yokoyama, K., Makino, K., Shinagawa, H., Honda, T. (2000). Journal of Clinical Microbiology 38:2156-2161. [TOP OF PAGE]

145. Phytoremediation of domestic wastewater for reducing populations of Escherichia coli and MS-2 coliphage. Neralla, S., Weaver, R.W. (2000). Environmental Technology 21:691-698. Sub-surfaceflow constructed wetlands enhance water quality of domestic wastewater by decreasing biochemical oxygen demand and the survival of enteric pathogens. Wetlands contain plants for aesthetic reasons and for phytoremediation. Glasshouse experiments were conducted during February and May to evaluate the role of four aquatic plant species, Cyperus alternifolius, Cyperus isocladus, Typha latifolia, and Iris sp. in reducing the populations of E. coli and MS-2 coliphage, indicators of bacterial and viral pathogens, respectively over 2-3 days retention. Plants were grown in 5-1 buckets containing washed gravel and domestic wastewater. Phytoremediation reduced populations of E. coli and MS-2 coliphage in February, but not in May. In February, plants reduced populations of E. coli by approximately 1.5 log 100ml-1 in 2 d, compared to 0.2 log 100 ml-1 reduction by the control without plants. In February, C. alternifolius, C. isocladus and T. latifolia decreased populations of MS-2 coliphage by approximately 1.5 log ml^-1 in 3 d compared to 0.4-log ml^-1 reduction by the control without plants and Iris sp. During May, C. isocladus reduced E. coli populations from 4.5 log 100 ml^-1 to 1.85 log 100 ml^-1 in 2 d while populations in other treatments remained approximately 0.5 log 100 ml^-1 higher. Survival of E. coli and MS-2 in May was confounded by high temperature in microcosms without plants because of lack of shading, which led to poorer survival of the indicators. Phytoremediation may be one mechanism through which populations of enteric microorganisms are reduced in wetlands and may be enhanced by selection of plants for the wetland. [TOP OF PAGE]

146. Prokaryotic gene therapy to combat multidrug resistant bacterial infection [editorial]. Norris, J.S., Westwater, C., Schofield, D. (2000). Gene Therapy 7:723-725. [TOP OF PAGE]

147. Investigation of the relationship between lysogeny and lysis of Lactococcus lactis in cheese using prophage-targeted PCR. O'Sullivan, D., Ross, R.P., Fitzgerald, G.F., Coffey, A. (2000). Appl. Environ. Microbiol. 66:2192-2198. The ability of lactococcal strains to lyse (and release intracellular enzymes) during cheese manufacture can be a very desirable trait and has been associated with improvement of flavor and acceleration of cheese ripening. Using a laboratory-scale cheese manufacturing assay, the autocatalytic behavior of 31 strains of Lactococcus lactis was assessed. In general, marked variation was observed between strains with a 20-fold difference between best and worst lysing strains based on the release of intracellular enzyme lactate dehydrogenase. In a parallel experiment, the genomes of these strains were examined for the presence of prophage integrase (int) sequences by using concerved primer sequences from known lysogenic phage. Results demonstrated that the lytic behavior of lactococcal starter strains significantly correlates with the presence of prophage sequences. These results highlight not only the contribution of prophage to starter cell lysis but also the potential of PCR as a useful screen to assess strains for this important industrial trait. [TOP OF PAGE]

148. A new bacteriophage, VHML, isolated from a toxin-producing strain of Vibrio harveyi in tropical Australia. Oakey, H.J., Owens, L. (2000). Journal of Applied Microbiology 89:702-709. Some strains of Vibrio harveyi are known to be pathogenic for fish and many invertebrates including crustaceans. Despite their importance, their modes of virulence have yet to be fully elucidated. Here, we present a previously unreported bacteriophage extracted from a toxin-producing strain of V. harveyi isolated from moribund prawn larvae in tropical Australia. Classification into the family Myoviridae was based upon morphological characteristics (an icosahedral head, a neck/collar region and a sheathed rigid tail) and nucleic acid characteristics (double-stranded linear DNA). We have termed the bacteriophage VHML (Vibrio Harveyi Myovirus Like). VHML is a temperate bacteriophage that has a narrow host range and shows an apparent preference for V. harveyi above other vibrios (63 Vibrio isolates tested) and other genera (10 other genera were tested). The conventional methods for phage concentration and extraction of nucleic acids from phage particles were not efficient and the alternative methods that were used are discussed. [TOP OF PAGE]

149. Independent contrasts succeed where ancestor reconstruction fails in a known bacteriophage phylogeny. Oakley, T.H., Cunningham, C.W. (2000). Evolution Int J Org Evolution 54:397-405. Methods of ancestor reconstruction are important tools for evolutionary inference that are difficult to test empirically because ancestral states are rarely known with certainty. We evaluated reconstruction methods for continuous phenotypic characters using taxa from an experimentally generated bacteriophage phylogeny. Except for one slowly evolving character, the estimated ancestral states of continuous phenotypic characters were highly inaccurate and biased, even when including a known ancestor at the root. This error was caused by a directional trend in character evolution and by rapid rates of character evolution. Computer simulations confirmed that such factors affect reconstruction of continuous characters in general. We also used phenotypic viral characters to evaluate two methods that attempt to estimate the correlation between characters during evolution. Whereas a nonphylogenetic regression was relatively inaccurate and biased, independent contrasts accurately estimated the correlation between characters with little bias. [TOP OF PAGE]

150. Lateral gene transfer and the nature of bacterial innovation. Ochman, H., Lawrence, J.G., Groisman, E.A. (2000). Nature 405:299-304. Unlike eukaryotes, which evolve principally through the modification of existing genetic information, bacteria have obtained a significant proportion of their genetic diversity through the acquisition of sequences from distantly related organisms. Horizontal gene transfer produces extremely dynamic genomes in which substantial amounts of DNA are introduced into and deleted from the chromosome. These lateral transfers have effectively changed the ecological and pathogenic character of bacterial species. [TOP OF PAGE]

151. Protection against bacteriophage contamination in industrial fermentation processes: Investigation and applications of phage resistance mechanisms in bacteria. Ogata, S., Eguchi, T., Doi, K. (2000). Virus (Nagoya) 50:17-26. [TOP OF PAGE]

152. Identification of virus-specific vesicles in Giardiavirus-infected Giardia lamblia. Ong, S.J., Tai, J.H. (2000). Chung-Hua Min Kuo Wei Sheng Wu Chi Mien I Hsueh Tsa Chih Chinese 33:9-13. Giardiavirus (GLV), which infects the parasitic protozoan Giardia lamblia, is a nonsegmented double-stranded (ds) ribonucleic acid (RNA) virus. We previously purified two distinct types of related GLV from infected G. lamblia, and showed differential export of one of the viruses from infected cells. In the present study, fractionation of cell lysate was performed, revealing the presence of viruses in the membranous fraction. Distribution of viral antigens in the infected cells was examined by immunocytochemistry. The signal was enriched in certain regions of the cytoplasm, suggesting that a portion of GLV is confined to certain cellular compartments. A significantly reduced signal was also detected in the nuclei. We directly observed the viruses in the infected cells by electron microscopy. Consistent with previous observations, virus-like particles were clearly observed in some membranous vesicles in the cytoplasm at 48 h postinfection, and virus-like particles were again seen in the cytoplasm and then in the nuclei toward the late phase of virus infection. The virus-associated vesicles and some electron-dense nuclear structures were only observed in virus-infected cells, suggesting that virus infection may induce ultrastructural alteration of G. lamblia. [TOP OF PAGE]

153. Genotypic variations of Shiga toxin-converting phages from enterohaemorrhagic Escherichia coli O157: H7 isolates. Osawa, R., Wada, A., Iyoda, S., Nakayama, S.I., Yamai, S., Watanabe, H. (2000). J. Med. Microbiol. 49:565-574. Pulsed-field gel electrophoresis (PFGE) analysis revealed that enterohaemorrhagic Escherichia coli (EHEC) O157:H7 strains had considerable variations in their genomes. This study investigated whether or not the molecular profile of Shiga toxin (Stx) 1- and Stx2-converting phages isolated from EHEC O157:H7 strains, derived from various sources in the USA and Japan, corresponded to the variations of host strains' genotypes as determined by PFGE. A total of 51 Stx-converting phages including 12 Stx1-converting phages and 37 Stx2-converting phages was isolated from seven USA isolates and 20 Japanese isolates. The average Dice coefficient values showed 44% similarity between phage DNAs in Stx2-converting phages digested with SmaI and 55% in Stx1-converting phages digested with HindIII, indicating considerable variation among phage DNA. In particular, restriction fragment length polymorphism (RFLP) patterns of Stx2-converting phage DNA varied according to the PFGE type of their host strain, which suggests that the phage genomes have altered their genotypic characteristics with those of host genomes. However, there are several exceptions: the RFLP patterns of some Stx2-converting phages were quite similar irrespective of the different genotypes of the host strains, indicating that horizontal transfer of Stx2-converting phage may also occur under some circumstances. [TOP OF PAGE]

154. A Stalinist Antibiotic Alternative. Osborne, L. (2000). New York Times Magazine (Sunday, February 6), ???-??? A hoary Soviet method for fighting infections may prove invaluable in an age of antibiotic resistance. Maybe that's why pharmaceutical companies are flocking to a remote laboratory in Tbilisi. [TOP OF PAGE]

155. Bacteriophage phiYeO3-12, specific for Yersinia enterocolitica serotype O:3, is related to coliphages T3 and T7. Pajunen, M., Kiljunen, S., Skurnik, M. (2000). J. Bacteriol. 182:5114-5120. Bacteriophage phiYeO3-12 is a lytic phage of Yersinia enterocolitica serotype O:3. The phage receptor is the lipopolysaccharide O chain of this serotype that consists of the rare sugar 6-deoxy-L-altropyranose. A one-step growth curve of phiYeO3-12 revealed eclipse and latent periods of 15 and 25 min, respectively, with a burst size of about 120 PFU per infected cell. In electron microscopy phiYeO3-12 virions showed pentagonal outlines, indicating their icosahedral nature. The phage capsid was shown to be composed of at least 10 structural proteins, of which a protein of 43 kDa was predominant. N-terminal sequences of three structural proteins were determined, two of them showing strong homology to structural proteins of coliphages T3 and T7. The phage genome was found to consist of a double-stranded DNA molecule of 40 kb without cohesive ends. A physical map of the phage DNA was constructed using five restriction enzymes. The phage infection could be effectively neutralized using serum from a rabbit immunized with whole phiYeO3-12 particles. The antiserum also neutralized T3 infection, although not as efficiently as that of phiYeO3-12. phiYeO3-12 was found to share, in addition to the N-terminal sequence homology, several common features with T3, including morphology and nonsubjectibility to F exclusion. The evidence conclusively indicated that phiYeO3-12 is the first close relative of phage T3 to be described. [TOP OF PAGE]

