Bacteriophage Ecology Group Reference Abstracts (1997)
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© Phage et al.	last updated on Wednesday, December 26, 2001

1. [A virus at the service of health]. Anonymous (1997). SERVIR 45:332 [TOP OF PAGE]

2. Attenuated poliovirus, bacteriophage, and bromide transport through a coarse-grained aquifer, western Montana. Anonymous (1997). p.138Microbial contamination of groundwater supply wells causes 50% of the outbreaks associated with waterborne disease each year. The transport of the bacteriophages MS2, PRD1, OX174, the attenuated enterovirus poliovirus type-1 (CHAT strain), and bromide in a cold water, sand and gravel aquifer was studied under natural gradient conditions near Missoula, MT. The average transport velocity for bromide was 25-30 m/d. Bacteriophages were observed at concentrations of 10 (super 3) PFU/ml 40.5 m from the injection well. After 8 hours of transport approximately 97% of the injected attenuated poliovirus and 35-79% of the bacteriophages adsorbed to the aquifer material. Although adsorption occurs, a portion of the viruses appears to act conservatively creating breakthrough curves similar to bromide, though with long tails. Virus were persistent, as seeded viruses were observed 185 days after injection. [TOP OF PAGE]

3. Antibiotic Resistance: Origins, Evolution, Selection and Spread. Anonymous (1997). John Wiley & Sons, [TOP OF PAGE]

4. Disinfection of human enteric viruses on fomites. Abad, F.X., Pinto, R.M., Bosch, A. (1997). FEMS Microbiol. Let. 156:107-111. The virucidal action of several commercially available disinfectant preparations was assayed against hepatitis A virus and human rotavirus dried on polystyrene. Overall, the level of virus disinfection achieved was very poor, usually inducing less than 3 log titre reduction. Suspension tests performed with the same disinfectants showed different virus inactivation rates, thus failing to provide a reliable indication of the actual virus disinfection on fomites. In our studies, bacteriophages of Bacteroides fragilis proved to be a simple, cheap and reliable screening tool for the evaluation of virus disinfection on non-porous surfaces. The same conclusion cannot be drawn for poliovirus. [TOP OF PAGE]

5. Bacteriophage ecology. Ackermann, H.-W. (1997). pp. 335-339. In In Martins, M.T., Sato, M.I.Z., Tiedje, J.M., Hagler, L.C.N., Döbereiner, J., and Sanchez, P.S. (eds.), Progress in Microbial Ecology (Proceedings of Seventh International Symposium on Microbial Ecology). Brazilian Society for Microbiology, Bacteriophage are classified into 12 families. Phages are tailed or cubic, filamentous or pleomorphic. Phages occur in all parts of the bacteria world and in every possible habitat. Phage titers may attain 8 log/ml in seawater and 9-10 log/ml in rumen fluid. Somatic coliphages, F-RNA and Bacteriodes fragilis phages indicate sewage and/or fecal contamination. Industrial microbiology provides particular environments in which phages proliferate. [TOP OF PAGE]

6. A catalogue of T4-type bacteriophages. Ackermann, H.-W., Krisch, H.M. (1997). Archives of Virology 142:2329-2345. The T4-type of bacteriophages is broadly defined on the basis of particle morphology. It occurs in enterobacteria (125 representatives), acinetobacters, aeromonads, pseudomonads, and vibrios (16 isolates). In addition, 18 apparently unrelated phages with prolate heads and contractile tails are found in a wide range of bacteria. A descriptive catalogue of these phages is presented. The T4-type probably originated in precursors of enterobacteria. [TOP OF PAGE]

7. Taxonomic changes in tailed phages of enterobacteria. Ackermann, H.W., DuBow, M.S., Gershman, M., Karska-Wysocki, B., Kasatiya, S.S., Loessner, M.J., Mamet-Bratley, M.D., Regue, M. (1997). Archives of Virology 142:1381-1390. Out of 136 new phages, 80 (59%) are classified into 23 species according to morphology and physicochemical properties. Six new species are described and species beta 4, from a previous classification scheme, is renamed T1. The morphology of 36 phage species is schematically represented. [TOP OF PAGE]

8. Characterization of a novel bacteriophage in Fusobacterium varium. Andrews, D.M., Gharbia, S.E., Shah, H.N. (1997). Clinical Infectious Diseases 25 Suppl 2:S287-S288 [TOP OF PAGE]

9. Virus retention by a hydrophilic triple-layer PVDF microporous membrane filter. Aranha-Creado, H., Oshima, K., Jafari, S., Howard, G.J., Brandwein, H. (1997). PDA JOURNAL OF PHARMACEUTICAL SCIENCE AND TECHNOLOGY 51:119-124. Retention of bacteriophages (phi 6, PR772, T1, and PP7) and mammalian viruses (poliovirus and influenza A virus) by a hydrophilic triple layer PVDF microporous membrane, the Ultipor VF grade DV50 membrane, was evaluated. Challenges of membrane discs or pleated filter cartridges were performed at concentrations of 10(6)-10(8) PFU/mL in one or more of the following carrier fluids: water, saline, gelatin (0.1%) in phosphate buffer, Dulbecco's Modified Eagle Medium (MEM), and MEM supplemented with 10% fetal bovine serum (MEM + 10). The data demonstrate a minimum log titer reduction (LTR) of 6 for viruses larger than 50 nm irrespective of the carrier fluid. Protein transmission levels of greater than 95% for IgG and albumin were achieved. For integral pleated filter cartridges, correlation between a nondestructive integrity test (using the forward flow integrity test method) and virus retention was demonstrated. The Ultipor VF grade DV50 filter can be applicable in the manufacture of biologicals and biopharmaceuticals, where high protein transmission and consistent viral titer reduction are desired. [TOP OF PAGE]

10. Abundance of bacteriophages of enteric bacteria in different freshwater environments. Araujo, R., Lasobras, J., Puig, A., Lucena, F., Jofre, J. (1997). Water Science and Technology 35:11-12. The abundances of somatic coliphages, F-specific phages and B. fragilis phages were measured in freshwater environments with different levels of faecal pollution. In samples with recent pollution of domestic origin the numbers of the three groups of phages were highly correlated. In this set of samples B. fragilis phages were significantly outnumbered by F-specific and these by somatic coliphages. In waters with intermediate levels of pollution, coliphages were more abundant than phages infecting B. fragilis. The levels of the three groups of phages, which were very low, were similar in waters with persistent faecal pollution indicating that B. fragilis phages were most resistant to natural inactivation processes. [TOP OF PAGE]

11. Abundance of bacteriophages of enteric bacteria in different freshwater environments. Araujo, R., Lasobras, J., Puig, A., Lucena, F., Jofre, J. (1997). Water Science & Technology 35:125-128. The abundances of somatic coliphages, F-specific phages and B. fragilis phages were measured in freshwater environments with different levels of faecal pollution. In samples with recent pollution of domestic origin the numbers of the three groups of phages were highly correlated. In this set of samples B. fragilis phages were significantly outnumbered by F-specific and these by somatic coliphages. In waters with intermediate levels of pollution, coliphages were more abundant than phages infecting B. fragilis. The levels of the three groups of phages, which were very low, were similar in waters with persistent faecal pollution indicating that B. fragilis phages were most resistant to natural inactivation process. [TOP OF PAGE]

12. Phages of enteric bacteria in fresh water with different levels of faecal pollution. Araujo, R.M., Puig.A., Lasobras, J., Lucena, F., Jofre, J. (1997). Journal of Applied Microbiology 82:281-286. Levels of somatic and F-specific coliphages, and phages infecting Bacteroides fragilis were measured in 257 samples collected in different freshwater environments with different levels and characteristics of faecal pollution. In samples with recent pollution of domestic origin, the numbers of the three groups of phages were highly correlated, thus showing that their excretion is fairly constant. In this set of samples somatic coliphages, which were the most abundant, and F-specific coliphages outnumbered significantly Bact. fragilis phages. Normalized lines of the numbers of the three groups of phages in water samples and their sediments show that they settle similarly. The correlation between the values of the three groups of phages was not observed in waters with intermediate levels of pollution. An increase in the relative numbers of coliphages with respect to numbers of phages infecting Bact. fragilis was observed. In waters with persistent faecal pollution a dramatic change was recorded in the relative numbers of the different groups of phages. Phages infecting Bact. fragilis suffered the lowest reduction in numbers. [TOP OF PAGE]

13. Bacteriophages of enteric bacteria in drinking water, comparison of their distribution in two countries. Armon, R., Araujo, R., Kott, Y., Lucena, F., Jofre, J. (1997). Journal of Applied Microbiology 83:627-633. The presence of bacteriophages infecting enteric bacteria was tested in more than 1500 drinking water samples in Israel and Spain. Bacteriophages tested were somatic coliphages, F-specific bacteriophages and Bacteroides fragilis bacteriophages. The three groups of bacteriophage were isolated in 100 ml water samples by the presence/absence test with similar frequencies, which ranged from 4.4% for somatic coliphages to 6.1% for bacteriophages infecting Bact. fragilis. In contrast, the frequency of isolation of bacteriophages was significantly higher than the frequency of isolation of faecal coliforms, which averaged only 1.9%. No significant differences were observed between the frequencies of isolation between the samples tested in Spain and those tested in Israel. The percentage of groundwater samples containing faecal coliforms and somatic coliphages was reduced significantly by chlorination, despite known deficiencies. However, there was no effect on the occurrence of F-specific bacteriophages and Bact. fragilis bacteriophages. [TOP OF PAGE]

14. Synchronized disruption of Escherichia coli cells by T4 phage infection. Asami, K., Xing, X.H., Tanji, Y., Unno, H. (1997). Journal of Fermentation and Bioengineering 83:511-516. For development of an autolytic Escherichia coli protein expression system, T4 bacteriophage (T4)-mediated E. coli disruption was investigated. At least two types of E. coli cell lysis, "lysis from without" (LO) and "lysis from within" (LI), are known to be induced by T4. The efficiency of cell disruption was monitored by the release of beta-galactosidase from the cells. In the case of multiplicity of infection (m.o.i.) of 100, the infected cells were lysed without proliferation of the progeny phage (LO). When the cells were infected at a m.o.i. of 5, slow cell lysis (LI) was observed. The beta-galactosidase activity detected in the supernatant of the culture subjected to LO was the same as that in the lysate produced by chloroform treatment or sonication of the T4-uninfected culture, but about twice that in the supernatant of the culture subjected to LI. At a m.o.i. of 0.01, delayed onset of cell lysis, called lysis inhibition (LIN), was observed. However, the cells in LIN state were simultaneously lysed upon shifting of the temperature from 37 degree C to 0 degree C, which was accompanied by an increase in extracellular beta-galactosidase activity. [TOP OF PAGE]

15. Using microcosms to study gene transfer in aquatic habitats. Ashelford, K.E., Fry, J.C., Day, M.J., Hill, K.E., Learner, M.A., Marchesi, J.R., Perkins, C.D., Weightman, A.J. (1997). FEMS Microbiol. Ecol. 23:81-94. Aquatic habitats are important potential sites for gene transfer between indigenous bacteria and released genetically engineered microorganisms (GEMs). Legislation governing GEM release, and other practical considerations, have resulted in microcosms, of varying complexity, being used to study gene transfer in aquatic environments. This article reviews these microcosms, with particular emphasis on the more complex designs and, where possible, compares gene transfer results obtained in them with in situ studies. We conclude that microcosms can give results that are consistent with those obtained in situ and thus can be relied upon to give realistic predictions of in situ behaviour. [TOP OF PAGE]

16. Isolation and Characterization of a New Lactobacillus delbrueckii ssp.bulgaricus Temperate Bacteriophage. Auad, L., de Ruiz Holgado, A.A.P., Forsman, P., Alatossava, T., Raya, R.R. (1997). J. Dairy Sci. 80:2706-2712. Lactobacillus delbrueckii ssp. bulgaricus strain CRL 539 was shown to be lysogenic and inducible with mitomycin C. The conditions were determined for an optimal induction of temperate bacteriophage lb539 with mitomycin C as well as the sensitivity of lb539 to physical and chemical agents. Electron microscopy of lysates revealed bacteriophage particles with an isometric head of 47 nm and a noncontractile tail of 159 nm. Phage lb539 was classified within Bradley's B1 phage group and the Siphoviridae family. The host range of lb539 encompassed mainly Lactobacillus delbrueckii ssp. lactis strains; strain LKT (CNRZ 700) was the most sensitive for detection of lb539 lysates induced by mitomycin C. The lb539 genome is a linear, double-stranded DNA molecule of approximately 35 kbp. The presence of submolar fragments in restriction enzyme digests suggests that lb539 DNA may contain a pac site. Dot-blot experiments showed that the lb539 genome hybridized with the genomes of phages mv4 and LL-H, which are type phages of group a of L. delbrueckii ssp. phages. Restriction enzyme patterns and morphological features showed lb539 to be distinct from mv4 and LL-H. [TOP OF PAGE]

17. Morphology and protein pattern of bacteriophages isolated from type strains of Bacillus thuringiensis. Azizbekyan, K.R., Kuzin, A.I., Shamshina, T.N., Dobrzhanskaya, E.O. (1997). Mikrobiologiya 66:242-246. The lysogeny of eleven type strains of Bacillus thuringiensis was studied. Eight new phages were isolated from the variants B. thuringiensis var. tochigiensis, yunnanensis, colmeri, shandongiensis, neoleonensis, silo, mexicanensis, and toguchini belonging to the B1 morphotype. The phages that were isolated from B. thuringiensis var. tochigiensis, yunnanensis, shandongiensis, and mexicanensis possessed transverse disks at their tails and were classified into one morphological group. The protein patterns of the isolated phages were determined. [TOP OF PAGE]

18. New thermal inducible Streptomyces phages isolated from tropical soils. Balan, A., Padilla, G. (1997). Brazilian Journal of Genetics 20:547-552. Two new Streptomyces phages, variant phiBP1 and variant phiBP2, were isolated from tropical soil samples. These phages presented a large host range and developed both lytic and lysogenic responses in different Streptomyces species tested. Variations in the incubation temperature showed to be important in the development of the replication cycle. Increasing incubation temperature from 30degree C to 42degree C induced the lytic response of variant phiBP2 and lysogenic of variant phiBP1 in the host strain Streptomyces sp. WL6. variant phiBP1 and variant phiBP2 have icosahedral heads with long tails and were characterized in relation to morphology, G + C content, genome size and adsorption curve. [TOP OF PAGE]

19. Bacteriophage and microsphere transport in saturated porous media; forced-gradient experiment at Borden, Ontario. Bales, R.C., Gerba, C.P., Lenczewski, M.E., Li, S., Yeh, T.C.J. (1997). Water Resources Research 33:639-648. [TOP OF PAGE]

20. Bacteriophage therapy and prophylaxis: Rediscovery and renewed assessment of potential. Barrow, P.A., Soothill, J.S. (1997). Trends in Genetics 5:268-271. Bacteriophages were discovered 82 years ago. Claims for their use in the treatment of infections were not confirmed by early controlled trials, and the success of antibiotics superseded this potential use. However, recent studies have shown interesting therapeutic effects that warrant further investigation and development. [TOP OF PAGE]

21. Novel approaches to control of bacterial infections in animals. Barrow, P.A. (1997). ACTA VETERINARIA HUNGARICA 45:317-329. Bacterial infections of poultry remain of great importance world-wide in terms of economic effects and public health. They include infections caused by Salmonella, Escherichia coli, Campylobacter and Pasteurella. Through the introduction of rigid hygienic measures it is possible to breed and rear poultry free of these pathogens. However, the cost to the industry would be prohibitive and economically disastrous. Biological measures have been introduced albeit in a relatively empirical way. Antibiotic therapy and prophylaxis is used extensively with the associated problems of development of resistance. Killed vaccines are used but are not usually very effective. Live vaccines are increasingly becoming acceptable and studies are under way to increase our understanding of the pathogenesis of these infections so that vaccine development may become less empirical. Work with live vaccines to be used against Salmonella has shown that they may be administered orally to newly-hatched chicks. The vaccine strain colonises the gut extensively and prevents re-infection by other Salmonella strains by a genus-specific mechanism which is similar to that which occurs during down-regulation of bacterial growth in stationary-phase nutrient broth cultures. The mechanism of this phenomenon is currently being studied. This approach may also be applied to control Campylobacter infections. Bacteria of the Pasteurella group and E. coli may produce septicaemic infections in poultry. Recent work with K1+ E. coli infections in mice has shown that virulent bacteriophages may be used to treat or prevent septicaemias and meningitides. This work has been extrapolated to chickens with a similar degree of success and it suggests that some infections of this sort in animals and man may be amenable to this approach. In-bred lines of chickens have been found to vary greatly in their susceptibility to systemic Salmonella infections. This is probably mediated by one gene and the effect is dominant and not linked to sex or MHC. The mouse natural resistance gene (NrampI) does not appear to contribute greatly to this effect. Differences in the extent of gut colonisation by Salmonella in in-bred and out-bred lines can also be detected. These results are very exciting and open up opportunities for disease control for the future. [TOP OF PAGE]

22. Phage resistance in Mycobacterium smegmatis. Barsom, E.K. (1997). University of Pittsburgh. Bacteriophage infection requires a specific initial interaction with the outer surface of bacterial hosts, followed by a secondary interaction with a membrane bound receptor upon which phage DNA is injected into the host cell. Phages of the mycobacteria encounter a cell wall that is rich in unusual lipid-and sugar-containing components which form a highly ordered barrier that must be passed to gain access to the membrane. This work describes a gene of Mycobacterium smegmatis which confers resistance to the mycobacteriophages L5 and D29. The phage-resistance phenotype arises not from mutation but from elevated expression of the wild type gene. The product of this multicopy phage-resistance (mpr) gene is a membrane protein which may alter the structure of the host cell wall or membrane, thereby inhibiting productive phage DNA injection. [TOP OF PAGE]

23. The record of horizontal gene transfer in Salmonella. Bäumler, A.J. (1997). Trends in Microbiology 5:318-322. The evolution of virulence in Salmonella is driven by horizontal gene transfer. This has given rise to highly flexible pathogens that are able to colonize new niches and extend their host range. Tracing the record of horizontal gene transfer can provide clues to the virulence factors that contribute to the formation of new pathovars. [TOP OF PAGE]

24. Transduction of antibiotic resistance including imipenem resistance by wild type phages from nosocomial strains of Pseudomonas aeruginosa. Blahova, J., Kralikova, K., Krcmery, V.S., Mlynarcik, D., Trupl, J. (1997). Acta Virol. 41:293-296. In this report we describe transduction of antibiotic resistance determinants by three wild type bacteriophages isolated from three Pseudomonas aeruginosa strains. The strains showed evident plaques of a lysis caused by a bacteriophage. The strains were identified as lysogenic among 31 imipenem (IMP)-resistant P. aeruginosa strains isolated at the National Institute of Oncology in Bratislava. The carbenicillin (CAR) resistance determinant was transduced by all the three phages to four P. aeruginosa recipients - PAO-1670, ML-M-88, ML-1292 and ML-1008. The gentamicin (GEN) resistance was transduced to ML-1 008 only. The kanamycin (KAN) resistance was transduced in the following systems (combinations): "phageAP-37 to M-88", "phage AP-38 to PAO-1670, ML-1292 and M-88", and "phage AP-40 to M-88". The IMP resistance determinant was transduced by all the three phages to P. aeruginosa recipient strains. All transductant colonies were tested for the presence of directly not selected but co-transduced resistance determinants. Whereas transductants selected on media with IMP were resistant to five antibiotics (IMP, CAR, streptomycin (STR), KAN and GEN), transductants selected on CAR, KAN, STR, or GEN were resistant to a block of four of these antibiotics but not to IMP. [TOP OF PAGE]

25. Effect of resource enrichment on a chemostat community of bacteria and bacteriophage. Bohannan, B.J.M., Lenski, R.E. (1997). Ecology 78:2303-2315. We determined the responses of a model laboratory community to resource enrichment and compared these responses to the predictions of prey-dependent and ratio-dependent food chain models. Our model community consisted of Escherichia coli B and bacteriophage T4 in chemostats supplied with different concentrations of glucose. We observed the following responses to enrichment: (1) a large and highly significant increase in the equilibrium population density of the predator, bacteriophage T4, (2) a small but significant increase in the equilibrium population density of the prey, E. coli, and (3) a large and highly significant decrease in the stability of both the predator and prey populations. These responses were better predicted by a prey-dependent model (altered to include a time delay between consumption and reproduction by predators) than by a ratio-dependent model. Enrichment had a large effect on evolutionary change in our system. Enrichment significantly decreased the amount of time required for mutants of E. coli that were resistant to predation by bacteriophage to appear in the chemostats. Enrichment also significantly increased the rate at which these bacteriophage-resistant mutants invaded the chemostats. These results were also better predicted by the prey-dependent model. Invasion by bacteriophage-resistant mutants had a large effect on the subsequent population dynamics of both predator and prey. Both the equilibrium density and stability of the E. coli population increased following invasion, and the population shifted from being primarily limited by predators to being primarily limited by resources. After invasion by the mutants, the T4 population decreased in equilibrium density, and.the population cycled with an increased period. These results were compared to the predictions of a ratio-dependent model and a prey-dependent model altered to include T4-resistant mutants. The dynamics of this community were better predicted by the modified prey-dependent model; however, this model was more complex mathematically than the simpler ratio-dependent model. [TOP OF PAGE]

26. Physico-chemical properties of three phages of Pseudomonas syringae. Boiko, A.L., Semchuk, L.I., Tokarchuk, L.V., Romashev, S.A. (1997). Ukrainskii Biokhimicheskii Zhurnal 69:133-137. The properties of DNA for 9B, 123, 788/8 phages lysing phytopathogenic Pseudomonas syringae bacteria have been analysed with results presented. It was ascertained that their genomes consist of GC-type two-chain DNA molecules having molecular weight 14-15 mDa. It was shown that sedimentation coefficient for all three phage DNA is identical and equals 26S. GC-base percentage was calculated for the phage genomes. Its value, according to results of sedimentation analysis and melting temperature, was the same for 9B (51%, 57%) and 123 (51%, 57%) and differed for 788/8 (53%, 60%). The molecular weight of DNA phages calculated from the sum of fragments, obtained after genome splitting with restriction endonucleases is in agreement with the data of sedimentation analysis. The distribution of phage DNA fragments if electrophoregrams suggests the presence of numerous common restriction sites in the phages' genomes. [TOP OF PAGE]

27. Lysogeny in Xanthomonas campestris pv. erythrinae and X. campestris pv. azadirachtae and the behaviour of harboured temperate phages. Borkar, S.G. (1997). Journal of Mycology and Plant Pathology 27:283-285. Lysogenic strains harbouring temperate phages were detected in plant pathogenic bacterium Xanthomonas campestris pv. erythrinae and X. c. pv. azadirachtae. Thus, X. c. pv. erythrinae and X. c. pv. azadirachtae are an addition to the previous list of bacterium harbouring temperate phages. The lysis of these lysogenic strains occurred during its stationary growth phase and under ordinary cultural condition. The phenomenon of lysogeny i.e. occurrence of lysis due to harboured temperature phages, was influenced by temperature. Temperate phages released from X. c. pv. erythrinae and X. c. pv. azadirachtae were non-host-specific. These could attack the bacterium X. c. pv. glycineae and lysed it in a lytic manner. This indicated the change in behaviour of these temperate phages. [TOP OF PAGE]

28. Large-scale production and plaque titration of European chlorella viruses. Bornemann, C., Follmann, H. (1997). Journal of Virological Methods 67:119-125. [TOP OF PAGE]

29. Microbiological quality of natural waters. Borrego, J.J., Figueras, M.J. (1997). Microbiologia (Madrid) 13:413-426. Several aspects of the microbiological quality of natural waters, especially recreational waters, have been reviewed. The importance of the water as a vehicle and/or a reservoir of human pathogenic microorganisms is also discussed. In addition, the concepts, types and techniques of microbial indicator and index microorganisms are established. The most important differences between faecal streptococci-and enterococci have been discussed, defining the concept and species included. In addition, we have revised the main alternative indicators used to measure the water quality. [TOP OF PAGE]

30. Microorganism removal in wastewater stabilisation ponds in Maracaibo, Venezuela. Botero, L., Montiel, M., Estrada, P., Villalobos, M., Herrera, L. (1997). Water Science and Technology 35:205-209. Waste stabilisation ponds are an efficient means of wastewater treatment in many parts of the world wherever suitable land is available at reasonable cost and solar energy is an abundant energy resource. This study evaluated the removal of total coliforms TC, faecal coliforms FC and coliphages C in waste stabilisation ponds functioning as a pilot system in the tropical climate of Maracaibo, Venezuela. Sampling points included raw sewage and each pond effluent. Turbidity, pH and temperature were recorded. The results for raw sewage show average levels of 4.1 times 10-6 TC, 2.8 times 10-6FC and 7.0 times 10-5 C/100mL. Temperature, pH and turbidity ranges between 26-31 degree C, 6.2-9.5 and 15-98 NTU respectively. Removal of microorganisms in the three systems ranged between 93-98%. Despite the high removal efficiency of microorganisms, the final effluents showed average counts of 5.4 times 10-4-1.4 times 10-5 TC, 5.2 times 10-4-1.3 times 10-5 FC and 1.6 times 10-4-4.7 times 10-4 C/100mL. This study shows that the microbiological quality of the final effluents did not achieve the WHO water quality requirement for FC (10-3/100mL); therefore, they cannot be used for irrigation. Additional treatments, such as slow sand filtration, are needed in order to improve the quality of the water. [TOP OF PAGE]

31. The site-specific integration system of the temperate Streptococcus thermophilus bacteriophage fSfi21 [In Process Citation]. Bruttin, A., Foley S, Brussow, H. (1997). Virology 237:148-158. The temperate bacteriophage fSfi21 integrates its DNA into the chromosome of Streptococcus thermophilus strains via site- specific recombination. Nucleotide sequencing of the attachment sites identified a 40-bp identity region which surprisingly overlaps both the 18- terminal bp of the phage integrase gene and the 11-terminal bp of a host tRNAArg gene. A 2.4-kb phage DNA segment, covering attP, the phage integrase, and a likely immunity gene contained all the genetic information for faithful integration of a nonreplicative plasmid into the attB site. A deletion within the int gene led to the loss of integration proficiency. A number of spontaneous deletions were observed in plasmids containing the 2.4-kb phage DNA segment. The deletion sites were localized to the tRNA side of the identity region and to phage or vector DNA with 3- to 6-bp-long repeats from the border region. A similar type of deletion was previously observed in a spontaneous phage mutant. [TOP OF PAGE]

