Bacteriophage Ecology Group
Reference Abstracts (1996)
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. Catalogue of Bacteria and Phages / American Type Culture Collection. Anonymous (1996). Rockville, MD.[TOP OF PAGE]

  2. Antiviral activity of water extract of Sanicula europaea L. leaves on the bacteria-bacteriophage system. Anonymous (1996). Turkish Journal of Biology 20:225-234. The effect of water soluble extract of Sanicula europaea L. leaves and two fractions separated from this extract through Sephadex gel filtration chromatography were tested on bacteriophage T2hr+ replication. The plant extract inhibited the adsorption and/or penetration of phage infection without any toxic effect on Escherichia coli B2 host bacteria. The heavy molecular weight fraction eluted first from the gel filtration column was found to be responsible for this inhibition. [TOP OF PAGE]

  3. The phagosome containing Legionella pneumophila within the protozoan Hartmannella vermiformis is surrounded by the rough endoplasmic reticulum. Abu, K.Y. (1996). Appl. Environ. Microbiol. 62:2022-2028. Legionella pneumophila is an intracellular parasite of protozoa and human phagocytes. To examine adaptation of this bacterium to parasitize protozoa, the sequence of events of the intracellular infection of the amoeba Hartmannella vermiformis was examined. The previously described uptake phenomenon of coiling phagocytosis by human monocytes was not detected. A 1 h postinfection with wild-type strain AA100, mitochondria were observed within the vicinity of the phagosome. At 2.5 h postinfection, numerous vesicles surrounded the phagosomes and mitochondria were in close proximity to the phagosome. At 5 h postinfection, the bacterium was surrounded by a ribosome-studded multilayer membrane. Bacterial multiplication was evident by 8 h postinfection, and the phagosome was surrounded by a ribosome-studded multilayer membrane until 15 h postinfection. The recruitment of organelles and formation of the ribosome-studded phagosome was defective in an isogenic attenuated mutant of L. pneumophila (strain AA101A) that failed to replicate within amoebae. At 20 h postinfection with wild-type strain AA100, numerous bacteria were present in the phagosome and ribosome were not detected around the phagosome. These data showed that, at the ultrastructural level, the intracellular infection of protozoa by L. pneumophila is highly similar to that of infection of macrophages. Immunocytochemical studies provided evidence that at 5 h postinfection the phagosome containing L. pneumophila acquired an abundant amount of the endoplasmic reticulum-specific protein (BiP). Similar to phagosomes containing heat-killed wild-type L. pneumophila, the BiP protein was not detectable in phagosomes containing the mutant strain AA101A. In addition to the absence of ribosomes and mitochondria, the BiP protein was not detected in the phagosomes at 20 h postinfection with wild-type L. pneumophila. The data indicated that the ability of L. pneumophila to establish the intracellular infection of amoebae is dependent on its capacity to reside and multiply within a phagosome surrounded by the rough endoplasmic reticulum. This compartment may constitute a rich source of nutrients for the bacteria and is probably recognized as cellular compartment. The remarkable similarity of the intracellular infections of macrophages and protozoa by L. pneumophila strongly supports the hypothesis that adaptation of the bacterium to the intracellular environment of protozoa may be the mechanism for its ability to adapt to the intracellular environment of human alveolar macrophages and causes pneumonia. [TOP OF PAGE]

  4. Frequency of morphological phage descriptions in 1995. Ackermann, H.-W. (1996). Archives of Virology 141:209-218. At least 4500 bacterial viruses have been examined in the electron microscope since 1959. About 4400 phages (96%) are tailed and only 162 phages (4%) are cubic, filamentous, or pleomorphic. Phages belong to 12 virus families and occur in about 130 bacterial genera. Phages are listed by morphotypes and host genera. Siphoviridae or phages with long, noncontractile tails include about 60% of tailed phages. [References: 21]. [TOP OF PAGE]

  5. Phage specific for Vibrio cholerae O139 Bengal. Albert, M.J., Bhuiyan, N.A., Rahman, A., Ghosh, A.N., Hultenby, K., Weintraub, A., Nahar, S., Kibriya, A.K., Ansaruzzaman, M., Shimada, T. (1996). Journal of Clinical Microbiology 34:1843-1845. From the stool of a Vibrio cholerae O139 Bengal-infected patient, a phage that specifically lysed capsulated V. cholerae O139 strains only was isolated. The phage is useful for the confirmatory diagnosis of V. cholerae O139 infection and for the differentiation of variants that lack the capsule. [TOP OF PAGE]

  6. Characterization of Mycobacterium smegmatis gene that confers resistance to phages L5 and D29 when overexpressed. Barsom, E.K., Hatfull, G.F. (1996). Molecular Microbiology 21:159-170. Bacteriophage infection requires a specific interaction with the outer surface of a bacterial host followed by interaction with the cell membrane and phage DNA injection. Phages of the mycobacteria encounter a cell wall that is rich in unusual lipid- and sugar-containing components which form a formidable barrier that must be passed to gain access to the membrane. We describe here a gene of Mycobacterium smegmatis that confers resistance to mycobacteriophages L5 and D29. The phage-resistance phenotype results not from mutation but from elevated expression of a wild-type gene. It appears that the product of this multicopy phage-resistance (mpr) gene may alter the structure of the host cell wall or membrane, thereby inhibiting productive phage DNA injection. [TOP OF PAGE]

