Bacteriophage Ecology Group
Reference Abstracts (1997)
Dedicated to the ecology and evolutionary biology of the parasites of unicellular organisms (UOPs)
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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