32. Characterization of the lysogeny DNA module from the temperate Streptococcus thermophilus bacteriophage f Sfi21. Bruttin, A., Desiere, F., Lucchini S, Foley S, Brussow, H. (1997). Virology 233:136-148. Phage f Sfi21, the only temperate Streptococcus thermophilus phage from our phage collection, showed extensive DNA homology with virulent phages from lytic group I. Southern blot hybridizations demonstrated that the f Sfi21- specific DNA was clustered in an approximately 6.6- kb-long region, the putative lysogeny module. Sequence analysis and database research identified an integrase within this module; orf 203 with homology to an anonymous orf 258 from the temperate lactococcal phage BK5-T; orf 127 and orf 122 with weak homology to the N- and C- terminal parts, respectively, of the cl-like repressor from lactococcal phages Tuc2009 and BK5-T; orf 75 with homology to a repressor protein from lambdoid phage 434 and an anti-repressor ant with homology to phage P1. The molecular arrangement of the predicted orfs in phage phi Sfi21 was very similar to that of the lactococcal phage BK5-T. The transition from f Sfi21-specific DNA into DNA shared with virulent phages was abrupt and flanked at one side by notable DNA repeats. Sequence analysis identified a holin protein to the left of the lysogeny module. A site-specific deletion of 2.4 kb, which reproducibly transformed f Sfi21 into a lytic phage, was localized in the lysogeny module. It was flanked at both sides by conspicuous DNA repeats. One repeat region reflected the DNA around the attP site, while the other reflected the putative genetic switch region between repressor and anti- repressor genes. S. thermophilus host Sfi1 transformed with a plasmid containing int and orf 203 showed resistance to superinfection by heterologous phages, but not by the homologous f Sfi21. Part of the int gene could be deleted without loss of this activity, while a deletion in orf 203 resulted in loss of the phage resistance. We speculate on the possibility of a bipartite immunity system for the control of lysogeny in f Sfi21. [TOP OF PAGE]

33. Molecular ecology of Streptococcus thermophilus bacteriophage infections in a cheese factory. Bruttin, A., Desiere, F., d'Amico N, Guerin JP, Sidoti J, Huni B, Lucchini S, Brussow, H. (1997). Appl. Environ. Microbiol. 63:3144-3150. A mozzarella cheese factory using an undefined, milk-derived Streptococcus thermophilus starter system was monitored longitudinally for 2 years to determine whether the diversity of the resident bacteriophage population arose from environmental sources or from genetic changes in the resident phage in the factory. The two hypotheses led to different predictions about the genetic diversity of the phages. With respect to host range, 12 distinct phage types were observed. With two exceptions, phages belonging to different lytic groups showed clearly distinct restriction patterns and multiple isolates of phages showing the same host range exhibited identical or highly related restriction patterns. Sequencing studies in a conserved region of the phage genome revealed no point mutations in multiple isolates of the same phage type, while up to 12% nucleotide sequence diversity was observed between the different phage types. This diversity is as large as that between the most different sequences from phages in our collection. These observations make unlikely a model that postulates a single phage invasion event and diversification of the phage during its residence in the factory. In the second stage of our factory study, a defined starter system was introduced that could not propagate the resident factory phage population. Within a week, three new phage types were observed in the factory while the resident phage population was decreased but not eliminated. Raw milk was the most likely source of these new phages, as phages with identical host ranges and restriction patterns were isolated from raw milk delivered to the factory during the intervention trial. Apparrently, all of the genetic diversity observed in the S. thermophilus phages isolated during our survey was already created in their natural environment. A better understanding of the raw-milk ecology of S. thermophilus phages is thus essential for successful practical phage control. [TOP OF PAGE]

34. Prophages and cryptic prophages. Campbell, A. (1997). pp. 23-29. In In de Bruin, F.J., Lupski, J.R., and Weinstock, G.M. (eds.), Bacterial Genomes, Physical Structure and Analysis. Chapman and Hall, New York. [TOP OF PAGE]

35. Conditionally replicating luciferase reporter phages: improved sensitivity for rapid detection and assessment of drug susceptibility of Mycobacterium tuberculosis. Carriere, C., Riska, P.F., Zimhony, O., Kriakov, J., Bardarov, S., Burns, J., Chan, J., Jacobs, W.J. (1997). Journal of Clinical Microbiology 35:3232-3239. TM4 is a lytic mycobacteriophage which infects mycobacteria of clinical importance. A luciferase reporter phage, phAE40, has been constructed from TM4 and was previously shown to be useful for the rapid detection and drug susceptibility testing of Mycobacterium tuberculosis. However, the lytic nature of the phage results in a loss of detectable light output and limits the sensitivity of detection. We describe several strategies aimed at improving the luciferase activity generated by TM4 luciferase phages, including (i) varying the position of the luciferase gene in the phage genome, (ii) isolating host-range mutants of the phage, and (iii) introducing temperature-sensitive mutations in the phage such that it will not replicate at the infecting temperature. Several new phages generated by these methods show increased intensity of luciferase production compared to the first-generation reporter phage phAE40, and one phage, phAE88, also demonstrates an enhanced duration of luciferase activity. This has allowed the detection of as few as 120 BCG cells and the determination of drug susceptibilities of M. tuberculosis in as little as 1 day. [TOP OF PAGE]

36. Isolation of Vibrio cholerae 0139 phages to develop a phage typing scheme. Chakrabarti, A.K., Ghosh, A.N., Sarkar, B.L. (1997). Indian Journal of Medical Research 105:254-257. Five V. cholerae 0139 phages isolated from different parts of India have been used for phage typing study. A strain isolated from Nagpur city (NPR-4) was used as the host for phage propagation. All but one of the 260 strains of V. cholerae 0139 were found to be typeable and could be clustered into 8 distinct phage types as revealed by lytic patterns. Phage type 1 was the predominant type (61.15%) followed by type 2 (18.46%). The strains isolated from Madras exhibited 7 out of 8 phage types. These newly isolated phages could be adopted for phage typing of V. cholerae 0139 strains as an epidemiological tool. [TOP OF PAGE]

37. Analysis of the DNA sequence, gene expression, origin of replication, and modular structure of Lactococcus lactis lytic bacteriophage. Chandry, P.S., Moore, S.C., Boyce, J.D., Davidson, B.E., Hillier, A.J. (1997). Molecular Microbiology 26:49-64. [TOP OF PAGE]

38. The advantage of sex in the RNA virus phi6. Chao, L., Tran, T.T. (1997). Genetics 147:953-959. When laboratory populations of the RNA bacteriophage phi6 are subjected to intensified genetic drift, they experience a decline in fitness. These experiments demonstrate that the average effect of mutations is deleterious, and they are used to suggest that Muller's ratchet can operate in these viruses. However, the operation of Muller's ratchet does not alone guarantee an advantage of sex. When phi6 populations were subjected to a series of bottlenecks of one individual and then crossed, the measured advantage of sex was not significant. To determine whether a small sample size, as opposed to allelism or another explanation, can account for the negative result, we repeated the phi6 experiments by crossing a larger set of populations. We found that bottlenecked populations of phi6 could recover fitness through mutations. However, hybrids produced by crossing the populations recovered an additional amount over the contribution of mutations. This additional amount, which represents an advantage of sex to phi6, was determined to be significantly greater than zero. These results provide indirect support for an advantage of sex through Muller's ratchet. However, we also use our experimental design and results to propose an alternative to Muller's ratchet as a model for the evolution of sex. [TOP OF PAGE]

39. Evolution of sex and the molecular clock in RNA viruses. Chao, L. (1997). Gene 205:301-308. Although there exist many hypotheses for the advantage of sexual reproduction, Muller's ratchet is one that has received recent attention as an explanation for the evolution of sex in RNA viruses. Muller's ratchet provides for an advantage of sex when the rate of deleterious mutations is high and population size is small. A small population size intensifies genetic drift, which can lead to the random loss of genomes that are free of deleterious mutations. Sex becomes advantageous because it can re-create, through genetic exchange, genomes with fewer or no mutations. RNA viruses may be subject to Muller's ratchet because they have very high mutation rates and they may experience genetic drift if their populations are forced through small bottlenecks during infection. This review discusses the results of laboratory studies examining the possibility of an advantage of sex through Muller's ratchet in RNA viruses. Data from studies of wild populations of RNA viruses are also considered, and a model is presented for how an observed pattern of molecular evolution (or the molecular clock) in wild populations may be explained by Muller's ratchet (or a similar process) and the addition of compensatory mutations to Ohta's model of evolution by slightly deleterious mutations. [TOP OF PAGE]

40. Generalized gene transfer by virus-like particles from marine bacteria. Chiura, H.X. (1997). Aquat. Microb. Ecol. 13:75-83. Spontaneous VLP (virus-like particle) production and VLP- mediated gene transfer into Escherichia coli AB1157 as recipient was demonstrated. Five marine isolates (Alc 096, Alc 233, Alc 252, Agrobacterium kieliense and Flavobacterium sp. 11604) were investigated for their potential to produce VLP as well as for the gene transfer capability of these VLPs to the E. coli recipient. These strains are classified as ubiquinone-10- possessing marine bacteria (Q10MB) in the 16s-rRNA Superfamily IV. VLPs were obtained from 100 h cultured broth of all strains examined. VLP-host ratio after 100 h growth culture was: Alc 233, 1.54; Alc 252, 1.26; Alc 096, 1.06; Flavobacterium sp. I1604, 0.69; and A. kieliense, 0.06. These ratios were smaller than those found in the marine environment. However, the spontaneously produced VLP number can be considered as high because the reported numbers are relatively low from coliphage lambda (0.005) and phage Mu ( apprx 0.0001). VLP-mediated gene transfer was examined using an auxotrophic mutant of E. coli (AB1157) with 4 amino acid deficiencies (leu, pro, his, arg) as recipient at multiplicity of infection (MOI) of 0.1. Through this treatment, VLPs showed lethal effect on the recipient. The survival rate of control was: Alc 096, 7%; Alc 252, 8%; A. kise and Flavobacterium sp. 11604) were investigated for their potential to produce VLP as weliense, 17%; Flavobacterium sp, I1604, 31%; and Alc 233,40%. At the same time, all the purified VLPs derived from these 5 strains successfully transferred genes to rescue genetic defects of the recipient. Overall average efficiency of VLP- mediated gene transfer at MOI of 0.1 was estimated to be between 2.62 times 10-3 and 3.58 times 10-5 per VLP particle. Loci of employed genetic markers were dispersed on the E. coli chromosome with mutual distance of 121, 1154, 1397 and 364 kb between them. Since VLPs from different sources showed similar gene transfer efficiency in respect to the genetic marker rescued, it is suggested that VLPs from Q10MB transferred genes as generalized transduction. These results indicate that the VLPs produced by certain marine bacteria may be an important element for both non- specific generalized horizontal gene transfer towards a broad range of bacterial hosts and population control in the marine environment. [TOP OF PAGE]

41. Characterization of bacteriophages from tox-containing, non-toxigenic isolates of Corynebacterium diphtheriae. Cianciotto, N.P., Groman, N.B. (1997). Microbial Pathogenesis 22:343-351. Non-toxigenic strains of Corynebacterium diphtheriae continue to cause disease within immunized populations. A subset of these corynebacteria carry the diphtheria toxin gene but in a cryptic form. To determine whether such strains might contribute to the re-emergence of functional toxin genes, the phages and tox mutations within three clone types were examined, tox-containing, beta-related phages were isolated from two of the strain types. The third isolate appeared to harbour a defective prophage. One of the tox- phages encoded truncated, yet enzymatically-active, forms of diphtheria toxin, suggesting that it had sustained a point mutation within the latter half of its toxin gene. In contrast, the other mutant phage did not elicit the production of either a cross-reacting material or an ADP-ribosylating activity. Complementation tests employing a series of double lysogens confirmed that the mutations responsible for the non-toxigenic phenotype of all of the phages were cis dominant. Given these findings, it is reasonable to hypothesize that tox+ genes can arise within human populations by either homologous recombination between two distinct tox- phages or spontaneous reversion within a single mutant allele. [TOP OF PAGE]

42. Reduced polysialic acid capsule expression in Escherichia coli K1 mutants with chromosomal defects in kpsF. Cieslewicz, M., Vimr, E. (1997). Molecular Microbiology 26:237-249. Neuroinvasive Escherichia coli K1 synthesizes and assembles a polysialic acid capsule virulence factor on the external leaflet of the outer membrane. This capsule functions in pathogenesis by blocking non-immune host defence mechanisms and acting as a relatively non-immunogenic molecular mimic of the polysialic acid chains found in high concentrations on neural cell adhesion molecules of the human embryo and neonate. The synthetic, regulatory and export components for capsule expression are encoded in three functionally distinct gene blocks or regions of the 20 kb kps 'pathogenicity island'. These regions are organized as two convergently transcribed operons inserted into the monocistronic tRNA gene, pheV. The six genes of the so-called region 1 operon are transcribed in the same direction as pheV, and at least four of these genes are required for polysialic acid export. Expression of this operon is thermoregulated by transcriptional control of its first gene, kpsF. To investigate the function of region 1 further, two independent chromosomal disruptions were engineered by inserting promoterless, terminatorless kanamycin or chloramphenicol resistance cassettes into the HindIII site of the kpsF coding sequence. The chromosomal insertions were regulated by temperature in the same way as the wild-type operon, demonstrating that this control mechanism remained intact in these mutants. Chemical, immunological and ultrastructural microscopical methods demonstrated that full-length polysialic acid chains were synthesized but not exported by the kpsF mutants. This phenotype was correlated with decreased plaque diameter when the mutants were infected with the capsule-specific bacteriophage K1F. The export defect could not be complemented in trans with kpsF+ containing its cis-regulatory region because of titration of an apparent positive regulator of region 1 expression, whereas complementation was observed with a plasmid expressing kpsF from a physiologically irrelevant promoter. An N-terminal polyhistidine peptide was attached to KpsF and used to purify the overproduced polypeptide. Antibodies raised against KpsF identified at least one of its paralogues in E. coli, GutQ, suggesting that KpsF and its homologues are membrane associated. The results indicate the requirement for a precise balance between region 1 components of the capsule export machinery, and that KpsF plays a positive role in the assembly, operation or regulation of this apparatus. [TOP OF PAGE]

43. INHIBITION OF LACTIC BACTERIOPHAGE PROLIFERATION BY HYDROLYZED BACTERIOPHAGE PEPTIDE (STREPTOCOCCUS LACTIS, PHAGE PEPTIDES). Clark-Safko, P.A. (1997). University of Kentucky. Bacteriophage infections are the number one problem in terms of milk, cheese, and quality losses, confronting the cheese industry today. Development of inhibitors that reduce plant phage titers would save the U.S. cheese industry millions of dollars. A peptide inhibitor was developed from bacteriophage c2 by hydrolyzing phage with 0.1% crude ficin at 26$/sp/circ$C for four hours. Ficin and cellular debris were removed using a 3000 mwco pressurized filter. Supernatant was dialyzed in 500 mwco dialysis tubing. Retentate was freeze-dried and stored at 2$/sp/circ$C. Commercial M17 medium (37.5g/L) with added peptide solids (2%) and CaCl$/sb2$ (0.1%) was used to test the susceptibility of Streptococcus lactis subsp. lactis C2 (4% inoculum) to c2 bacteriophage ($1/times10/sp7$ pfu/ml added 1 h after culture inoculum). Absorbance readings (600 nm) were taken every twenty minutes until a loss in absorbance curve was observed. Culture grown in the presence of phage peptide was slightly inhibited (P $<$.07). Time from phage inoculation to point where absorbency decrease began was longer (P $<$.01) when phage peptide was added to M17 medium. Inhibition of ml3 and whey cocktail phages was tested in M17 medium with and without c2 peptide. Time to lysis for phage m13 was not extended (p $>$.88). However, C2 time to lysis was extended (p $<$.15) when infected with whey cocktail phage. Phage peptide was added (1, 2, and 3%) to M17 medium containing C2 culture to determine the optimum peptide concentration for blocking phage proliferation without inhibiting culture growth. Time from phage inoculation to point of loss in absorbency (clearing) rapidly decreased when 1% peptide was added to the medium compared to the control. Time of growth before loss in absorbency was extended (compared to 1% medium) 60 min and 100 min when 2% and 3% peptide was added, respectively. The clearing curve (1% peptide medium) of C2 culture was uncharacteristic of most clearing curves and a large flocculation was observed at the bottom of the test broth suggesting that the culture had agglutinated. The c2 phage peptides delayed phage proliferation but enhanced culture agglutination problems. Therefore the c2 phage peptides may not be the best source of peptides to inhibit phage proliferation. [TOP OF PAGE]

44. Application and evaluation of the phage resistance- and bacteriocin-encoding plasmid pMRC01 for the improvement of dairy starter cultures. Coakley, M., Fitzgerald, G., Ros, R.P. (1997). Appl. Environ. Microbiol. 63:1434-1440. The conjugative 63-kb lactococcal plasmid pMRC01 encodes bacteriophage resistance and production of and immunity to a novel broad-spectrum bacteriocin, designated lacticin 3147 (M.P. Ryan, M.C. Rea, C. Hill, and R.P. Ross, Appl. Environ. Microbiol. 62:612-619, 1996). The phage resistance is an abortive infection mechanism which targets the phage-lytic cycle at a point after phage DNA replication. By using the genetic determinants for bacteriocin immunity encoded on the plasmid as a selectable marker, pMRC01 was transferred into a variety of lactococcal starter cultures to improve their phage resistance properties. Selection of resulting transconjugants was performed directly on solid media containing the bacteriocin. Since the starters exhibited no spontaneous resistance to the bacteriocin as a selective agent, this allowed the assessment of the transfer of the naturally occurring plasmid into a range of dairy starter cultures. Results demonstrate that efficient transfer of the plasmid was dependent on the particular recipient strain chosen, and while high-frequency transfer (10(-3) per donor) of the entire plasmid to some strains was observed, the plasmid could not be conjugated into a number of starters. In this study, transconjugants for a number of lactococcal starter cultures which are phage resistant and bacteriocin producing have been generated. This bacteriocin-producing phenotype allows for control of nonstarter flora in food fermentations, and the phage resistance property protects the starter cultures in industry. The 63-kb plasmid was also successfully transferred into Lactococcus lactis MG1614 cells via electroporation. [TOP OF PAGE]

45. Parallel molecular evolution of deletions and nonsense mutations in bacteriophage T7 [letter]. Cunningham, C.W., Jeng, K., Husti, J., Badgett, M.R., Molineux, I.J., Hillis, D.M., Bull, J.J. (1997). Molecular Biology and Evolution 14:113-116. [TOP OF PAGE]

46. Bacteriophages and bacteriocins. Day, M. (1997). In Barlow and Duerden (eds.), Topley and Wilson's Microbiology and Microbial Infections. [TOP OF PAGE]

47. A novel plasmid-encoded phage abortive infection system from Lactococcus lactis biovar. diacetylactis [published erratum appears in FEMS Microbiol Lett 1997 Mar 15;148(2):273]. Deng, Y.M., Harvey, M.L., Liu, C.Q., Dunn, N.W. (1997). FEMS Microbiol. Let. 146:149-154. A 16-kb plasmid (pND859) was identified from Lactococcus lactis biovar. diacetylactis UK12922 which encodes phage resistance to the small isometric phage 712 when tested in L. lactis LM0230. The gene encoding phage abortive infection, designated abi-859, was localized on a 1.2-kb region which consists of an open reading frame (ORF) of 846 bp preceded by a potential ribosome-binding site and a putative promoter region. A helix-turn-helix region typical of DNA-binding motifs was identified near the N-terminal of the abi-859 product, suggesting a possible interaction with the phage DNA. [TOP OF PAGE]

48. A novel plasmid-encoded phage abortive infection system from Lactococcus lactis biovar. diacetylactis. Deng, Y.M., Harvey, M.L., Liu, C.Q., Dunn, N.W. (1997). FEMS Microbiol. Let. 146:149-154. A 16-kb plasmid (pND859) was identified from Lactococcus lactis biovar. diacetylactis UK12922 which encodes phage resistance to the small isometric phage 712 when tested in L. lactis LM0230. The gene encoding phage abortive infection, designated abi-859, was localized on a 1.2-kb region which consists of an open reading frame (ORF) of 846 bp preceded by a potential ribosome-binding site and a putative promoter region. A helix-turn-helix region typical of DNA-binding motifs was identified near the N-terminal of the abi-859 product, suggesting a possible interaction with the phage DNA. [TOP OF PAGE]

49. Distribution of Vibrio vulnificus phage in oyster tissues and other estuarine habitats. Depaola, A., McLeroy, S., McManus, G. (1997). Appl. Environ. Microbiol. 63:2464-2467. Phages lytic to Vibrio vulnificus were found in estuarine waters, sediments, plankton, crustacea, molluscan shellfish, and the intestines of finfish of the U.S. Gulf Coast, but no apparent relationship between densities of V. vulnificus and its phages was observed. Phage diversity and abundance in molluscan shellfish were much greater than in other habitats. V. vulnificus phages isolated from oysters did not lyse other mesophilic bacteria also isolated from oysters. Both V. vulnificus and its phages were found in a variety of oyster tissues and fluids with lowest densities in the hemolymph and mantle fluid. These findings suggest a close ecological relationship between V. vulnificus phages and molluscan shellfish. [TOP OF PAGE]

50. A highly conserved DNA replication module from Streptococcus thermophilus phages is similar in sequence and topology to a module from Lactococcus lactis phages. Desiere, F., Lucchini S, Bruttin, A., Zwahlen MC, Brussow, H. (1997). Virology 234:372-382. A highly conserved DNA region extending over 5 kb was observed in Streptococcus thermophilus bacteriophages. Comparative sequencing of one temperate and 26 virulent phages demonstrated in the most extreme case an 18% aa difference for a predicted protein, while the majority of the phages showed fewer, if any aa changes. The relative degree of aa conservation was not homogeneous over the DNA segment investigated. Sequence analysis of the conserved segment revealed genes possibly involved in DNA transactions. Three predicted proteins (orf 233, 443, and 382 gene product (gp)) showed nucleoside triphosphate binding motifs. Orf 443 gp showed in addition a DEAH box motif, characteristically found in a subgroup of helicases, and a variant zinc finger motif known from a phage T7 helicase/primase. Tree analysis classified orf 443 gp as a distant member of the helicase superfamily. Orf 382 gp showed similarity to putative plasmid DNA primases. Downstream of orf 382 a noncoding repeat region was identified that showed similarity to a putative minus origin from a cryptic S. thermophilus plasmid. Four predicted proteins showed not only high degrees of aa identity (34 to 63%) with proteins from Lactococcus lactis phages, but their genes showed a similar topological organization. We interpret this as evidence for a horizontal gene transfer event between phages of the two bacterial genera in the distant past. [TOP OF PAGE]

51. Microbiological quality of coastal sea water of Alexandria, Egypt. Divizia, M., Ruscio, V., Donia, D., el, G.E., Elcherbini, E., Gabrieli, R., Gamil, F., Kader, O., Zaki, A., Renganathan, E., Pana, A. (1997). Annali di Igiene 9:289-294. The aim of the present study was to evaluate the quality of the seawater in Alexandria, Egypt. Samples were collected in 6 different points: Kayet Bay, El Shatby, Camp Cesar, Sporting, Beir Massoud and El Max. In total, 24 samples were analyzed. For each point the analysis included estimation of the following parameters: Esherichia coli, total coliform and fecal streptococci, Yersinia, Shigella, Salmonella, bacteriophages and enteric viruses. Just one sample (El Max) was positive for the presence of Salmonella, neither Shigella or Yersinia were isolated from any of the analyzed points. E. coli was identified in 10 samples while the ratio between total coliform and fecal streptococci showed variable results with the exception of El Max that resulted constantly high. Three samples were positive for the presence of enteric viruses: El Shatby, Beir Massoud and Sporting. The analysis of phages showed a variable pollution values. [TOP OF PAGE]

52. Bacteriophage-triggered defense systems: phage adaptation and design improvements. Djordjevic, G.M., Klaenhammer, T.R. (1997). Appl. Environ. Microbiol. 63:4370-4376. A novel bacteriophage defense system, based on an inducible suicide gene, was challenged with a lactococcal bacteriophage to investigate the potential for phage adaptation. The defense system was encoded by pTRK414H, a high-copy-number replicon encoding a tightly regulated phi 31p trigger promoter fused to the lethal LlaIR+ restriction endonuclease cassette. Repeated transfers of Lactococcus lactis NCK690(pTRK414H) in the presence of phi 31 selected for phage phi 31 derivatives which were markedly less sensitive to phi 31p-LlaIR(+)-encoded restriction than the parental phage, phi 31. The efficiency of plaquing (EOP) on L. lactis NCK690(pTRK414H) was 10(-4) for phi 31 versus 0.4 for the derived phages. The mutant phages remained fully sensitive to LlaIR+ restriction, suggesting an alteration in the recognition or firing of the phi 31p promoter. Sequencing over the promoter region in four mutant phages revealed the identical C-to-A transversion, generating a Phe-to-Leu substitution, in a transcriptional activator of the phi 31p promoter, designated ORF2. The mutant phages were analyzed for their ability to induce the native phi 31p promoter element fused to a lacZst reporter gene. Compared to the parental phage, phi 31, lower levels of beta-galactosidase activity were induced throughout the lytic cycle, indicating that the strength at which the mutant phages activated the phi 31p promoter was altered. Based on these observations, improvements were made in promoter strength and restriction activity in an attempt to elevate the effectiveness of the phage-triggered suicide system. When the phi 31p-LlaIR+ cassette was paired with other abortive defense systems, Per31 and AbiA, the EOP of phi 31 was reduced to < 10(-10) and the level of phage in the culture was lowered below the detection limits of the assay. [TOP OF PAGE]

53. Development of a triggered suicide system for bacteriophage defense (Guanylyl transferase, restriction endonuclease, phage inducible, Lacotococcus lactis). Djordjevic, G.M. (1997). North Carolina State University. A novel bacteriophage defense mechanism was developed for Lactococcus lactis where an inducible phage promoter was used to activate bacterial suicide system after the infection. The $Lla[/rm IR]/sp+$ restriction endonuclease was exploited as a lethal gene of a phage-inducible suicide system. When expressed from a constitutive promoter, the $Lla[/rm IR]/sp+$ endonuclease was lethal across a wide range of Gram-positive bacteria, including L. lactis. Lethality of the $Lla[/rm IR]/sp+$ was exploited to develop several novel, positive selection cloning vectors for these organisms. A middle, phage-inducible promoter $/rm(/phi31P)$ from lytic lactococcal bacteriophage $/phi31$ was cloned upstream of the $Lla[/rm IR]/sp+$ on the high-copy plasmid (pTRK414H). When L. lactis (pTRK414H) was infected with $10/sp7$ pfu/ml phage in broth culture, at multiplicity of infection (MOI) of 0.1, no lysis was observed and the culture developed normally. The efficiency of plaquing (EOP) for $/phi31$ on L. lactis (pTRK414H) was lowered to $10/sp[-4].$ Center of infection assays revealed that 85% of the infected L. lactis (pTRK414H) cells did not release progeny phage. The burst size of $/phi31$ in L. lactis (pTRK414H) was 41, four-fold lower than in control cells. The $/phi31[/rm P]/Lla[/rm IR]/sp+$ cassette also inhibited four $/phi31$-recombinant derivatives, at levels at least ten-fold greater than $/phi31.$ However, mutant phages could be enriched that were less sensitive to the $/phi31[/rm P]/Lla[/rm IR]/sp+$-encoded restriction. These phages were altered in the strength and timing of $/phi31/rm P$ induction. The efficiency of the $/phi31[/rm P]/Lla[/rm IR]/sp+$-based suicide system was improved by increasing promoter strength, providing a restriction enhancer llaIC, and by combination with other abortive defenses, per31 and abiA. When either per31 or abiA were combined with llaIC and $/phi31[/rm P]/Lla[/rm IR]/sp+,$ the EOP was reduced to $[<]10/sp[-10]$ and virulent phages were eliminated from the infected population. Broader application of bacterial suicide systems depends on the availability of phage-specific promoters. A rapid method for isolation of these promoters, based on the 'capping' activity of the vaccinia virus guanylyltransferase, was developed in this study and used successfully to identify a phage-specific promoter from lytic lactococcal bacteriophage sk1. [TOP OF PAGE]