  7. An oligonucleotide hybridization assay for the identification and enumeration of F-specific RNA phages in surface water. Beekwilder, J., Nieuwenhuizen, R., Havelaar, A.H., van Duin, J. (1996). J. Appl. Bacteriol. 80:179-186. F-specific RNA phages can be used as model organisms for enteric viruses to monitor the effectiveness of sewage treatment, and to assess the potential contamination of surface water with these viruses. In this paper a method is described which identifies RNA phages quantitatively by a plaque hybridization assay. Oligonucleotide probes were developed that can assign phages to their phylogenetic subgroups. Such a distinction is important, since some subgroups preferentially occur in sewage of human origin, while others tend to be associated with animal wastewater. The method has been tested on a large number of isolates and represents an improvement in time and reliability over the previously used serological classification. [TOP OF PAGE]

  8. Similarities and dissimilarities of phage genomes. Blaisdell, B.E., Campbell, A.M., Karlin, S. (1996). Proc. Natl. Acad. Sci. USA 93:5854-5859. Genomic similarities and contrasts are investigated in a collection of 23 bacteriophages, including phages with temperate, lytic, and parasitic life histories, with varied sequence organizations and with different hosts and with different morphologies. Comparisons use relative abundances of di-, tri-, and tetranucleotides from entire genomes. We highlight several specific findings. (i) As previously shown for cellular genomes, each viral genome has a distinctive signature of short oligonucleotide abundances that pervade the entire genome and distinguish it from other genomes. (ii) The enteric temperate double-stranded (ds) phages, like enterobacteria, exhibit significantly high relative abundances of GpC = GC and significantly low values of TA, but no such extremes exist in ds lytic phages. (iii) The tetranucleotide CTAG is of statistically low relative abundance in most phages. (iv) The DAM methylase site GATC is of statistically low relative abundance in most phages, but not in P1. This difference may relate to controls on replication (e.g., actions of the host SeqA gene product) and to MutH cleavage potential of the Escherichia coli DAM mismatch repair system. (v) The enteric temperate dsDNA phages form a coherent group: they are relatively close to each other and to their bacterial hosts in average differences of dinucleotide relative abundance values. By contrast, the lytic dsDNA phages do not form a coherent group. This difference may come about because the temperate phages acquire more sequence characteristics of the host because they use the host replication and repair machinery, whereas the analyzed lytic phages are replicated by their own machinery. (vi) The nonenteric temperate phages with mycoplasmal and mycobacterial hosts are relatively close to their respective hosts and relatively distant from any of the enteric hosts and from the other phages. (vii) The single-stranded RNA phages have dinucleotide relative abundance values closest to those for random sequences, presumably attributable to the mutation rates of RNA phages being much greater than those of DNA phages. [TOP OF PAGE]

  9. Effect of effluent quality and temperature on the persistence of viruses in soil. Blanc, R., Nasser, A. (1996). Water Science and Technology 33:237-242. [TOP OF PAGE]

  10. Dynamics of virus abundance in coastal seawater. Bratbak, G., Heldal, M., Thingstad, T.F., Tuomi, P. (1996). FEMS Microbiol. Ecol. 19:263-269. The short term dynamics of virus abundance in coastal sea water was investigated by frequent sampling of open ecosystems and of water incubated in bottles in situ. Sampling intervals were 6-10 min. The viral abundance showed significant temporal fluctuations both in situ and in the bottles and it changed in some cases by a factor of 2-4 within 10-20 min. Laboratory incubations showed that production and release of viruses were not induced or stimulated by nutrient addition, high light intensities or transient increase in temperatures ca. 10 degree C. Our interpretation of these results is that they result from synchronous lysis and release of virus particles from bacterial hosts and a rapid disintegration of these particles when released in sea water. [TOP OF PAGE]

  11. Viral control of Emiliania huxleyi blooms? Bratbak, G., Wilson, W., Heldal, M. (1996). J. Mar. Syst. 9:75-81. [TOP OF PAGE]

  12. One infection cures another. Brown, P. (1996). New Scientist ???, ???-??? [TOP OF PAGE]

  13. Virus-like particles in a summer bloom of Emiliania huxleyi in the North Sea. Brussaard, C.P.D., Kempers, R.S., Kop, A.J., Riegman, R., Heldal, M. (1996). Aquat. Microb. Ecol. 10:105-113. [TOP OF PAGE]

  14. Site-specific spontaneous deletions in three genome regions of a temperate Streptococcus thermophilus phage. Bruttin, A., Brussow, H. (1996). Virology 219:96-104. Site-specific spontaneous deletions were observed with high frequency in three regions of the genome of the temperate Streptococcus thermophilus phage f SFi21. Deletion sizes were 750 bp (type 1), 2.7 kb (type 2), and 1 kb (type 3). Combinations of types 1 and 3 and 2 and 3 were observed. The mutants grew lytically although with reduced burst sizes. Type 2 mutants lost the capacity to lysogenize host cells. Upon serial passage, the deletion mutants overgrew the wild-type phage. No direct or inverted DNA repeats were associated with type 1 or 2 deletion sites. Several independent phage isolates showed deletions at identical nucleotide positions, suggesting a site- specific recombination system. Sequencing of an Xbal restriction fragment covering the type 1 deletion predicted a single long open reading frame (ORF) showing a high degree of amino acid similarity with two proteins from bacteriophage P1 implicated in its immunity control (KiIA, Ant). Type 1 deletion leads to a loss of the conserved C-terminal part of this ORF. [TOP OF PAGE]