156. Isolation of bacteriophages specific to a fish pathogen, Pseudomonas plecoglossicida, as a candidate for disease control. Park, S.C., Shimamura, I., Fukunaga, M., Mori, K.I., Nakai, T. (2000). Appl. Environ. Microbiol. 66:1416-1422. Two types of bacteriophage specific to Pseudomonas plecoglossicida, the causative agent of bacterial hemorrhagic ascites disease in cultured ayu fish (Plecoglossus altivelis), were isolated from diseased ayu and the rearing pond water. One type of phage, which formed small plaques, was tentatively classified as a member of the family Myoviridae, and the other type, which formed large plaques, was classified as a member of the family Podoviridae. All 27 strains of P. plecoglossicida examined, which were isolated from diseased ayu from geographically different areas in 1991 to 1999, exhibited quite similar sensitivities to either type of phage. One strain of P. plecoglossicida was highly virulent for ayu, and the 50% lethal dose (LD(50)) when intramuscular injection was used was 10(1.2) CFU fish(-1); in contrast, phage-resistant variants of this organism were less virulent (LD(50), >10(4) CFU fish(-1)). Oral administration of phage-impregnated feed to ayu resulted in protection against experimental infection with P. plecoglossicida. After oral administration of P. plecoglossicida cells of this bacterium were always detected in the kidneys of control fish that did not receive the phage treatment, while the cells quickly disappeared from the phage-treated fish. Bacterial growth in freshwater was lower in the presence of phage, and the number of phage PFU increased rapidly. These results suggest that it may be possible to use phage to control the disease caused by P. plecoglossicida. [TOP OF PAGE]

157. Analysis of two-stage continuous operation of Escherichia coli containing bacteriophage lambda vector. Park, S.H., Park, T.H. (2000). Bioprocess Engineering 23:557-563. Bacteriophage lambda containing a cloned-gene is stably maintained in Escherichia coli in the lysogenic state while it is replicated and it overproduces a recombinant protein product in the lytic state. The host cell is eventually lysed in the lytic state. The kinetics of cell lysis and production induction were studied and are reported in this article through model equations. In two-stage continuous operation, the first tank is maintained in the lysogenic state for cell growth and cloned-gene stability while the second tank is in the lytic state for the overproduction of cloned-gene product. Individual cells in the second tank have different extent of the induction for product formation, since each has a different residence time. The different residence time for individual cells was taken into account using a population model. The numerical results show good agreement with the experimental data for the prediction of dilution rate in the second tank which gives the maximum product concentration. [TOP OF PAGE]

158. Ecology of bacteriophages in nature. Paul, J.H., Kellogg, C.A. (2000). pp. 211-246. In In Hurst, C.J. (ed.), Viral Ecology. Academic Press, San Diego. [no abstract?]. [TOP OF PAGE]

159. Rapid movement of wastewater from on-site disposal systems into surface waters in the Lower Florida Keys. Paul, J.H., McLaughlin, M.R., Griffin, D.W., Lipp, E.K., Stokes, R., Rose, J.B. (2000). Estuaries 23:662-668. Viral tracer studies have been used previously to study the potential for wastewater contamination of surface marine waters in the Upper and Middle Florida Keys. Two bacteriophages, the marine bacteriophage phiHSIC and the Salmonella phage PRD1, were used as tracers in injection well and septic tank studies in Saddlebunch Keys of the Lower Florida Keys and in septic tank studies in Boot Key Harbor, Marathon, of the Middle Keys. In Boot Key Harbor, both phages were detected in a canal adjacent to the seeded septic tank within 3 h 15 min of the end of the seed period. The tracer was then detected at all sampling sites in Boot Key Harbor, including one on the opposite side of U.S. Highway 1 in Florida Bay, and at an Atlantic Ocean beach outside Boot Key Harbor. Rates of migration based on first appearance of the phage ranged from 1.7 to 57.5 m h-1. In Saddlebunch Keys, phiHSIC and PRD1 were used to seed a residential septic tank and a commercial injection well. The septic tank tracer was not found in any surface water samples. The injection well tracer was first detected at a site most distant from the seed site, a channel that connected Sugarloaf Sound with the Atlantic Ocean. The rate of tracer migration from the injection well to this channel ranged from 66.8 to 141 m h-1. Both tracer studies showed a rapid movement of wastewater from on-site sewage treatment and disposal systems in a southeasterly direction toward the reef tract and Atlantic Ocean, with preferential movement through tidal channels. These studies indicate that wastewater disposal systems currently in widespread use in the Florida Keys can rapidly contaminate the marine environment. [TOP OF PAGE]

160. Induction of vaginal Lactobacillus phages by the cigarette smoke chemical benzo[a]pyrene diol epoxide. Pavlova, S.I., Tao, L. (2000). Mutation Research 466:57-62. Because smoking increases a woman's risk of contracting bacterial vaginosis (BV), which is manifested by a reduction of vaginal lactobacilli and an overgrowth of anaerobic bacteria, chemicals contained in cigarette smoke were analyzed in vitro to determine their role in reducing lactobacilli. The result showed that trace amounts of benzo[a]pyrene diol epoxide (BPDE), which can be found in vaginal secretion of women who smoke, significantly increased phage induction in lactobacilli. This finding implies that smoking may reduce vaginal lactobacilli by promoting phage induction. [TOP OF PAGE]

161. Phage therapy: The peculiar kinetics of self-replicating pharmaceuticals. Payne, R.J.H., Phil, D., Jansen, V.A.A. (2000). Clinical Pharmacology and Therapeutics 68:225-230. The specter of antibiotic-resistant bacteria has provoked renewed interest in the possible use of bacteriophages to control bacterial infections. We argue that clinical application of phage therapy has been held back by a failure to appreciate the extent to which the pharmacokinetics of self-replicating agents differ from those of normal drugs. For self-replicating pharmaceutical agents, treatment outcome depends critically on various density-dependent thresholds, often with apparently paradoxical consequences. An ability to predict these thresholds and associated critical time points is a necessity if phage therapy is to become clinically practicable. [TOP OF PAGE]

162. Bacterial plasmids: Parasitic organisms? Permaul, K., Pillay, B., Pillay, D. (2000). South African Journal of Science 96:555-556. The presence of plasmids in bacteria raises the question whether these plasmids constitute a stable characteristic of the host cell or whether they are parasitic genetic nomads whose stay in any bacterial population is incidental and transient. While it is still largely true that specific plasmids are associated with certain host bacteria, the existence of broad-host-range conjugative plasmids lifts this restriction on their lifestyle. It is becoming apparent from analyses of DNA sequences of plasmids that they comprise DNA segments from various sources. This apparent sequestering of DNA by plasmids and the presence of separate evolutionary lineages by the various subgroups of plasmids suggests that they could be regarded as bacterial parasites, a concept that is the focus of this article. [TOP OF PAGE]

163. Efficacy of four conjugal lactococcal phage resistance plasmids against phage in commercial Lactococcus lactis subsp. cremoris cheese starter strains. Pillidge, C.J., Collins, L.J., Ward, L.J.H., Cantillon, B.M., Shaw, B.D., Timmins, M.J., Heap, H.A., Polzin, K.M. (2000). International Dairy Journal 10:617-625. The efficacy of four lactococcal phage resistance plasmids (pNP40, pMU1311, pDI60 and pKP100) against phage was assessed after their conjugal transfer to four commercial Lactococcus lactis subsp. cremoris cheese starter strains and to the plasmid-free strain L. lactis subsp. cremoris MG1363. In MG1363, only pNP40 conferred resistance to prolate phages c2 and 643. Highest levels of resistance to small isometric phages in MG1363 occurred when pNP40 was stacked together with pMU1311 or pDI60. In the four starter strains, the plasmids conferred varying levels of resistance to small isometric phages. Growth and acidification rates in milk of most transconjugants derived from the starter strains decreased, but this was not always due to loss of plasmid-encoded cell wall proteinase (lactocepin) activity. Only one transconjugant grew during repeated subculture in milk with addition of factory wheys containing phages. This and the presence of bacteriocins encoded on pMU1311 and pDI60 limited application of the plasmids to protect L. lactis subsp. cremoris starters against phages in industry. However, some of the plasmids could be useful in extending the industry life of starters where fast acid production is not required or where bacteriocin production is acceptable. [TOP OF PAGE]

164. Phage therapy—advantages over antibiotics? Pirisi, A. (2000). Lancet 356:1418 As antibiotic-resistant bacteria continue to threaten standard therapies against bacterial infections, a new breed of antimicrobials may be on the horizon. Many researchers believe that bacteriophages—viruses that only infect bacteria—are a promising potential therapy for bacterial disease treatment. [TOP OF PAGE]

165. Comparative study of temperate bacteriophages isolated from Yersinia. Popp, A., Hertwig, S., Lurz, R., Appel, B. (2000). Systematic and Applied Microbiology 23:469-478. 170 Yersinia strains belonging to various species were investigated for the presence of temperate bacteriophages. By induction with mitomycin C seven phages were isolated from Y. enterocolitica strains and one phage from a Y. frederiksenii strain. The phages were characterized on the basis of their morphology, host range, genome size, DNA homology, and protein composition. They belong to different phage families and reveal narrow to moderate wide host ranges. Some of the isolated phages were able to infect pathogenic as well as nonpathogenic strains of Y. enterocolitica. The genomes of all isolated phages were found to be composed of double stranded DNA ranging from about 40 to 60 kb. In addition to the analysed phages, a number of putative phages were induced in strains of Y. frederiksenii, Y. kristensenii, Y. intermedia, and Y. mollaretii. The putative phages were identified by isolation of phage DNA from cell free lysates but could not be propagated on indicator strains. Southern hybridization experiments revealed relationships between phages belonging to different families. Moreover, DNA homologies were observed between phages isolated from nonpathogenic Yersinia strains and a phage which was isolated from a pathogenic Y. enterocolitica serogroup O:3 strain. [TOP OF PAGE]

166. Selection of tumor-specific internalizing human antibodies from phage libraries. Poul, M.A., Becerril, B., Nielsen, U.B., Morisson, P., Marks, J.D. (2000). J. Mol. Biol. 301:1149-1161. Antibody internalization into the cell is required for many targeted therapeutics, such as immunotoxins, immunoliposomes, antibody-drug conjugates and for targeted delivery of genes or viral DNA into cells. To generate directly tumor-specific internalizing antibodies, a non-immune single chain Fv (scFv) phage antibody library was selected on the breast tumor cell line SKBR3. Internalized phage were recovered from within the cell and used for the next round of selection. After three rounds of selection, 40 % of clones analyzed bound SKBR3 and other tumor cells but did not bind normal human cells. Of the internalizing scFv identified, two (F5 and C1) were identified as binding to ErbB2, and one (H7) to the transferrin receptor. Both F5 and H7 scFv were efficiently endocytosed into SKBR3 cells, both as phage antibodies and as native monomeric scFv. Both antibodies were able to induce additional functional effects besides triggering endocytosis: F5 scFv induces downstream signaling through the ErbB2 receptor and H7 prevents transferrin binding to the transferrin receptor and inhibits cell growth. The results demonstrate the feasibility of selecting internalizing receptor-specific antibodies directly from phage libraries by panning on cells. Such antibodies can be used to target a variety of molecules into the cell to achieve a therapeutic effect. Furthermore, in some instances endocytosis serves as a surrogate marker for other therapeutic biologic effects, such as growth inhibition. Thus, a subset of selected antibodies will have a direct therapeutic effect. [TOP OF PAGE]

167. Epidemiologic Subtyping of Escherichia coli Serogroup O157 Strains Isolated in Ontario by Phage Typing and Pulsed-Field Gel Electrophoresis. Preston, M.A., Johnson, W., Khakhria, R., Borczyk, A. (2000). Journal of Clinical Microbiology 38:2366-2368. [TOP OF PAGE]