54. A triggered-suicide system designed as a defense against bacteriophages. Djordjevic, G.M., O'Sullivan, D.J., Walker, S.A., Conkling, M.A., Klaenhammer, T.R. (1997). J. Bacteriol. 179:6741-6748. A novel bacteriophage protection system for Lactococcus lactis based on a genetic trap, in which a strictly phage-inducible promoter isolated from the lytic phage phi31 is used to activate a bacterial suicide system after infection, was developed. The lethal gene of the suicide system consists of the three-gene restriction cassette LlaIR+, which is lethal across a wide range of gram-positive bacteria. The phage-inducible trigger promoter (phi31P) and the LlaIR+ restriction cassette were cloned in Escherichia coli on a high-copy-number replicon to generate pTRK414H. Restriction activity was not apparent in E. coli or L. lactis prior to phage infection. In phage challenges of L. lactis(pTRK414H) with phi31, the efficiency of plaquing was lowered to 10(-4) and accompanied by a fourfold reduction in burst size. Center-of-infection assays revealed that only 15% of infected cells released progeny phage. In addition to phage phi31, the phi31P/LlaIR+ suicide cassette also inhibited four phi31-derived recombinant phages at levels at least 10-fold greater than that of phi31. The phi31P/LlaIR+-based suicide system is a genetically engineered form of abortive infection that traps and eliminates phages potentially evolving in fermentation environments by destroying the phage genome and killing the propagation host. This type of phage-triggered suicide system could be designed for any bacterium-phage combination, given a universal lethal gene and an inducible promoter which is triggered by the infecting bacteriophage. [TOP OF PAGE]

55. Survival and transport of selected bacterial pathogens and indicator viruses under sandy aquifer conditions. Dowd, S.E., Pillai, S.D. (1997). Journal of Environmental Science and Health Part A Environmental Science and Engineering & Toxic and Hazardous Substance Control 32:2245-2258. Microbial contamination of groundwater is a serious threat to public health along the US-Mexico border. The survival and transport of two pathogenic bacteria (Salmonella typhimurium and Klebsiella sp.) and two indicator viruses (bacteriophages PRD-1 and MS-2), were studied using microcosms to determine the behavior of pathogenic microorganisms in the Rio Grande alluvium, which underlies the border between the United States and Mexico. Culturable populations of Salmonella typhimurium declined rapidly ( gt 5 log units) to below detection limits within 12 days, while as much as 10-3 CFU/mL of Klebsiella sp. was viable even after 30 days. Less than 1% of the surviving Salmonella sp. population was viable based on microscopic viability assays. The population of both MS-2 and PRD-1 also declined rapidly ( gt 6 log units) to below detection limits within 10 days. Salmonella sp. exhibited a relatively greater "straining" or adsorption than the Klebsiella sp. under saturated conditions in a 0.2 m column. Even after flushing with 6 pore volumes, as much a 10-3 CFU/mL of both bacterial genera were obtainable from the columns, Both MS-2 and PRD-1 exhibited little straining or adsorption within the 0.2 m columns. [TOP OF PAGE]

56. Natural protection of spring and well drinking water against surface microbial contamination. II. Indicators and monitoring parameters for parasites. Edberg, S.C., Leclerc, H., Robertson, J. (1997). CRITICAL REVIEWS IN MICROBIOLOGY 23:179-206. Recent outbreaks of cryptosporidiosis and reports of other newly described para-sitic diseases associated with drinking water transmission prompted a reevaluation of source water monitoring criteria for public health protection. The field of microbial indicators was reviewed and each candidate sentinel evaluated in terms of its sensitivity, specificity, and technical feasibility. In addition, a clear distinction was made between source water monitoring and monitoring in the distribution system. Of all potential candidate microbial sentinels, Escherichia coli is deemed the most efficacious for public health protection. Based on a conservative estimate of its half-life in groundwater for 8 d, it is recommended that at least two samples be obtained during this half-life. In addition to E. coli, two water quality indicator sentinels, which are not necessarily direct public health threats, should also be monitored at the same frequency. These are the total coliform group and the enterococci. If E. coli is present in any source water sample, the borehole and any directly connected borehole should be embargoed. If either total coliforms or enterococci are detected, only that individual borehole should be taken off line and not used until the situation is remediated and the cause of the fecal contamination eliminated. Clostridium perfringens spores serve as a useful long-lived indicator. However, their perseverance in a sample should not be considered a direct public health threat because spores may far outlive pathogens. As a parasite indicator, C. perfringens should have the same importance as a positive coliform or enterococcus analysis. Coliphages do not yet fulfill enough of the criteria to be routinely employed. Biological monitoring should be coupled with physicochemical monitoring to establish a long-term history of the source. Because all natural waters vary in the amounts of heterotrophic plate count bacteria, test methods should be employed that are refractory to them. A combination of rigorous source protection plus extraordinary source monitoring serve as sufficient multiple barriers for parasite protection. [TOP OF PAGE]

57. Characterization of filamentous phages of Vibrio cholerae O139 and O1. Ehara, M., Shimodori, S., Kojima, F., Ichinose, Y., Hirayama, T., Albert, M.J., Supawat, K., Honma, Y., Iwanaga, M., Amako, K. (1997). FEMS Microbiol. Let. 154:293-301. We have analyzed our collection of Vibrio cholerae O139 strains to determine whether filamentous phages are produced in their culture supernatants, and whether any replicative form of DNA is detectable in cell lysates. Two types of filamentous phage, designated fs1 (6.4 kb) and fs2 (8.5 kb), were found in strains of Vibrio cholerae O139, fs1 was commonly produced from clinical isolates of Vibrio cholerae O1. Infectious particles (filamentous phages) were inducible by subculture, mitomycin C, and cultivation in a ligated ileal loop of a rabbit. Type 4 fimbriae of Vibrio cholerae O1 sensitive to D-glucose and D-mannose were suggested to be receptors for fs1 and fs2. The genome of fs1 was revealed to encode a potential new enterotoxin homologous to zonula occludens toxin. Clarification of the relation of type 4 fimbriae and these filamentous phages will provide a new understanding of the colonization of Vibrio-cholerae O1 and O139. Thus the presence of a new enterotoxin encoded by the genome of filamentous phage like fs1 may clarify the pathogenesis of cholera toxin negative clinical isolates of Vibrio cholerae O1 and non-O1. Our findings combined with the earlier report by Ehara et al. [Microbio. Immunol. 37 (1993) 679-688] suggest that type 4 fimbriae of Vibrio cholerae O1 are important for the development of an effective vaccine against cholera. [TOP OF PAGE]

58. Phenotypic and genetic characterization of the bacteriophage abortive infection mechanism AbiK from Lactococcus lactis. Emond, E., Holler, B.J., Boucher, I., Vandenbergh, P.A., Vedamuthu, E.R., Kondo, J.K., Moineau, S. (1997). Appl. Environ. Microbiol. 63:1274-1283. The natural plasmid pSRQ800 isolated from Lactococcus lactis subsp. lactis W1 conferred strong phage resistance against small isometric phages of the 936 and P335 species when introduced into phage-sensitive L. lactis strains. It had very limited effect on prolate phages of the c2 species. The phage resistance mechanism encoded on pSRQ800 is a temperature-sensitive abortive infection system (Abi). Plasmid pSRQ800 was mapped, and the Abi genetic determinant was localized on a 4.5-kb EcoRI fragment. Cloning and sequencing of the 4.5-kb fragment allowed the identification of two large open reading frames. Deletion mutants showed that only orf1 was needed to produce the Abi phenotype. orf1 (renamed abiK) coded for a predicted protein of 599 amino acids (AbiK) with an estimated molecular size of 71.4 kDa and a pl of 7.98. DNA and protein sequence alignment programs found no significant homology with databases. However, a database query based on amino acid composition suggested that AbiK might be in the same protein family as AbiA. No phage DNA replication nor phage structural protein production was detected in infected AbiK+ L. lactis cells. This system is believed to act at or prior to phage DNA replication. When cloned into a high-copy vector, AbiK efficiency increased 100-fold. AbiK provides another powerful tool that can be useful in controlling phages during lactococcal fermentations. [TOP OF PAGE]

59. Intracellular kinetics of a growing virus: A genetically-structured simulation for bacteriophage T7. Endy, D., Kong, D., Yin, J. (1997). Biotechnology and Bioengineering 55:375-389. Viruses have evolved to efficiently direct the resources of their hosts toward their own reproduction. A quantitative understanding of viral growth will help researchers develop antiviral strategies, design metabolic pathways, construct vectors for gene therapy, and engineer molecular systems that self-assemble. As a model system we examine here the growth of bacteriophage T7 in Escherichia coli using a chemical-kinetic framework. Data published over the last three decades on the genetics, physiology, and biophysics of phage T7 are incorporated into a genetically structures simulation that accounts for entry of hte T7 genome into its host, expression of T7 genes, replication of T7 DNA, assembly of T7 procapsids, and packaging of T7 DNA to finally produce intact T7 progeny. Good agreement is found between the simulated behavior and experimental observations for the shift in transcription capacity from the host to the phage, the initation times of phage protein synthesis, and the intracellular assembly of both wild-type phage and a fast-growing deletion mutant. The simulation is utilized to predict the effect of antisense molecules targetted to different T7 mRNA. Further, a postulated mechanism for the down regulation of T7 transcription in vivo is quantitatively examined and shown to agree with available data. The simulation is found to be a useful tool for exploring and understanding the dyanamics of virus growth at the molecular level. [TOP OF PAGE]

60. Development and application of a genetically-structured simulation for bacteriophage T7. Endy, D. (1997). Darmouth College. [TOP OF PAGE]

61. Mycobacteriophage L5 infection of Mycobacterium bovis BCG: implications for phage genetics in the slow-growing mycobacteria. Fullner, K.J., Hatfull, G.F. (1997). Molecular Microbiology 26:755-766. Mycobacteriophage L5 is a well-characterized temperate phage that forms stable lysogens in Mycobacterium smegmatis. The host range of L5 is, however, unclear because previous reports suggested that it does not infect slow-growing mycobacteria such as Mycobacterium tuberculosis and bacille Calmette-Guerin (BCG). Moreover, luciferase reporter phage derivatives of L5 failed to produce light from BCG, suggesting that infection is blocked at or before the stage of DNA injection. In this study, we demonstrate that L5 infection of slow growing mycobacteria specifically requires a high concentration of Ca2+, conditions that differs from those required for infection of M. smegmatis by L5 and for infection of BCG by the closely related phage D29. In addition, we show that there are specific genetic determinants of L5 that confer the ability to infect slow growing mycobacteria, without altering infection of M. smegmatis. These observations extend the use of phage L5 for the diagnosis and analysis of tuberculosis and other mycobacterial diseases. [TOP OF PAGE]

62. Chlorella virus PBCV-1 encodes a homolog of the bacteriophage T4 UV damage repair gene denV. Furuta, M., Schrader, J.O., Schrader, H.S., Kokjohn, T.A., Nyaga, S., McCullough, A.K., Lloyd, R.S., Burbank, D.E., Landstein, D., Lane, L., Van Etten, J.L. (1997). Appl. Environ. Microbiol. 63:1551-1556. The bacteriophage T4 denV gene encodes a well-characterized DNA repair enzyme involved in pyrimidine photodimer excision. We have discovered the first homologs of the denV gene in chlorella viruses, which are common in fresh water. This gene functions in vivo and also when cloned in Escherichia coli. Photodamaged virus DNA can also be photoreactivated by the host chlorella. Since the chlorella viruses are continually exposed to solar radiation in their native environments, two separate DNA repair systems, one that functions in the dark and one that functions in the light, significantly enhance their survival. [TOP OF PAGE]

63. Polymorphism of bacteriophage T7. Gabashvili, I.S., Khan, S.A., Hayes, S.J., Serwer, P. (1997). J. Mol. Biol. 273:658-667. For viruses made of nucleic acid and protein, the structure of the protein outer shell has, in the past, been found to be uniquely determined by the viral genome. However, here, non-denaturing agarose gel electrophoresis of bacteriophage T7 reveals two states of the mature T7 capsid; the conditions of growth are found to alter the population by T7 of these two electrophoretically defined states. Both states have been previously observed for a genetically altered T7 and they are observed here for wild-type T7. The average electrical surface charge density of a bacteriophage particle (delta) determines its state; the delta of particles in both states is negative. For a given condition of growth, the population of these two states is influenced by the extent to which the major T7 outer shell protein, p10A, is accompanied by its minor readthrough variant, p10B. Comparison of the two electrophoretic states reveals the following. (1) No difference in radius is present in the outer shell (+/-2%). (2) As the pH of electrophoresis is either increased or decreased from neutrality, the state becomes more highly populated for which delta is greater in magnitude (state 1). By changing the pH, some T7 particles are made to change state. (3) Particles in state 1 adsorb less quickly to host cells than do the particles in the alternative state (state 2). This latter observation suggests the hypothesis that state 1 evolved to reduce the probability of re-initiating an infection when conditions are not favorable for growth. This hypothesis is supported by the observation that, as conditions of growth become apparently more unfavorable, progeny increasingly populate state 1. [TOP OF PAGE]

64. Evaluation of the wastewater treatment plant of Rome airport. Gabrieli, R., Divizia, M., Donia, D., Ruscio, V., Bonadonna, L., Diotallevi, C., Villa, L., Manzone, G., Pana, A. (1997). Water Science and Technology 35:193-196. The wastewater plant of Rome airport, which receives all the sewage from the airport as well as the cess from aeroplanes, was analysed for microbiological parameters. From the bacteriological point of view, in the water and sludge samples the densities of the faecal indicator of pollution and the presence of Salmonella spp and Vibrio cholerae as bacteriological pathogens were determined. At the same time, samples were analysed for the presence of enteric viruses and phages. Overall, the mean reduction of the faecal coliforms was 96%, E. coli 92% and faecal streptococci 99%. Salmonella spp was identified in all but one of the final effluents and V. cholerae in 2/10. Enteric viruses were identified in all but one of the raw waters and in three samples of final effluent. Bacteriophages (somatic coliphage, F-plus phage and B40-8), were found in all the samples but irregularly. Phages and enteric viruses were also found in the prefilter membranes used for prefiltering the raw water samples. [TOP OF PAGE]

65. Kluyvera bacteriophage Kvp1: a new member of the Podoviridae family phylogenetically related to the coliphage T7. Gadaleta, P., Zorzopulos, J. (1997). Virus Research 51:43-52. A DNA containing bacteriophage, Kvp1, was isolated from the water of a very polluted river, the Matanza river, near the central district of Buenos Aires City. This bacteriophage infects bacteria belonging to the Kluyvera cryocrescens species (strain 21 g) isolated from the same river. Kvp1 is a lytic bacteriophage and its propagation characteristics are: burst size 30, latent period 13 min and rise period 10 min. Morphologically, Kvp1 is a small icosahedral bacteriophage, 59.1 nm in diameter, which possesses a short wedge-shaped tail. Its buoyant density in ClCs is 1.517 g/cm3. Kvp1 DNA is linear, double stranded and approximately 40,000 bp in size. The viral particle is composed of at least nine proteins. SDS-PAGE patterns of these proteins and of those produced during the host infection, in addition to its morphological and genomic characteristics, suggested that Kvp1 is similar to the coliphage T7. Molecular cloning, sequencing and computer-assisted analysis of Kvp1 DNA fragments confirmed the relationship to the coliphage. Taking this into account, the partial sequence of the phage RNA polymerase was used to construct phylogenetic relationships between Kvp1 and other related phages. To our knowledge, Kvp1 is the first bacteriophage described which uses as host a member of the Kluyvera bacterial genus. [TOP OF PAGE]

66. Bacteriophage resistance in Lactococcus lactis engineered by replacement of a gene for a bacteriophage receptor. Garbutt, K.C., Kraus, J., Geller, B.L. (1997). Journal of Dairy Science 80:1512-1519. The objective of this study was to construct a foodgrade, phage-resistant strain of Lactococcus lactis by replacing a specific chromosomal gene with an allele that had been mutated in vitro. Lactococcus lactis contains a chromosomal gene (pip) that is requited for infection by bacteriophages of the c2 species. A nonsense mutation in pip was constructed in vitro. The wild-type pip on the chromosome of strain LM2301 was exchanged for the mutated pip. The exchange left no antibiotic resistance genes or nonlactococcal DNA in the engineered strain (JK101). JK101 was resistant to the same phages as a strain that contains a spontaneous mutation in pip. JK101 grew as well as the pip+ isogenic strain did in minimal or rich media. [TOP OF PAGE]

67. Bacteriophages of Streptococcus pneumoniae: A molecular approach. Garcia, P., Martin, A.C., Lopez, R. (1997). Microbial Drug Resistance-Mechanisms Epidemiology & Disease 3:165-176. We have characterized four families of pneumococcal phages with remarkable morphological and physiological differences. Dp-1 and Cp-1 are lytic phages, whereas HB-3 and EJ-1 are temperate phages. Interestingly, Cp-1 and HB-3 have a terminal protein covalently linked to the 5' ends of their lineal DNAs. In the case of Dp-1, we have found that the choline residues of the teichoic acid were essential components of the phage receptors. We have also developed a transfection system using mature DNAs from Dp-4 and Cp-1. In the latter case, the transfecting activity of the DNA was destroyed by treatment with proteolytic enzymes, a feature also shared by the genomes of several small Bacillus phages. DNA replication was investigated in the case of Dp-4 and Cp-1 phages. The terminal protein linked to Cp-1 DNA plays a key role in the peculiar mechanism of DNA replication that has been coined as protein-priming. Recently, the linear 19,345-bp double-stranded DNA of Cp-1 has been completely sequenced, several of its gene products have been analyzed, and a complete transcriptional map has been elaborated. Most of the pneumococcal lysins exhibit an absolute dependence of the presence of choline in the cell wall substrate for activity, and phage lysis requires, as reported for other systems, the action of a second phage-encoded protein, the holin, which presumably forms some kind of lesion in the membrane. The two lytic gene cassettes, from EJ-1 and Cp-1 phages, have been cloned and expressed in heterologous and homologous systems. The finding that some lysogenic strains of Streptococcus pneumoniae harbor phage remnants has provided important clues on the interchanges between phage and bacteria and supports the view of the chimeric origin of phages. [TOP OF PAGE]

68. Identification of a RecA homolog (RecA-LP) on the conjugative lactococcal phage resistance plasmid pNP40: Evidence of a role for chromosomally encoded RecA-L in abortive infection. Garvey, P., Rince, A., Hill, C., Fitzgerald, G.F. (1997). Appl. Environ. Microbiol. 63:1244-1251. The determinants for two bacteriophage resistance mechanisms, AbiE and AbiF, are separated by approximately 3,300 nucleotides on the lactococcal plasmid pNP40 (P. Garvey, G. F. Fitzgerald, and C. Hill, Appl. Environ. Microbiol. 61:4321-4328, 1995). DNA sequence analysis of the intervening region led to the identification of two open reading frames (ORFs) which are transcribed in the opposite direction to the Abi determinants. One of these ORFs encodes a recA homolog (designated recA-LP). This is the first report of a recA-like determinant located to a plasmid. The second ORF (orfU) shares homology with the umuC gene of the SOS response. Analysis of a number of lactococcal strains confirmed the presence of recA-LP-like sequences in at least two other lactococcal strains. The proximity of the recA and umuC homologs suggested a possible role in the phage resistance encoded by the Abi determinants. However, no evidence was obtained to demonstrate a function for either ORF in the expression of either AbiE or AbiF. Nor could the recA-LP gene restore resistance to mitomycin in a recA-deficient lactococcal strain, VEL1122. Interestingly, it was shown that the chromosomally encoded recA is necessary for complete expression of the AbiF phenotype, confirming a role for RecA in this abortive infection system. [TOP OF PAGE]

69. Release and bioavailability of C, N, P, Se, and Fe following viral lysis of a marine chrysophyte. Gobler, C.J., Hutchins, D.A., Fisher, N.S., Cosper, E.M., Sanudo Wilhelmy, S.A. (1997). Limnol. Oceanogr. 42:1492-1504. [TOP OF PAGE]

70. Persistence of MS-2 and PRD-1 bacteriophages in an ultrapure water system. Governal, R.A., Gerba, C.P. (1997). JOURNAL OF INDUSTRIAL MICROBIOLOGY AND BIOTECHNOLOGY 18:297-301. The persistence of bacteriophages MS-2 and PRD-1 was evaluated in tap water, in reverse osmosis (RO) permeate, and in three locations within an ultrapure water system; ultrapure samples included pre- and post-UV sterilization and post-mixed bed ion exchange tank. The inactivation rates for MS-2 were calculated as log10 reduction per hour and per day: k = -(log 10 Ct/C0)/t. PRD-1 was found to persist with no significant loss of infectivity in all water purity environments evaluated. Inactivation of MS-2 was dependent on water quality and pH. Short-term inactivation rates for chlorinated tap water, post-RO, pre-UV, post-UV and post-ion exchange sample locations were 0.028, 0.455, 0.231, 0.191 and 0.168 log10 h-1, respectively. Long-term inactivation rates for chlorinated tap water, post-RO, pre-UV, post-UV and post-ion exchange sample locations were 0.485, 0.911, 0.605, 0.632 and 0.684 log10 day-1, respectively. Since phages were found to remain intact as well as to lyse in the ultrapure water environment, the phages have the potential to contaminate the ultrapure water environments of the microelectronics, pharmaceutical and power generation industries in both colloidal and dissolved form. Further work is proceeding to generate standardized and cost-effective methods to detect viruses in water environments. [TOP OF PAGE]

71. Bacteriophage T4 development depends on the physiology of its host Escherichia coli. Hadas, H., Einav, M., Fishov, I., Zaritsky, A. (1997). Microbiology 143:179-185. Several parameters of phage T4 adsorption to and growth in Escherichia coli B/r were determined. All changed monotonously with the bacterial growth rate (mu), which was modified by nutritional conditions. Adsorption rate was faster at higher mu values, positively correlated to cell size, and increased by pretreatment with low penicillin (Pn) concentrations; it was directly proportional to total cellular surface area, indicating a constant density of T4 receptors on cell envelopes irrespective of growth conditions. Parameters of phage development and cell lysis were mu-dependent. The rate of phage release and burst size increased, while the eclipse and latent periods decreased with increasing mu. Differentiation between the contribution of several physiological parameters to the development of T4 was performed by manipulating the host cells. A competitive inhibitor of glucose uptake, methyl alpha-D-glucoside, was exploited to reduce the growth rate in the same effective carbon source. Synchronous cells were obtained by the 'baby- machine' and large cells were obtained by pretreatment with low Pn concentrations. Lysis was delayed by superinfection, and DNA content and concentration were modified by growing a thy mutant in limiting thymine concentrations. The results indicate that burst size is not limited by cell size or DNA composition, nor directly by the rate of metabolism, but rather by the rates of synthesis and assembly of phage components and by lysis time. The rates of synthesis and assembly of phage components seem to depend on the content of the protein-synthesizing system and lysis time seems to depend on cellular dimensions. [TOP OF PAGE]

72. Microorganisms as tracers in groundwater injection and recovery experiments: a review. Harvey, R.W. (1997). FEMS Microbiol. Rev. 20:461-472. [TOP OF PAGE]

73. Presence of bacteriophage Aa phi 23 correlates with the population genetic structure of Actinobacillus actinomycetemcomitans. Haubek, D., Willi, K., Poulsen, K., Meyer, J., Kilian, M. (1997). European Journal of Oral Sciences 105:2-8. Several bacteriophages associated with the oral bacterium Actinobacillus actinomycetemcomitans have been identified. Lysogeny might affect the virulence of this bacterium, which has been implicated in the etiology of juvenile and adult periodontitis. We have determined the presence of bacteriophage Aa phi 23-related DNA sequences among 185 A. actinomycetemcomitans strains belonging to 2 well-characterized collections and have related the findings to the population genetic structure of the collections. 2 cloned Aa phi 23-specific DNA probes were used in Southern blot hybridization experiments to detect homologous sequences in whole-cell DNA of the strains. DNA from 65 (35%) of the 185 strains hybridized to either of the DNA probes. The majority (74%) of the hybridizing strains showed an identical hybridization pattern, indicating presence of phage Aa phi 23. Whole-cell DNA from the remaining hybridizing strains hybridized to the probes with different patterns, indicating that DNA sequences related to but different from phage Aa phi 23 occur in these strains. The majority (81%) of the strains which harbored phage Aa phi 23 were of serotype a, whereas serotype d strains appeared to be resistant to infection with this phage. There was a clear correlation between hybridization patterns and genetic subdivisions based on our previous population genetic analyses of A. actinomycetemcomitans. However, there was no significant correlation between occurrence of Aa phi 23 among A. actinomycetemcomitans strains and the periodontal status of the patients from whom the isolates were obtained, suggesting that this bacteriophage does not significantly influence the virulence of A. actinomycetemcomitans. [TOP OF PAGE]

74. Infection and removal of L-forms of Listeria monocytogenes with bred bacteriophage. Hibma, A.M., Jassim, S.A., Griffiths, M.W. (1997). Int. J. Food Microbiol. 34:197-207. Phage breeding was employed to produce a bacteriophage (Listeria monocytogenes phage ATCC 23074-B1) which was specific for L-forms of L. monocytogenes. The bred phage was compared to its unbred parent for lytic activity and specificity. It was also tested for its ability to prevent L-form biofilm formation on stainless steel and compared with an organic acid (lactic) at L-form biofilm inactivation on stainless steel. The bred phage lysed only L-forms of L. monocytogenes in broth culture and only plaqued on L-form lawns. Likewise, the unbred phage performed similarly with classical cell-walled culture and lawns. The bred phage successfully inhibited L-form biofilm formation on stainless steel and was as successful as lactic acid (130 ppm) at inactivating pre-formed L-form biofilms. Both reduced viable cell numbers by 3-long cycles over a 6 h period. It appears that phage breeding technology may be an attractive alternative to chemical sanitizers which lack specificity and can be toxic. [TOP OF PAGE]