  15. Towards a universal virus database - progress in the ICTVdB. Buechen-Osmond, C., Dallwitz, M. (1996). Archives of Virology 141:392-399. [TOP OF PAGE]

  16. Bacteriophages
    Genome homology and superinfection immunity between temperate and virulent Lactobacillus delbrueckii bacteriophages. Campbell, A.M., Fields, B.N., Knipe, D.M., Howley, P.M., Alatossava, T., Forsman, P., Ritzenthaler, P. (1996). Fundamental virology, Third edition. 140:2261-2268    . The presence of short homologous DNA segments along the genomes of the temperate phage mv4 and the virulent phages LL-H, LL-K and JCL1032 of Lactobacillus delbrueckii was demonstrated with Southern hybridizations. One of these segments, the 2817 nt MIS element of phage mv4, was further characterized by nucleotide sequence analysis and by superinfection immunity studies. [TOP OF PAGE]

  17. Bacteriophages. Campbell, A.M. (1996). pp. 2325-2338. In In Neidhardt, F.C. (ed.), Escherichia coli and Salmonella Cellular and Molecular Biology. ASM Press, Washington, D.C. [TOP OF PAGE]

  18. Comparative removal of coliphage and poliovirus from secondary wastewater during soil aquifer treatment (SAT). Carrol, S.M., Gerba, C.P., Quanrud, D.M., Arnold, R.G. (1996). pp. 1001-1005. In AnonymousSan Diego, CA. [TOP OF PAGE]

  19. Sequences homologous to yeast mitochondrial and bacteriophage T3 and T7 RNA polymerases are widespread throughout the eukaryotic lineage. Cermakian, N., Ikeda, T.M., Cedergren, R., Gray, M.W. (1996). Nucleic Acids Research 24:648-654. Although mitochondria and chloroplasts are considered to be descendants of eubacteria-like endosymbionts, the mitochondrial RNA polymerase of yeast is a nucleus-encoded, single-subunit enzyme homologous to bacteriophage T3 and T7 RNA polymerases, rather than a multi-component, eubacterial-type alpha-2-beta- beta' enzyme, as encoded in chloroplast DNA. To broaden our knowledge of the mitochondrial transcriptional apparatus, we have used a polymerase chain reaction (PCR) approach designed to amplify an internal portion of phage T3/T7-like RNA polymerase genes. Using this strategy, we have recovered sequences homologous to yeast mitochondrial and phage T3/T7 RNA polymerases from a phylogenetically broad range of multicellular and unicellular eukaryotes. These organisms display diverse patterns of mitochondrial genome organization and expression, and include species that separated from the main eukaryotic line early in the evolution of this lineage. In certain cases, we can deduce that PCR-amplified sequences, some of which contain small introns, are localized in nuclear DNA. We infer that the T3/T7-like RNA polymerase sequences reported here are likely derived from genes encoding the mitochondrial RNA polymerase in the organisms in which they occur, suggesting that a phage T3/T7-like RNA polymerase was recruited to act in transcription in the mitochondrion at an early stage in the evolution of this organelle. [TOP OF PAGE]

  20. Characterization of Vibrio cholerae EIT or typing phage D10. Chakrabarti, B.K., Si, K., Chattopadhyay, D. (1996). J. Gen. Virol. 77 ( Pt 11):2881-2884. The Vibrio cholerae EITor typing phage D10 was characterized. The adsorption kinetics of the phage on V. cholerae MAK757 strain were biphasic in nature. Intracellular growth was characterized by an eclipse period, latent period and burst size which were 20 min, 25 min and 80 particles per cell respectively. The phage yield was dependent on the concentration and time of addition of DNA synthesis inhibitors such as nalidixic acid and novobiocin, and RNA synthesis inhibitors such as rifampicin. The 32+/-0.2 kb linear double-stranded DNA molecule has unique termini. A restriction map of the phage DNA was constructed with the enzymes BamHI, HindIII and PstI. [TOP OF PAGE]

  21. Selection of the phage-resistant mutant strains of E.coli M 17 and study of their main biological properties. Chanishvili, N., Tediashvili, M., Eliashvili, T., Zviadadze, N., Porchkhidze, K., Alavidze, Z., Giorkhelidze, D., Kvatadze, N., Natroshvili, G., Giorkhelidze, R., Adamia, R., Chanishvili, T. (1996). Proceedings of the Georgian Academy of Sciences 22:203-209. [TOP OF PAGE]