168. Detection of phages infecting Bacteroides fragilis HSP40 using a specific DNA probe. Puig, M., Jofre, J., Girones, R. (2000). Journal of Virological Methods 88:163-173. Nine bacteriophage isolates of Bacteroides fragilis, obtained from urban sewage and pig faeces samples using four different host strains (HSP40, RYC4023, RYC2056 and RYC3318), were compared on the basis of morphology, host range, DNA restriction patterns, DNA hybridisation and protein composition. All the phages are siphovirus and, as judged from cleavage by restriction endonucleases, their genome is composed of double-stranded DNA of similar size ( approximately 51-kb). Host range analysis differentiated two types of phages: (1) phages that clearly infect B. fragilis strains HSP40 (B40-2, B23-1, B23-2, B23-3 and B23-4, of which B40-8 is the phage type); and (2) the group of bacteriophages that were not infectious for HSP40 (B56-1, B56-2 and B18-1). Similarity in DNA restriction patterns and protein characteristics was found in the HSP40 infectious phages. Anti-B40-8 serum recognised only the proteins of the phages of this type. Although all phages showed similar major protein sizes, minor specific bands were detected. Bacteriophages B56-1, B56-2 and B18-1 showed heterogeneity in their DNA restriction profiles although some degree of DNA-DNA homology between all genomes was observed. Southern blot analysis with phage B40-8 DNA based probes identified a 1.5-kb DNA region homologous for all HSP40 phage isolates, but absent in the genome of the other phage isolates that did not infect this bacterial strain. The homologous region was used as a specific probe to specifically detect B. fragilis HSP40 phages. [TOP OF PAGE]

169. Occurrence of coliphages in fish and aquaculture farms. Rao, B.M., Surendran, P.K. (2000). Fishery Technology 37:146-149. Coliphages were detected in water samples collected from brackish water and fresh water fish farms. Coliphages were also detected in the farmed fresh water fish, common carp and marine fish, oil sardine, from local market. Coliphage levels obtained were as follows:- water from brackish water fish farm 3 pfu.ml-1, water from fresh water fish farm 23 pfu.ml-1, fresh water fish 240 pfu.g^-1 and marine fish 3500 pfu.g^-1. [TOP OF PAGE]

170. Genomic sequence and analysis of the atypical temperate bacteriophage N15. Ravin, V., Ravin, N., Casjens, S., Ford, M.E., Hatfull, G.F., Hendrix, R.W. (2000). J. Mol. Biol. 299:53-73. N15 is a temperate bacteriophage that forms stable lysogens in Escherichia coli. While its virion is morphologically very similar to phage lambda and its close relatives, it is unusual in that the prophage form replicates autonomously as a linear DNA molecule with closed hairpin telomeres. Here, we describe the genomic architecture of N15, and its global pattern of gene expression, which reveal that N15 contains several plasmid-derived genes that are expressed in N15 lysogens. The tel site, at which processing occurs to form the prophage ends is close to the center of the genome in a similar location to that occupied by the attachment site, attP, in lambda and its relatives and defines the boundary between the left and right arms. The left arm contains a long cluster of structural genes that are closely related to those of the lambda-like phages, but also includes homologs of umuD', which encodes a DNA polymerase accessory protein, and the plasmid partition genes, sopA and sopB. The right arm likewise contains a mixture of apparently phage- and plasmid- genes including genes encoding plasmid replication functions, a phage repressor, a transcription antitermination system, as well as phage host cell lysis genes and two putative DNA methylases. The unique structure of the N15 genome suggests that the large global population of bacteriophages may exhibit a much greater diversity of genomic architectures than was previously recognized. [TOP OF PAGE]

171. Comparative analysis of Chlamydia bacteriophages reveals variation localized to a putative receptor binding domain. Read, T.D., Fraser, C.M., Hsia, R.C., Bavoil, P.M. (2000). Microbial and Comparative Genomics 5:223-231. Three recently discovered ssDNA Chlamydia-infecting microviruses, phiCPG1, phiAR39, and Chp2, were compared with the previously characterized phage from avian C. psittaci, Chp1. Although the four bacteriophages share an identical arrangement of their five main genes, Chp1 has diverged significantly in its nucleotide and protein sequences from the other three, which form a closely related group. The VP1 major viral capsid proteins of phiCPG1 and phiAR39 (from guinea pig-infecting C. psittaci and C. pneumoniae, respectively) are almost identical. However, VP1 of ovine C. psittaci phage Chp2 shows a high rate of nucleotide sequence change localized to a region encoding the "IN5" loop of the protein, thought to be a potential receptor-binding site. Phylogenetic analysis suggests that the ORF4 replication initiation protein is evolving faster than the other phage proteins. phiCPG1, phiAR39, and Chp2 are closely related to an ORF4 homolog inserted in the C. pneumoniae chromosome. This sequence analysis opens the way toward understanding the host-range and evolutionary history of these phages. [TOP OF PAGE]

172. Transmission of viruses via contact in a household setting: Experiments using bacteriophage phiX174 as a model virus. Rheinbaben, F., Schuenemann, S., Gross, T., Wolff, M.H. (2000). Journal of Hospital Infection 46:61-66. Contamination of the environment with pathogens is the prerequisite for contact infections. The aim of this study was to elucidate how viruses can be transmitted from a primary contact person to further individuals. Bacteriophage phiX174 was chosen as a model virus. In its stability phiX174 is comparable with the most resistant human pathogenic viruses, e.g. polio- or parvoviruses. About 10⁷ pfu were applied to exposed contact points such as door handles or the hands of volunteers. After touching of these handles and common social contacts like hand shaking, re-isolation rates were determined from the hands of our test persons. Contaminated door handles and skin surfaces were found to be efficient sources for potential infection. At least 14 persons could be contaminated by horizontal spread, one after the other by touching the same door handle. Successive transmission from one person to another could be followed up to the sixth contact person. These results were confirmed under everyday life conditions in a flat shared by four students. The transmission could not be prevented by the usual standards of hand hygiene, practised in this household. phiX174 could be reisolated after 24 h from the hands of all persons tested even after normal use and cleaning of their hands. This might be improved by the use of liquid soap dispensers. [TOP OF PAGE]

173. Screening environmental samples for source-specific bacteriophage hosts using a method for the simultaneous pouring of 12 petri plates. Ricca, D.M., Cooney, J.J. (2000). Journal of Industrial Microbiology & Biotechnology. 24:124-126. A simple apparatus was developed to allow 12 petri plates to be poured simultaneously by hand. It was used when screening bacterial isolates from sewage and dog feces for their ability to detect phages from these sources. This was done to assess the ease with which source-specific phage hosts can be isolated from these sources of fecal pollution. Host bacteria that consistently detected phages from sewage were easily isolated from sewage. These bacterial isolates did not detect phages from dog feces. Host bacteria were not isolated from dog feces even after screening hundreds of colonies from fecal samples from six dogs. [TOP OF PAGE]

174. The passage and propagation of fecal indicator phages in birds. Ricca, D.M., Cooney, J.J. (2000). Journal of Industrial Microbiology & Biotechnology. 24:127-131. The presence of F-specific phages in the diet of birds influenced the presence of these fecal indicators in their feces. F-specific phage concentrations in the feces of Canada geese and pigeons, which are normally low, increased greatly the same day coliphage MS2 was added to their diets. F-specific phage concentrations decreased to the original low levels a week after the phage-spiked feed was removed. Geese kept in pens that were cleaned regularly to reduce fecal-oral contamination had significantly lower somatic coliphage concentrations in their feces than wild geese had in their feces. Somatic coliphage concentrations in feces of feral pigeons were typically low with an occasional fecal sample having high numbers of either one of the two types of phages seen in this population of birds. Sometimes many birds had high numbers of only one type of phage in their feces. This lasted only a day and was probably due to fecal contamination of the feeding pans by the pigeons. The degree to which birds are a source of phage indicators of fecal pollution can change in a short period of time. Thus the presence of contaminated feeding sites should be considered before ruling out animals as a possible source of fecal indicators. F-specific phages may be useful tracers for modeling viral transmission and tracking feeding habits in birds. [TOP OF PAGE]

175. Bacterial monopolists: the bundling and dissemination of antimicrobial resistance genes in gram-positive bacteria. Rice, L.B. (2000). Clinical Infectious Diseases 31:762-769. Antibiotic resistance is the unavoidable result of our placing selective pressure on the microbial community. Advances in molecular biology techniques in the past 2 decades have allowed us to greatly improve our understanding of the mechanisms by which resistance emerges and disseminates among human pathogenic bacteria. Gram-positive bacteria employ a diverse array of elements, including plasmids, transposons, insertion sequences, and bacteriophages, to disseminate resistance. An understanding of these mechanisms and their prevalence can improve our ability to treat clinical infections in hospitalized patients, as well as to predict and control the spread of resistant bacteria in the nosocomial environment. [TOP OF PAGE]

176. Dynamics of bacterial community composition and activity during mesocosm diatom blooms. Riemann, L., Steward, G.F., Azam, F. (2000). Appl. Environ. Microbiol. 66:578-587. Bacterial community composition, enzymatic activities, and carbon dynamics were examined during diatom blooms in four, 200 liter laboratory seawater mesocosms. The objective was to determine whether the dramatic shifts in growth rates and ectoenzyme activities, which are commonly observed during the course of phytoplankton blooms and their subsequent demise, could result from shifts in bacterial community composition. Nutrient enrichment of metazoan-free seawater resulted in diatom blooms dominated by Thalassiosira sp. which peaked nine days after enrichment ( 24 g chl a l^-1). At this time bacterial abundance abruptly decreased from 2.8 to 0.75 x 10⁶ ml^-1 and analysis of bacterial community composition, by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified, 16S rRNA gene fragments, revealed a disappearance of three dominant phylotypes. Increased viral and flagellate abundance suggested that both lysis and grazing could have played a role in the observed phylotype-specific mortality. Subsequently, new phylotypes appeared and bacterial production, abundance and enzyme activities shifted from being predominantly associated with the <1.0 m size-fraction towards the >1.0 m size-fraction indicating a pronounced microbial colonization of particles. Sequencing of DGGE bands suggested that the observed rapid and extensive colonization of particulate matter was mainly by specialized ??Proteobacteria and Cytophagales-related phylotypes. These particle-associated bacteria had high growth rates as well as high cell specific aminopeptidase, ??glucosidase and lipase activities. Rate measurements as well as bacterial population dynamics were almost identical among the mesocosms indicating that the observed bacterial community dynamics were systematic and repeatable responses to the manipulated conditions. [TOP OF PAGE]

177. Temporal variability of viruses, bacteria, phytoplankton and zooplankton in the western English Channel off Plymouth. Rodriguez, F., Frenandez, E., Head, R.N., Harbour, D.S., Bratbak, G., Heldal, M., Harris, R.P. (2000). Journal of the Marine Biological Association of the United Kingdom 80:575-586. The temporal distribution of autotrophic and heterotrophic components of the planktonic community was studied from samples collected weekly at station L4, located to the south of Plymouth, UK, from October 1992 to January 1994. Phytoplankton succession followed the typical pattern of temperate waters. the development of a summer Gyrodinium aureolum bloom being the most prominent feature. Bacterial numbers were significantly correlated with temperature during autumn and winter, whereas resource availability and predation, including viruses, appear to be the most important controlling factors in spring and summer. High mesozooplankton densities, mainly copepods, were observed throughout most of the study associated with a series of diatom blooms, and also during autumn when low phytoplankton biomass was measured. This data set was analysed in order to build up conceptual trophodynamic models whereby the role of biological communities on the cycling of organic matter could be inferred. The results obtained in this study provide empirical evidence supporting the existence of a succession of trophic organization patterns in a coastal temperate environment. Classical models (herbivorous or microbial webs) appeared episodically whereas transition models (multivorous web) dominated throughout most of the seasonal cycle. [TOP OF PAGE]