75. Pathogenesis and virulence of Rhodococcus equi. Hondalus, M.K. (1997). Veterinary Microbiology 56:257-268. Inhalation of the soil-borne organism, Rhodococcus equi, can lead to a chronic and severe pyogranulomatous pneumonia in young horses and immunocompromised people. In addition, ulcerative colitis is a common sequela to infection in foals, and dissemination from the lung to other body sites is not uncommon in either the horse or man. Although the facultative intracellular bacterium is susceptible to neutrophil-mediated killing, it is able to resist innate macrophage defenses and establish residence within the intracellular environment of that phagocyte. Definitive virulence factors of R. equi have not yet been determined, but potential candidates include capsular polysaccharide, the exoenzyme cholesterol oxidase, cell wall mycolic acids, and the products encoded by a virulence-associated plasmid. The ability to replicate within the macrophage is associated with virulence, and correlates in animals with the possession of a large plasmid and expression of the plasmid-encoded, surface-expressed lipoprotein, VapA. All strains of R. equi isolated from horses with clinical disease possess a large plasmid and express VapA antigens. In addition, bacterial clearance and granuloma development in mice is linked to plasmid possession and VapA expression. Plasmid containing strains replicate within the tissues of the mouse, whereas plasmid-cured strains are rapidly cleared. At present, the function of the VapA protein is unknown. In contrast to what is observed in the foal, only a small percentage of R. equi strains isolated from humans with rhodococcal disease express VapA antigens, although a high proportion of others express a related protein which is associated with reduced virulence and is also plasmid-encoded. In a limited number of plasmid-negative human isolates, virulence has been linked to beta-lactam resistance, and preliminary evidence suggests that the phenotype may be phage encoded. It is likely that the immune status of the patient can influence whether a particular strain of R. equi is able to produce clinical disease, and certainly experimental infection in mice has confirmed that an intact cellular immune response is necessary for clearance of the organism. [TOP OF PAGE]

76. Purification and characterization of VSH-1, a generalized transducing bacteriophage of Serpulina hyodysenteriae. Humphrey, S.B., Stanton, T.B., Jensen, N.S., Zuerner, R.L. (1997). J. Bacteriol. 179:323-329. Serpulina hyodysenteriae B204 cells treated with mitomycin (20 microg of mitomycin/ml of culture broth) lysed and released bacteriophages. Bacteriophage particles, precipitated by using polyethylene glycol and purified by CsC1 density gradient ultracentrifugation, had a buoyant density of 1.375 g/cm3 and consisted of a head (45-nm diameter) and an ultrastructurally simple (noncontractile) tail (64 by 9 nm) composed of at least 13 proteins with molecular masses ranging between 13 and 101 kDa. The purified bacteriophage has been designated VSH-1 (VSH for virus of S. hyodysenteriae). VSH-1 was incapable of lytic growth on any of five intestinal spirochete strains, representing three Serpulina species. VSH-1 nucleic acid was determined to be approximately 7.5 kb in size and to be linear, double-stranded DNA based on differential staining with acridine orange, DNase I sensitivity, electrophoretic mobility, and contour length as measured by electron microscopy. Phage DNA digested by the restriction enzymes SspI, AseI, EcoRV, and AflII gave electrophoretic banding patterns nearly identical to those of digested chromosomal DNA from S. hyodysenteriae. Additionally, VSH-1 DNA fragments hybridized with probes complementary to S. hyodysenteriae chromosomal genes nox and flaA1. When purified bacteriophages induced from cultures of S. hyodysenteriae A203 (deltaflaA1 593-762::cat) were added to growing cells of strain A216 (deltanox 438-760::kan), transductants (Cmr Kmr) were obtained at a frequency of 1.5 x l0(-6) per phage particle (enumerated by electron microscopy). These findings indicate that induced VSH-1 virions package DNA of S. hyodysenteriae and are capable of transferring host genes between cells of that spirochete. To our knowledge, this is the first report of genetic transduction of a spirochete. [TOP OF PAGE]

77. The characterisation of Brucella strains isolated from marine mammals. Jahans, K.L., Foster, G., Broughton, E.S. (1997). Veterinary Microbiology 57:373-382. Small Gram negative coccobacilli isolated from seals, porpoises, dolphins and from an otter road casualty were identified as Brucellae by their colonial and cell morphology, staining characteristics, biochemical activity, agglutination by monospecific antisera and susceptibility to lysis by Brucella specific bacteriophage. Their characterisation, including metabolic profiles, is described. These strains could not be assigned to recognised nomen species of the genus Brucella and it is suggested that they comprise a new nomen species to be called B. maris (sp. nov., type strain 2/94). It is further suggested the nomen species be subdivided into three biovars corresponding to their CO2 requirement, metabolic activity on galactose, dominant antigen and animal host. [TOP OF PAGE]

78. Isolation and characterization of bacteriophage BCJA1, a novel temperate bacteriophage active against the alkaliphilic bacterium, Bacillus clarkii. Jarrell, K.F., Vydykhan, T., Lee, P., Agnew, M.D., Thomas, N.A. (1997). Extremophiles 1:199-206. The isolation and characterization of a novel bacteriophage active against the obligately alkaliphilic bacterium Bacillus clarkii is described. The bacteriophage, designated BCJA1. is a member of the Siphoviridae family with a B1 morphology. It possesses an isometric head, which measures 65 nm between opposite apices, and a noncontractile tail of 195 nm length. It had a buoyant density of 1.518 g/ml and an estimated particle mass of 37 x 10(7) daltons. BCJA1 was stable over the pH range of 6-11. A one-step growth experiment conducted at pH 10 demonstrated a latent period of about 40 min and a burst size of approximately 40. The purified bacteriophage appeared to consist of 10 proteins with the major head and tail proteins likely to be of molecular weight 36500 and 28000, respectively. The genome size was estimated to be between 32.1 and 34.8 kb. The percent G + C content of purified bacteriophage DNA was 45.6. The wildtype bacteriophage is temperate but a clear plaque mutant was isolated. [TOP OF PAGE]

79. Automatic typing of bacterial isolates. Jason, D., Jason, A.C. (1997). Letters in Applied Microbiology 25:431-434. [TOP OF PAGE]

80. Sorption of viruses during flow through saturated sand columns. Jin, Y., Yates, M.V., Thompson, S.S., Jury, W.A. (1997). Environmental Science & Technology 31:548-555. Viruses pathogenic to humans have been found in wells and drinking water due to the improper placement of wastewater disposal operations (e.g., septic tanks, wastewater infiltration basins) and inadequate removal of the organisms as the wastewater percolated through the soil. In order to develop well head protection criteria that are protective of public health, it is necessary to understand the mechanisms that control virus retention and removal in porous media. In this study, we report the results of a series of experiments on virus transport through sand columns (9.2 cm in diameter and 10.5 or 20 cm long) under saturated flow conditions. Two bacteriophages, MS-2 and vphi-X-174, were used in the experiments. Virus solution was applied to the lower end of the column as a constant input, and samples were collected at the effluent end. A virus transport and fate model, partially calibrated with the transport parameters obtained from Br- tracer experiments, was used to evaluate the retention and inactivation characteristics of the viruses. We found that, while MS-2 was not sorbed by the Ottawa sand, a significant amount of the applied vphi-X-174 was retained but not inactivated in the columns. This was probably due to the difference in their isoelectric points. Retention of vphi-X-174 exhibited trends consistent with first-order attenuation that increased with residence time; however, the sand was found to have a finite sorption capacity for vphi-X-174. This study also demonstrates that virus sorption can be evaluated more effectively with a well-controlled column flow system than by the commonly used batch equilibration method. [TOP OF PAGE]

81. Elution and reconcentration of coliphages in water from positively charged membrane filters with urea-arginine phosphate buffer. Jothikumar, N., Cliver, D.O. (1997). Journal of Virological Methods 65:281-286. Coliphages in drinking water and waste water samples have been adsorbed onto positively charged membrane filters, eluted with urea-arginine phosphate buffer (UAPB), reconcentrated, and detected with Escherichia coli C (ATCC 13706). The proposed membrane filter-based UAPB method for concentration and detection of coliphages compares favorably with the beef extract elution and reconcentration procedure and also with the proposed coliphage detection procedure described in Standard Methods for the Examination of Water and Wastewater. The higher recovery of coliphages with UAPB elution from positively charged membrane filters is attributed to testing the whole volume of concentrated sample, rather than partial analysis of the sample as in the procedure described in Standard Methods for the Examination of Water and Wastewater, especially when the titre is very low. [TOP OF PAGE]

82. Photocatalysis-dependent inactivation of Lactobacillus phage PL-1 by a ceramics preparation. Kakita, Y., Kashige, N., Miake, F., Watanabe, K. (1997). TEMP 99/06/16 61:1947-1948. A ceramics preparation (Cleansand-205), which was coated with a mixture of the oxides of Si, Al, Ti, and Ag, was found to inactivate Lactobacillus phage PL-1 suspended in a buffer solution. The inactivation of phage was dependent on the amounts of Cleansand-205 added, and the reaction obeyed almost first-order reaction kinetics. The phage inactivation was considerably accelerated by the presence of light. [TOP OF PAGE]

83. Identification of Pseudomonas aeruginosa genes required for epithelial cell injury. Kang, P.J., Hauser, A.R., Apodaca, G., Fleiszig, S.M.J., Wiener-Kronish, J., Mostov, K., Engel, J.N. (1997). Molecular Microbiology 24:1249-1262. We have developed a simple, reproducible and rapid genetic screen for Pseudomonas aeruginosa-induced epithelial cell cytotoxicity in cultures of MDCK cells. This screen was used to isolate isogenic transposon-tagged non-cytotoxic mutants of a cytotoxic and lung-virulent strain of P. aeruginosa (PA103). The transposon-insertion site was determined by using an inverse polymerase chain reaction followed by DNA-sequence analysis. On the basis of phenotype and sequence analysis, these mutants fell into four classes. One class had absent or defective pill, based on their resistance to phage PO4 and/or loss of twitching motility (twt-). A second class exhibited decreased adherence. A third class of mutants exhibited probable defects in the machinery or targets of type III protein secretion. A final class of mutants exhibited decreased but not absent cytotoxicity. This class included members of the first three classes as well as other mutants. These results suggest that localized cytotoxicity is likely to require several steps and several components, including pill and other (unidentified) extracellular proteins. The type III protein-secretion apparatus appears to be involved in this process. [TOP OF PAGE]

84. Effect of irradiation on microbial indicators during sewage treatment. Karem, H.A., Waite, T.D. (1997). Egyptian Journal of Microbiology 32:169-182. A study was carried out to assess the changes in the population densities of certain microbial groups of hygienic significance throughout the successive steps of treatment in Virginia Key Wastewater Plant in Miami, Florida. Samples were taken from raw sewage, oxygenated tanks, settling tanks and chlorinated effluent. Total count, total coliform, Streptococcus faecalis, Aeromonas hydrophila and coliphage increased or remained unchanged after oxygenation but they sharply decreased in settling and chlorination tanks were more than 99% reduction in counts initially present was observed. A. hydrophila and coliphage were still detected in the final effluent. With respect to the last stages of treatment till the production of dewatered sludge, all tested groups of microorganisms attained their highest values in the effluent of concentration tanks. They decreased throughout the steps of treatment till the dewatered sludge to attain 106-105 cfu/g for total counts and total coliform as well as 104 cfu/g for A. hydrophila and faecalis and 103 pfu/g for coliphage. An experiment was conducted to study the efficiency of gamma radiation in reducing the microbial load in the chlorinated effluent and dewatered sludge. In the chlorinated effluent, total counts, S. faecalis and coliphage were eliminated with 2kGy while 1 kGy was sufficient to eradicate total coliform and A. hydrophila. Regarding the dewatered sludge, a dose of 6 kGy was quite sufficient to decrease total counts from 6X 105 to few cells/g Whereas, total coliform, S. faecalis and A. hydrophila could not be detected after irradiation with 2.4 and 1 Kgy respectively. Coliphage was eliminated after irradiation with 6 kGy. Another experiment was carried out to compare the effect of gamma radiation with that of electron beam on microbial groups in chlorinated effluent. At any given dose of radiation, gamma rays proved to be of more lethal than electron beam for all types of organisms. [TOP OF PAGE]

85. The abundance of planktonic viruses in antarctic lakes. Kepner, R., Galchenko, V., Wharton, R. (1997). pp. 241-252. In In Lyons, W.B., Howard-Williams, C., and Hawes, I. (eds.), Ecosystem Processes in Antarctic Ice-Free Landscapes. Balkema Press, Rotterdam. [TOP OF PAGE]

86. Virus-like, double-stranded RNAs in the parasitic protozoan Cryptosporidium parvum. Khramtsov, N.V., Woods, K.M., Nesterenko, M.V., Dykstra, C.C., Upton, S.J. (1997). Molecular Microbiology 26:289-300. We have discovered and analysed two novel, linear extrachromosomal double-stranded RNAs (dsRNAs) within oocysts of major north Amercian isolates of Cryptosporidium parvum, a parasitic protozoan that infects the gastrointestinal tract of a variety of mammals, including humans. These dsRNAs were found to reside within the cytoplasm of sporozoites, and were not detected in other species of the genus. cDNAs representing both dsRNA genomes were cloned and sequenced, 1786 and 1374 nt, and each encoded one large open reading frame (ORF). The deduced protein sequence of the larger dsRNA (L-dsRNA) had homology with viral RNA-dependent RNA polymerases (RDRP), with more similarity to polymerases from fungi than those from other protozoa. The deduced protein sequence from the smaller dsRNA (S-dsRNA) had limited similarity with mitogen-activated c-June NH2 terminal protein kinases (JNK) from mammalian cells. Attempts to visually identify or purify virus-like particles associated with the dsRNAs were unsuccessful. Sensitivity of the dsRNAs to RNase A also suggests that the dsRNAs may be unencapsidated. A RDRP activity was identified in crude extracts from C. parvum sporozoites and products of RNA polymerase activity derived in vitro were similar to the dsRNAs purified directly from the parasites. [TOP OF PAGE]

87. Evaluation of an upwelling injection field polishing system for elimination of fecal coliforms and enteric viruses (F+ RNA phage). Kilgen, M.B., Melancon, E., Rush, K., Malone, R. (1997). Hilton Head Island, SC (USA). 1. Int. Conf. on Shellfish Restoration. 1900.A natural system using a sand/soil bed in an upwelling injection field polishing system was designed and implemented to remove fecal coliforms, Escherichia coli and enteric viruses from wastewater effluents of coastal dwellings. This system treats wastewater by injecting it 15' down into a salt sand/soil marsh adjacent area. This forces it to flow upward through a salt or brackish water sand bed by the hydrostatic pressure difference between the fresh wastewater influent and the salt water. As the fresh wastewater is pushed upward through the sand bed, suspended or particle-associated microorganisms are either adsorbed by the sand surface or die-off, thus reducing their impact to the surrounding estuary. The injection system proved to be extremely effective in removing fecal coliforms, E. coli, and RNA F+ coliphage from secondary treatment wastewater influents. Initial loads of fecal coliforms from this injection influent were reduced by 2-4 logs from about 10 super(3-4) or higher to 10 super(1), 10 super(0), or in most cases, to undetectable levels. The system also removed 6 liters of concentrated (10 super(8) pfu/ml) MS2 RNA F+ phage seeded directly into the injection line as a model for human Norwalk and hepatitis type A enteric viruses. Overall, this upwelling injection system for salt or brackish water coastal dwellings is extremely effective in removing not only the fecal coliform and E. coli load from partially treated sewage effluents, but also the enteric viruses which are of greatest concern in actual human health risk. (DBO). [TOP OF PAGE]

88. Prevalence of Salmonella in municipal sewage treatment plant effluents in southern California. Kinde, H., Adelson, M., Ardans, A., Little, E.H., Willoughby, D., Berchtold, D., Read, D.H., Breitmeyer, R., Kerr, D., Tarbell, R., Hughes, E. (1997). Avian Diseases 41:392-398. Effluents from 12 sewage treatment plants in southern California were examined for Salmonella using a Moore swab technique. Eight of the 12 plants were positive for Salmonella when sampled at the chlorination/dechlorination site (inside the plant). Effluents from 11 of 12 sewage treatment plants were positive for Salmonella when samples were analyzed downstream of the chlorination/dechlorination site, before effluents merge with the receiving stream (outside the plant). Two of the three control sites, an urban runoff, a raw potable water reservoir, and two other sites were also positive for Salmonella. A total of 683 Salmonella isolations were represented by 11 serogroups and 54 serotypes from 26 of 32 sampling sites. Effluents from three treatment plants and one control site (raw potable water resevior) yielded Salmonella enteritidis phage type 4, in addition to other serotypes. [TOP OF PAGE]

89. Shuffling of structural elements in filamentous bacteriophages. Kishchenko, G., Dmakowski, L. (1997). Proteins Structure Function and Genetics 27:405-409. All class II filamentous bacteriophage coat proteins contain a conserved, 12-amino acid sequence highly homologous to the loop portion of the EF-hand Ca-2+-binding motif. The Pf3 coat protein contains two regions of homology to this sequence. The 12-amino acid sequence corresponds to a region of the Pf1 coat protein whose structure is controversial. In some models of the virus structure, this region is alpha-helical. In others, it forms a loop that folds back on itself. The similarity of this region to the loop in the helix-loop-helix Ca-2+-binding motif suggests that it takes on a loop structure in the virion. Each filamentous phage lacks at least one residue normally involved in Ca-2+-coordination, consistent with the relatively weak Ca-2+ binding properties of the filamentous phages. Consideration of the structure of the coat protein in the membrane and in the virus particle indicates that the protein may be more effective in binding cations in its membrane-bound form than in the virus particle. This suggests that release of cations from this loop may be an obligate step during assembly of the proteins into the virus particle. [TOP OF PAGE]

90. Rapid evolution of translational control mechanisms in RNA genomes. Klovins, J., Tsareva, N.A., De, S., Berzins, V., Van, D. (1997). J. Mol. Biol. 265:372-384. We have introduced 13 base substitutions into the coat protein gene of RNA bacteriophage MS2. The mutations, which are clustered ahead of the overlapping lysis cistron, do not change the amino acid sequence of the coat protein, but they disrupt a local hairpin, which is needed to control translation of the lysis gene. The mutations decreased the phage titer by four orders of magnitude but, upon passaging, the virus accumulated suppressor mutations that raised the fitness to almost wild-type level. Analysis of the pseudorevertants showed that the disruption of the local hairpin, controlling expression of the lysis gene, had apparently been so complete that its restoration by chance mutations could not be achieved. Instead, alternative foldings initiated by the starting mutations were further stabilized and optimized. Strikingly, in the pseudorevertants analyzed, translational control of the lysis gene had been restored. This feat was accomplished by, on average, four suppressor mutations that generally occurred at codon wobble positions. We also introduced 11 mutations in a hairpin more upstream in the coat protein gene and not implicated in lysis control. Here the titer dropped by three logs, but pseudorevertants with a fitness close to wild-type were soon generated. These pseudorevertants again were the result of the optimization of alternative foldings induced by the mutations. The transition of the secondary structure from wild-type to pseudorevertant could be visualized by structure probing. Our study shows that the folding of the RNA is an important phenotypic property of RNA viruses. However, its distortion can easily be overcome by optimizing alternative base-pairings. These new structures are not qualitatively equivalent to the original one, since they do not successfully compete with the wild-type. [TOP OF PAGE]

91. Repeatability of microbial evolution in simple experimental arrangements. Korona R, Koron (1997). Wiadomosci Ekologiczne 43:97-116. Repeatability of evolution is determined by the relative strength of three factors: random processes, such as mutation and genetic drift, natural selection and, finally, by historical and constructional limitations. These factors can be studied experimentally if large populations of microorganisms are applied. The extent and repeatability of adaptation may be then estimated in direct comparisons between replicate populations and their common ancestor. It is commonly observed that the fitness increase in separate populations of bacteria is similar when strictly homogeneous environment is applied (Fig. 2). This lack of fitness differentiation is caused by the presence of common constraints to further adaptation, and not by acquiring the same genetical changes. When the environment is stressful or non-homogeneous (Fig. 3), then not only genetic basis of fitness, but also the extent of fitness increase is different in different populations. Experimental evolution of microorganisms is fairly repeatable when more then one populations are evolving together (Fig. 4). For example, an organic compound is digested in steps with different clones specializing in different stages. In bacteria-fage systems the improved resistance of host cells to the parasite is accompanied by decrease of their fitness in phage-free environment. Therefore both sensitive and resistant bacterial populations may persist under frequency-dependent selection. Generally, particular adaptive changes can be different in separate populations, but the overall results may be comparable if the shape of selective landscape is determined by some universal constraints or clearly defined ecological roles. [TOP OF PAGE]

92. Bacterial capsules: No barrier against Bdellovibrio. Koval, S.F., Bayer, M.E. (1997). Microbiology (Reading) 143:749-753. Bdellovibrio bacteriovorus 109J attached to both capsulated and noncapsulated Escherichia coli K29 cells. Electron microscopy revealed penetration of the thick polysaccharide capsule without any major disintegration of the neighbouring capsular matrix. The capsule remained intact during bdelloplast formation and lysis was unaffected by capsulation of the prey cell. This study shows that, in contrast to its effect on bacteriophage penetration and its protective activities against immune defence mechanisms, the capsule of E. coli does not serve as a barrier against invasion by B. bacteriovorus. [TOP OF PAGE]

93. [The study on biology of bacteriophages and their usage in the treatment of bacterial diseases and on the influence of different bacteriophages on cytokine production by leukocytes in human peripheral blood]. Kozminska, J., Weber-Dabrowska, B., Mulczyk, M. (1997). OTOLARYNGOLOGIA POLSKA 51 Suppl 25:195-198. The authors showed the examinations of the biology bacteriophages and using them in the treatment of the bacteriology infection and influence difference bacteriophages in producing cytokinins by leukocytes of human peripheral blood. [TOP OF PAGE]

94. Relationship between the morphology of bacteriophages and their persistence in the environment. Lasobras, J., Muniesa, M., Frias, J., Lucena, F., Jofre, J. (1997). Water Science and Technology 35:129-132. The aim of the present work was to observe the morphological differences between bacteriophages from sewage, recent pollution and those from samples that contain microorganisms that have survived either natural inactivation or water treatment processes (persistent pollution). We studied the occurrence of bacteriophages infecting Bacteroides fragilis (HSP40 strain), F-specific coliphages (HS(pFamp)R strain) and somatic coliphages (CN13 strain). Phages isolated with B. fragilis in both wastewater and samples with persistent pollution were in all cases members of the Siphoviridae group with flexible tails. With E. coli strain HS(pFamp)R we detected F-specific coliphages in 97.7% wastewater. However, the percentage decreased to 76.9% in samples with persistent pollution. In the case of somatic coliphages in wastewater, detected using E. coli CN13, 91% of the phages belonged to the Myoviridae family and a 6% to the Siphoviridae family. In contrast, in persistent pollution samples, an increase of Siphoviridae phages was observed (up to 26.4%). [TOP OF PAGE]

95. Two groups of bacteriophages infecting Streptococcus thermophilus can be distinguished on the basis of mode of packaging and genetic determinants for major structural proteins. Le Marrec, C., van Sinderen, D., Walsh, L., Stanley, E., Vlegels, E., Moineau, S., Heinze, P., Fitzgerald, G., Fayard, B. (1997). Appl. Environ. Microbiol. 63:3246-3253. A comparative study of 30 phages of Streptococcus thermophilus was performed based on DNA restriction profiles, DNA homology, structural proteins, packaging mechanisms, and host range data. All phages exhibited distinct DNA restriction profiles, with some phages displaying similarly sized restriction fragments. DNA homology was shown to be present among all 30 phages. The phages could be divided into two groups on the basis of their packaging mechanism as was derived from the appearance of submolar DNA fragments in restriction enzyme digests and the presence (cos-containing phages) or absence (pac-containing phages) of cohesive genomic extremities. Interestingly, the 19 identified cos-containing phages possessed two major structural proteins (32 and 26 kDa) in contrast to the remaining 11 pac-containing phages, which possessed three major structural proteins (41, 25, and 13 kDa). Southern hybridization demonstrated that all pac-containing phages tested contain homologs of the genes encoding the three major structural proteins of the pac-containing phage O1205, whereas all cos-containing phages tested exhibit homology to the gene specifying one of the structural components of the cos-containing phage phi 7201. Fifty-seven percent of the phages (both cos and pac containing) possessed the previously identified 2.2-kb EcoRI fragment of the temperate S. thermophilus phage Sfi18 (H. Brussow, A. Probst, M. Fremont, and J. Sidoti, Virology 200:854-857, 1994). No obvious correlation was detected between grouping based on packaging mechanism and host range data obtained with 39 industrial S. thermophilus strains. [TOP OF PAGE]

96. Protozoa in open reservoirs. Lechevallier, M.W., Norton, W.D., Atherholt, T.B. (1997). American Water Works Association Journal 89:84-96. The impact of storage of potable water in open reservoirs was assessed by examining inlet and effluent water samples from six open finished water reservoirs used by four New Jersey utilities. Water quality parameters investigated included Giardia cysts, Cryptosporidium oocysts, total and fecal coliforms, bacteriophage, heterotrophic plate count bacteria, turbidity, particle counts, chlorine residuals and other parameters. Fifteen percent of inlet samples and 25 percent of effluent samples contained the organisms. When data for cysts and oocysts were combined, the difference in concentrations between the inlet and effluent was statistically significant, even when results were adjusted for analytical recovery efficiency. Although the concentration of protozoa increased following open reservoir storage, the analytical method used does not permit assessment of the organisms' public health significance. Nearly all of the cysts and oocysts were empty or contained undiscernible internal structures, suggesting the health risk is low. The implication of these findings for watershed control programs is discussed. [TOP OF PAGE]

97. Negative effects of chemical mutagenesis on the adaptive behavior of vesicular stomatitis virus. Lee, C.H., Gilbertson, D.L., Novella, I.S., Huerta, R., Domingo, E., Holland, J.J. (1997). J. Virol. 71:3636-3640. Changes in adaptability of vesicular stomatitis virus (VSV) upon treatment with chemical mutagens have been investigated. Results showed no improvement in virus viability or adaptability at any given level of mutagenesis. In fact, increasing inhibition of virus production and adaptability was observed with increasing levels of mutagenesis. This was true for all tested VSV variants replicating either in changing or constant host cell environments. Results also showed that mutagen-treated RNA virus populations which had undergone severe fitness declines were able to recover lost fitness completely after several large-population passages in BHK21 cells. The present findings illustrate the highly optimized states of RNA viruses and their potential to adapt readily. These results are significant for the possible development of specific antiviral agents designed to be mutagenic. [TOP OF PAGE]

98. The effects of methylene blue and oxygen concentration on the photoinactivation of Q beta bacteriophage. Lee, D., Foux, M., Leonard, E.F. (1997). PHOTOCHEMISTRY AND PHOTOBIOLOGY 65:161-165. Concentration effects of methylene blue (MB) and oxygen on the photoinactivation rate of Q beta bacteriophage were examined. The effect of initial virus concentration was verified on the similar f2 phage. The inactivation rate, kappa, is an increasing function of MB and O2 concentration and shows saturation with respect to MB concentration. Thus the results suggest that MB must adsorb to Q beta sites and oxygen must be present for photoinactivation to occur. The inactivation rate is independent of the initial number of phage particles present before inactivation, indicating that inactivation does not depend upon interaction among viral particles or on surface effects. The results indicate that at least two different viral phenotypes exist within the wild-type Q beta and f2 populations: one susceptible and the other resistant. [TOP OF PAGE]