  22. Genetic diversity in marine algal virus communities as revealed by sequence analysis of DNA polymerase genes. Chen, F., Suttle, C.A., Short, S.M. (1996). Appl. Environ. Microbiol. 62:2869-2874. Algal-virus-specific PCR primers were used to amplify DNA polymerase gene (pol) fragments (683 to 689 bp) from the virus-sized fraction (0.02 to 0.2 mu m) concentrated from inshore and offshore water samples collected from the Gulf of Mexico. Algal-virus-like DNA pol genes were detected in five samples collected from the surface and deep chlorophyll maximum. PCR products from an offshore station were cloned, and the genetic diversity of 33 fragments was examined by restriction fragment length polymorphism and sequence analysis. The five different genotypes or operational taxonomic units (OTUs) that were identified on the basis of restriction fragment length polymorphism banding patterns were present in different relative abundances (9 to 34%). One clone from each OTU was sequenced, and phylogenetic analysis showed that all of the OTUs fell within the family Phycodnaviridae. Four of the OTUs fell within a group of viruses (MpV) which infect the photosynthetic picoplankter Micromonas pusilla. The genetic diversity among these genotypes was as large as that previously found for MpV isolates from different oceans. The remaining genotype formed its own clade between viruses which infect M. pusilla and Chrysochromulina brevifilum. These results imply that marine Virus communities contain a diverse assemblage of MpV-like viruses, as well as other unknown members of the Phycodnaviridae. [TOP OF PAGE]

  23. Evolutionary relationships among large double-stranded DNA viruses that infect microalgae and other organisms as inferred from DNA polymerase genes. Chen, F., Suttle, C.A. (1996). Virology 219:170-178. In order to examine genetic relatedness among viruses that infect microalgae, DNA polymerase gene (DNA pol) fragments were amplified and sequenced from 13 virus clones that infect three genera of distantly related microalgae (Chlorella strains NC64A and Pbi, Micromonas pusillia and Chrysochromulina spp.). Phylogenetic trees based on DNA pol sequences and hybridization of total genomic DNA showed similar branching patterns. Genetic relatedness calculated from the hybridization and sequence data showed good concordance (r = 0.90), indicating that DNA pol sequences can be used to determine genetic relatedness and infer phylogenetic relationships among these viruses. The phylogenetic tree inferred from the deduced amino acid sequences of DNA pol from 24 dsDNA viruses, including phycodnaviruses, herpesviruses, poxviruses, baculoviruses, and African swine fever virus corresponded well with groupings based on the International Committee on Taxonomy of Viruses. Microalgal viruses are more closely related to each other than to the other dsDNA viruses and form a distinct phyletic group, suggesting that they share a common ancestor and belong to the Phycodnaviridae. Moreover, the Phycodnaviridae are more closely related to the Herpesviridae than to other virus families for which DNA pol sequences are available. [TOP OF PAGE]

  24. Extended host range of a beta-related corynebacteriophage. Cianciotto, N.P., Groman, N.B. (1996). FEMS Microbiol. Let. 140:221-225. Unlike most beta-related phages isolated from Corynebacterium diphtheriae, phage 782 readily plaqued on strains of C. ulcerans and C. pseudotuberculosis. The extended host range of phage 782 was not, however, due to a greater ability of the phage to absorb to bacterial surfaces. Using chemical mutagenesis, a number of phage mutants were isolated which had diminished capacities to infect C. ulcerans, suggesting the existence of a locus (ehr) for extended host range. Pre-lysogenization of C. ulcerans strains with phage 782, but not its mutant form or other beta-related phages, rendered them susceptible to infection by previously excluded phages. An examination of recombinant between phages 782, pie, and beta localized ehr to a 7 kilobase region of DNA including attP. The data are compatible with the notion that ehr encodes an anti-restriction function. [TOP OF PAGE]

  25. The diversity of bacteria, eukaryotic cells and viruses in an oligotrophic lake. Corpe, W.A., Jensen, T.E. (1996). Applied Microbiology and Biotechnology 46:622-630. An in situ transmission electron microscopic study of biomass samples concentrated from oligotrophic lake water revealed a variety of virus-infected microbial cells and many free viruses and virus-like particles. The most abundant group of microorganisms in screened and filtered water-column samples were 2 mu-m or less in diameter, and included representatives of several oligotrophic genera, Prosthecomicrobium, Ancyclobacter, Caulobacter and Hyphomicrobium. Among the prokaryotic host cells, which included both heterotrophs and autotrophs, on the basis of electron microscope observations. approximately 17% were infected with bacteriophage or bore adherent phage particles on their surfaces. Several bacterial morphotypes were observed among the prokaryotic hosts. Water samples passed through a 20-mu-m Nitex screen allowed us to concentrate and examine the larger host cells as well, including several species of single-celled algae and two amoeba species. The infected algal cells included those Chlorella-like in appearance, photosynthetic flagellates and others that could not be positively identified. About one-third of the eukaryotic cells were infected by viruses that were larger (150-200 nm) and structurally more complex than bacteriophages (50-60 nm). None of the viruses have been isolated, but when 0.2 mu-m filtrate from a biomass sample was spotted onto lawns of four representative heterotrophs and a Chlorella, the clearing observed was taken as evidence of lysis. Cyanobacterial lawns showed no plaques. Thin sections of two amoeba showed food vacuoles containing what appeared to be virus particles of a type seen in certain prokaryotic and eukaryotic cells in the biomass. [TOP OF PAGE]

  26. Biotechnology of lactic acid bacteria with special reference to bacteriophage resistance. Daly, C., Fitzgerald, G.F., Davis, R. (1996). Antonie van Leeuwenhoek 70:99-110. Lactic acid bacteria play an important role in many food and feed fermentations. In recent years major advances have been made in unravelling the genetic and molecular basis of significant industrial traits of lactic acid bacteria. Bacteriophages which can infect and destroy lactic acid bacteria pose a particularly serious threat to dairy fermentations that can result in serious economic losses. Consequently, these organisms and the mechanisms by which they interact with their hosts have received much research attention. This paper reviews some of the key discoveries over the years that have led us to our current understanding of bacteriophages themselves and the means by which their disruptive influence may be minimized. [References: 118]. [TOP OF PAGE]