178. The complete genomic sequence of the marine phage Roseophage SIO1 shares homology with nonmarine phages. Rohwer, F., Segall, A., Steward, G., Seguritan, V., Breitbart, M., Wolven, F., Azam, F. (2000). Limnol. Oceanogr. 45:408-418. Viruses are ubiquitous components of the marine environment, frequently reaching concentrations of 10⁷-10⁸ viruses per milliliter of surface seawater The majority of these viral particles are bacteriophages (phages). Although the oceans are probably the largest pool of bacteriophages on the planet, the evolutionary relationships of marine phages to phages from other environments are unknown. To address this issue, we have completely sequenced the genome of the lytic marine phage, Roseophage SIO1, that infects the heterotrophic marine bacterium Roseobacter SIO67. This phage has an isometric capsid with a diameter of approximately 43 nm, a short tail, a buoyant density of 1.49 g cm^-3 in CsCl, and a 39,906-bp dsDNA genome. Sequence similarities and relative positions within the genome suggest that three of the open reading frames (ORFs) are homologous to the primase, DNA polymerase, and endodeoxyribonuclease I proteins of coliphages T3 and T7. The results are consistent with the mosaic theory of phage evolution and indicate a genetic link between marine and nonmarine phages. Additionally, basic life histories of marine phages can be elucidated by comparison of complete genomes to those of other extensively studied phages (e.g., lambda, T4, T7). The DNA replication machinery of Roseophage SIO1 shows a clear homology with that of coliphages T3 and T7, suggesting that the process of DNA replication may be similar among these phages. The Roseophage SIO1 genome also encodes four predicted proteins involved in phosphate metabolism (RP PhoH, RP ribonucleotide reductase, RP Thy1, and RP endodeoxyribonuclease I) suggesting that phosphate recycling is important to Roseophage SIO1's life cycle. Other interesting clues about Roseophage SIO1's life history come from the absence of certain expected protein regions. For example, we have not been able to identify the Roseophage SIO1 structural proteins (e.g., capsid proteins) by homology to other phages. It is also conspicuous that the Roseophage SIO1 genome lacks a recognizable RNA polymerase, an essential component of T3 and T7 life cycles. Analysis of the Roseophage SIO1 genome shows that marine and nonmarine phages are genetically related but basic life histories may be significantly different. [TOP OF PAGE]

179. Progressive specific immune attrition after primary, secondary and tertiary immunizations with bacteriophage PHI X174 in asymptomatic HIV-1 infected patients. Rubinstein, A., Mizrachi, Y., Bernstein, L., Shliozberg, J., Golodner, M., Liu, G.Q., Ochs, H.D. (2000). AIDS (Hagerstown). 14:F55-F62 Background: Antibody responses to immunization are often compromised in patients infected by HIV-1, and the use of childhood immunization in affected children is controversial. We investigated whether multiple immunizations with a T cell-dependent neoantigen, bacteriophage PHI X174, induce selective immune attrition and post-vaccination viremia. Methods: Seventeen asymptomatic, antiretroviral therapy-naive HIV-1-infected patients with a CD4 cell count of 450 cells/mul or greater were immunized in 1990/1991 with three intravenous doses of bacteriophage PHI X174. Group 1 received zidovudine (ZDV) during the primary and secondary immunization. Group 2 received ZDV exclusively during the tertiary immunization. Bacteriophage-specific antibodies of the IgM and IgG class, lymphocyte phenotypes (CD4+, CD8+, CD4+DR+, CD8+DR+, CD4+CD45RO+ and CD4+45RA+, CD4+CD45RO+DR+) and HIV-1 plasma viremia were measured sequentially. Results: In both patient groups the primary, secondary and tertiary antibody responses, as expressed by geometric mean antibody titres and IgM to IgG switch, were impaired. Booster immunizations resulted in a progressive attrition of specific antibody responses to bacteriophage. Antibodies to tetanus toxoid remained stable. The HIV-1 viral loads, which were evaluated in archived specimens from eight patients, increased after immunization but returned to baseline appoximately 4 weeks later. The humoral immune attrition and increases in plasma viremia were blunted by concomitant short courses of ZDV. Discussion: Multiple boosters of immunizations in asymptomatic treatment-naive HIV-1-infected patients may result in a specific immune attrition and vaccine-induced viremia. Short-term monotherapy with ZDV may have blunted these adverse effects. Hyperimmunization of HIV-1-infected patients may be detrimental unless accompanied by antiretroviral therapy. [TOP OF PAGE]

180. Removal efficiencies of indicator micro-organisms in the Al-Khobar wastewater treatment plant. Saleem, M., Bukhari, A.A., Al-Malack, M.H. (2000). Environmental Engineering Science 17:227-232. Increasing population and developmental needs of Saudi Arabia underline the need for an increase in the reuse of treated wastewater. However, treated wastewater contains a large number of pathogens that requires proper treatment before reuse. Little information is available on the treatment efficiency of wastewater treatment plants operating in this region. A 1-year study was conducted at the Al-Khobar wastewater treatment plant to investigate the removal efficiency of five indicator micro-organisms, namely, Standard Plate Count, total coliform, fecal coliform, coliphage, and Clostridium perfringens. The raw sewage, secondary effluent, and chlorinated effluent were analyzed weekly for the detection and enumeration of these indicator micro-organisms. High-percent removal of Standard Plate Count, total coliform, and fecal coliform (98 to 99%) was observed after secondary treatment compared to coliphage removal of 83.6% and Clostridium perfringens removal of 55.5%, whereas, after chlorination, Standard Plate Count, total coliform, and fecal coliform were removed up to 99.7% compared to coliphage reduction of 52% and Clostridium perfringens removal of only 42%, showing a high resistance against chlorination. The insight gained from this study may be applied to other similar treatment plants. [TOP OF PAGE]

181. Comparison of methods for detecting genotypes of F-specific RNA bacteriophages and fingerprinting the origin of faecal pollution in water samples. Schaper, M., Jofre, J. (2000). Journal of Virological Methods 89:1-10. The performance of Salmonella typhimurium WG49 and Escherichia coli HS(pFamp)R was compared on detecting the different genotypes of F-specific RNA bacteriophages by plaque hybridisation. The sensitivity of this assay was also compared with the sensitivity of RT-PCR followed by Southern blotting for detecting F-specific RNA bacteriophages belonging to genotype III in water. S. typhimurium WG49 detected slightly higher numbers of F-specific RNA bacteriophages than E. coli HS(pFamp)R both in mixtures of pure culture bacteriophage suspensions and in water samples. There were no differences between the two host strains with regard to detection of the four genotypes of F-specific RNA phages both in mixtures of pure culture bacteriophage suspensions and in environmental samples. In urban sewage samples, the host strains detected genotypes II and III as the predominant F-RNA bacteriophages. Plaque transfer to a N(+) hybond membrane and posterior hybridisation was easier using S. thyphimurium WG49 as the host strain. The efficiency of detection in sewage of genotype III F-specific RNA bacteriophages by RT-PCR was inferior to that of plaque hybridisation with the assay conditions described below. Hybridisation of plaques obtained on WG49 seems to be the most sensitive method to study the distribution of genotypes of F-specific RNA bacteriophages in water samples. [TOP OF PAGE]

182. Removal of microorganisms by deep well injection. Schijven, J.F., Medema, G., Vogelaar, A.J., Hassanizadeh, S.M. (2000). Journal of Contaminant Hydrology 44:301-327. The removal of bacteriophages MS2 and PRD1, spores of Clostridium bifermentans (R5) and Escherichia coli (WR1) by deep well injection into a sandy aquifer, was studied at a pilot field site in the southeast of the Netherlands. Injection water was seeded with the microorganisms for 5 days. Breakthrough was monitored for 93 days at 4 monitoring wells with their screens at a depth of about 310 m below surface. Within the first 8 m of soil passage, concentrations of MS2 and PRD1 were reduced by 6 log10, that of R5 spores by 5 log10 and that of WR1 by 7.5 log10. Breakthrough of MS2 and R5 could also be followed at greater distances from the injection well. Concentrations of MS2 were reduced only by about 2 log10 in the following 30 m, and reduction of concentrations of R5 was negligible. Apparently, attachment was greater during the first 8 m of aquifer passage. At the point of injection, the inactivation rate coefficient of free MS2 was found to be 0.081 day-1, that of free PRD1 0.060 day-1, and that of E. coli strain WR1 0.063 day-1. In injection water that had passed 8 m of soil, inactivation of MS2 phages was found to be less than in water from the injection well: 0.039 day-1. Probably, the higher inactivation rate of MS2 in water from the injection well may be ascribed to the activity of aerobic bacteria. Inactivation of the R5 spores was not significant. From geochemical mass balances, it could be deduced that within the first 8 m distance from the injection well, ferric oxyhydroxides precipitated as a consequence of pyrite oxidation, but not at larger distances. Ferric oxyhydroxides provide positively charged patches onto which fast attachment of the negatively charged microorganisms may take place. The non-linear logarithmic reduction of concentrations with distance may therefore be ascribed to preferable attachment of microorganisms to patches of ferric oxyhydroxides that are present within 8 m distance from the injection point, but not thereafter. Declogging of the injection well introduced hydrodynamic shear that remobilized MS2, which was then transported farther downstream. [TOP OF PAGE]

183. Food and water contamination by human pathogenic viruses. Scipioni, A., Daube, G., Thiry, E. (2000). Annales de Medecine Veterinaire 144:207-221. Food and water contamination by human viruses is a great health problem. These viruses are shed in stools. Norwalk-like viruses, hepatitis E virus, poliovirus, echovirus, hepatitis A virus, rotavirus, astrovirus, enteric adenovirus and parvovirus B19 have been described. The most important ones are Norwalk-like viruses, rotavirus and hepatitis A virus as reported in epidemiological surveys. The most frequently implicated foods are shellfish (bivalve mollusks) harvested from waters contaminated with human sewage, as well as water itself. The other source of infection is the handling of food in poor hygienic conditions. In this case contaminated foods are vegetables, sandwiches, fruits, pastries that are soiled. The detection of viruses in foods is difficult for several reasons: Virus-food interactions make difficult the concentration and the purification of viruses, several virus species are difficult or unable to grow in cell culture, furthermore viruses are present in the sample in very low amounts. Molecular techniques are therefore the methods of choice for detecting these viruses, especially the polymerase chain reaction which is often described. Another possibility consists in a fecal viral indicator. Bacteriophages seem to be the most promising in this respect. [TOP OF PAGE]