99. Bacteriophages are a better indicator of illness rates than bacteria amongst users of a white water course fed by a lowland river. Lee, J.V., Dawson, S.R., Ward, S., Surman, S.B., Neal, K.R. (1997). Water Science and Technology 35:165-170. An examination was made of the risk factors for gastrointestinal illness (GI) and other symptoms among canoeists and rafters using an artificial white-water canoe slalom course fed by a lowland river. The investigation was made by carrying out cohort studies of users on several days throughout one year. On each day water samples were collected for the determination of Escherichia coli, enterococci (faecal streptococci), F-specific RNA bacteriophage, sulphite reducing clostridia, culturable enteroviruses and turbidity. Of 755 questionnaires distributed, 473 (63%) were returned. The relative risks of GI and other symptoms were determined by logistic regression analyses. The variables associated with an increased risk of GI-illness were swallowing water, unintentional swimming in the course, eating and drinking before getting changed and the levels of F-specific RNA bacteriophages. Being a regular user was associated with a decreased risk of GI-illness. This study demonstrates the value of F-specific RNA bacteriophages as an index of risk from recreational use of a fresh water environment. [TOP OF PAGE]

100. Antiserum inhibition of propagating viruses. Lee, Y., Eisner, S.D., Yin, J. (1997). Biotechnology & Bioengineering 55:542-546. The design and implementation of controlled environments to continuously culture and evolve viruses provides a means to track how their populations respond to natural and designed anti-viral agents. We have previously demonstrated how the growth of viruses in spreading plaques enables detection and characterization of their evolutionary dynamics. Using plaques of phage T7 growing on E. coli as a model system, we observe here that velocities of propagation can be readily controlled by the level of anti-viral antiserum incorporated into the propagation medium. Further, we develop a simple analytic expression for the radial velocity of propagation in terms of the microscopic rates of viral amplification, Fickian diffusion of the virions and their neutralization by antiserum. Our analysis captures the essential dependence of propagation velocity on antiserum concentration. This study provides an ex vivo foundation for exploring how medically relevant viruses escape suppression by the immune system. [TOP OF PAGE]

101. Optimization of the detection of bacteriophages induced from Listeria sp. Lemaitre, J.P., Delcourt, A., Rousset, A. (1997). Letters in Applied Microbiology 24:51-54. It is necessary to isolate new phages in order to improve the rate of typeability of Listeria monocytogenes strains. We propose a method which increases the detection of induced phages in the presence of inhibitory substances synthesized or liberated by the cells during phage production. Of the 29 phages isolated, 11 (38%) were detected by the spot-on-the-lawn technique and 18 (62%) were revealed by the soft-agar technique. To increase the rate of phage detection, both techniques appear useful. Listeria cultures were subjected to phage typing procedures utilizing these newly isolated phages and the French International set of phages. It appears that the newly isolated phages are good tools for the differentiation of Listeria strains. Among them, one phage seems to be complementary to the French International set. [TOP OF PAGE]

102. An improved plaque assay for poor plaque-producing temperate lactococcal bacteriophages. Lillehaug, D. (1997). Journal of Applied Microbiology 83:85-90. This report summarizes the results from an effort to optimize the double-agar plate assay for visualization of the plaques made by six temperate bacteriophages induced from industrial strains of Lactococcus lactis. Among the several parameters found to influence the plaque assay, the effect of incorporating glycine into the growth medium was most striking, resulting in extensive increase in the plaque size of all of the 13 phage-host pairs tested. Notable effects on the plaque size of other factors such as the procedure for sterilization of the agar medium, the volume and softness of the top and bottom layers, and the number and growth stage of the bacterial cells added to the lawn, were also observed. By exploiting these findings in an optimized procedure for plaque assaying, several indicator strains were identified which were unable to support the development of plaques on standard double-agar plates. Since bacterial hosts usually are identified by their ability to support the development of plaques, this observation suggests that the severe difficulty experienced in identifying lactococcal starter strains that are sensitive towards a temperate phage, partly is a problem of methodology. [TOP OF PAGE]

103. Fate of liberated viral RNA in wastewater determined by PCR. Limsawat, S., Ohgaki, S. (1997). Appl. Environ. Microbiol. 63:2932-2933. The use of a PCR method to investigate the fate of liberated Q-beta coliphage RNA in wastewater suggests that a positive PCR result indicates the recent presence of potentially viable virus in the sample and therefore increases the public health significance of the result. [TOP OF PAGE]

104. Konwersja lizogenna jako czynnik wpylwajacy na zjawisko tolerancji na wankomycyne u Staphylococcus aureus [Lysogenic conversion as a factor influencing tolerance to vancomycin in Staphylococcus aureus]. lMynarczyk, G., lMynarczyk, A., Zabicka, D., Jeljaszewicz, J. (1997). Medycyna Doswiadczalna i Mikrobiologia 49:13-17. The standard, non-lysogenic, bacteriophage-free S. aureus NCTC 8325-4 strain was lysogenized with 15 different, obtained in our laboratory staphylokinase-converting bacteriophages belonging to serological groups A, B and F. MIC and MBC of vancomycin as well as the ratio of MBC to MIC were evaluated for all 15 lysogenic derivatives. The obtained results were compared with those for maternal strain. In the case of eight strains the ratio MBC/MIC showed the presence of tolerance to vancomycin (MBC/MIC > or = 32). Four of the vancomycin-tolerant derivatives were lysogenized with bacteriophages belonging to the serological group A, two were members of group B and two belonged to group F. [TOP OF PAGE]

105. Evaluation of luciferase reporter bacteriophage A511::luxAB for detection of Listeria monocytogenes in contaminated foods. Loessner, M.J., Rudolf, M., Scherer, S. (1997). Appl. Environ. Microbiol. 63:2961-2965. A511::luxAB is a recombinant derivative of a broad-host-range bacteriophage specific for the genus Listeria, transducing bacterial bioluminescence into infected cells. In this study, we have evaluated its use for rapid and easy testing of contaminated foods and environmental samples for the presence of viable Listeria cells, in comparison to the standard plating procedure. With a short preenrichment step of 20 h, the system was capable of detecting very low initial contamination rates in several foods artificially contaminated with Listeria monocytogenes Scott A cells. In ricotta cheese, chocolate pudding, and cabbage, less than one cell per g of food could be clearly identified by comparing the light emission of phage-infected samples to that of controls without lux phage. In foods having a large and complex microbial background flora, such as minced meat and soft cheese, at least 10 cells per g were necessary to produce a positive bioluminescence signal. Of 348 potentially contaminated natural food and environmental samples, 55 were found to be Listeria positive by the lux phage method. The standard plating procedure detected 57 positive samples. Some differences were observed with respect to the individual samples, i.e., the lux phage procedure detected more positive samples among the dairy products and environmental samples, whereas the plating procedure revealed more contaminated meat and poultry samples. Overall, both methods performed similarly, i.e., were equally sensitive. However, the minimum time required for detection of Listeria with the luciferase phage assay was 24 h, which is much shorter than the 4 days needed by the standard plating method. Furthermore, a most probable number technique with three parallels, based on the use of A511::luxAB for differentiation of positive and negative tubes, is described. The method enables rapid enumeration of low levels of Listeria cells in several foods tested, against the background of a competing microflora. [TOP OF PAGE]

106. Three Bacillus cereus bacteriophage endolysins are unrelated but reveal high homology to cell wall hydrolases from different bacilli. Loessner, M.J., Maier, S.K., Daubek-Puza, H., Wendlinger, G., Scherer, S. (1997). J. Bacteriol. 179:2845-2851. The ply genes encoding the endolysin proteins from Bacillus cereus phages Bastille, TP21, and 12826 were identified, cloned, and sequenced. The endolysins could be overproduced in Escherichia coli (up to 20% of total cellular protein), and the recombinant proteins were purified by a two-step chromatographical procedure. All three enzymes induced rapid and specific lysis of viable cells of several Bacillus species, with highest activity on B. cereus and B. thuringiensis. Ply12 and Ply21 were experimentally shown to be N-acetylmuramoyl-L-alanine amidases (EC 3.5.1.28). No apparent holin genes were found adjacent to the ply genes. However, Ply21 may be endowed with a signal peptide which could play a role in timing of cell lysis by the cytoplasmic phage endolysin. The individual lytic enzymes (PlyBa, 41.1 kDa; Ply21, 29.5 kDa, Ply12, 27.7 kDa) show remarkable heterogeneity, i.e., their amino acid sequences reveal only little homology. The N-terminal part of Ply21 was found to be almost identical to the catalytic domains of a Bacillus sp. cell wall hydrolase (CwlSP) and an autolysin of B. subtilis (CwlA). The C terminus of PlyBa contains a 77-amino-acid sequence repeat which is also homologous to the binding domain of CwlSP. Ply12 shows homology to the major autolysins from B. subtilis and E. coli. Comparison with database sequences indicated a modular organization of the phage lysis proteins where the enzymatic activity is located in the N-terminal region and the C-termini are responsible for specific recognition and binding of Bacillus peptidoglycan. We speculate that the close relationship of the phage enzymes and cell wall autolysins is based upon horizontal gene transfer among different Bacillus phages and their hosts. [TOP OF PAGE]

107. Studies of divergence between "early" genes of defective phages in different strains of soil bacilli related to Bacillus subtilis 168. Lotareva, O.V., Poluektova, E.U., Prozorov, A.A. (1997). Genetika 33:752-756. The extent of xre gene divergence was studied in nine soil bacillar strains with different degrees of relationship to Bacillus subtilis 168. This gene product is a repressor of defective phages. Bac. subtilis 168 recipient strains were transformed by DNA from these bacillar strains for the xhi-1479 marker and ten markers of amino acid and nitrous bases metabolism. The efficiency of soil strain DNA hybridization with Bac. subtilis 168 DNA was assessed. Eight strains were close to Bac. subtilis 168 with respect to the efficiency of heterotransformation for all markers and hybridization, and one strain (1621) strongly differed from other strains with regard to these parameters. As determined by the degree of differences in heterotransformation for all markers, the nucleotide sequence of the xre gene diverged in the evolution process at a rate similar to that of the nucleotide sequences of the housekeeping genes. All examined genes were shown to have similar selective value. [TOP OF PAGE]

108. Wastewater reclamation at Lake Arrowhead, California: An overview. Madireddi, K., Babcock, R.W.Jr., Levine, B., Huo, T.L., Khan, E., Ye, Q.F., Neethling, J.B., Suffet, I.H., Stenstrom, M.K. (1997). Water Environment Research 69:350-362. A demonstration pilot study was conducted in Lake Arrowhead, Calif, to determine the feasibility of reclaiming municipal secondary effluent for indirect potable reuse and stabilizing the lake level during periods of extended drought. The lake, which is the sole drinking water source for the community, was severely affected during the long drought from 1985 to 1991. A 12000-L/d pilot plant was constructed and tested for nearly 3 years. The pilot plant included denitrification followed by alum coagulation/flocculation/sedimentation, sand filtration, primary ozonation, granular activated carbon (GAC) filtration, ultrafiltration (UF)/nanofiltration (NF), reverse osmosis (RO), and final ozone disinfection. A comprehensive analytical testing program was devised to monitor product water quality as well as to compare it with the lake water. Phosphorus and turbidity in the product water were consistently below detection limits (0.02 mg/L and 0.1 nephelometric turbidity unit (NTU), respectively). Product water total organic carbon (TOC) and conductivity levels were 1-2 mg/L and 20-40 mu-Mho/cm, respectively, which were approximately 25%-50% and 30%-50% of the lake concentration. Challenge testing revealed nearly complete removal of pathogenic material (an approximate 21-22 log removal of bacteriophage and 8-10 log removal of Giardia and Cryptosporidium). Trace organic chemical analysis of volatile and base neutral organic compounds indicated that it is possible to produce reclaimed water that is superior to the lake water. Only nitrogen (N) removal did not meet expectations for the entire period. It is anticipated that better process control will ensure meeting the nitrogen product water goals for full scale treatment. [TOP OF PAGE]

109. Viricidal activity and mechanisms of action of biocides. Maillard, J.Y., Russell, A.D. (1997). Science Progress 80:287-315. [TOP OF PAGE]

110. Characterization of a bacteriophage for Carnobacterium divergens NCFB 2763 by host specificity and electron microscopy. Manchester, L.N. (1997). Letters in Applied Microbiology 25:401-404. Carnobacterium divergens NCFB 2763 was used to isolate bacteriophage cd1 from minced beef using an enrichment isolation technique. Host specificity testing showed that it exhibited lytic activity only against a limited number of C. divergens strains. No activity was observed against any of the C. gallinarum, C. mobile, C. piscicola, Enterococcus durans, Lactobacillus plantarum and Lact. brevis strains tested. Anaerobic incubation had no effect on the host specificity pattern. Bacteriophage cd1 belongs to Bradley group B and on average the head was 94 nm, the tail 103 x 23 nm and the base plate 48 x 32 nm. [TOP OF PAGE]

111. Effect of microbravity on Escherichia coli and MS-2 bacteriophage disinfection by iodinated resins. Marchin, G.L., Silverstein, J., Brion, G.M. (1997). Acta Astronautica . Experiments on chemical disinfection by iodinated resins were conducted on STS 50 (USML-1), which flew a 13-day mission during 1992. Fluid processing apparatus containing microorganisms and iodinated resins was assembled in either Manhattan, Kansas, or Boulder, Colorado, and loaded on-board the Space Shuttle for the mission. Pentaiodide resin was more effective than the triiodide resin against Escherichia coli. Both resins were more effective bactericides at unit gravity than microgravity because of co-sedimentation of bacteria and iodinated resin beads. In bacteriophage experiments, the triiodide resin reduced the viable titer of MS-2 by nine logs. The bulk of viable phage surviving chemical disinfection was associated with precipitant formation in the fluid processing apparatus. [TOP OF PAGE]

112. Mechanism of bacteriophage SfII-mediated serotype conversion in Shigella flexneri. Mavris, M., Manning, P.A., Morona, R. (1997). Molecular Microbiology 26:939-950. We have isolated the lysogenic bacteriophage SfII, which mediates glucosylation of Shigella flexneri O-antigen, resulting in expression of the type II antigen. SfII belongs to group A of the Bradley classification and has a genome size of 42.3kb. DNA sequencing of a 4 kb BamHI subclone identified four open reading frames (ORFs), of which only two were found to be necessary for serotype conversion. These genes were named bgt, which encodes a putative bactoprenol glucosyl transferase, and gtrII, encoding the putative type II antigen determining glucosyl transferase. These genes are adjacent to the integrase gene (int) and attachment site (attP), which are highly homologous to those of Salmonella bacteriophage P22. Another ORF encoded a highly hydrophobic protein of 120 amino acids with homologues in Escherichia coli, Salmonella bacteriophage P22 and S. flexneri. Previous studies identified gtrX, the glucosyl transferase gene, of bacteriophage SfX, which also glucosylates the O-antigen specifically. We determined that gtrX-mediated expression of the group 7,8 antigen also requires bgt. This allowed us to identify gtrII as being the serotype antigen II determining glucosyl transferase. Southern hybridization and polymerase chain reaction (PCR) analyses indicated that bgt homologues exist in the genomes of all S. flexneri serotypes and in E. coli K-12, whereas gtrII was only detected in strains of serotype 2. Transposon TnphoA-derived chromosomal mutations of bgt and gtrII in S. flexneri serotype 2a were isolated and characterized. [35S]-methionine labelling and the use of a T7 RNA polymerase expression system identified a protein of 34kDa corresponding to Bgt. However, GtrII, which has a predicted molecular weight of 55 kDa, was not detected. We propose that the function of Bgt is to transfer the glucose residues from the UDP-glucose onto bactoprenol and GtrII then transfers the glucose onto the O-antigen repeat unit at the rhamnose III position. The chromosomal organization of these serotype-converting genes, when compared with their homologues in E. coli K-12 chromosome and the P22 bacteriophage genome, were very similar. This suggests that the regions encode similar functions in these organisms and have a similar evolutionary origin. [TOP OF PAGE]

113. Factors influencing colloid transport in fractured shale saprolite. McKay, L.D., Cumbie, D.H., Harton, A.D. (1997). Abstracts with Programs - Geological Society of America 29:307-308. Recent field and laboratory tracer experiments show that colloid-sized particles can be transported by flowing groundwater in fractured and weathered shales at rates of tens of meters per day, under gradients that are representative of natural conditions. This suggests that contaminants such as pathogenic microorganisms or radionuclides attached to naturally-occurring colloids could be transported rapidly in these materials. A series of colloid transport experiments were performed in 29-35 cm long undisturbed columns of fractured shale saprolite taken near Oak Ridge National Laboratory using bacteriophage and fluorescent latex microspheres as analogs for colloidal contaminants. The first series of experiments found that colloid retention is strongly influenced by flow rate, with greater losses occurring at lower flow rates. At the lowest flow rate, 0.0075 m/d (gradient of 0.001), loss rates for the bacteriophage MS-2 and PRD-1 were 99.9 and 93%, respectively. At the highest flow rate, 0.96 m/d (gradient of 0.1), loss rates for the same bacteriophage were only 43 and 52%. The second series of experiments found that colloid loss rates are also strongly dependent on particle diameter. Six different sizes of carboxylate-coated latex microspheres (0.05 to 4.23 microns) were pumped through another undisturbed saprolite column at a constant flow rate of 0.15 m/d (gradient of 0.08). In this case there was an optimum size for transport (approximately 0.5 to 1.0 microns), with the larger sizes experiencing loss rates up to 1800 times greater than for the optimum size and the smaller sizes experiencing loss rates up to 10 times greater than for the optimum. Preliminary analysis suggests that the greater loss of the larger sizes was due to gravitational settling and physical straining in small pore throats. Losses of the smaller than optimum sizes are believed related to their higher diffusion coefficients. Faster diffusion could lead to more frequent collisions with the fracture walls or to greater transport into relatively immobile pore water in the matrix or in small aperture regions within the fractures. A new series of experiments is currently underway to examine the influence of geochemical perturbations (ionic strength and pH) on colloid transport in fractured shale saprolite. [TOP OF PAGE]

114. Genetic studies of erythrogenic toxin carrying temperate bacteriophages of Streptococcus pyogenes. McShan, W.M., Ferretti, J.J. (1997). Advances in Experimental Medicine & Biology 418:971-973. [TOP OF PAGE]

115. Genetic diversity in temperate bacteriophages of Streptococcus pyogenes: Identification of a second attachment site for phages carrying the erythrogenic toxin A gene. McShan, W.M., Ferretti, J.J. (1997). J. Bacteriol. 179:6509-6511. Bacteriophage T12, the prototypic bacteriophage of Streptococcus pyogenes carrying the erythrogenic toxin A gene (speA), integrates into the bacterial chromosome at a gene for a serine tRNA (W. M. McShan, Y.-F. Tang, and J. J. Ferretti, Mol. Microbiol. 23:719-728, 1997). This phage is a member of a group of related temperate phages, and we show here that not all speA-carrying phages in this group use the same attachment site for integration into the bacterial chromosome. Additionally, other phages in the group use the same serine tRNA gene attachment site as phage T12 and yet do not carry speA. The evidence suggests that recombination between phage genomes has been an important means of generating diversity and disseminating virulence-associated genes like speA. [TOP OF PAGE]

116. Assessment of the exposure of swimmers to microbiological contaminants in fresh waters. Medema, G.J., Van Asperen, I.A., Havelaar, A.H. (1997). Water Science and Technology 35:157-163. As part of a prospective cohort study among triathletes to determine a relationship between the microbiological quality of fresh bathing water and the risk of acquiring an intestinal infection, the exposure of the triathletes to microbiological contaminants was assessed. Waters were collected at seven triathlons (swimming course 1-1.5 km) held in the summer of 1993 and 1994 to have a range of water qualities. All were influenced by sewage effluents, most also by agricultural run-off. Samples were collected several weeks before the event to establish a sampling programme (1993) and during the actual exposure of the triathletes (1993 and 1994) and examined for thermotolerant coliforms alone (samples preceding the event) and for E. coli, faecal enterococci, Staphylococcus aureus, F-specific RNA phages, enteroviruses (1993 and 1994) and for thermophilic Campylobacter, Salmonella, Aeromonas, Plesiomonas shigelloides and Pseudomonas aeruginosa (1993). The samples taken in the weeks before the exposure showed significant differences in thermotolerant coliform concentration between locations, depths and times. Also during swimmer exposure, significant differences occurred in microorganism levels at the different sampling points over the swimming course. As the triathletes swam as a group, they were exposed to approximately the same water at the same time. The geometric mean concentration was used to characterise each site. In the epidemiological study, the risk of an intestinal infection correlated with the concentration of thermotolerant coliforms and E coli but not with the other parameters. The geometric mean concentration of thermotolerant coliforms at the triathlons ranged from 11-330/100mL and 54-1,200/100mL E. coli. Ranking of the seven sites by faccal pollution level, based on the geometric mean concentration of a faecal indicator, resulted in a different ranking for each indicator. At the fresh water sites studied, only the ratio between the geometric mean density of E. coli and thermotolerant coliforms was constant. The ratio between the other parameters related to faecal pollution (faccal enterococci, F-specific RNA phages, enteroviruses) varied considerably. Water quality standards relating to faccal pollution can only be based on parameters that show a significant correlation with risk of intestinal illness. [TOP OF PAGE]

117. Is group selection a factor modulating the virulence of RNA viruses? Miralles, R., Moya, A., Elena, S.F. (1997). Genet. Res. 69:165-172. RNA viruses consist of populations of extremely high genetic heterogeneity called quasispecies. Based on theoretical considerations, it has been suggested that the unit of selection in such complex genetic populations is not the single viral particle but a set of genetically related particles which form the quasispecies. In the present study we carried out a set of experiments with the vesicular stomatitis virus (VSV) dealing with the evolution of life-history characters under selection acting at two factors either in the same or in opposite directions. The two factors at which selective pressure is applied are the individual and the group. We show evidence that group selection modulates the virulence of VSV populations, in opposition to an unlimited increase in virulence by competitive optimization promoted by individual selection. The results are of relevance for understanding the evolution of parasite virulence. [TOP OF PAGE]

118. Investigations of the marine lysogenic bacterium H24. III. Growth of bacteria and production of phage under nutrient-limited conditions. Moebus, K. (1997). Mar. Ecol. Prog. Ser. 148:241-250. The marine lysogenic bacterium H24, known for the genetic instability of the resident wild-type phage vphi-H24 when propagated in nutrient-rich medium, was investigated by cultivation in synthetic seawater enriched to various levels of yeast extract plus peptone (YEP, in a relation of 1:5). The incidence of phage mutants was found to depend on a balanced equilibrium of nutrient concentration (tested between 0.6 and 600 mg l-1) and the bacterial yield allowed by it on the one hand, and on the duration of incubation and the dilution between successive subcultures on the other. With too-low dilutions used in combination with high nutrient levels, non-virulent mutants, followed by virulent ones, increased in concentration until breakdown of the bacterial population occurred. Conversely, extinction of populations was caused by combinations of low nutrient levels with too-high dilution after too-short incubation periods. At low nutrient levels phage mutants did not influence the outcome of the experiments. However, at nutrient concentrations of 6 and 0.6 mg l-1 release of particles of wild- type phage vphi-H24 was found to be initiated by nutrient addition inducing sufficiently rigorous bacterial multiplication. The latter findings are discussed as an ecologically promising explanation of how phage-host systems are maintained in nature. The number of free virions of vphi-H24, which always remained low, rapidly decreased in the cultures but survived in cell-free filtrates stored in the refrigerator for the same period of time. The loss of virions in cultures is attributed to infection of H24- wt cells which due to their immunity remained unharmed. Pseudolysogenization was indicated at nutrient levels of 600 and 60 mg YEP l-1, but not at lower YEP concentrations. [TOP OF PAGE]

119. Investigations of the marine lysogenic bacterium H24. II. Development of pseudolysogeny in nutrient-rich broth culture. Moebus, K. (1997). Mar. Ecol. Prog. Ser. 148:229-240. Development of pseudolysogeny by 3 strains of H24 was investigated in nutrient-rich liquid cultures. At first, H24(L10), a cured derivative of the lysogenic wild-type strain H24-wt, and the non-virulent mutant vphi-H24-2 of the wild-type phage vphi- H24 residing in H24-wt, were used as a model phage-host system. Attempts to separate phage from pseudolysogenized cells failed due to abundantly present 'cellular PFU'. These release phage particles only after removal of the pseudolysogeny-inducing agent and do so for a period of time. Therefrom it is concluded that phage production in 'cellular PFU' was halted at various stages during the development of pseudolysogeny, depending on time of infection. Observations made with 2 lysogenic H24 strains during the course of several successive cultures are in general agreement with results obtained with the model phage-host system. The nature of the pseudolysogeny-inducing agent remains unknown. Based on information from the literature, the possibility of polysaccharide depolymerase being involved is discussed. [TOP OF PAGE]

120. Investigations of the marine lysogenic bacterium H24. I. General description of the phage-host system. Moebus, K. (1997). Mar. Ecol. Prog. Ser. 148:217-228. General features of the marine lysogenic bacterium H24 are described. The bacterial strain was isolated from a sample of North Sea water in 1978. After several consecutive transfers on seawater agar slants spontaneous plaque-formation was observed in 1981. When H24 was grown in bouillon the cultures were found to contain plaque forming units (PFU) at any number between zero and 10-9 ml-1, indicating that spontaneous plaque formation was due to mutational events. Cultures with the highest contents of PFU hardly differed in turbidity from cultures lacking PFU. These observations are ascribed to pseudolysogeny, i.e. immunity of cells against the phage present. When a cured derivative, H24(L10), became available the wild-type phage vphi-H24 residing in H24 was isolated and shown to re-lysogenize H24(L10). It also enabled differentiation between virulent and non-virulent mutants. Mutants of vphi-H24 were found to induce pseudolysogeny. Upon streaking on nutrient agar, material from colonies of pseudolysogenized cells produced various kinds of colonies representing clones of fully sensitive cells, of pseudolysogenized cells, or of mixtures of both. This and accompanying papers report the first intensive studies of a marine lysogenic bacterium. [TOP OF PAGE]

121. Growth and phage resistance of Anabaena sp. strain PCC 7120 in the presence of cyanophage AN-15. Mole, R., Meredith, D., Adams, D.G. (1997). Journal of Applied Phycology [J. Appl. Phycol. ] 9:339-345. The cyanophage AN-15 was found to have a requirement for either 1 mM calcium or 1 mM magnesium ions to maintain viral stability, whereas 1 mM calcium ions alone were essential for the infection process to proceed in Anabaena sp. strain PCC 7120. Following prolonged incubation, phage-resistant cells were detected at a high frequency (approximately 10 super(-5)) in lysates, as either renewed growth in liquid cultures, or as colonies in confluently lysed lawns. Southern hybridisation failed to detect AN-15 DNA in any of the resistant strains, implying that resistance is unlikely to be due to the presence of temperate phages. A high rate of spontaneous mutation is therefore likely to be the cause of resistance. Two classes of resistant cells were identified; those in which AN-15 failed to attach to host cells, and those in which attachment occurred, but subsequent replication was defective. However, it was possible to overcome phage resistance by the isolation of spontaneous mutants of AN-15, capable of infecting phage-resistant cells. These observations imply that if cyanophages are to be assessed as a means of controlling cyanobacterial blooms in freshwater bodies, the ionic (notably calcium) concentration of the water must be considered, together with the possible need to employ alternative cyanophage strains if resistance to the original one arises. [TOP OF PAGE]