  27. Gene transfer by transduction. Day, M.J., Marchesi, J.R. (1996). p. ?-? In Ackermans, A.D.L., van Elsas, J.D., and Bruijn, F.J. (eds.), Molecular Microbial Ecology Manual. Kluwer Acad. Pub., [TOP OF PAGE]

  28. GENETIC AND MUTATIONAL STUDIES OF THE ABIA PHAGE RESISTANCE GENE IN LACTOCOCCUS LACTIS AND IDENTIFICATION OF A PHAGE GENOMIC REGION INVOLVED IN SENSITIVITY TO ABIA. Dinsmore, P.K. (1996). North Carolina State University. The objectives of this study were to determine the effect of abiA gene copy number on phage resistance, to characterize an essential region in the AbiA protein, and to identify the phage gene(s) involved in sensitivity to AbiA. To analyze the effect of abiA gene copy number on phage resistance, abiA was cloned into four plasmids. Efficiencies of plaquing of four phages representing three species decreased dramatically with increasing copy number of abiA. The phages surviving on strains containing the high copy number abiA plasmid were insensitive to AbiA upon further propagation. A heptad leucine repeat motif was discovered in the AbiA protein which resembled the leucine zipper motif responsible for dimerization of many proteins. The leucine residues of the five heptad leucine repeats were substituted with hydrophobic, charged, and helix-breaking residues to determine the requirements at these positions. Results indicated that hydrophobicity is generally required at these positions, and that the predicted alpha-helix in this region is necessary for phage resistance. The data indicated that the AbiA heptad leucine repeats are required for resistance of L. lactis to phages from three species. This suggests that the active form of AbiA could consist of a homo- or heterodimer. A short region rich in basic amino acids lies downstream of the leucine repeat motif in AbiA. Mutagenesis of three of the five basic residues in the region indicated that the basic residues are important in AbiA function, but not simply due to their positive charge. An open reading frame involved in inhibition of $/phi$31 by AbiA was identified. A point mutation (G to T) was localized in $/phi$31A. This mutation lies within an open reading frame, ORF245, and results in an arginine to leucine substitution in the predicted amino acid sequence. The cloned mutant ORF245 complements the wild-type $/phi$31, reducing its sensitivity to AbiA. This confirmed that the mutation in ORF245 is responsible for AbiA-insensitivity in $/phi$31A and implies that ORF245 is involved in inhibition of $/phi$31 by AbiA. Analysis of the region upstream of ORF245 in $/phi$31 revealed two more open reading frames. ORF71 is followed by an 84-bp untranslated region that contains two inverted repeats. ORF169 follows the 84-bp region, and ORF245 begins immediately after the end of ORF169. This entire region (1732 bp) was sequenced from $/phi$31 and from the AbiA-insensitive phages $/phi$31A and $/phi$31B. The study of this region of phage $/phi$31 has identified phage factors that interact with AbiA and are likely to be involved in the sensitivity of this phage to AbiA. (Abstract shortened by UMI.). [TOP OF PAGE]

  29. Basic concepts in RNA virus evolution. Domingo, E., Escarmis, C., Sevilla, N., Moya, A., Elana, S.F., Quer, J., Novella, I.S., Holland, J.J. (1996). FASEB Journal 10:859-864. A hallmark of RNA genomes is the error-prone nature of their replication and retrotranscription. The major biochemical basis of the limited replication fidelity is the absence of proofreading/repair and postreplicative error correction mechanisms that normally operate during replication of cellular DNA, In spite of this unique feature of RNA replicons, the dynamics of viral populations seems to follow the same basic principles that classical population genetics has established for higher organisms. Here we review recent evidence of the profound effects that genetic bottlenecks have in enhancing the deleterious effects of Muller's ratchet during RNA virus evolution, The validity of the Red Queen hypothesis and of the competitive exclusion principle for RNA viruses are viewed as the expected result of the highly variable and adaptable nature of viral quasispecies. Viral fitness, or ability to replicate infectious progeny, can vary a million-fold within short time intervals. Paradoxically, functional and structural studies suggest extreme limitations to virus variation, Adaptability of RNA viruses appears to be based on the occupation of very narrow portions of sequence space at any given time. [TOP OF PAGE]

  30. Tracing the interaction of bacteriophage with bacterial biofilms using fluorescent and chromogenic probes. Doolittle, M.M., Cooney, J.J., Caldwell, D.E. (1996). J. Indust. Microbiol. 16:331-341. Phages T4 and E79 were fluorescently-labeled with rhodamine isothiocyanate (RITC), fluoroscein isothiocyanate (FITC), and by the addition of 4'6-diamidino-2-phenylindole (DAPI) to phage- infected host cells of Escherichia coli and Pseudomonas aeruginosa. Comparisons of electron micrographs with scanning confocal laser microscope (SCLM) images indicated that single RITC-labeled phage particles could be visualized. Biofilms of each bacterium were infected by labeled phage. SCLM and epifluorescence microscopy were used to observe adsorption of phage to single-layer surface-attached bacteria and thicker biofilms. The spread of the recombinant T4 phage, YZA1 (containing an rII-LacZ fusion), within a lac E. coli biofilm could be detected in the presence of chromogenic and fluorogenic homologs of galactose. Infected cells exhibited blue pigmentation and fluorescence from the cleavage products produced by the phage-encoded beta-galactosidase activity. Fluorescent antibodies were used to detect non-labeled progeny phage. Phage T4 infected both surface-attached and surface-associated E. coli while phage E79 adsorbed to P. aeruginosa cells on the surface of the biofilm, but access to cells deep in biofilms was somewhat restricted. Temperature and nutrient concentration did not affect susceptibility to phage infection, but lower temperature and low nutrients extended the time-to- lysis and slowed the spread of infection within the biofilm. [TOP OF PAGE]