184. Twenty-three years of Klebsiella phage typing: a review of phage typing of 12 clusters of nosocomial infections, and a comparison of phage typing with K serotyping. Sechter, I., Mestre, F., Hansen, D.S. (2000). Clin Microbiol Infect 6:233-238. OBJECTIVE: To review phage typing of 12 clusters of nosocomial Klebsiella infections which occurred between 1974 and 1997, and to compare phage typing and K serotyping. Materials and methods A total of 489 clinical and laboratory Klebsiella isolates were phage typed using 110 different phage preparations and K typed by counter current immunoelectrophoresis against 77 K antisera. RESULTS: A total of 152 phage types (PT) and 82 K types were found. Thirty-six phage types and 14 K types were represented only by the reference type strains. Of the remaining 68 K types, 60 could be subdivided into from two to 10 phage types. Ten out of 12 clusters of nosocomial Klebsiella infections could be verified as outbreaks by phage typing, whereas two clusters were found to be accumulations of sporadic cases. K typing performed retrospectively confirmed these results. In addition, for a subset of 104 epidemiologically unrelated isolates, O typing and pulsed field gel electrophoresis typing data were available. Based on these results the discriminative power of phage typing was found to be comparable with that of K typing, but phage types were less stable and reproducible. CONDITIONS: In an outbreak situation, phage typing was found to be very useful, although it seems less suitable for long-term surveillance purposes. [TOP OF PAGE]

185. Features of sanitary and hygienic work conditions of operators servicing purification structures of cardboard printing enterprise. Semyonova, V.V., Figurovsky, A.P., Vasilyev, O.D., Zakharov, A.P., Zhigalov, V.A., Shevtchenko, N.N., Goik, V.G. (2000). Meditsina Truda i Promyshlennaya Ekologiya 42-44. [TOP OF PAGE]

186. [Isolation and comparative study of a group of temperate bacteriophages of rhizospheric pseudomonads Pseudomonas putida]. Shaburova, O.V., Burkal'tseva, M.V., Pleteneva, E.A., Krylov, V.N. (2000). Genetika 36:915-919. We have isolated several new temperate bacteriophages for rhizosphere pseudomonads Pseudomonas putida. Examination of these phages, along with two previously isolated temperate phages PP56 and PP71 of P. putida PpG1 (biovar A), allowed us to classify them into four species on the basis of DNA cross-homology; relative genomic size; and, to a certain extent, the morphology of phage particles. Two of these species are represented by nonidentical variants. No transposable phages were found among these two new species. Three phage species cause various-types of lysogenic conversion manifested in growth suppression of other phage species. This seems to account for the fact that the temperate phage of rhizosphere pseudomonads are seldom encountered. The new phages described can be used for selection of phage-resistant bacterial forms exhibiting antifungal activity that are commercially produced and used for treatment of seeds of cultivated plants. [TOP OF PAGE]

187. Discovery, purification, and characterization of a temperate transducing bacteriophage for Bordetella avium. Shelton, C.B., Crosslin, D.R., Casey, J.L., Ng, S., Temple, L.M., Orndorff, P.E. (2000). J. Bacteriol. 182:6130-6136. We discovered and characterized a temperate transducing bacterlophage (Ba1) for the avian respiratory pathogen Bordetella avium. Ba1 was initially identified along with one other phage (Ba2) following screening of four strains of B. avium for lysogeny. Of the two phage, only Ba1 showed the ability to transduce via an allelic replacement mechanism and was studied further. With regard to host range, Ba1 grew on six of nine clinical isolates of B. avium but failed to grow on any tested strains of Bordetella bronchiseptica, Bordetella hinzii, Bordetella pertussis, or Bordetella parapertussis. Ba1 was purified by CsCl gradient centrifugation and was found to have an icosahedral head that contained a linear genome of approximately 46.5 kb (contour length) of double-stranded DNA and a contractile, sheathed tail. Ba1 readily lysogenized our laboratory B. avium strain (197N), and the prophage state was stable for at least 25 generations in the absence of external infection. DNA hybridization studies indicated the prophage was integrated at a preferred site on both the host and phage replicons. Ba1 transduced five distinctly different insertion mutations, suggesting that transduction was generalized. Transduction frequencies ranged from approximately 2 X 10-7 to 1 X 10-8 transductants/PFU depending upon the marker being transduced. UV irradiation of transducing lysates markedly improved transduction frequency and reduced the number of transductants that were lysogenized during the transduction process. Ba1 may prove to be a useful genetic tool for studying B. avium virulence factors. [TOP OF PAGE]

188. Denaturing gradient gel electrophoresis resolves virus sequences amplified with degenerate primers. Short, S.M., Suttle, C.A. (2000). BioTechniques 28:20 [TOP OF PAGE]

189. The genome sequence of the plant pathogen Xylella fastidiosa. Simpson, A.J.G., et al (2000). Nature 406:151-157. Xylella fastidiosa is a fastidious, xylem-limited bacterium that causes a range of economically important plant diseases. Here we report the complete genome sequence of X. fastidiosa clone 9a5c, which causes citrus variegated chlorosis-a serious disease of orange trees. The genome comprises a 52.7% GC-rich 2,679,305-base-pair (bp) circular chromosome and two plasmids of 51,158 bp and 1,285 bp. We can assign putative functions to 47% of the 2,904 predicted coding regions. Efficient metabolic functions are predicted, with sugars as the principal energy and carbon source, supporting existence in the nutrient-poor xylem sap. The mechanisms associated with pathogenicity and virulence involve toxins, antibiotics and ion sequestration systems, as well as bacterium-bacterium and bacterium-host interactions mediated by a range of proteins. Orthologues of some of these proteins have only been identified in animal and human pathogens; their presence in X. fastidiosa indicates that the molecular basis for bacterial pathogenicity is both conserved and independent of host. At least 83 genes are bacteriophage-derived and include virulence-associated genes from other bacteria, providing direct evidence of phage-mediated horizontal gene transfer. [TOP OF PAGE]

190. The interactions of peptides with the innate immune system studied with use of T7 phage peptide display [see comments]. Sokoloff, A.V., Bock, I., Zhang, G., Sebestyen, M.G., Wolff, J.A. (2000). Mol Ther 2:131-139. The icosahedral T7 phage (diameter approximately 65 nm) displaying random peptides at the carboxy-terminus of the phage coat proteins was used as a model for drug and gene delivery vehicles containing peptide ligands. We found that displayed peptides were recognized by natural antibodies and induced complement activation. Strikingly, the phage inactivation by complement was peptide-specific that implied the existence of numerous natural antibodies with different peptide specificity. Selection of phage that avoided inactivation by complement allowed the identification of peptides that protected the phage by binding to serum proteins. In rat blood, peptides with carboxy-terminal lysine or arginine residues protected the phage against complement-mediated inactivation by binding C-reactive protein. In human serum, a number of protective peptides with tyrosine residues were selected. The recognition of displayed peptides by natural antibodies appears to represent a universal mechanism for activation of complement at sites that contain identical or homologous proteins with exposed carboxy-termini. [TOP OF PAGE]

191. Identification of four loci isolated from two Streptococcus thermophilus phage genomes responsible for mediating bacteriophage resistance. Stanley, E., Walsh, L., van, d.Z., Fitzgerald, G.F., van, S.D. (2000). FEMS Microbiol. Let. 182:271-277. Sequence data derived from the Streptococcus thermophilus phages phiO1205 and phi7201 indicated that each of these phages contains a distinct DNA region dedicated to replication. Southern blotting experiments showed that phages infecting S. thermophilus may be divided into at least two groups, each containing the presumptive replication functions of either fO1205 (group I) or f7201 (group II). Specific regions from the putative replication module of each of the two phages were examined for their ability to provide phage resistance. [TOP OF PAGE]

192. Tissue-specific gene expression identifies a gene in the lysogenic phage Gifsy-1 that affects Salmonella enterica serovar typhimurium survival in Peyer's patches. Stanley, T.L., Ellermeier, C.D., Slauch, J.M. (2000). J. Bacteriol. 182:4406-4413. In vivo expression technology was used to identify Salmonella enterica serovar Typhimurium genes that are transcriptionally induced when the bacteria colonize the small intestines of mice. These genes were subsequently screened for those that are transcriptionally inactive during the systemic stages of disease. This procedure identified gipA, a gene that is specifically induced in the small intestine of the animal. The gipA gene is carried on the lambdoid phage Gifsy-1. Consistent with the expression profile, the sole defect conferred by a gipA null mutation is in growth or survival in a Peyer's patch. The gipA strain is wild type in its ability to initially colonize the small intestine and invade the intestinal epithelium. The mutant also survives and propagates at wild-type levels during the systemic stages of disease. The gipA open reading frame is homologous to a family of putative insertion sequence elements, although our evidence shows that transposition is not required for gipA function in the Peyer's patch. These results suggest that the bacteria sense and respond to the particular environment of the Peyer's patch, a critical site for the replication of Salmonella serovar Typhimurium. [TOP OF PAGE]

193. Analysis of marine viral assemblages. Steward, G.F., Azam, F. (2000). pp. 159-165. In In Bell, C.R., Brylinski, M., and Johnson-Green, P. (eds.), Microbial Biosystems: New Frontiers. Atlantic Canada Society for Microbial Ecology, ??? Viruses are the numerically dominant microbes in every oceanic environment from the surface into the sediments. A liter of surface seawater from a typical mesotrophic area contains 10¹⁰ of them, about ten times more than bacteria. While total counts of viruses are becoming easier to make, we still know very little about the viruses that comprise a given assemblage. Infectivity assays are extremely useful and still the best way to assay for infectious viruses for any particular host. However, this approach requires that each potential host organism be cultured, making it impractical if not impossible to completely characterize natural assemblages. Morphological studies have been enlightening, but are time consuming and difficult to do quantitatively. Here we report a fingerprinting approach to characterize natural viral assemblages. In this approach, viruses are concentrated and intact viral genomes are separated based on their size via pulsed-field gel electrophoresis. The number of distinguishable bands provides a minimum estimate of the number of different viruses, while band position and staining intensity reveal the genome size distribution within the assemblage. With this technique we have detected spatial and temporal differences, as well as many similarities, in viral assemblages among a variety of marine habitats. Current efforts are directed toward combining this technique with other methods of fractionation and sequence analysis to allow both morphological and genetic description of uncultivated marine viruses. Direct investigation of dominant or particularly widespread viruses may ultimately provide clues as to which marine organisms contribute most to the viral pool, and which organisms are likely to be significantly influenced by viral mortality. [TOP OF PAGE]

194. Genome size distributions indicate variability and similarities among marine viral assemblages from diverse environments. Steward, G.F., Montiel, J.L., Azam, F. (2000). Limnol. Oceanogr. 45:1697-1706. Pulsed field gel electrophoresis (PFGE) was used to determine the size distributions of virus-like DNA in seawater from diverse environments (Arctic Ocean, Ross Sea, Coastal Pacific Ocean, and Northern Adriatic Sea). Changes in DNA banding patterns indicated that shifts in the viral assemblage composition occurred on the order of = 2 d during an intense dinoflagellate bloom in coastal Pacific waters. Different DNA banding patterns from diverse locations also indicated spatial variability in composition, but all of the samples analyzed had similar features. Size frequency distributions for virus-like genomes (VLGs) were multi-modal with major peaks occurring around 31-36 kilobases (kb) and 58-63 kb. The smallest discrete band resolved was 26 kb and the largest was >200 kb and the overall mean virus-like genome size was 50 ± 4 kb (mean ± sd, n = 30). On average, in surface seawater, > 90% of the VLGs occurred in the 26-69 kb size range and at least half were between 28 to 45 kb. This first extensive survey of viral genome sizes in seawater indicates that most marine viruses have physical properties similar to other known viruses. The distributions revealed that the vast majority of the detected VLGs have sizes typical of bacteriophages while only a few percent were in the size range of known algal viruses. [TOP OF PAGE]