122. The genome of the pseudo T-even bacteriophages, a diverse group that resembles T4. Monod, C., Repoila, F., Kutateladze, M., Tétart, F., Krisch, H.M. (1997). J. Mol. Biol. 267:237-249. Polymerase chain reaction analysis of a large collection of bacteriophages with T-even morphology revealed four phages that are distantly related to all the others. The genomes of these pseudo T-even phages hybridized under stringent conditions to only a limited portion of the T4 genome that encodes virus head, head-to-tail joining and contractile tail genes. Except for this region, no extensive hybridization was detected between most pairs of the different pseudo T-even genomes. Sequencing of this conserved region of the pseudo T-even phage RB49 revealed substantial nucleotide sequence divergence from T4 (approximately 30% to 40%), and random genomic sequencing of this phage indicated that more than a third of its sequences had no detectable homology to T4. Among those sequences related to the T- even genes were virion structural components including the constituents of the phage base plate. Only a few sequences had homology to T4 early functions; these included ribonucleotide diphosphatase reductase, DNA ligase and the large subunit of DNA topoisomerase. The genomes of the pseudo T-even phage were digested by restriction enzymes that are unable to digest the T- even DNAs which contain glucosylated hydroxymethyl-cytosine residues. This suggests that only limited nucleotide modifications must be present in the pseudo T-even genomes. Conservation of much of the morphogenetic region of these diverse phage genomes may reflect particularly strong sequence constraints on these gene products. However, other explanations are considered, including the possibility that the various morphogenetic segments were acquired by the pseudo T-even genomes by modular evolution. These results support the notion that phage evolution may proceed within a network of both closely and distantly related genomes. [TOP OF PAGE]

123. Viruses scout evolution's path [news]. Morell, V. (1997). Science 278:1563-1564. [TOP OF PAGE]

124. Sex frees viruses from genetic 'ratchet'. Morell, V. (1997). Science 278:1562-1563. [TOP OF PAGE]

125. The disinfection of wastewater by ultraviolet light. Moreno, B., Goni, F., Fernandez, O., Martinez, J.A., Astigarraga, M. (1997). Water Science and Technology 35:233-235. The aim of this study, carried out over four months at a pilot wastewater treatment facility, was to evaluate ultraviolet treatment of final effluent discharged into bathing waters in coastal zones. Minimum disinfection values were fixed with a target limit of 10-3cfu/100mL for faecal coliforms (FC) in line with the present and proposed European Directive on bathing water quality. Disinfection performance with different sources of water was studied and the disinfectant capacity with other indicator microorganisms was also checked. The target limit was reached using doses around 30mW.s/cm-2. Results of this study suggest that the applied UV dose is influenced by transmittance and the microbiological load. The most sensitive indicators were faecal coliforms, the specific F-RNA bacteriophages being the most resistant. From the results obtained in these experiments, UV treated water is suitable for being discharged into bathing zones, being an alternative to submarine discharges, avoiding high investment and maintenance costs. [TOP OF PAGE]

126. Bacteria in oligotrophic environments: Starvation-survival lifestyle. Morita, R.Y. (1997). Chapman and Hall Ltd., London.From the "Chapman and Hall Microbiology Series," this volume presents and reviews information regarding physiologic, ecologic, and metabolic status of bacteria in oligotrophic conditions. It highlights the marine environment but addresses terrestrial and freshwater conditions as well. It comprises ten chapters intended for microbial ecologists, general and marine microbiologists, chemists, limnologists, and soil biochemists. A historical background on bacterial starvation-survival phenomenon and oligotrophic environments is presented. The survival of bacteria in rocks, coal, frozen minerals, and soil is discussed as well as fossil bacteria. The bioavailability of organic matter in the environment, nutritional bioavailability for microbes, and metabolic activity on oligotrophic environments are addressed. Various bacterial groups including plant pathogens, chemolithotrophs, prosthecate bacteria, and fish pathogens are examined. The physiology of the starvation-survival process, nutrient transport, and molecular genetics of bacterial starvation are discussed. Prophage inductions, modeling natural environments, and maintaining energy in the environment are also evaluated. Incorporated into the chapters of this text are numerous figures, charts, tables, and graphs. An extensive reference list and an index are provided at the end of the work. [TOP OF PAGE]

127. Characterization of the locus encoding the Streptococcus pneumoniae type 19F capsular polysaccharide biosynthetic pathway. Morona, J.K., Morona, R., Paton, J.C. (1997). Molecular Microbiology 23:751-763. We have previously reported the nucleotide sequence of the first six genes of the Streptococcus pneumoniae type 19F capsular polysaccharide biosynthesis locus (cps19f). In this study we used plasmid insertion/rescue and inverse polymerase chain reaction (PCR) to clone an additional 10 kb downstream region containing the remainder of the cps19f locus, which was then subjected to sequence analysis. The cps19f locus is located in the S. pneumoniae chromosome between dexB and aliA, and consists of 15 open reading frames (ORFs), designated cps19fA to cps19fO, that appear to be arranged as a single transcriptional unit. Insertion-duplication mutants in seven out of the nine new ORFs have been constructed in a smooth type 19F strain, all of which resulted in a rough (nonencapsulated) phenotype, confirming that the operon is essential for capsule production. Comparison with sequence databases has allowed us to propose functions for 12 of the cps19f gene products, and a biosynthetic pathway for type 19F capsular polysaccharide. T7 expression studies confirmed that cps19fH, cps19fK, cps19fL, cps19fM and cps19fN directed the production of polypeptides of the expected size in Escherichia coli. The function of the cps19fK product was confirmed by its ability to complement a mutation in nfrC (rffE) in E. coli, as judged by restoration of sensitivity to bacteriophage N4. Interestingly, the last four genes of the locus (cps19fL-O) exhibit very strong homology (up to 70% amino acid identity) to a portion of the Shigella flexneri rfb gene cluster encoding biosynthesis of dTDP-rhamnose. When expressed in E. coli, cps19fL-O were capable of complementing a mutation deleting the respective Shigella flexneri homologues. Southern hybridization analysis indicated that cps19fA and cps19fB were the only cps genes found in all 16 S. pneumoniae serotypes/groups tested. The region from cps19fG to cps19fK was found only in members of serogroup 19, and, within this, cps19fl was unique to type 19F. [TOP OF PAGE]

128. A novel mechanism of virus-virus interactions: bacteriophage P2 Tin protein inhibits phage T4 DNA synthesis by poisoning the T4 single-stranded DNA binding protein, gp32. Mosig, G., Yu, S., Myung, H., Haggard-Ljungquist, E., Davenport, L., Carlson, K., Calendar, R. (1997). Virology 230:72-81. P2 prophages have been known to inhibit DNA replication and growth of T-even phages. We show here that this inhibition is due to poisoning of the T-even single-stranded DNA binding protein gp32 by the product of the nonessential P2 tin gene. Synthesis of Tin protein from a gene cloned in a multicopy plasmid is necessary and sufficient to completely prevent de novo DNA replication and growth of wild-type T2 or T4 phage. We isolated more than 20 independent mutants that render T-even phages resistant to poisoning by the P2 Tin protein. In all of these mutants, which we call asp, Asp codon 163 of gene 32 is changed to a Gly or Asn codon. The mutant alleles are recessive; i.e., when wild-type and asp mutants coinfect the same host cells, most DNA replication is poisoned by P2 Tin protein. To explain our results, we propose that the P2 Tin protein interacts with T-even gp32 at position 163 and distorts the helical filament of gene 32 protein on single-stranded DNA. Thereby Tin protein inhibits either assembly or function, or both, of the T4 replisome. The inhibition of late gene expression by P2 Tin protein may be an indirect consequence of inhibition of DNA replication. [TOP OF PAGE]

129. Isolation of a virus infectious to the harmful bloom causing microalga Heterosigma akashiwo. Nagasaki, K., Yamaguchi, M. (1997). Aquat. Microb. Ecol. 13:135-140. [TOP OF PAGE]

130. Beating bacteria: New ways to fend off antibiotic pathogens. Nemecek, S. (1997). Scientific American 276(2 (February)), 38 (24?)-39 (25?). . . . One such approach relies on bacteria-attacking viruses, called bacteriophages. . . Neither, however, has garnered significant support among the medical community as a whole, which doubts the efficacy of such procedures . . . [TOP OF PAGE]

131. Virus decay and its cause in coastal waters. Noble, R.T., Fuhrman, J.A. (1997). Appl. Environ. Microbiol. 63:77-83. Mesocosms filled with 80 liters of coastal seawater from Santa Monica, California, were used twice (June and November) to budget bacterial production and loss, as well as to assess the relative significance of viral lysis and protist grazing in bacterial mortality. Bacterial abundance was similar to 6 x 10 super(9) cells/liter in June and 2 x 10 super(9) in November, with viral abundances similar to 2 x 10 super(10) particles/liter in June and 1.5 x 10 super(10) in November. Incorporation of [ super(3)H]thymidine and leucine yielded essentially identical production estimates and allowed calculation on total bacterial mortality in these closed systems. Bacterial growth rates were 1-2/d in June and 1-3/d in November. Three independent lines of evidence indicated that bacterial mortality attributed to grazing by protists was about equal to that attributed to viruses: size fractionation of disappearance of labeled DNA, with a 50% reduction after protists were removed; comparison of protist grazing rates estimated with fluorescently labeled bacteria and virus production-based bacterial lysis rates, with 40-50% of the total ascribed to viruses; and model-based interpretation of the 3.3-4.6% of bacteria visibly infected with assembled intracellular viruses, suggesting that 24-66% of loss is due to infection. Redundant production and loss measurements as well as the independent loss process estimates agreed within similar to 30%, yielding a reasonably balanced budget. We believe the loss of bacteria to viruses reflects a significant dissipation of energy in this ecosystem and that viruses and protists contribute similarly to bacterial mortality. [TOP OF PAGE]

132. Serratia entomophila bacteriophages: host range determination and preliminary characterization. O'Callaghan, M., Jackson, T.A., Glare, T.R. (1997). Canadian Journal of Microbiology 43:1069-1073. Eight bacteriophages specific to Serratia entomophila, a commercially available bacterial pathogen of the New Zealand grass grub (Costelytra zealandica), were characterized by host range determination, morphology and restriction endonuclease patterns of DNA. Phages were originally isolated from grass grub larvae and fermenter broth where phages had disrupted large-scale production of S. entomophila. Seven of the phages (CW1-CW5, BC, and BT) had heads similar in size (approximately 60 x 60 nm) and long noncontractile tails (185 x 10 nm). Phage AgRP8 (P8) had a smaller head and a short tail structure. Restriction endonuclease analysis divided the phages into four groups: CW2, CW4, CW5, BC, and BT gave identical patterns, white CW1, CW3, and P8 each gave different patterns. Six distinct phage groups were distinguished by host range determination, after screening phages against 70 bacterial isolates: CW1, CW2/CW4, CW3, CW5, BC/BT, and P8. While confirming the indicated groupings by DNA analysis, it was possible to distinguish between some of the phages in the largest group: CW2/4 could be distinguished from CW5 and BC/BT. Screening of soil bacterial isolates of S. entomophila against nondiluted phages will aid in monitoring the establishment and persistence of strains applied for biological control of the grass grub. [TOP OF PAGE]

133. Stability of CII is a key element in the cold stress response of bacteriophage lambda infection. Obuchowski, M., Shotland, Y., Koby, S., Giladi, H., Gabig, M., Wegrzyn, G., Oppenheim, A.B. (1997). J. Bacteriol. 179:5987-5991. Bacteria are known to adapt to environmental changes such as temperature fluctuations. It was found that temperature affects the lysis-lysogeny decision of lambda such that at body temperature (37 degree C) the phage can select between the lytic and lysogenic pathways, while at ambient temperature (20 degree C) the lytic pathway is blocked. This temperature-dependent discriminatory developmental pathway is governed mainly by the phage CII activity as a transcriptional activator. Mutations in cII or point mutations at the PRE promoter lead to an over-1,000- fold increase in mature-phage production at low temperature while mutations in cI cause a smaller increase in phage production. Interference with CII activity can restore lytic growth at low temperature. We found that at low temperature the stability of CII in vivo is greatly increased. It was also found that phage DNA replication is blocked at 20 degree C but can be restored by supplying O and P in trans. It is proposed that CII hampers transcription of the rightward p-R promoter, thus reducing the levels of the lambda O and P proteins, which are necessary for phage DNA replication. Our results implicate CII itself or host proteins affecting CII stability as a "molecular thermometer". [TOP OF PAGE]

134. Occurence of virus-like particles in the Dead Sea. Oren, A., Bratbak, G., Heldal, M. (1997). Extremophiles 1:143-??? [TOP OF PAGE]

135. On the origin of spontaneous somatic mutations and sectored plaques detected in transgenic mice. Paashuis, L., Zhang, X.B., Heddle, J.A. (1997). Mutation Research 373:277-284. The use of transgenic mice with bacterial genes that can be readily recovered and analysed for mutation has made it possible to measure mutant frequencies in many tissues. The mutations are detected by packaging the murine DNA into lambda phage and then growing these phage on a bacterial lawn under conditions such that the mutants are distinguishable from nonmutants. In the lacI mouse assay, the mutant plaques are blue whereas the nonmutant plaques are clear. The mutations detected in the lacI Big Blue Mouse are, in principle, a mixture of mutations that arose in the mouse (in vivo mutations), mutations that arose in the bacterium from lesions pre-existing in the murine DNA (ex vivo mutations), and mutations that arose during growth of the phage on the bacteria (in vitro mutations). It has been suggested that plaque morphology can be used to visually distinguish in vivo mutations (which would be seen as wholly blue plaques) from ex vivo mutations (which would produce blue and white sectored plaques) and in vitro mutations (which would produce sectored or pin-point plaques). We show here that this is not the case: ex vivo mutations produce plaques that are a homogeneous blue. By superinfection of bacteria with mutant and nonmutant phage and by in vitro mutagenesis, we found that blue plaques may contain large proportions of nonmutant phage. None of the mutant plaques seen after in vitro mutagenesis were sectored but most contained nonmutant phage. In addition, we show that most spontaneous mutations from the small intestine, which has a higher than normal mutant frequency, arose in vivo, since 17 of 17 mutants were homogeneous mutants. Sectored plaques must arise in some way other than by ex vivo mutation, like perhaps by the confluence of a mutant and nonmutant plaque. [TOP OF PAGE]

136. The detection of enteroviruses in large volume concentrates of recreational waters by the polymerase chain reaction. Pallin, R., Wyn-Jones, A.P., Place, B.M., Lightfoot, N.F. (1997). Journal of Virological Methods 67:57-67. A rapid and simple method was developed to detect enteroviruses in large-volume water samples. It relies on the adsorption of the virus capsids to silica particles under acidic conditions, allowing their recovery by relatively gentle centrifugation. Different reagents used in enterovirus concentration and detection were seeded with Coxsackievirus B5 and used to optimise the recovery method, which was then used to detect the enteroviruses from seeded and unseeded 101 seawater samples in one PCR tube rather than in up to 50 sub-sample volumes, demonstrating its use for routine environmental monitoring. Concentrates from 36 recreational water samples from three sites in N.E. England were analysed for enteroviruses by regular and the new method semi-nested PCR, and infectivity in cell culture. Some of the samples were also analysed for faecal indicator bacteria and F-specific bacteriophage. The results showed a marked increase in detection sensitivity when the whole sample concentrate was assayed as compared with a small volume aliquot. [TOP OF PAGE]

137. Control of Erwinia amylovora by mixtures of bacteriophage. Palmer, E.L., Fernando, W.G.D., Jones, A.L. (1997). Phytopathology 87:S73-S74 [TOP OF PAGE]

138. [Diagnosis of Yersinia pestis]. Pan, T.M., Chien, S.H., Wang, T.K., Tsai, J.L., Horng, C.B. (1997). Chung-Hua Min Kuo Wei Sheng Wu Chi Mien I Hsueh Tsa Chih Chinese 30:43-50. There is no plaque case report in Taiwan since 1952. However, it is necessary to set up a laboratory system to investigate the distribution of Yersinia pestis in the natural environment to implement the public policy for preventing plague. Besides the traditional methods; e.g. culture, microscopic observation, biochemical characteristics, anti-F1 antigen detection by slide agglutination, immunofluorescence, and phage lytic assay, PCR was used as rapid screening test in our study. These laboratory methods were used to examine whether the flea samples harvested in King-Men island carry Y. pestis. The results showed that the flea index per mouse was high but no Y. pestis was detected in the fleas. [TOP OF PAGE]

139. A Mycobacterum smegmatis mutant with a defectivbe inositol monophosphate phosphatase gene homolog has altered cell envelope permeability. Parish, T., Liu, J., Nikaido, H., Stoker, N.G. (1997). J. Bacteriol. 179:7827-7833. [TOP OF PAGE]

140. A virulent bacteriophage of Lactococcus garvieae (formerly Enterococcus seriolicida) isolated from yellowtail Seriola quinqueradiata. Park, K.H., Matsuoka, S., Nakai, T., Muroga, K. (1997). Diseases of Aquatic Organisms 29:145-149. A virulent bacteriophage, designated PLgY, was detected from cultures of Lactococcus garvieae (formerly Enterococcus seriolicida) isolated from diseased yellowtail Seriola quinqueradiata. The phage had an isometric head measuring 50 to 60 nm, a thin flexible tail of 7 times 140-180 nm, and the genome consisting of double stranded DNA, indicating that PLgY is a member of the family Siphoviridae. Of 26 strains of L. garvieae examined, 24 were sensitive to the phage but 2 strains of L. garvieae and another 22 strains of bacteria including fish and shellfish-pathogenic bacteria were not. Lysis of L. garvieae cells due to the phage infection was dependent on culture temperature and occurred at between 17 and 29 degree C. Although an infection experiment of young yellowtail revealed that the 2 phage-insensitive L. garvieae strains were less virulent than 2 phage-sensitive strains, there was no correlation between phage sensitivity and antigenic variation. [TOP OF PAGE]

141. Evidence for groundwater and surface marine water contamination by waste disposal wells in the Florida Keys. Paul, J.H., Rose, J.B., Jiang, S.C., Xhou, X., Cochran, P., Kellogg, C., Kang, J.B., Griffin, D., Farrah, S., Lukasik, J. (1997). Water Res. 31:1448-1454. [TOP OF PAGE]

142. Coliphage and indigenous phage in Mamala Bay, Oahu, Hawaii. Paul, J.H., Rose, J.B., Jiang, S.C., London, P., Xhou, X., Kellogg, C. (1997). Appl. Environ. Microbiol. 63:133-138. Public concern over the discharge of primarily treated sewage by two offshore outfalls in Mamala Bay, Oahu, prompted a multidisciplinary study to determine the impact of such activities on the water quality in the bay and at adjacent recreational beaches. As part of this study, we determined the abundance of coliphage as an indicator of fecal pollution along with total viral direct counts and phages infective for Vibrio parahaemoltyicus 16 at stations in Mamala Bay in four quarterly samplings over 13 months. Coliphage ( lt 1 to 1.2 times 10^-3/liter) were found during each quarterly sampling along an offshore transect to the Sand Island waste treatment facility outfall. The nonpoint coastal stations (Pearl Harbor, Ala Wai Canal, and Ke'ehi Lagoon) had high levels of coliphage during the storm event sampling in February 1994 but much lower levels or none when sampled during dry weather. Coliphage were absent at all samplings at Waikiki Beach and at the control station off Diamond Head. Viral direct counts in eutrophic coastal stations (Pearl Harbor, Ke'ehi Lagoon, Ala Moana Beach, and Ala Wai canal) averaged 10^-9/liter, while counts at offshore stations ranged from 9 times 10^-7 to 1 times 10^-9 viruses/liter, values similar to those for other marine environments. Vibriophage were found mainly in eutrophic coastal environments (Ala Wai Canal, Pearl Harbor, and Ke'ehi Lagoon) and at the Sand Island Transect stations D1 and D2. The greatest abundance was found during the storm event (February 1994) sampling. These results suggest that the Sand Island outfall influenced the water quality of the immediate surrounding waters but had little effect on the quality of the recreational beaches. Nonpoint discharge sources appeared to be more important in the distribution of fecal indicators in the coastal zone. [TOP OF PAGE]

143. Bacteriophage B103: complete DNA sequence of its genome and relationship to other Bacillus phages. Pecenkova, T., Benes, V., Paces, J., Vlcek, C., Paces, V. (1997). Gene 199:157-163. The genome of Bacillus subtilis bacteriophage B103 consists of double-stranded linear DNA 18,630 bp long. The DNA was sequenced, and the sequence was compared with DNA sequences of closely related phages, namely the members of the phage f29 family. Among them, phage Nf was shown to be the most closely related to B103. Comparisons of several open reading frames (ORFs) among the family members helped to identify genes 1 and 5. A cluster of ORFs between genes 16 and 17 contains two ORFs with partial homology with two f29 ORFs located in the same region. There are three more ORFs in this region of B103 with good ribosome binding sites (RBS) and optimal codon usage that are not homologous to any of the f29 ORFs. The function of these five ORFs remains unexplained. It was shown that major promoters characterized in f29 are retained in B103. Where many substitutions occur in the vicinity of a promoter, at least the - 10 and -35 boxes are conserved. [TOP OF PAGE]

144. Phage-mediated transfer of tetracycline resistance in Staphylococcus aureus isolated from cattle in Brazil. Periera, M.S., Barreto, V.P., Siqueira-Junior, J.P. (1997). Microbios 92:147-155. Tetracycline-resistant strains of Staphylococcus aureus isolated from cattle in Brazil, were used as prospective donors for the transfer of resistance to laboratory strains, using mixed-culture and filter-mating protocols. Three lysogenic donors transferred tetracycline resistance in both mixed culture and during filter mating. In contrast, when a non-lysogenic strain was used as prospective donor, transfer was not detected using either mating protocol. In order to evaluate the involvement of phage, successful transfer experiments were repeated with the addition of sodium citrate, which sequestered calcium ions. Mixed-culture and filter-mating protocols did not result in the transfer of resistance. These results support the notion that transfer of the resistance determinant under both sets of conditions described here involve the same bacteriophage-mediated mechanism. Although transfer of tetracycline resistance was detected, without any attempt to create specialized transduction agents or to extract phages, the co-transfer of additional resistance markers indicated that it could not be conventional transduction. [TOP OF PAGE]

145. Transport and recovery of bacteriophage PRD1 in a sand and gravel aquifer: Effect of sewage-derived organic matter. Pieper, A.P., Ryan, J.H., Harvey, R.W., Amy, G.L., Illangasekare, T.H., Metge, D.W. (1997). Environmental Science & Technology 31:1163-1170. To test the effects of sewage-derived organic matter on virus attachment, 32P-labeled bacteriophage PRD1, linear alkylbenzene sulfonates (LAS), and tracers were injected into sewage- contaminated (suboxic, elevated organic matter) and uncontaminated (oxic, low organic matter) zones of an iron oxide- coated quartz sand and gravel aquifer on Cape Cod, MA. In the uncontaminated zone, 83% of the PRD1 were attenuated over the first meter of transport by attachment to aquifer grains. In the contaminated zone, 42% of the PRD1 were attenuated over the first meter of transport. Sewage-derived organic matter contributed to the difference in PRD1 attenuation by blocking attachment sites in the contaminated zone. At greater distances down-gradient (to a total transport distance of 3.6 m), a near-constant amount of PRD1 continued to break through, suggesting that aquifer grain heterogeneities allowed a small amount of reversible attachment. Injection of an LAS mixture (25 mg L-1), a common sewage constituent, remobilized 87% of the attached PRD1 in the contaminated zone, but only 2.2% in the uncontaminated zone. LAS adsorption promoted virus recovery in the contaminated zone by altering the PRD1-surface interactions; however, the amount of LAS adsorbed was not sufficient to promote release of the attached PRD1 in the uncontaminated zone. [TOP OF PAGE]

146. Cloning and characterization of bacteriophage-like DNA from Haemophilus somnus homologous to phages P2 and HP1. Pontarollo, R.A., Rioux, C.R., Potter, A.A. (1997). J. Bacteriol. 179:1872-1879. In an attempt to identify and characterize components of a heme uptake system of Haemophilus somnus, an Escherichia coli cosmid library of H. somnus genomic DNA was screened for the ability to bind hemin (Hmb+). The Hmb+ phenotype was associated with a 7,814-bp HindIII fragment of H. somnus DNA that was subcloned and sequenced. Thirteen open reading frames (orfs) were identified, all transcribed in one direction, and transposon mutagenesis identified orf7 as the gene associated with the Hmb+ phenotype. Orf7 (178 amino acids) has extensive homology with the lysozymes of bacteriophages P-A2, P21, P22, PZA, phi-29, phi-vML3, T4, or HP1. The orf7 gene complemented the lytic function of the K gene of phage P2 and the R gene of phage lambda. A lysozyme assay using supernatants from whole-cell lysates of E. coli cultures harboring plasmid pRAP501 or pGCH2 (both of which express the orf7 gene product) exhibited significant levels of lysozyme activity. The orf6 gene upstream of orf7 has the dual start motif common to the holins encoded by lambdoid S genes, and the orf6 gene product has significant homology to the holins of phages HP1 and P21. When expressed from a tac promoter, the orf6 gene product caused immediate cell death without lysis, while cultures expressing the orf7 gene product grew at normal rates but lysed immediately after the addition of chloroform. Based on this data, we concluded that the Hmb+ phenotype was an artifact resulting from the expression of cloned lysis genes which were detrimental to the E. coli host. The DNA flanking the cloned lysis genes contains orfs that are similar to structural and DNA packaging genes of phage P2. Polyclonal antiserum against Orf2, which is homologous to the major capsid precursor protein (gpN) of phage P2, detected a 40,000-M(r) protein expressed from pRAP401 but did not detect Orf2 in H. somnus, lysates. The phage-like DNA was detected in the serum-susceptible preputial strains HS-124P and HS-127P but was absent from the serum-resistant preputial strains HS-20P and HS-22P. Elucidation of a potential role for this cryptic prophage in the H. somnus life cycle requires more study. [TOP OF PAGE]