  31. Evolution of fitness in experimental populations of vesicular stomatitis virus. Elena, S.F., Gonzalez-Candelas, F., Novella, I.S., Duarte, E.A., Clarke, D.K., Domingo, E., Holland, J.J., Moya, A. (1996). Genetics 142:673-679. The evolution of fitness in experimental clonal populations of vesicular stomatitis virus (VSV) has been compared under different genetic (fitness of initial clone) and demographic (population dynamics) regimes. In spite of the high genetic heterogeneity among replicates within experiments, there is a clear effect of population dynamics on the evolution of fitness. Those populations that went through strong periodic bottlenecks showed a decreased fitness in competition experiments with wild type. Conversely, mutant populations that were transferred under the dynamics of continuous population expansions increased their fitness when compared with the same wild type. The magnitude of the observed effect depended on the fitness of the original viral clone. Thus, high fitness clones showed a larger reduction in fitness than low fitness clones under dynamics with included periodic bottleneck. In contrast, the gain in fitness was larger the lower the initial fitness of the viral clone. The quantitative genetic analysis of the trait ''fitness'' in the resulting populations shows that genetic variation for the trait is positively correlated with the magnitude of the change in the same trait. The results are interpreted in terms of the operation of MULLER's ratchet and genetic drift as opposed to the appearance of beneficial mutations. [TOP OF PAGE]

  32. Study of biological properties of CB phages isolated from the cells of the industrial strain E. coli M17. Eliashvili, T., Tediashvili, M., Chanishvili, N., Zviadadze, N., Kvachadze, L., Adamia, R., Chanishvili, T. (1996). Selected articles, G. Eliava Institute of BMV 9:152-154. [TOP OF PAGE]

  33. Relationships between indicators, pathogens and water quality in an estuarine system. Ferguson, C.M., Coote, B.G., Ashbolt, N.J., Stevenson, I.M. (1996). Water Res. 30:2045-2054. This study examined water and sediment samples for a range of indicator and pathogenic microorganisms from six sites in an urban estuary, Sydney, Australia. Water quality was affected by rainfall and sewage overflows which were associated with significant increases in the concentration of faecal coliforms, faecal streptococci, Clostridium perfringens spores, F-RNA bacteriophage, Aeromonas spp., Giardia and Cryptosporidium spp. However, in sediments, only faecal coliform concentrations were significantly increased by rainfall, although sewage overflow again resulted in increased concentrations of faecal coliforms, faecal streptococci, C. perfringens spores and Aeromonas. Isolation of Salmonella appeared to coincide with wet weather events and occasionally identical serotypes were detected in sediments at several locations within the estuary. However, isolations of enteric virus were sporadic and did not appear to be exclusively related to wet weather events. C. perfringens was identified as the most useful indicator of faecal pollution and was the only indicator significantly correlated to the presence of pathogenic Giardia (r = 0.41, p lt 0.05) and the opportunistic bacterial genus Aeromonas (r = 0.39, p lt 0.05). F-RNA bacteriophage was not significantly correlated with any of the pathogens examined. [TOP OF PAGE]

  34. A method for the enumeration of Aeromonas bacteriophage in aquatic environments. Flint, K.P. (1996). Letters in Applied Microbiology 22:244-248. Bacteriophage were isolated against type strains and environmental isolates of Aeromonas hydrophila, Aeromonas sobria and Aeromonas caviae. A most probable number method for estimating the number of bacteriophage in a water sample was devised and tested using some of these isolates. The maximum number of bacteriophage against all three type strains were found in water from below a sewage effluent outfall. This corresponds to the increased numbers of each species of bacterium also found in this water sample. High numbers of bacteriophage against Aer. hydrophila were also found in the lake sample examined. Bacteriophage against Aer. caviae were rare in water samples other than those contaminated with sewage effluent. [TOP OF PAGE]

  35. An evaluation of retention time and short-circuiting in waste stabilisation ponds using Serratia marcescens bacteriophage as a tracer. Frederick, G.L., Lloyd, B.J. (1996). Water Science and Technology 33:49-56. [TOP OF PAGE]

  36. Sensitization of bacteria to danofloxacin by temperate prophages. Froshauer, S., Silvia, A.M., Chidambaram, M., Sharma, B., Weinstock, G.M. (1996). Antimicrobial Agents and Chemotherapy 40:1561-1563. Danofloxacin (CP-76,136) is in a class of agents that inhibit DNA gyrase and trigger induction of the SOS response and temperate bacteriophages. Killing studies against the bovine pathogen Pasteurella haemolytica demonstrated that danofloxacin exhibits particularly rapid killing kinetics. Here, lysogenic Escherichia coli bearing lambda is found to be more sensitive to danofloxacin than nonlysogenic E. coli. Danofloxacin exposure also induced a prophage in P. haemolytica. The potency of danofloxacin against lysogens in likely enhanced by this prophage induction. [TOP OF PAGE]