195. Use of bioluminescent Salmonella for assessing the efficiency of constructed phage-based biosorbent. Sun, W., Brovko, L., Griffiths, M. (2000). Journal of Industrial Microbiology & Biotechnology 25:273-275. A bacteriophage-based biosorbent for Salmonella enteritidis was constructed, and bacterial bioluminescence was used for assessment of the efficiency of cell capture. A strain of S. enteritidis with bioluminescent phenotype was constructed by transformation with plasmid pT7 carrying the entire lux operon from Photorhabdus luminescens. The relation between relative light output (RLU) and colony-forming units (CFU/ml) of the bioluminescent strain was established. The bacteriophage specific to S. enteritidis was biotinylated, and the biotinylation procedure was optimized based on the maximum retention of phage infectivity. The biotinylated phages were then coated onto streptavidin-labeled magnetic beads, and were used to capture the bioluminescent S. enteritidis cells. Our preliminary results showed that the number of cells captured by constructed biosorbent was five times higher than that of the control, magnetic beads coated with nonbiotinylated phage, indicating the capture is specific. [TOP OF PAGE]

196. The ecology, evolutionary and geochemical consequences of viral infection of cyanobacteria and eukaryotic algae. Suttle, C.A. (2000). pp. 248-286. In In Hurst, C.J. (ed.), Viral Ecology. Academic Press, New York. [TOP OF PAGE]

197. Cyanophages and their role in the ecology of cyanobacteria. Suttle, C.A. (2000). pp. 563-589. In In Whitton, B.A. and Potts, M. (eds.), The Ecology of Cyanobacteria: Their Diversity in Time and Space. Kluwer Academic Publishers, Boston. [TOP OF PAGE]

198. Elements of a theory for the mechanisms controlling abundance, diversity, and biogeochemical role of lytic bacterial viruses in aquatic systems. Thingstad, T.F. (2000). Limnol. Oceanogr. 45:1320-1328. Mechanisms controlling virus abundance and partitioning of loss of bacterial production between viral lysis and protozoan predation are discussed within the framework of an idealized Lotka-Volterra-type model. This combines nonselective protozoan predation with host-selective viral lysis of bacteria. The analysis leads to a reciprocal relationship between bacterial diversity and viruses, in which coexistence of competing bacterial species is ensured by the presence of viruses that "kill the winner," whereas the differences in substrate affinity between the coexisting bacterial species determine viral abundance. The ability of the model to reproduce published observations, such as an approximate 1:10 ratio between bacterial and viral abundance, and the ability of viral lysis to account for 10-50% of bacterial loss are discussed. [TOP OF PAGE]

199. Use of the bacteriophage F44 for the search of the Erwinia horticola external suppressors. Tovkach, F.I., Gorb, T.E. (2000). Biopolimery i Kletka 16:64-68. Earlier we have separated from E. horticola the phage F44 which has the wide area of the host bacteria. The hydroxylamine has been used to obtain the F44 nonsense mutants. The selection of F44 nonsense mutants has been carried out on Escherichia coli having the definite suppressor (supE44/su2+). We have isolated spontaneous revertants His+ from the auxotrophic mutants of the E. horticola His- which have been obtained by the treatment of 2-aminopurine and hydroxylamine. The selection of the E. horticola suppressor strains has been based on the sensitivity of the revertants to the F44 and to its nonsense mutants. We have concluded that the use of the F44 as the test system for the search of the E. horticola external suppressors is sufficiently effective for such poorly investigated bacteria as Erwinia. [TOP OF PAGE]

200. Cost of host radiation in an RNA virus. Turner, P.E., Elena, S.F. (2000). Genetics 156:1465-1470. Although host radiation allows a parasite to expand its ecological niche, traits governing the infection of multiple host types can decrease fitness in the original or alternate host environments. Reasons for this reduction in fitness include slower replication due to added genetic material or modifications, fitness trade-offs across host environments, and weaker selection resulting from simultaneous adaptation to multiple habitats. We examined the consequences of host radiation using vesicular stomatitis virus (VSV) and mammalian host cells in tissue culture. Replicate populations of VSV were allowed to evolve for 100 generations on the original host (BHK cells), on either of two novel hosts (HeLa and MDCK cells), or in environments where the availability of novel hosts fluctuated in a predictable or random way. As expected, each experimental population showed a substantial fitness gain in its own environment, but those evolved on new hosts (constant or fluctuating) suffered reduced competitiveness on the original host, However, whereas evolution on one novel host negatively correlated with performance on the unselected novel host, adaptation in fluctuating environments led to fitness improvements in both novel habitats. [TOP OF PAGE]

201. A new Mesorhizobium loti HAMBI 1129 phage isolated from Polish soil. Turska-Szewczuk, A., Russa, R. (2000). Current Microbiology 40:341-343. Phage A1 isolated from the rhizosphere of Lotus corniculatus was studied. It had a very narrow host range, as it was active only against Mesorhizobium loti HAMBI 1129. Phage A1 was classified as belonging to C Bradley's group bacteriophages. The latent period of A1 was 120-130 min and a burst size 13-17 particles per cell. The nature of the phage receptor was examined. Lipopolysaccharide from the phage-sensitive strain inactivated phage A1 in contrast to LPS from the phage-resistant bacteria. Purified LPS obtained from M. loti HAMBI 1129 had a high receptor activity with PhI(50) value of 0. 025 microgram/ml. [TOP OF PAGE]

202. Characterization of AbiR, a novel multicomponent abortive infection mechanism encoded by plasmid pKR223 of Lactococcus lactis subsp. lactis KR2. Twomey, D.P., De, U.P., McKay, L.L., O'Sullivan, D.J. (2000). Appl. Environ. Microbiol. 66:2647-2651. The native lactococcal plasmid pKR223 encodes two distinct phage resistance mechanisms, a restriction and modification (R/M) system designated LlaKR2I and an abortive infection mechanism (Abi) which affects prolate-headed-phage proliferation. The nucleotide sequence of a 16,174-bp segment of pKR223 encompassing both the R/M and Abi determinants has been determined, and sequence analysis has validated the novelty of the Abi system, which has now been designated AbiR. Analysis of deletion and insertion clones demonstrated that AbiR was encoded by two genetic loci, separated by the LlaKR2I R/M genes. Mechanistic studies on the AbiR phenotype indicated that it was heat sensitive and that it impeded phage DNA replication. These data indicated that AbiR is a novel multicomponent, heat-sensitive, "early"-functioning Abi system and is the first lactococcal Abi system described which is encoded by two separated genetic loci. [TOP OF PAGE]

203. Fate of indigenous bacteriophage in a membrane bioreactor. Ueda, T., Horan, N.J. (2000). Water Res. 34:2151-2159. Indigenous bacteriophage was isolated from a conventional sewage treatment plant treating a largely domestic wastewater. The isolated phage was T-even-like with a mean size of 200 nm and was used to indicate the viral removal efficiency achieved in a bench-scale membrane bioreactor (MBR). The MBR incorporated three flat microfiltration membrane modules which were polyethylene with a pore size of 0.4 mum. When treating settled domestic sewage, the MBR achieved an overall removal rate of phage of 2.3-5.9 log across the treatment process. The membrane alone demonstrated a poor phage removal efficiency, but removal efficiency increased as the filtration resistance was increased. It was proposed therefore, that the biofilm accumulating on the surface of the membrane made a major contribution to phage removal. The MBR demonstrated an almost complete removal of faecal coliforms and faecal streptococci (up to 7 log). By comparison a full-scale treatment plant treating the same settled sewage and incorporating tertiary treatment, achieved only up to 2 log removal of the same excreted phage and bacteria. [TOP OF PAGE]

204. Construction of lux bacteriophages and the determination of specific bacteria and their antibiotic sensitivities. Ulitzur, S., Kuhn, J. (2000). Methods in Enzymology 305:543-557. [TOP OF PAGE]

205. Targeting of phage display vectors to mammalian cells. Uppala, A., Koivunen, E. (2000). Combinatorial Chemistry & High Throughput Screening 3:373-392. Phage display libraries offer a strategy to isolate peptide ligands to target proteins and to define potential interaction sites between proteins. Recent studies have indicated a novel utility for phage display in that bacteriophage engineered to express peptide ligands to specific cell surface receptors are internalized by mammalian cells. Thus, reporter genes such as green fluorescent protein and lacZ harbored in the phage genome can be delivered to mammalian cells using targeting peptides displayed on the surface of phage. There is also the possibility to generate novel types of peptide libraries expressed intracellularly using a phage capable of inducing expression of its coding genes in human cells. [TOP OF PAGE]

206. Killing of flies in electrocuting insect traps releases bacteria and viruses. Urban, J.E., Broce, A. (2000). Current Microbiology 41:267-270. Electrocuting insect traps (EIT) are popular devices frequently used by homeowners and food handlers attempting to localize the control of flying insects, including the ubiquitous house fly (Musca domestica L.). The traps contain a visual attractant and a high-voltage metal grid. Upon contact with the grids, the insects are disintegrated by the high voltage. As part of a systematic evaluation of EITs and their role in infectious disease spread, we quantitated spread of bacteria and a bacterial virus during electrocution of house flies. We loaded flies with Serratia marcescens or with the Escherichia coli phage PHIX174 and placed sprayed or fed flies into a room containing an EIT. While flies were being electrocuted, liberated particles and bacteria were assayed via agar plates or via air filtration samplers. Sprayed flies released one of every 10,000 of the added bacteria or viruses, and fed flies released one of every 1,000,000 of the consumed bacteria or viruses. Results of our studies suggest EITs could play a role in the spread of infectious disease agents, but the potential is influenced by the insect's route of contamination. [TOP OF PAGE]

207. Genus Chlorovirus (Phycodnaviridae). Van Etten, J.L. (2000). p. ???-??? AnonymousThe Springer Index of Viruses. Springer-Verlag, Berlin. [TOP OF PAGE]

208. Clay minerals protect bacteriophage PBS1 of Bacillus subtilis against inactivation and loss of transducing ability by UV radiation. Vettori, C., Gallori, E., Stotzy, G. (2000). Canadian Journal of Microbiology 46:770-773. The effect of UV radiation on the survival of and transduction by phage PBS1 of Bacillus subtilis, free or adsorbed on the clay minerals montmorillonite (M) and kaolinite (K), was studied. After free or clay-associated phage (similar to 10(7) PFU.mL(-1)) was irradiated with UV light (254 nm) for 0, 1, 2, 5, 10, and 30 min and then allowed to infect B. subtilis FB300 (thiB4 metA29 argF4 Rfm(r)), the phage was titered, and Met(+) transductants were enumerated on selective media. After 1 min of irradiation, the titer of free and clay-associated phage decreased significantly (similar to 1.6 times for free phage, and similar to 4.9 and 6.8 times for M and K, respectively), whereas the transduction frequency increased significantly (similar to 3 times for free phage and similar to 1.4 and 2.2 times for M and K, respectively). The titer and transduction frequency of clay-associated phage remain essentially constant between 1 and 10 min of irradiation, whereas the titer of free phage decreased by similar to 1 order of magnitude after 5 min of irradiation. When free phage was irradiated for 10 min, the titer and transduction frequency decreased by similar to 2 and 0.5 orders of magnitude, respectively, whereas 30 min of irradiation was necessary to obtain comparable decreases with clay-associated phage. These results indicated that phages are protected to some extent from UV radiation when adsorbed on clay minerals. [TOP OF PAGE]