147. Evolutionary Ecology of Parasites - From Individuals to Communities. Poulin, R. (1997). ???, ???[TOP OF PAGE]

148. Blending of pure strains for safer and enhanced flavouring in the dairy industry. Prigent, J.R. (1997). Comptes Rendus de l'Academie d'Agriculture de France 83:67-82. One of the main objectives for food cultures manufacturers is to be able to propose, for each of the different cheeses making technologies, blends of pure strains giving, in a reproducible and safe way, pasteurized milk cheeses with typical characters as high as those of raw milk cheeses. The first step in the process is to get the right quality curd in any given technological conditions, i.e., the right pH evolution versus time for the temperature profile of this technology in the precise time allowed by the process (reproducibility better than 15 minutes). This is done by the use of blends of pure strains of lactic acid bacteria carefully selected and screened for their functional properties. Furthermore, these blends must be resistant to phage attacks. The curd obtained in the first step of the process is then the substrate for ripening cultures: molds, yeasts, many species of bacteria. These microorganisms are specially selected for their functionalities: neutralizing, flavouring, texturizing, coloring, coating properties. Interactions between microorganisms are very important: synergies with useful flora and antagonisms towards undesirable microorganisms. The whole problem is rather complex: a significant simplification may however be obtained by the use of direct vat inoculation giving a better reproducibility than intermediate subcultures. [TOP OF PAGE]

149. Advances in the study of marine viruses. Proctor, L.M., Lita, M. (1997). Microscopy Research and Technique 37:136-161. Free viruses are abundant in the world's oceans. With this realization has come renewed interest in marine viruses and the role viruses play in structuring marine planktonic communities, primarily members of the microbial assemblage. The principal means of studying marine viruses has been by electron microscopy. This review discusses the use of microscopy to study free viruses and compares the ultrastructure of free viruses with bacteriophages and viruses which have been cultured from marine hosts. ¶ Many of the free viruses are smaller than typical cultured bacteriophages, which suggests that either many native phages are smaller than cultured phages or that many of the free viruses may be members of those phage families with smaller size classes or, in some cases, that many free viruses may be eukaryotic viruses. Some of the forms currently considered free viruses may be "defective phage" or "phage ghosts," noninfectious particles produced by bacteria, or virus-sized inorganic/organic colloids and warrant further study. ¶ Gross virus ultrastructure cannot be used as the sole criterion for determining marine virus diversity, since, as with many microbes, many unrelated viruses have similar morphological characters. Determination of DNA or RNA content as well as studies of protein and DNA relatedness of marine viruses will be needed if we are to understand the complexity of marine virus assemblages. Another important direction for future work is the need for marine bacteriophage/host and virus/host systems in order to study the biology of virus infection. Microsc. Res. Tech. 37:136–161, 1997. [TOP OF PAGE]

150. Bacteriophages infecting various Bacteroides fragilis strains differ in their capacity to distinguish human from animal faecal pollution. Puig, A., Jofre, J., Araujo, R. (1997). Water Science and Technology 35:359-362. Strain HSP40 of Bacteroides fragilis (ATCC 51477) selectively detects bacteriophages excreted by humans. We have tested 115 strains of B. fragilis, isolated from humans, as hosts for the detection of bacteriophages in urban sewage. Only 10% of the strains examined gave counts that were similar to or higher than those given by B. fragilis HSP40. To determine their specificity, some of these were examined for their ability to detect phages in faeces from various animal species and in slaughterhouse wastewater. Two groups of strains were distinguished. The first allowed the recovery of phages in samples with human and animal faecal pollution. The second, which included HSP40, detected phages preferentially in samples with human faecal pollution. [TOP OF PAGE]

151. Viral spread within ageing bacterial populations. Ramirez, E., Villverde, A. (1997). Gene 202:147-149. The viral spread within isolated host populations has been studied throughout the growth of P22-infected Salmonella cell colonies. By using an integration mutant of this bacteriophage, horizontal and vertical transmission have been analyzed independently. The data obtained show that both strategies are not simultaneous but consecutive during the colony development. Lytic cycles are tightly repressed during the exponential cell growth but stimulated in independent colonies with remarkable synchrony when the cell division rate decreases. The coincidence of the viral outburst and the decay of bacterial replicative fitness is a new example of the extreme viral competence in exploiting the host cells as dissemination vehicles for viral genomes. [TOP OF PAGE]

152. Filtration of recombinant Norwalk virus particles and bacteriophage MS2 in quartz sand: Importance of electrostatic interactions. Redman, J.A., Grant, S.B., Olson, T.M., Hardy, M.E., Estes, M.K. (1997). Environmental Science & Technology 31:3378-3383. Norwalk virus is known to be transmitted through groundwater, yet the environmental factors that facilitate its interstitial transport in subsurface systems are not yet clear. This paper investigates the filtration and surface charge of recombinant Norwalk virus (rNV) particles that are morphologically and antigenically similar to live Norwalk strains but lack nucleic acid and are therefore noninfectious. In contrast to bacteriophage MS2, a common surrogate for waterborne viral pathogens, the surface charge of rNV particles and their filtration in packed beds of quartz sand are strongly influenced by pore water pH over the environmentally important range of pH 5-7. From a mechanistic perspective, these results suggest that the physicochemical filtration of the Norwalk virus is highly dependent on the nature and magnitude of electrostatic interactions that develop between the virus and filter media. Furthermore, because MS2 and the rNV particles differ significantly with respect to their electrostatic properties, MS2 may not mimic the subsurface filtration of Norwalk virus in natural systems. [TOP OF PAGE]

153. Natural milk cultures for the production of Argentinian cheeses. Reinheimer, J.A., Binetti, A.G., Quiberoni, A., Bailo, N.B., Rubiolo, A.C., Giraffa, G. (1997). Journal of Food Protection 60:59-63. Samples (32) of natural milk cultures used in the Santa Fe, Argentina, area for soft and semihard cheese production were examined. The microbial composition (including lactic acid microflora characterization) and technological parameters (acidifying and proteolytic activities) were evaluated. The cultures contained mainly thermophilic lactic acid bacteria, identified as Streptococcus salivarius subsp. thermophilus (96.8% of the total strains) and Enterococcus spp. The strains showed a low proteolytic activity. The isolates of S. salivarius subsp. thermophilus exhibited a widespread phage resistance. The nonlactic microflora comprised coliforms, yeasts, spore-forming bacteria and lactate fermentative bacteria. The samples showed an acidity level from 0.38 to 0.69% lactic acid (pH from 4.25 to 5.75). The acidifying activity was optimal at 45 degree C. The advantages and disadvantages of the employment of natural milk starters are discussed. [TOP OF PAGE]

154. The role of pseudolysogeny in bacteriophage-host interactions in a natural freshwater environment. Ripp, S., Miller, R.V. (1997). Microbiology 143:2065-2070. Bacteriophages occur in high numbers in environmental ecosystems and are thus significant mediators of microbial survival and activities. However, interactions between microbial populations and phages in situ have been largely ignored. Current understanding of the process relies on studies performed with well-fed, laboratory-grown host bacteria. The purpose of the experiments reported here was to determine bacteriophage-host interactions under environmentally relevant conditions of nutrient limitation. These studies have revealed the importance of a phenomenon called pseudolysogeny in the maintenance of viral genetic material for extended periods of time in natural ecosystems. Pseudolysogeny is a form of phage-host cell interaction in which the nucleic acid of the phage resides within its starved host in an unstable, inactive state. It is hypothesized that pseudolysogeny occurs due to the cell's highly starved condition. In such cells, there is insufficient energy available for the phage to initiate genetic expression leading to either a true temperate response or to the lytic response. However, upon nutrient addition, the pseudolysogenic state is resolved, resulting in either the establishment of true lysogeny or the initiation of the lytic production of progeny virions. The pseudolysogenic state may explain the long-term survival of viruses in unfavorable environments in which the infective half- life of their virions is relatively short. [TOP OF PAGE]

155. Specific identification of Mycobacterium tuberculosis with the luciferase reporter mycobacteriophage: use of p-nitro-alpha-acetylamino-beta-hydroxy propiophenone. Riska, P.F., Jacobs, W.J., Bloom, B.R., McKitrick, J., Chan, J. (1997). Journal of Clinical Microbiology 35:3225-3231. We have previously described a luciferase reporter mycobacteriophage (LRP) assay that can detect Mycobacterium tuberculosis and characterize mycobacterial drug susceptibility patterns within 24 to 48 h in positive cultures. One drawback of this LRP protocol is the ability of the recombinant mycobacteriophage phAE40 to infect a variety of Mycobacterium species, thus limiting its specificity for the detection of M. tuberculosis. In this study, we have (i) explored the host range of phAE40, (ii) developed a modified LRP assay that exploits the selective inhibitory effect of the compound p-nitro-alpha-acetylamino-beta-hydroxy propiophenone (NAP) against members of the M. tuberculosis complex to differentiate between the tubercle bacillus and other mycobacterial species, and (iii) tested over 300 samples, including primary clinical isolates and drug-resistant strains of M. tuberculosis, demonstrating the ability of the NAP-modified LRP assay to identify M. tuberculosis complex organisms with high degrees of sensitivity and specificity. [TOP OF PAGE]

156. Comparison of PCR and plaque assay for detection and enumeration of coliphage in polluted marine waters. Rose, J.B., Zhou, X., Griffin, D.W., Paul, J.H. (1997). Appl. Environ. Microbiol. 63:4564-4566. [TOP OF PAGE]

157. Adsorption of viruses in water environment onto solid surfaces. Sakoda, A., Sakai, Y., Hayakawa, K., Suzuki, M. (1997). Water Science and Technology 35:107-114. Recently the contamination of water environment involving rivers, lakes, the sources of drinking water, etc. by viruses has been paid attention to as a new threat. The behavior of the viruses found in water environment is not well understood so far. However, it is suspected in general that the viruses are adsorbed onto solid surfaces such as suspended solids and sediment and keep their activities for a long time. Most likely, it is true that the adsorption of the viruses onto solid surfaces is one of the major factors controlling their transport and survival in water environment. In this work, the adsorption equilibrium relations of model viruses in water environment and their activities on solid surfaces were investigated. The Escherichia coli phage such as Q beta , fr, MS2 and T4 were employed for experiments as model viruses, and cellulose and its derivatives, kaolin, carbon black, etc. were chosen as model solid surfaces. All the adsorption isotherms of model viruses on model surfaces were successfully written as the linear expression by the Henry equation in the concentration range of 10 super(2)-10 super(7) [PFU/ml]. The resultant Henry constants were correlated with the total acidity of the solid surfaces. Stability of the model viruses was completely different when they were adsorbed on the solid surfaces and when they were suspended in water. The viruses adsorbed on the solid surfaces were significantly stable compared with the suspended ones regardless of the surface properties. It is suggested that the shrinkage of the virus is one of the important survival factors and the adsorption onto solid surfaces enhances their activities. [TOP OF PAGE]

158. Studies on phage pattern, antibiotic sensitivity and enterotoxigenicity of Staphylococcus aureus strains isolated from fish and factory workers. Sanjeev, S., Stephen, J., Gopakumar, K. (1997). In James, D.G. (ed.), Asia Pacific Fishery Commission. Summary report of and papers presented at the tenth session of the Working Party of Fish Technology and Marketing. Colombo, Sri Lanka. FAO fisheries report, Rome. The phage pattern, antibiotic sensitivity and enterotoxigenicity of 89 strains of Staphylococcus aureus isolated from fish products and workers engaged in fish processing in India were studied. Phage typing was carried out with a basic set consisting of 23 phages. Forty out of 89 (44.9%) S. aureus strains were found phage typable. 45% of typable strains were lysed by phages of group 3 and 25% were lysed by phages of group 2. Phages of group 1 and unallocated phages were found to lyse 5% of the strains. Twenty percent of the strains were lysed by phages of the mixed group. The results of antibiotic sensitivity tests revealed that phage typable strains were less sensitive to penicillin and ampicillin compared to untypable strains. 87.5% of the typable strains and 87.8% of the untypable strains produced enterotoxins A, B, C, D and E either singly or in combinations. None of the strains lysed by phages of group 1 produced toxins. Maximum toxin production was observed by strains lysed by phages of group 3. [TOP OF PAGE]

159. Characterization of host-range mutants of cyanophage N-1. Sarma, T.A., Kaur, B. (1997). Acta Virol. 41:245-250. Fifteen host-range (h) mutants of cyanophage N-1 were characterized with reference to their efficiency of plating, time of appearance, morphology and size of plaques on Nostoc muscorum and its three phage-resistant (Nm 1/N-1, Nm 2/N-1 and Nm 8/N-1) mutants. While phage N-1 did not adsorb to the three phage-resistant mutants, the h mutants differed one from the other in having lower or higher adsorption rate constants on N. muscorum or the phage-resistant mutants. The inability of majority of h mutants isolated on Nm 1/N-1 to grow in Nm 8/N-1 was shown to be due to a failure of adsorption. The h mutants also differed one from the other in their reversion (back mutation) frequencies. The lethal doses (LD sub(37)) required to kill 37% of free phage particles after UV-irradiation, heating and ethylenediamine tetraacetate (EDTA) treatment greatly varied. Most of the h mutants were found to be considerably more sensitive to UV and thermic inactivation than N-1 while they were resistant to EDTA. The h mutants except five of them were unable to multiply at 40 degree C. The significance of these features is discussed. [TOP OF PAGE]

160. Sequence comparison of the genes for immunity, DNA replication, and cell lysis of the P22-related Salmonella phages ES18 and L. Schicklmaier, P., Schmeiger, H. (1997). Gene 195:93-100. [TOP OF PAGE]

161. Bacteriophage infection and multiplication occur in Pseudomonas aeruginosa starved for 5 years. Schrader, H.S., Schrader, O., Walker, J.J., Wolf, T.A., Nickerson, K.W., Kokjohn, T.A. (1997). Canadian Journal of Microbiology 43:1157-1163. Bacteriophages specific for Pseudomonas aeruginosa and Escherichia coli were examined for their ability to multiply in stationary phase hosts. Four out of five bacteriophages tested, including E. coli bacteriophage T7M, were able to multiply in stationary phase hosts. The bacteriophage ACQ had a mean burst size of approximately 1000 in exponential phase P. aeruginosa hosts and 102 in starved hosts, with corresponding latent periods that increased from 65 to 210 min. The bacteriophage UT1 had a mean burst size of approximately 211 in exponential phase P. aeruginosa hosts and 11 in starved hosts, with latent periods that increased from a mean of 90 min in exponential phase hosts to 165 min in starved hosts. Bacteriophage multiplication occurred whether or not the hosts had entered stationary phase, either because the cultures had been incubated for 24 h or were starved. Significantly, bacteriophage multiplication occurred in P. aeruginosa, which had been starved for periods of 24 h, several weeks, or 5 years. Only one P. aeruginosa virus, BLB, was found to be incapable of multiplication in stationary phase hosts. These results reveal that starvation does not offer bacterial hosts refuge from bacteriophage infection and suggest that bacteriophages will be responsible for significant bacterial mortality in most natural ecosystems. [TOP OF PAGE]

162. Formation of submicron colloidal particles from marine bacteria by viral infection. Shibata, A., Kogure, K., Koike, I., Ohwada, K. (1997). Mar. Ecol. Prog. Ser. 155 (not 154):303-307. We tested the hypothesis that viral lytic infection leads to the formation of submicron-sized colloidal particles originating from marine bacteria. Laboratory experiments were performed using a marine bacterium, Vibrio alginolyticus, and its infectious phage. A particle counter was used to determine abundance and size distribution of particles. We found that the non-living submicron sized particles (size range from 0.38 to 0.7 mum in diameter) increased rapidly along with a decrease of bacteria and an increase of phage, indicating that these particles are cell debris originating from bacteria. These particles were stained faintly by acridine orange but were not countable due to the amorphous shape. These results show that amorphous submicron particles are produced by viral lysis of bacteria. This process may be one of the major pathways of colloid formation associated with microbial food webs in the sea. [TOP OF PAGE]

163. Morphological features of a filamentous phage of Vibrio cholerae O139 Bengal. Shimodori, S., Iida, K., Kojima, F., Takade, A., Ehara, M., Amako, K. (1997). Microbiology and Immunology 41:757-763. A filamentous phage was isolated from carrier strain AI-1841 of Vibrio cholerae 0139 Bengal and thus was termed fs phage. The phage was measured to be approximately 1 microm in length and 6 nm in width. One end of the phage was slightly tapered and had a fibrous appendage. The plaques developed on strain AI-4450 of V. cholerae 0139 were small and turbid. The phage grew in strain AI-4450 and reached a size of 10(8) to 10(9) pfu/ml at 5 hr after infection without inducing any lysis of the host bacteria. The group of phages attached on rod-shaped materials like fimbriae of this bacteria, with their fibrous appendages at the pointed end, were often found in the phage-infected culture. The anti-fimbrial serum effectively inhibited the infection of fs phage to the host strain AI-4450. We thus concluded that the phage can be adsorbed on fimbriae with a fibrous appendage on the pointed end of the phage filament. [TOP OF PAGE]

164. Viruses in aquatic ecosystems. A review. Sime-Ngando, T. (1997). Annee Biologique 36:181-210. Even though the contribution of water ecosystems for disseminating enteric viral pathogens has been known for decades, the importance of wild virions iii structuring aquatic communities and food webs has only come to light relatively recently. Evidences of viral infections in both pro-and eukaryotic phytoplankton, as well as in heterotrophic bacterio-and protozooplankton, have recently brought marine biologists to question the impact of viroplankton on processes such as (1) the mortality of microorganisms, (2) the nutrition of heterotrophic protists, (3) the promotion of genetic material exchanges among microbial populations, (4) the maintenance of species diversity, (5) the induction of planktonic aggregates, and (6) the cycling of organic matter in aquatic ecosystems. In this paper, all these points are reviewed and discussed, in the light of recent contributions to the ecology of aquatic viruses, for evidence of the impact of viruses on both steady and non-steady state processes in fresh- and salt-waters.
     
     Viruses are ubiquitous, abundant and dynamic components of pelagic ecosystems. They are, undoubtedly, more diversified than the phage -like morphotypes that are generally characterized by the presence of an icosa- or octahedral head and a tail, via observations under electron microscope. The diversity of planktonic viruses is further enhanced from the genetic viewpoint, and likely implies the diversity of sensible hosts. Genetically related marine phages are likely widely distributed in the space (i. e. without significant geographical segregation), suggesting prevalence of a reduced competition among viral ''populations'', and that the main biotic limiting factor for a viral ''species'' production is the density of the sensible host. Some viral ''species'', known from marine systems, typically harbor knob-like projections and long spines (i. e. previously not noted from non.-aquatic habitats), which are suggested to increase the efficiency of hitting a specific host, especially in oligotrophic waters. Despite the general scarcity of viral isolates that lyse ciliated protozooplankton and metazoan zooplankton, it is becoming increasingly evident that most of the pelagic pro-and eukaryotic organisms are subject to infectious attacks from ambient ''free-living'' viruses.
     
     Quantitatively, recent total counts from the plankton generally fall in between 10(4) and 10(8) viruses ml(-1), with seasonal high densities in spring and summer, and a lowering tendency in abundance from the coastal to the open marine systems, and from the surface to the depth waters, likely in relation to temperature and the organic matter load. it was recently shown that lytic infection, rather than induction of lysogeny, is responsible for the majority of bacteriophage production in the plankton, especially in the coastal marine and surface waters and during blooming events, where the threshold-product level of virus x bacteria numbers of greater than or equal to 10(12) for the start of a viral-lytic activity is generally achieved. Closed linear relationships have been reported between viroplankton dynamics and bacteria, algae and nutrients. Because of the preponderance of allochtonous organic matter and cyanobacterial cells in lakes as compared to oceanic systems, the virus-to-bacteria ratio in lakes are significantly higher than in marines systems, although there is little trend in the virus-to-bacteria ratio with increasing trophy, and despite the occurrence of more bacteria per unit chlorophyll in lake samples.
     
     The functional impact of virions on the structure and metabolism of planktonic communities is more important than their quantitative importance, as viruses represent only a minor fraction of the total planktonic biomass. The viral-induced mortality of microbial communities in marine systems is estimated to represent about 30 and < 10 pour 100 of the mortality of bacterio- and phytoplankton, respectively. Based on one study, the contribution of viruses to bacterial mortality in lakes seems considerably lower than in marine systems. The greatest impact of viruses on aquatic communities is likely through hazardous (i.e. non-steady state) processes which are difficult to quantified, such as the promotion of genetic material exchanges among populations and the maintenance of species diversity. The lytic pressure from virulent viruses may act as a ''nonstop'' inductor of modifications in the genetic heritage of host-organisms, thereby increasing the potential of these hosts to share their habitat with homologous species, i.e. with similar nutritional requirements.
     
     It has recently been shown that lysates resulting from phage infection can caused a significant increase in metabolic activity of noninfected bacterioplankton community, but the growth efficiency of these noninfected hosts decreased in the presence of viruses, likely because of the increase in bacterial energy demand associated with extracellular degradation of polymers that are prevalent in viral lysates. This seems to verify the hypothesis on a substantial contribution of the lytic activity from viruses, to the cycling of organic matter in aquatic systems. Viral lytic production may indeed (1) reduce the bacterial biomass contribution to the transfer of metabolic energy on to higher-order consumers, (2) result in an increase of bacterial secondary production in the absence of an increase in the ambient primary production, and (3) increase competition between bacterial exo- or ectoenzyme activity and the feeding activity of protozoa on high molecular weight polymers (including viruses), although ingestion of viruses by protists seems to be of less importance in the carbon flows through the microbial food web in pelagic systems.
     
     However, almost all studies on the ecology of pelagic viruses are done during a limited period of year, mainly in marine waters situated in temperate zones. The data discussed in this paper are thus to be considered as preliminary data. Nevertheless, viruses undoubtedly influence to various degrees the biological processes in aquatic ecosystems. The quantitative assessment of their functional impact is thus required for incorporation into models that simulate flues of matter, nutrients and energy in aquatic systems. This task is to be include on the agenda of both marine and freshwater biologists, as a high priority concern for the near future. [TOP OF PAGE]

165. Transport of bacteria and bacteriophages in irrigated effluent into and through an alluvial gravel aquifer. Sinton, L.W., Finlay, R.K., Pang, L., Scott, D.M. (1997). Water, Air, and Soil Pollution 98:17-42. The movement of bacteria and bacteriophages into and through an alluvial gravel aquifer was investigated at a bordered strip effluent irrigation scheme near Christchurch, New Zealand. irrigation of one set of strips resulted in the contamination, by faecal coliform bacteria, and somatic and F-RNA coliphages, of two bores, approximately 60 m and 445 m downstream of the centre of the strips. F-RNA coliphages showed the greatest attenuation between the soil surface and the first bore, and faecal coliforms the least. Estimates of percolation times through the 13 m vadoze zone (based on times to peak concentration in the groundwater) ranged from 1.6 to 10.5 hr, with travel times for the bacteriophages being 1.4-3.4 times longer than for the bacteria. Injection of oxidation pond effluent containing rhodamine WT dye into the first bore resulted in contamination of the second bore (385 m downstream) by the dye, F-RNA coliphages, and faecal coliforms. In a second experiment, injection (into the same bore) of a mixture of phage MS-2, the bacterial tracer Escherichia coli J6-2, and rhodamine WT dye, produced a similar result in the downstream bore and in a newly-installed bore, 401 m downstream. In both injection experiments, the phages exhibited the shortest times to peak concentrations in the downstream bore(s), followed by the bacteria, and then the dye. Attenuation of the bacteria and phages was similar, but the microbes exhibited 100-fold greater reduction than the dye. Flow direction and longitudinal dispersivity were determined in a preliminary analysis using an idealised 2-D dispersion model. This information, and other measured and reported data, were then used as inputs in a 3-D dispersion model. The predicted concentration curves were matched to the observed curves by trial and error adjustment of the decay constant (lambda). The best curve fits were obtained with lambda values higher than those reported elsewhere. It is suggested that many of the reported microbial decay values underestimate microbial reductions in groundwater because they do not account for other removal mechanisms, such as filtration, sedimentation and irreversible adsorption. [TOP OF PAGE]

166. The relationship between HP1 and S2 bacteriophages of Haemophilus influenzae. Skowronek, K., Baranowski, S. (1997). Gene (Amsterdam) 196:139-144. Comparison of the nucleotide sequences of the left arms of two Haemophilus influenzae phages, S2 and HP1 is presented. They exhibit a characteristic mosaic pattern of homologous and non-homologous regions. The homology extends over the attP site and int, orf 5 to 9, rep and the 3' part of cI genes. Two major non-homologous regions were detected. One is found between the int and cI genes; the other spans the region of promoters and the cox gene. Variations in the region of the promotors which is involved in the choice between a lysogenic and a lytic pathway and some divergences in the cI coding sequences are probably responsible for the observed immunity differences between the two phages. Distinctions in the distribution of consensus sequences for an integration host factor (IHF) and integrase-binding sites and promoters are described. These data offer an explanation of the relationship between three types of S2/HP1 phages. It allows in turn a final settlement of the nomenclature variation in the literature. The results presented, which are similar to those obtained for other phage groups, suggest that the mosaic structure of phage genomes is a normal outcome of phage divergence. [TOP OF PAGE]

167. Rod-shaped virus-like particles in intestinal contents of pheasant chicks. Smid, B., Valicek, L., Kudrna, J. (1997). Zentralblatt Fur Veterinarmedizin - Reihe B 44:445-447. Rod-shaped virus-like particles (RSV), 55-85 nm in length and 18 nm in diameter, with 5 to 10 segments or helical turns, were demonstrated in the intestinal contents of young diarrhoeic pheasants by examination of a fresh sample. The origin of RSV seems to be splitting tails of bacteriophages. [TOP OF PAGE]

168. Induction of a temperate marine cyanophage by heavy metal. Sode, K., Oonari, R., Oozeki, M. (1997). J. Mar. Biotechnol. 5:178-180. The activity of a temperate marine cyanophage, ms-1, of Synechococcus sp. NKBG 042902, was induced by Cu²⁺. This induction was specific to Cu²⁺ and dependent upon Cu²⁺ concentration. Cr, Pb, Co, and Zn were not effective as inducers. These results suggested that Cu²⁺ is a significant inducer for lysogenic cyanobacterial cells and consequently will be a potential trigger for changes in the cyanobacterial population in the marine environment. [TOP OF PAGE]

169. Control of bacterial spot of tomato in transplant production using h-mutant bacteriophage and a hrp- strain of Xanthomonas campestris pv. vesicatoria. Somodi, G.C., Jones, J.B., Jackson, L.E. (1997). Phytopathology 87:S92 [TOP OF PAGE]

170. Genetics of host-parasite interactions. Sorci, G., Moller, A.P., Boulinier, T. (1997). Trends in Ecology & Evolution 12:196-200. [TOP OF PAGE]