  37. Host-dependent modification/restriction and therapeutic potential of Pseudomonas phage. Gachechiladze, K.K., Adamia, R.S., Balardshishvili, N.S., Chanishvili, T.G., Kruger, D.H. (1996). Jerusalem (Israel). Xth International Congress of Virology. 1996.[TOP OF PAGE]

  38. The lactococcal plasmid pNP40 encodes a third bacteriophage resistance mechanism, one which affects phage DNA penetration. Garvey, P., Hill, C., Fitzgerald, G.F. (1996). Appl. Environ. Microbiol. 62:676-679. The lactococcal plasmid pNP40 mediates insensitivity to vphi-c2 by an early-acting phage resistance mechanism in addition to the previously identified abortive infection system. AbiF, in the Lactococcus lactis subsp. lactis MG1614 background. A second abortive infection determinant on pNP40, AbiE, does not confer resistance to vphi-c2. The early-acting mechanism on pNP40 does not prevent phage adsorption nor does it appear to operate by restriction/modification. Phage DNA was not detected in pNP40-containing cells until 30 min following exposure to vphi-c2 compared with 5 min in a sensitive host; however, electroporation of phage DNA into resistant hosts resulted in the release of phage progeny from a dramatically elevated number of cells compared with conventionally infected hosts. It appears therefore that pNP40 encodes a novel phage resistance mechanism which blocks DNA penetration specifically for vphi-c2. [TOP OF PAGE]

  39. Biological effectiveness of environmental radiation in aquatic systems, measurements by T7-phage sensor. Gaspar, S., Berces, A., Ronto, G., Grof, P. (1996). Journal of Photochemistry and Photobiology. B - Biology 32:183-187. Bacteriophage T7 as a biologic sensor was used for systematic underwater UV measurements at the freshwater lake Gyekenyes, Hungary. Time evolution of the biologically effective doses, cumulated daily doses and daily dose-profiles have been measured. Measurements were undertaken not only in different water depths but also just above the water surface (15 cm height) and also on the beach. The exponential attenuation of daily doses in water has been confirmed. Although the values of H-T7 above the water's surface and on the beach were similar, the dose rates were very different between the two sides of the water surface, i.e. just under it (at 5 cm depth) and above it (at 15 cm height). Their ratio has been found to be dependent on the zenith angle with a maximum of not more than 0.5, and at higher zenith angles it is about 0.3. [TOP OF PAGE]

  40. Lytic systems in lactic acid bacteria and their bacteriophages. Gasson, M.J. (1996). Antonie van Leeuwenhoek 70:147-159. Lytic systems of lactic acid bacteria and their bacteriophages are reviewed with an emphasis on molecular characterization. Details of enzyme biochemistry and the cloning and analysis of lytic genes are presented, with coverage of lactococcal prolate headed bacteriophages, lactococcal isometric bacteriophages, Lactobacillus bacteriophages and lactococcal autolysins. Some comments on the importance of autolysis in cheese ripening are included and the biotechnological exploitation of cloned and characterized lytic genes is presented. [References: 63]. [TOP OF PAGE]

  41. Influence of the contaminating phages on the propagation process of the industrial strain E-coli M17, held in industrial conditions. Goderdzishvili, M., Kvatadze, N., Eliashvili, T., Tediashvili, M., Chanishvili, N., Zviadadze, N., Chanishvili, T. (1996). Selected articles, G. Eliava Institute of BMV 9:56-59. [TOP OF PAGE]

  42. A moderately halophilic Vibrio from a Spanish saltern and its lytic bacteriophage. Goel, U., Kauri, T., Ackermann, H.W., Kushner, D.J. (1996). Canadian Journal of Microbiology 42:1015-1023. A number of bacteria and their phages were isolated from a saltern near Alicante, Spain. One isolate, Vibrio B1, a moderate halophile that is probably a strain of Vibrio costicola, was host to a lytic phage, UTAK. Studies of the host bacterium included the effects of salt concentrations on the action of a number of inhibitory agents. Phage UTAK has a head, a tail, and a baseplate. It contains 80 kbp of double-stranded DNA with no unusual bases. It was stable for long periods in the absence of high salt concentrations and even in distilled water. Salt concentrations had little effect on adsorption of UTAK to its host but resulted in considerable changes in burst size. It appears that phages of halophilic and salt-tolerant eubacteria, and also of some marine bacteria, have much lower salt requirements for stability than the phages of halophilic archaebacteria. Our results suggest that ionic controls of phage replication in these eubacteria may differ from those of growth. [TOP OF PAGE]