209. Construction of mini-Tn10luxABcam/Ptac-ATS and its use for developing a bacteriophage that transduces bioluminescence to Escherichia coli O157:H7. Waddell, T.E., Poppe, C. (2000). FEMS Microbiol. Let. 182:285-289. Mini-Tn10luxABcam/Ptac-ATS was constructed in order to develop a luciferase-transducing bacteriophage for detecting Escherichia coli O157:H7. The transposon was designed to deliver a 3.6-kb insertion that confers n-decanal-dependent bioluminescence and resistance to chloramphenicol and was constructed using mini-Tn10cam/Ptac-ATS in the plasmid pNK2884 and luxAB from Vibrio harveyi. PhiV10, a temperate bacteriophage infecting common phage types of Escherichia coli O157:H7, was mutagenized as a prophage in E. coli O157:H7 strain R508. PhiV10::luxABcamA1-23 was rescued from the strain by propagating it on a strain lacking the bacteriophage and the vector containing the transposon. The bacteriophage transduced n-decanal-dependent bioluminescence to E. coli O157:H7 strain R508 that was measurable approximately 1 h post infection. [TOP OF PAGE]

210. Models of experimental evolution: The role of genetic chance and selective necessity. Wahl, L.M., Krakauer, D.C. (2000). Genetics 156:1437-1448. We present a theoretical framework within which to analyze the results of experimental evolution. Rapidly evolving organisms such as viruses, bacteria, and protozoa can be induced to adapt to laboratory conditions on very short human time scales. Artificial adaptive radiation is characterized by a list of common observations; we offer a framework in which many of these repeated questions and patterns can be characterized analytically. We allow for stochasticity by including rare mutations and bottleneck effects, demonstrating how these increase variability in the evolutionary trajectory. When the product Np, the population size times the per locus error rate, is small, the rate of evolution is limited by the chance occurrence of beneficial mutations; when Np is large and selective pressure is strong, the rate-limiting step is the waiting time while existing beneficial mutations sweep through the population. We derive the rate of divergence (substitution rate) and rate of fitness increase for the case when Np is large and illustrate our approach with an application to an experimental data set. A minimal assumption of independent additive fitness contributions provides a good fit to the experimental evolution of the bacteriophage phiX174. [TOP OF PAGE]

211. An explosive antisense RNA strategy for inhibition of a lactococcal bacteriophage. Walker, S.A., Klaenhammer, T.R. (2000). Appl. Environ. Microbiol. 66:310-319. The coding regions of six putative open reading frames (ORFs) identified near the phage phi31 late promoter and the right cohesive end (cos) of lactococcal bacteriophage phi31 were used to develop antisense constructs to inhibit the proliferation of phage phi31. Two middle-expressed ORFs (ORF 1 and ORF 2) and four late-expressed ORFs (ORF 3 through ORF 6) were cloned individually between the strong Lactobacillus P6 promoter and the T7 terminator (T(T7)) to yield a series of antisense RNA transcripts. When expressed on a high-copy-number vector from a strong promoter, the constructs had no effect on the efficiency of plaquing (EOP) or the plaque size of phage phi31. To increase the ratio of antisense RNA to the targeted sense mRNA appearing during a phage infection, the antisense cassettes containing the late-expressed ORFs (ORF 3 through ORF 6) were subcloned to pTRK360, a low-copy-number vector containing the phage phi31 origin of replication, ori31. ori31 allows for explosive amplification of the low-copy-number vector upon phage infection, thereby increasing levels of antisense RNA transcripts later in the lytic cycle. In addition, the presence of ori31 also lowers the burst size of phage phi31 fourfold, resulting in fewer sense, target mRNAs being expressed from the phage genome. The combination of ori31 and P6::anti-ORF 4H::T(T7) resulted in a threefold decrease in the EOP of phage phi31 (EOP = 0.11 +/- 0.03 [mean +/- standard deviation]) compared to the presence of ori31 alone (EOP = 0.36). One-step growth curves showed that expression of anti-ORF 4H RNA decreased the percentage of successful centers of infection (75 to 80% for ori31 compared to 35 to 45% for ori31 plus anti-ORF 4H), with no further reduction in burst size. Growth curves performed in the presence of varying levels of phage phi31 showed that ori31 plus anti-ORF 4H offered significant protection to Lactococcus lactis, even at multiplicities of infection of 0.01 and 0.1. These results illustrate a successful application of an antisense strategy to inhibit phage replication in the wake of recent unsuccessful reports. [TOP OF PAGE]

212. Morphology and general characteristics of phages specific for Astragalus cicer rhizobia. Wdowiak, S., MALEK, W., Gr, adka, M. (2000). Current Microbiology 40:110-113. Three newly isolated phages, K1, K2, and C1, specific for A. cicer rhizobia were characterized by their morphology, host range, rate of adsorption, restriction endonuclease patterns, and DNA molecular weights. All three phages were classified to the morphological group B of Bradley's (Siphoviridae family) on the basis of presence of hexagonal in outline heads and long noncontractile tails. Phages K1, K2, and C1 are related by host range and restriction endonuclease patterns. The molecular weights of phage DNAs estimated from restriction enzyme digests were in the range from 64.6 kb to 68.5 kb. [TOP OF PAGE]

213. Bacteriophage therapy of bacterial infections: An update of our institute's experience. Weber-Dabrowska, B., Mulczyk, M., Gorski, A. (2000). Archivum Immunologiae et Therapiae Experimentalis 48:547-551. 1307 patients with suppurative bacterial infections caused by multidrug-resistant bacteria of different species were treated with specific bacteriophages (BP). BP therapy was highly effective; full recovery was noted in 1123 cases (85.9%). In 134 cases (10.9%) transient improvement was observed and only in 50 cases (3.8%) was BP treatment found to be ineffective. The results confirm the high effectiveness of BP therapy in combating bacterial infections which do not respond to treatment with the available antibiotics. [TOP OF PAGE]

214. Effective phage therapy is associated with normalization of cytokine production by blood cell cultures. Weber-Dabrowska, B., Zimecki, M., Mulczyk, M. (2000). Archivum Immunologii et Therapiae Experimentalis 48:31-37. The aim of this study was to investigate the effect of phagotherapy on tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) serum levels and the ability of blood cells to produce these cytokines in culture. Fifty one patients with long-term, suppurative infections of various tissues and organs were enrolled. The ability of cells to secrete cytokines was tested using whole blood cell cultures, unstimulated or stimulated with lipopolysaccharide (LPS) from E. coli. In addition, cytokine serum levels were determined. Measurement of cytokine activity was performed using bioassays. We showed that TNF-alpha, but not IL-6 serum levels, were regulated upon division of patients into categories exhibiting initial: low, moderate and high cytokine levels. The low spontaneous production of IL-6 by blood cell cultures was elevated significantly on day 21 of phage therapy, whereas high release of this cytokine was inhibited. No such correlation was observed with LPS-induced IL-6 production in cell cultures when cells from low-, moderately- or highly-reactive patients were studied. Phage therapy modified TNF release according to the initial ability to produce that cytokine: it reduced TNF production in high responders and increased it in low responders. Patients infected only with Gram-positive bacteria demonstrated analogous changes in the spontaneous and LPS-induced TNF-alpha production as in the whole studied group. A similar kind of regulation was observed in TNF-alpha and LPS-induced production, i.e. low production was significantly elevated, high strongly inhibited, and moderate only slightly affected. In summary, we demonstrated for the first time that effective phage therapy can normalize TNF-alpha serum levels and the production of TNF-alpha and IL-6 by blood cell cultures. [TOP OF PAGE]

215. Design and evolution of artificial M13 coat proteins. Weiss, G.A., Sidhu, S.S. (2000). J. Mol. Biol. 300:213-219. Using simple design and selective pressure, we have evolved an artificial M13 bacteriophage coat protein. M13 coat proteins first reside in the bacterial inner membrane and subsequently surround the DNA core of the assembled virus. The artificial coat protein (ACP) was designed and evolved to mimic both functions of the natural M13 coat proteins, but with an inverted orientation. ACP is a non-functional coat protein because it is not required for the production of phage particles. Instead, it incorporates into a phage coat which still requires all the natural coat proteins for structural integrity. In contrast with other M13 coat proteins, which can display polypeptides as amino-terminal fusions, ACP permits the carboxy-terminal display of large polypeptides. The results suggest that viruses can co-opt host membrane proteins to acquire new coat proteins and thus new functions. In particular, M13 bacteriophage can be engineered for new functions, such as carboxy-terminal phage display. Copyright 2000 Academic Press. [TOP OF PAGE]

216. Experimental evolution recapitulates natural evolution. Wichman, H.A., Scott, L.A., Yarber, C.D., Bull, J.J. (2000). Philosophical Transactions of the Royal Society of London B Biological Sciences 355:1677-1684. Genomes of the closely related bacteriophages phiX174 and S13 are 5386 bases long and differ at 114 nucleotides, affecting 28 amino acids. Both parental phages were adapted to laboratory culture conditions in replicate lineages and analysed for nucleotide changes that accumulated experimentally. Of the 126 experimental substitutions, 90% encoded amino-acid changes, and 62% of the substitutions occurred in parallel in more than one experimental line. Furthermore, missense changes at 12 of the experimental sites were at residues differing between the parental phages; in ten cases the phiX174 experimental lineages were convergent with the S13 parent, or vice versa, at both the nucleotide and amino-acid levels. Convergence at a site was even obtained in both directions in three cases. These results point to a limited number of pathways taken during evolution in these viruses, and also raise the possibility that much of the amino-acid variation in the natural evolution of these viruses has been selected. [TOP OF PAGE]

217. Quantification of algal viruses in marine samples. Wilhelm, S.W., Poorvin, L. (2000). Methods in Microbiology 30:???-??? [TOP OF PAGE]

218. Bacterial carbon production in Lake Erie is influenced by viruses and solar radiation. Wilhelm, S.W., Smith, R.E.H. (2000). Canadian Journal of Fisheries and Aquatic Sciences 57:317-326. Bacterial production is an integral recycling mechanism that facilitates carbon flow through aquatic food webs. Factors influencing bacterial activity therefore impact carbon flow. Although ecologists consider grazing and dissolved organic carbon flux to be the major regulators of bacterial activity, we explored two other important pressures. Virus-like particle abundance ranged from 3.7 to 37.9 x 10¹⁰ L-1 in samples collected during August 1997 and July 1998. Bacterial abundance during these periods ranged from 1.8 to 4.6 x 10⁹ L-1. Based on electron microscopic analysis, viruses in Lake Erie would have been responsible for 12.1 to 23.4 % of bacterial mortality and, in quasi-steady state conditions, a comparable loss of bacterial productivity. In the central basin, solar radiation was also demonstrated to regulate bacterial productivity. Ultraviolet radiation (UVR, 295-400 nm) was shown to inhibit bacterial productivity according to a cumulative exposure kinetic model, and biological weighting functions were derived to enable calculation of time- and depth-integrated photoinhibition. The daytime photoinhibitory loss of bacterial carbon production was estimated to be 14 to 30% over the upper 5 m, primarily due to UVR > 320 nm. Viruses and sunlight are therefore of comparable importance as regulators of bacterial activity in this system. [TOP OF PAGE]