171. Isolation, cloning and characterisation of the abiI gene from Lactococcus lactis subsp. lactis M138 encoding abortive phage infection. Su, P., Harvey, M., Im, H.J., Dunn, N.W. (1997). Journal of Biotechnology 54:95-104. Plasmid pND852 (56 kb) encodes nisin resistance and was isolated from Lactococcus lactis ssp lactis (L. lactis) M138 by conjugation to L. lactis LM0230. It conferred strong resistance to the isometric-headed phage phi 712 and partial resistance to the prolate-headed phage phi c2. A 2.6 kb HpaII fragment encoding phage resistance was cloned into the streptococcal/Bacillus hybrid vector pGB301 to generate pND817. The mechanism of phage resistance encoded by pND817 involved abortive infection and this was illustrated by a reduction in burst size from 166 to 6 at 30 degrees C and from 160 to 90 at 37 degrees C. Partial resistance was therefore retained at 37 degrees C. DNA sequencing revealed that the abortive infection was encoded by a single open reading frame (ORF), designated abiI, encoding a 332 amino acid protein. Neither abiI nor the predicted product showed significant homology to any existing sequence in the GenBank database. Frame shift mutation at the unique EcoRI site within the ORF resulted in loss of the Abi+ phenotype, confirming that the ORF is responsible for the encoded phage resistance. [TOP OF PAGE]

172. Quantification of bacteriophages of Bacteroides fragilis in environmental water samples of Seine river. Sun, Z.P., Levi, Y., Kiene, L., Dumoutier, N., Lucena, F. (1997). Water 96:175-183. Virus detection in drinking water is very important to protect human health. The different methodologies for analysing human pathogenic virus are very time consuming and expensive, so until now only a few specialised laboratories carried out this analysis. Detection of bacteriophages may be possible by examining the aquatic virus, with advantages of easy and cheap. The bacteriophages of Bacteroides fragilis have been proven as specifically present in human faeces and have relationships with water contaminated by enterovirus. Our study, for the first time in France, discovered the B. fragilis phages present in sample of sewage, Seine river and raw water for water supply. Our results also presented that B. fragilis phages may be a better indicator for water bacteriology compared with classical bacteriological indicators in water treatment. On the other hand, our results demonstrated that MPN method (Most Probable Number) has more advantages than that of PFU (Plaque Forming Units). [TOP OF PAGE]

173. Accumulation of degradable DOC in surface waters: Is it caused by a malfunctioning microbial loop? Thingstad, T.F., Hagstrom, A., Rassoulzadegan, F. (1997). Limnol. Oceanogr. 42:398-404. Recent literature indicates that dissolved organic carbon (DOC) may accumulate in productive surface waters. Such accumulation will allow export of DOC to the aphotic zone by diffusion and downwelling. As an alternative to models based on low degradability, we here propose a mechanism where bacterial carbon consumption is restricted due to food web mechanisms controlling both growth and biomass of the bacteria: growth rate is kept low by bacteria-phytoplankton competition for mineral nutrients, and biomass is kept low by bacterial predators. With such a mechanism, otherwise degradable material may accumulate and become subject both to chemical transformation and vertical transport. The steady-states of a model describing the interactions between heterotrophic bacteria, phytoplankton, and bacterivorous protozoa is used to explore how the balance between DOC production and consumption shifts along a gradient from oligotrophy to eutrophy. [TOP OF PAGE]

174. Theoretical models for control of bacterial growth rate, abundance, diversity and carbon demand. Thingstad, T.F., Lignell, R. (1997). Aquat. Microb. Ecol. 13:19-27. Our conceptual understanding of the role of heterotrophic bacteria in pelagic ecosystems and in ocean biogeochemical cycles is closely Linked to our understanding of how their growth rate, abundance, and diversity is controlled. Here we discuss consequences of the simplifying assumption that there are only 5 potentially important interactions between heterotrophic bacteria and their biological and chemical environment. We consider 3 possible types of growth rate limitation: (1) organic carbon, (2) inorganic phosphate, and (3) organic and inorganic nitrogen; and 2 types of cell losses: (1) predation by heterotrophic flagellates, or (2) lysis by infectious viruses. Incorporating this into simple food web structures, we discuss 4 classes of models, 2 based on carbon limitation and 2 based on mineral nutrient limitation of bacterial growth rate. Bacterial abundance is assumed to be controlled by protozoan predation in all cases. For each class, we derive expressions describing bacterial carbon demand, and discuss the control of bacterial carbon demand, growth rate and diversity. It is shown how models predicting an ecosystem production of dissolved organic carbon (DOG) exceeding bacterial carbon demand may be constructed assuming either a low degradability of the DOG, or mineral nutrient limitation of bacterial growth rate. For 2 classes of models, infectious viruses are shown to affect neither growth rate nor abundance of the steady state bacterial community. For all 4 classes of models, viruses are suggested to control diversity of the steady state bacterial community. [TOP OF PAGE]

175. Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus: A review. Torriani, S., Vescovo, M., Dicks, L.M.T. (1997). Annali di Microbiologia ed Enzimologia 47:28-52. Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus are thermophilic lactic acid bacteria of particular interest to the dairy industry. For the protosymbiotic relationship that exists between strains of these bacteria, they are largely used in association as starter cultures for the production of several fermented foods. Due to their commercial significance, many basic and applied research has led to an increasing understanding of the essential characteristics and technological properties of strains belonging to these species. This review deals with some recent knowledge on taxonomy, metabolic and genetic characteristics of S. thermophilus and L. delbrueckii subsp. bulgaricus. The antagonistic activity and bacteriophage resistance of these species are also discussed. [TOP OF PAGE]

176. Generalized transduction in the potato blackleg pathogen Erwinia carotovora subsp. atroseptica by bacteriophage phi M1. Toth, I.K., Mulholland, V., Cooper, V., Bentley, S., Shih, Y.L., Perombelon, M.C.M., Salmond, G.P.C. (1997). Microbiology-UK 143:2433-2438. Using enrichment methods, a new bacteriophage (phi M1) was isolated, which is capable of generalized transduction in Erwinia carotovora subsp. atroseptica (Eca) strain SCRI1043. phi M1 is probably a virulent phage and contains double-stranded DNA of approximately 43 kb. Transduction frequencies for a number of chromosomal markers and plasmid pHCP2 were established, and conditions for transduction optimized. UV irradiation of the lysates prior to transduction enhanced the transduction frequency. phi M1 infected over 25% of Eca strains tested and so may be useful both for the genetic analysis of a number of Eca isolates and for the transductional transfer of selectable markers between strains. [TOP OF PAGE]

177. Bacterial population dynamics in a meromictic lake. Tuomi, P., Torsvik, T., Heldal, M., Bratbak, G. (1997). Appl. Environ. Microbiol. 63:2181-2188. Polyclonal antibodies against nine different bacteria isolated from Lake Saelenvannet in western Norway were produced, and the population dynamics of these strains in the lake were monitored through two spring seasons by immunofluorescence staining. The total counts of bacteria varied over time and space from 1.5 times 10-6 to 1.5 times 10-3 cells ml-1. The counts of specific bacteria were in the range of 10-3 to 10-4 cells ml-1 or less; in sum, they generally made up less than 1% of the bacterial community. Some populations showed significant changes in abundance, with blooms lasting 1 to 3 weeks. The rate of change (increase and decrease) in abundance during blooms was estimated to be 0.2 to 0.6 day-1. The average virus-to-bacteria ratio was 50, and there was a significant correlation between the abundances of virus and bacteria. Both protozoan grazing and lytic virus infection were assessed as possible mechanisms driving the variations in bacterial population density. [TOP OF PAGE]

178. Evaluation of seven experimental phages for inclusion in the international phage set for the epidemiological typing of Listeria monocytogenes. van der Mee-Marquet, N., Loessner, M., Audurier, A. (1997). Appl. Environ. Microbiol. 63:3374-3377. The purpose of our study was to evaluate the inclusion of seven experimental phages into the international phage set for subtyping Listeria monocytogenes. The seven additional phages included the broad-host-range virulent Myoviridae phage A511 (M. J. Loessner, Appl. Environ. Microbiol. 57:1912-1918, 1991), three temperate phages from the Danish subsystem for typing serotype 1/2 strains (12682, 6223, and 5775) (P. Gerner-Smidt, V.T. Rosdahl, and W. Frederiksen, APMIS 101:160-167, 1993), and three temperate phages isolated by this laboratory in France (9425, 1313, and 197). A panel of 395 Listeria monocytogenes isolates (including 180 that were non-phage typeable by the international set) were used in the study for a comparison of the lytic spectra of the various bacteriophages. These results showed that the inclusion of five of the experimental phages contributed greatly to the overall typeability and discriminatory power of the system, especially for strains within serogroup 1/2. [TOP OF PAGE]

179. Identification and characterization of a DNA region involved in the export of capsular polysaccharide by Actinobacillus pleuropneumoniae serotype 5a. Ward, C.K., Inzana, T.J. (1997). Infect. Immun. 65:2491-2496. Actinobacillus pleuropneumoniae synthesizes a serotype-specific capsular polysaccharide that acts as a protective barrier to phagocytosis and complement-mediated killing. To begin understanding the role of A. pleuropneumoniae capsule in virulence, we sought to identify the genes involved in capsular polysaccharide export and biosynthesis. A 5.3-kb XbaI fragment of A. pleuropneumoniae serotype 5a J45 genomic DNA that hybridized with DNA probes specific for the Haemophilus influenzae type b cap export region was cloned and sequenced. This A. pleuropneumoniae DNA fragment encoded four open reading frames, designated cpxDCBA. The nucleotide and predicted amino acid sequences of cpxDCBA contained a high degree of homology to the capsule export genes of H. influenzae type b bexDCBA, Neisseria meningitidis group B ctrABCD, and, to a lesser extent, Escherichia coli K1 and K5 kpsE and kpsMT. When present in trans, the cpxDCBA gene cluster complemented kpsM::TnphoA or kpsT::TnphoA mutations, determined by enzyme immunoassay and by restored sensitivity to a K5-specific bacteriophage. A cpxCB probe hybridized to genomic DNA from all A. pleuropneumoniae serotypes tested, indicating that this DNA was conserved among serotypes. These data suggest that A. pleuropneumoniae produces a group II family capsule similar to those of related mucosal pathogens. [TOP OF PAGE]

180. Comparison of epifluorescence and transmission electron microscopy for counting viruses in natural marine waters. Weinbauer, M.G., Suttle, C.A. (1997). Aquat. Microb. Ecol. 13:225-232. [TOP OF PAGE]

181. Photoreactivation compensates for UV damage and restores infectivity to natural marine virus communities. Weinbauer, M.G., Wilehelm, S.W., Suttle, C.A., Garza, D.R. (1997). Appl. Environ. Microbiol. 63:2200-2205. We investigated the potential for photoreactivation to restore infectivity to sunlight-damaged natural viral communities in offshore (chlorophyll a, lt 0.1 mu-g liter-1), coastal (chlorophyll a, ca. 0.2 mu-g liter-1), and estuarine (chlorophyll a, ca. 1 to 5 mu-g liter-1) waters of the Gulf of Mexico. In 67% of samples, the light-dependent repair mechanisms of the bacterium Vibrio natriegens restored infectivity to natural viral communities which could not be repaired by light-independent mechanisms. Similarly, exposure of sunlight-damaged natural viral communities to gt 312-nm-wavelength sunlight in the presence of the natural bacterial communities restored infectivity to 21 to 26% of sunlight-damaged viruses in oceanic waters and 41 to 52% of the damaged viruses in coastal and estuarine waters. Wavelengths between 370 and 550 nm were responsible for restoring infectivity to the damaged viruses. These results indicate that light-dependent repair, probably photoreactivation, compensated for a large fraction of sunlight-induced DNA damage in natural viral communities and is potentially essential for the maintenance of high concentrations of viruses in surface waters. [TOP OF PAGE]

182. Occurrence of temperate bacteriophages in different Actinobacillus actinomycetemcomitans serotypes isolated from periodontally healthy individuals. Willi, K., Sandmeier, H., Asikainen, S., Saarela, M., Meyer, J. (1997). Oral Microbiology & Immunology 12:40-46. The occurrence of temperate bacteriophages was studied in 34 isolates of Actinobacillus actinomycetemcomitans derived from 27 periodontally healthy Finnish individuals both by lysis/plaque assays and by DNA hybridizations. In addition the serotype, the ribotype and the arbitrarily primed polymerase chain reaction (AP-PCR) profile were determined for each A. actinomycetemcomitans strain. Fourteen isolates showed hybridization patterns very similar to that of a known lysogen when probed with the genome of the previously characterized temperate phage Aa-vphi-23. Only 6 of these 14 strains had produced lysis or single plaques on suitable indicator strains. Phage Aa-vphi-247 derived from one of these lysogens was indistinguishable from Aa-vphi-23 by electron microscopy, and the genomes showed highly related DNA hybridization patterns. The remaining 20 isolates exhibited hybridization patterns very different from that of Aa-vphi-23 DNA. Seven of these strains also gave lysis or single plaques, suggesting that 21 of the 34 strains were lysogenic. These data indicate that the prophages per se do not represent a virulence factor exclusively associated with periodontal disease. Presence of an Aa-vphi-23-related prophage correlated with serotype a and AP-PCR type 1 of the bacterial host. This may indicate that Aa-vphi-23 and related phages have a limited host range. [TOP OF PAGE]

183. Transduction of antibiotic resistance markers among Actinobacillus actinomycetemcomitans strains by temperate bacteriophages Aa phi 23. Willi, K., Sandmeier, H., Kulik, E.M., Meyer, J. (1997). Cellular & Molecular Life Sciences 53:904-910. Actinobacillus actinomycetemcomitans (Aa) strain ST1 carries the tetracycline (Tc) resistance transposon Tn916 and the Aa phi ST1 prophage, which is closely related to temperate bacteriophage Aa phi 23. High titre phage preparations were obtained from this strain by mitomycin C induction and were used to transduce the TcR determinant to the TcS recipient strains ZIB1001 and ZIB1015 (MIC 2 micrograms Tc/ml). TcR transductants (MIC > or = 32 micrograms Tc/ml) were detected at frequencies of 3 x 10(-6) to 5 x 10(-8) per pfu. All TcR transductants examined contained the entire Tn916 inserted at several different locations within the Aa genome. They appear to have resulted from generalized transduction. In addition both bacteriophages, Aa phi 23 and Aa phi ST1, were capable of transducing the chloramphenicol (Cm) resistance marker of plasmid pKT210 (transduction frequencies of 2 x 10(-5) to 3 x 10(-7) per pfu) to the recipient strain ZIB1001 (MIC 8 micrograms Cm/ml). Eleven CmR ZIB1001 transductants (MIC > or = 100 micrograms Cm/ml) studied carried a plasmid indistinguishable from pKT210 by restriction analyses. In view of the high prevalence of this phage family, and the increasing use of tetracycline in periodontitis therapy, these findings may have clinical importance. [TOP OF PAGE]

184. Evaluation of a new rapid bacteriophage-based method for the drug susceptibility testing of Mycobacterium tuberculosis. Wilson, S.M., Al Suwaidi, Z., McNerney, R., Porter, J., Drobniewski, F. (1997). Nature Medicine 149:487-495. [TOP OF PAGE]

185. Lysogenic and lytic viral production in marine microbial communities. Wilson, W.H., Mann, N.H. (1997). Aquat. Microb. Ecol. 13:95-100. It is now well established that viruses are an abundant component of marine ecosystems and they are being increasingly recognised and accepted as important contributors to element cycling within the microbial loop. However, some of the key questions regarding the ecological significance of viruses in the marine environment still remain largely unanswered. Thus, particular interest is currently focused on the extent to which lytic production or lysogeny predominates and the nature of factors in the marine environment, particularly nutrient availability and multiplicity of infection (MOI), which might influence the lysis/lysogeny 'decision'. The present evidence is still insufficient to unambiguously assess the relative ecological significance of lysogeny versus lysis and progress in this area will rely on the development and application of new techniques. This review attempts to collect recent information relating to this central question, focusing particularly on those viruses which infect the bacterioplankton and nano- and picophytoplankton. [TOP OF PAGE]

186. Characterization of Natronobacterium magadii phage PHI-Ch1, a unique archaeal phage containing DNA and RNA. Witte, A., Baranyi, U., Klein, R., Sulzner, M., Luo, C., Wanner, G., Krueger, D.H., Lubitz, W. (1997). Molecular Microbiology 23:603-616. A novel archaeal bacteriophage, PHI-Ch1, was isolated from a haloalkalophilic archaeon Natronobacterium magadii upon spontaneous lysis. The phage-cured strain N. magadii (L13) was used to demonstrate infectivity of phage PHI-Ch1. The turbid-plaque morphology and the fact that N. magadii cells isolated from plaques were able to produce phage indicated that PHI-Ch1 is a temperate phage. The phage morphology resembles other members of Myoviridae-infecting Halobacterium species. In solution below 2 M NaCl, the phage lost its morphological stability and infectivity. One- and two-dimensional SDS-PAGE of phage particles revealed at least four major and five minor proteins with molecular masses ranging from 15 to 80 kDa and acidic isoelectric points. Southern blot analysis of chromosomal DNA of a lysogenic N. magadii strain showed that PHI-Ch1 exists as a chromosomally integrated prophage. The phage particles contain both double-stranded, linear DNA (approx. 55 kbp) as well as several RNA species (80-700 nucleotides). Hybridization of labelled RNA fragments to total DNA from N. magadii and PHI-Ch1 showed that the virion-associated RNA is host encoded. Part of the phage DNA population is modified and restriction analysis revealed evidence for adenine methylation. Phage PHI-Ch1 is the first virus described for the genus Natronobacterium, and the first phage containing DNA and RNA in mature phage particles. [TOP OF PAGE]

187. Replication of coliphage Q-beta as affected by host cell number, nutrition, competition from insusceptible cells and non-FRNA coliphages. Woody, M.A., Cliver, D.O. (1997). Journal of Applied Microbiology 82:431-440. F-specific RNA (FRNA) coliphages, which infect Escherichia coli by attachment to F pili, might serve as indicators of human enteric viruses in groundwater, provided these phages do not replicate in groundwater and replicate only to a limited extent in wastewater. Several factors that could influence phage replication in either of these environments were examined. Q-beta did not replicate when host cells were fewer than 10-4 cfu ml-1. Replication selected for insusceptible cells when Q-beta was incubated with its E. coli host. Loss of Q-beta, presumably by inactivation, occurred in autoclaved on-site and urban wastewater, autoclaved groundwater, and in filter-sterilized spent LB broth. Replication did not occur in LB broth diluted with sterile saline to 1% of its original strength, which indicates that replication of FRNA coliphages cannot occur in such nutrient-poor environments as wastewater and groundwater. Competition from non-FRNA coliphages and insusceptible cells tended to reduce Q-beta replication, as predicted, but phage yields unexpectedly increased significantly when Enterococcus faecalis was added to cultures. [TOP OF PAGE]

188. Virus a la sauce Yo-Pro: microwave-enhanced staining for counting viruses by epifluorescence microscopy. Xenopoulos, M.A., Bird, D.F. (1997). Limnol. Oceanogr. 42:1648-1650. [TOP OF PAGE]

189. Microwave enhanced staining for counting viruses by epifluorescence microscopy. Xenopoulos, M.A., Bird, D.F. (1997). Limnol. Oceanogr. 42:1648-1650. [TOP OF PAGE]

190. Lipopolysaccharide dependence of cyanophage sensitivity and aerobic nitrogen fixation in Anabaena sp. strain PCC 7120. Xu, X., Khudyakov, I., Wolk, C.P. (1997). Journal of Bacteriology [J. BACTERIOL. ] 179:2884-2891. Fox super(-) mutants of Anabaena sp. strain PCC 7120 are unable to fix dinitrogen in the presence of oxygen. A fragment of the DNA of Anabaena sp. was cloned by complementation of a spontaneous Fox super(-), cyanophage-resistant mutant, R56, and characterized. Random insertion of transposon Tn5 delimited the complementing DNA to a 0.6-kb portion of the cloned fragment. Sequencing of this region and flanking DNA showed one complete open reading frame (ORF) similar to the gene rfbP (undecaprenyl-phosphate galactosephosphotransferase) and two partial ORFs similar to genes rfbD (GDP-D-mannose dehydratase) and rfbZ (first mannosyl transferase), all of which are active in the synthesis of the O antigen unit of the lipopolysaccharide (LPS) component of the outer membrane of gram-negative bacteria. In a transposon (Tn5-1087b)-induced, Fox super(-), cyanophage-resistant mutant, B14, the transposon was found within the same rfbP-like ORF. The three ORFs were insertionally inactivated with the omega cassette or with Tn5::omega. Only the insertions in the rfbZ- and rfbP-like ORFs led to resistance to cyanophages A-1(L) and A-4(L) and to a Fox super(-) phenotype. Electrophoretic analysis showed that interruption of the rfbZ- and rfbP-like ORFs resulted in a change in or loss of the characteristic pattern of the lengths of the LPS, whereas interruption of the rfbD-like ORF merely changed the distribution of the lengths of the LPS to one with a greater prevalence of low molecular weights. According to electron microscopy, interruption of the rfbP-like ORF may have led to aberrant deposition of the layers of the heterocyst envelope, resulting in increased leakage of oxygen into the heterocyst. The results suggest that modified LPS may prevent cyanophage infection of Anabaena sp. vegetative cells and the formation of a functional heterocyst envelope. [TOP OF PAGE]

191. Parasitic action of marine bdellovibrios on prawn-pathogenic bacteria and other bacteria. Yang, S., Huang, Q. (1997). Journal of Xiamen University. Natural science/Xiamen Daxue Xuebao. Xiamen [J. Xiamen Univ. [Nat. Sci. ]/Xiamen Daxue Xuebao] 36:449-453. Parasitic action of marine bdellovibrios on 25 strains of prawn-pathogenic bacteria E. coli, B. subtilis, Pseudomonas aeruginosa, and Staphylococcus aureus were determined by selection technique of double agar plates containing host. The results showed that the bdellovibrio strains tested had different host range, and that all the bacterial strains tested for host capability were capable of being host cells for some bdellovibrio strains, forming obvious plaques in the double agar plates. Morphology and phage process of bdellovibrios were investigated by phase contract microscopy and electron microscopy. [TOP OF PAGE]

192. Transfection of Actinomyces spp. by genomic DNA of bacteriophages from human dental plaque. Yeung, M.K., Kozelsky, C.S. (1997). Plasmid 37:141-153. Bacteriophages that produced turbid or clear zones of lysis in strains of Actinomyces were isolated from 22 of 124 samples of fresh human dental plaque. All human and nonhuman strains of Actinomyces viscosus or Actinomyces naeslundii tested in this study were sensitive to infection by one or more of these phages. In contrast, none of the Actinomyces odontolyticus, Actinomyces israelii, or Actinomyces bovis strains tested were susceptible. Results of restriction endonuclease analyses indicated that the genomes of these phages consisted of double-stranded DNA molecules ranging in size between 16 and 60 kbp. Sequence homology under hybridization conditions of high stringency was observed among a few of the isolated phages. A lysogenized isolate of A. viscosus MG-1 was obtained following infection with a temperate phage, designated vphi-225. Results of Southern blot analyses indicated that vphi-225 replicated as a plasmid in the lysogenized strain. Genomic DNA from several lytic phages was used to establish conditions for transfection by electroporation of strains of Actinomyces spp. Efficiencies of DNA transfer ranged from 10-2 to 10-5 plaque-forming units per microgram of DNA were obtained under optimal transfection conditions. The results of these studies demonstrate that transfer of genetic information in Actinomyces spp. can be achieved by transfection. [TOP OF PAGE]

193. A simple method for enumerating bacteriophages in soil. Yin, X., Zeph, L.R., Stotzky, G. (1997). Canadian Journal of Microbiology 43:461-466. A plaque technique that uses antibiotic-resistant bacteria growing on antibiotic-containing agar for the assay lawn resulted in significantly better recovery of bacteriophages PI of Escherichia coli and F116 of Pseudomonas aeruginosa from nonsterile soil than standard membrane filtration or centrifugation techniques. Adsorption of the phages on soil particles appeared to be involved in their recovery and survival in soil. [TOP OF PAGE]

194. Can immobilization of Bacillus megaterium cells in alginate beads protect them against bacteriophages? Zayed, G. (1997). Plant and Soil 197:1-7. The ability of free and alginate-immobilized cells of Bacillus megaterium to dissolve tricalcium phosphate as well as their susceptibility to phages were compared in pure liquid cultures and in pot experiment with maize. In both liquid culture and cultivated soil, alginate-immobilized cells of B. megaterium exhibited much higher efficiency in increasing the availability of phosphorus than the free cells. Bacteriophages of B. megaterium were found to be common in soil. Specific bacteriophages, in the presence of free cells of B. megaterium, completely inhibited the phosphate-dissolving activity of the bacteria in pure liquid culture and markedly decreased their number in rhizosphere of maize plants. The phosphorus content of maize plants inoculated with free cells of B. megaterium decreased in the presence of their specific bacteriophages, whereas, when alginate-immobilized cells were used as inoculum, no effect of the presence of bacteriophages on phosphate-dissolving activity of bacterial cells was detected. [TOP OF PAGE]

195. Isolation and characterization of an attenuated strain of Pseudomonas aeruginosa AC869, a 3,5-dichlorobenzoate degrader. Zhou, X., George, S.E., Frank, D.W., Utley, M., Gilmour, I., Krogfelt, K.A., Claxton, L.D., Laux, D.C., Cohen, P.S. (1997). Appl. Environ. Microbiol. 63:1389-1395. Pseudomonas aeruginosa AC869, a 3,5-dichlorobenzoate degrader, is a mouse pathogen and has a reported 50% lethal dose (LD-50) of 1.05 times 10-7 CFU when given intranasally to C3H/HeJ mice (S. E. George, M. J. Kohan, M. I. Gilmour, M. S. Taylor, H. G. Brooks, J. P. Creason, and L. D. Claxton, Appl. Environ. Microbiol. 59:35853591, 1993). AC869 was serotyped as 06 when grown in CD-1 mouse cecal and lung mucus but could not be assigned an O serotype when grown in Luria broth (LB). After growth in mouse cecal mucus, a less virulent mutant, AC869-11, was isolated from AC869 by using bacteriophage E79, which adsorbs to the O side chain of lipopolysaccharide (LPS). AC869-11 produced significantly less 0 antigen on its LPS than AC869 when grown in mouse lung and cecal mucus. The mutant also produced half the amount of exoenzyme S and 16-fold less extracellular protease than AC869 and was more sensitive than its parent to a number of antibiotics when grown either in LB or in mouse lung mucus. AC869-11 had a ninefold higher LD-50 than AC869 in CD-1 mice when administered intranasally. AC869-11 was found in the lungs, small intestine, cecum, and large intestine in numbers at least 100-fold below AC869, 3 h after intranasal exposure of mice to a sublethal dose of the two strains. Moreover, AC869-11 induced a decreased pulmonary inflammatory response relative to AC869. In contrast to AC869, AC869-11 did not translocate to the mesenteric lymph nodes, liver, and spleen following a sublethal dose. Despite attenuation, AC869-11 grew as well as AC869 with 3,5-dichlorobenzoate as the sole carbon and energy source. However, although AC869-11 survived in 3,5-dichlorobenzoate-contaminated soil as well as AC869 for 1 week, it failed to survive as well thereafter. These results suggest the possibility that mutations that lead to pulmonary attenuation of P. aeruginosa in mice also lead to weakness in the environment, despite such mutants maintaining the ability to degrade toxic substances under laboratory conditions. [TOP OF PAGE]

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