219. Virioplankton: viruses in aquatic ecosystems. Wommack, K.E., Colwell, R.R. (2000). Microbiology and Molecular Biology Reviews 64:69-114. [no abstract]. [TOP OF PAGE]

220. Phage conversion of exfoliative toxin A production in Staphylococcus aureus. Yamaguchi, T., Hayashi, T., Takami, H., Nakasone, K., Ohnishi, M., Nakayama, K., Yamada, S., Komatsuzawa, H., Sugai, M. (2000). Molecular Microbiology 38:694-705. The staphylococcal exfoliative toxins (ETs) are extracellular proteins that cause splitting of human skin at the epidermal layer during infection in infants. Two antigenically distinct toxins possessing identical activity have been isolated from Staphylococcus aureus, ETA and ETB. The gene for ETA (eta) is located on the chromosome, whereas that for ETB is located on a large plasmid. The observation that relatively few clinical isolates produce ETA suggests that the eta gene is acquired by horizontal gene transfer. In this study, we isolated a temperate phage (phiETA) that encodes ETA and determined the complete nucleotide sequence of the phiETA genome. phiETA has a head with a hexagonal outline and a non-contractile and flexible tail. The genome of phiETA is a circularly permuted linear double-stranded DNA, and the genome size is 43 081 bp. Sixty-six open reading frames (ORFs) were identified on the phiETA genome, including eta, which was found to be located very close to a putative attachment site (attP). phiETA converted ETA non-producing strains into ETA producers. Southern blot analysis of chromosomal DNA from clinical isolates suggested that phiETA or related phages are responsible for the acquisition of eta genes in S. aureus. [TOP OF PAGE]

221. Effect of alcohols on Escherichia coli phages. Yamashita, M., Murahashi, H., Tomita, T., Hirata, A. (2000). Biocontrol Science 5:9-16. Escherichia coli T even phages were ethanol sensitive, whereas T odd phages were ethanol tolerant. The phagecidal activities of alcohols on ethanol sensitive phages (T even, lambda) were revealed to be in the order n-propanol>ethanol>methanolmchgtn-butanolmchgtn-hexanolmchgtn-octanol . However, the those(?) of alcohols on ethanol tolerant phages (T odd) were revealed to be in the order methanol>ethanol>n-propanol. The phagecidal activity of n-butanol (hydrophobic) increased when small amounts of hydrophilic methanol (10 and 20%, v/v), ethanol (10 and 20%, v/v) were added. The relative solubility curves of glucose plus Sudan III (hydrophobicity-hydrophilicity indication) were similar to the lambda phagecidal activity curves of ethanol solution. The denaturation of ethanol-sensitive phage proteins with ethanol solution (50 or 70%, v/v) on SDS-polyacrylamide gel electrophoresis was associated with a loss of the plaque-forming activity of the phage. These phenomena were not observed for the ethanol-tolerant phages treated with ethanol solutions. The head proteins of ethanol-tolerant phage have a greater number of hydrophobic amino acids and fewer hydrophilic amino acids, whereas those of ethanol sensitive phage have a greater number of hydrophilic amino acids and fewer hydrophobic amino acids. From these data, we suggest that the hydrophobicity-hydrophilicity balance of the alcohol solution and the phage surface proteins affects the loss of the plaque-forming activity of E. coli phages by alcohol. [TOP OF PAGE]

222. An exfoliative toxin A-converting phage isolated from Staphylococcus aureus strain ZM. Yoshizawa, Y., Sakurada, J., Sakurai, S., Machida, K., Kondo, I., Masuda, S. (2000). Microbiology and Immunology 44:189-191. Exfoliative toxin A (ETA) causes staphylococcal scalded-skin syndrome in children. The gene for ETA was believed to be coded by the chromosomal DNA. We isolated temperate phages from an ETA-producing strain, ZM, using a restriction minus strain, 1039, as an indicator. One of the prophages, designated phi-ZM-1 mediated lysogenic conversion of ETA. The polymerase chain reaction assay of the eta gene revealed that phage phi-ZM-1 carries the structural gene for ETA. [TOP OF PAGE]

223. Phages will out: strategies of host cell lysis. Young, R., Wang, I.-N., Roof, W.D. (2000). Trends in Microbiology 8:120-128. Most phages accomplish host lysis using a muralytic enzyme, or endolysin, and a holin, which permeabilizes the membrane at a programmed time and thus controls the length of the vegetative cycle. By contrast, lytic single-stranded RNA and DNA phages accomplish lysis by producing a single lysis protein without muralytic activity. [TOP OF PAGE]

224. Characterization of the distal tail fiber locus and determination of the receptor for phage AR1, which specifically infects Escherichia coli O157:H7. Yu, S.L., Ko, K.L., Chen, C.S., Chang, Y.C., Syu, W.J. (2000). J. Bacteriol. 182:5962-5968. Phage AR1 is similar to phage T4 in several essential genes but differs in host range. AR1 infects various isolates of Escherichia coli O157:H7 but does not infect K-12 strains that are commonly infected by T4. We report here the determinants that confer this infection specificity. In T-even phages, gp37 and gp38 are components of the tail fiber that are critical for phage-host interaction. The counterparts in AR1 may be similarly important and, therefore, were characterized. The AR1 gp37 has a sequence that differs totally from those of T2 and T4, except for a short stretch at the N terminus. The gp38 sequence, however, has some conservation between AR1 and T2 but not between AR1 and T4. The sequences that are most closely related to the AR1 gp37 and gp38 are those of phage Ac3 in the T2 family. To identify the AR1-specific receptor, E. coli O157:H7 was mutated by Tn10 insertion and selected for an AR1-resistant phenotype. A mutant so obtained has an insertion occurring at ompC that encodes an outer membrane porin. To confirm the role of OmpC in the AR1 infection, homologous replacement was used to create an ompC disruption mutant (RM). When RM was complemented with OmpC originated from an O157:H7 strain, but not from K-12, its AR1 susceptibility was fully restored. Our results suggest that the host specificity of AR1 is mediated at least in part through the OmpC molecule. [TOP OF PAGE]

225. Interspecies lysogenization in staphylococci: Transfer of enterotoxin a converting bacteriophage from clinical strain of Staphylococcus aureus to Staphylococcus intermedius. Zabicka, D., Mlynarczyk, G., Luczak, M. (2000). Medycyna Doswiadczalna i Mikrobiologia 52:317-326. The ability of lysogenization was examined of 50 S. intermedius strains and of 77 strains belonging to 14 different species of coagulase-negative staphylococci using 8 enterotoxin A converting bacteriophages isolated from S. aureus. All the examined bacteriophages showed lytic activity against at least 1 of 11 susceptible strains of S. intermedius to them. Lytic activity towards coagulase-negative staphylococci was observed for 6 of 8 examined bacteriophages. Two bacteriophages were active against 1 of 9 examined S. capitis strains, one against 1 of 11 examined S. haemolyticus strains, four against 1 of 6 examined S. lugdunensis strains, three against 1 of 6 examined S. warneri strains and one against 1 of 5 examined S. xylosus strains. Lysogenization with bacteriophage f421-1 able to convert positively enterotoxin A and staphylokinase and negatively beta-haemolysin of one S. intermedius strain was successful. S. intermedius lysogenized with variant phi421-1 was able to produce both enterotoxin A and staphylokinase and lost ability to produce beta-haemolysin. Our results showed a broad lytic spectrum and interspecies host range of some S. aureus bacteriophages and the ability of interspecies transfer of bacteriophages between S. aureus and S. intermedius. [TOP OF PAGE]

226. Antirestriction. Zavilgelsky, G.B. (2000). Molekulyarnaya Biologiya (Moscow) 34:854-862. A review was made of the analysis of current data on the molecular mechanisms of antirestriction. Systems of inhibition of the enzymes of restriction-modification type 1 in bacteriophages and conjugative plasmids were examined, as well as the phenomenon of "DNA restriction weakening" and its mechanism in the bacteria of Escherichia coli. The principle of "protein mimicry of nucleic acids" as a new mechanism of regulating biological processes in the cell in its application to Ard proteins-antirestrictors was discussed. [TOP OF PAGE]

227. Quinolone antibiotics induce Shiga toxin-encoding bacteriophages, toxin production, and death in mice. Zhang, X., McDaniel, A.D., Wolf, L.E., Keusch, G.T., Waldor, M.K., Acheson, D.W. (2000). J. Infect. Dis. 181:664-670. Shiga toxin-producing Escherichia coli (STEC) cause significant disease; treatment is supportive and antibiotic use is controversial. Ciprofloxacin but not fosfomycin causes Shiga toxin-encoding bacteriophage induction and enhanced Shiga toxin (Stx) production from E. coli O157:H7 in vitro. The potential clinical relevance of this was examined in mice colonized with E. coli O157:H7 and given either ciprofloxacin or fosfomycin. Both antibiotics caused a reduction in fecal STEC. However, animals treated with ciprofloxacin had a marked increase in free fecal Stx, associated with death in two-thirds of the mice, whereas fosfomycin did not. Experiments that used a kanamycin-marked Stx2 prophage demonstrated that ciprofloxacin, but not fosfomycin, caused enhanced intraintestinal transfer of Stx2 prophage from one E. coli to another. These observations suggest that treatment of human STEC infection with bacteriophage-inducing antibiotics, such as fluoroquinolones, may have significant adverse clinical consequences and that fluoroquinolone antibiotics may enhance the movement of virulence factors in vivo. [TOP OF PAGE]

228. Prophage, phiPV83-pro, carrying panton-valentine leukocidin genes, on the Staphylococcus aureus P83 chromosome: comparative analysis of the genome structures of phiPV83-pro, phiPVL, phi11, and other phages. Zou, D., Kaneko, J., Narita, S., Kamio, Y. (2000). Biosci. Biotech. Biochem. 64:2631-2643. Staphylococcus aureus P83 has Panton-Valentine leukocidin (PVL)-like genes, lukM and lukF-PV. Here, lukM and lukF-PV genes were found on the genome of a prophage, which was designated as phiPV83-pro. The precise genome size was 45,636 bp with att core sequences of 10 base pairs. Sixty-four ORFs were identified on the phiPV83-pro genome, including two extra operons, lukM-lukF-PV and orfs63-64. The lukM-lukF-PV cluster was located 2.1 kb upstream of the attL site. The most striking feature of the phiPV83-pro genome was a constituent of at least 4 regions from phi11, phiPVL, and other phages, i.e., (i) att sites identical with those of phi11, (ii) a cos sequence and the genes encoding packaging and head proteins of phiPVL (occupied half region of phiPV83-pro), and (iii) the other two regions which showed no significant similarity with known phages (occupied about 40% of phiPV83-pro). Furthermore, two insertion sequences, ISSA1 and ISSA2 were integrated into attL site and orf44, respectively. PhiPV83-pro was not induced as phage particles from S. aureus P83 regardless of its treatment with mitomycin C. The insertion of ISSA1 into the attL site was one of the reasons of the failure of the induction of the phage particles by mitomycin C treatment of the strain P83. [TOP OF PAGE]

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