  43. A bacteriophage of Rhodopseudomonas blastica. Gorham, H., Dow, C.S. (1996). Microbiology 142:979-983. A bacteriophage, vphi-BHG1, was isolated from a small eutrophic pond from which its host, Rhodopseudomonas blastica, was originally obtained. it is a lytic bacteriophage specific for R. blastica which also causes non-specific lysis of Rhodobacter sphaeroides 8253. vphi-BHG1 has an icosahedral head of 62 nm diameter and a short 39 nm tail. Caesium chloride density gradient centrifugation of infected cell lysates gave a single bacteriophage band at a density of 1.385 g cm-3, but also occasionally a second band was observed at a lower density. No differences were apparent between bacteriophage taken from either of the two bands. vphi-BHG1 contained double-stranded DNA with a size of 48 kb and a G+C content of 50-6 mol %. The bacteriophage adsorbed to both photosynthetically and chemoheterotrophically grown R. blastica at an identical rate of 1.39 times 10-9 ml-1 min-1. One-step growth curves and kinetic studies of the bacteriophage under these physiological regimes showed no differences in the latent and rise periods and only slight changes in the burst size. Adsorption of this bacteriophage is cell-surface specific and attachment only occurs to the 'older' pole of the budding reproductive cell. [TOP OF PAGE]

  44. Viral lysis and bacterivory as prokaryotic loss factors along a salinity gradient. Guixa-Boixareu, N., Calderon-Paz, J.I., Heldal, M., Bratbak, G., Pedros-Alio, C. (1996). Aquat. Microb. Ecol. 11:215-227. We estimated prokaryotic mortality due to viruses and bacterivores through salinity gradients in 2 solar salterns. In each saltern system, successive ponds provided steady state environments with a range of salinities from 37 to 372ppt. Prokaryotic and viral abundance increased with salinity, reaching about 10 super(8) prokaryotic cells ml super(-1) and 10 super(9) virus-like particles (VLP) ml super(-1) at salinities higher than 250ppt. Prokaryotic doubling times became longer than 2 d above 250ppt salinity until the end of the gradient. Bacterivory accounted for all the production at lower salinities but it was found to be zero at the highest salinities. The percentage of visibly infected cells was not different among the ponds where infected cells could be detected and it was always lower than 4%. From the percentage of infected cells and using conversion factors from the literature we estimated rates of prokaryotic mortality due to viral lysis: about 0.6 to 2 x 10 super(6) prokaryotes m super(-1) were lysed daily by the viruses in the salterns. This number represented a low percentage of prokaryotic abundance and production compared to the prokaryotic losses due to bacterivores (0.2 to 4 x 10 super(7) bacteria ml super(-1) d super(-1)). However, viral production reached values higher than 10 super(8) VLP ml super(-1) d super(-1) above 250ppt salinity, due to the large burst size (200 viruses cell super(-1)) found in a particular morphotype of prokaryotes, the square archaea. These archaea represented more than 25% of the prokaryotic assemblage above 250ppt salinity. At this point they became the prokaryotic morphotype with the largest percentage of infected cells (1 to 10% of square archaea with visible phages inside). A lemon-shaped virus (similar to one described for some other groups of archaea) was found infecting square archaea, its abundance increased in the saltiest ponds together with that of the square archaea. In this system viruses did not exert a strong control over the prokaryotic abundance and growth rate. [TOP OF PAGE]

  45. Factors influencing the loss of bacteria in preserved seawater samples. Gundersen, K., Bratbak, G., Heldal, M. (1996). Mar. Ecol. Prog. Ser. 137:305-310. Several time course storage experiments with preserved seawater samples were con ducted to study the loss of bacterial cells as a function of storage lime. The number of bacteria decreased by 24 to 50% within 7 to 29 d in samples preserved with 2.5% glutaraldehyde (final cone.). A comparison between epifluorescence and electron microscope counts showed that the decrease was not due to filtration artefacts. Only 0.4 to 0.6% of the bacterial cells were found to be attached to the walls of the sample containers after 1 yr of storage. There was no positive correlation between the frequency of virus-infected cells at the start of the storage experiments and the loss of bacteria as a function of storage time. Numbers of bacteria declined by only 5% the first 9 d in samples preserved in glutaraldehyde and stored at -20 degrees C. By adding phenolmethylsulfonylfluoride (PMSF), a protease inhibitor, prior to the addition of glutaraldehyde, the loss of bacterial cells only 17 to 18% over a 30 to 35 d period. Our study shows that protease activity may be a major cause of bacterial loss in glutaraldehyde preserved samples. [TOP OF PAGE]

  46. Analysis of the enzymatic cleavage (beta elimination) of the capsular K5 polysaccharide of Escherichia coli by the K5-specific coliphage: reexamination. Hanfling, P., Shashkov, A.S., Jann, B., Jann, K. (1996). J. Bacteriol. 178:4747-4750. The capsular K5 polysaccharide of Escherichia coli is the receptor of the capsule-specific coliphage K5, which harbors an enzyme that degrades the capsular K5 polysaccharide to a number of oligosaccharides. Analysis of the degradation products using gel permeation chromatography, the periodate-thiobarbituric acid and bicinchoninic acid reactions, and nuclear magnetic resonance spectroscopy showed that the major reaction products are hexa-, octa-, and decasaccharides with 4,5-unsaturated glucuronic acid (delta4,5GlcA) at their nonreducing end. Thus, the bacteriophage enzyme is a K5 polysaccharide lyase and not, as we had reported previously, an endo-N-acetylglucosaminidase. [TOP OF PAGE]

  47. Abundance of viruses in deep oceanic waters. Hara, S., Koike, I., Terauchi, K., Kamiya, H., Tanoue, E. (1996). Mar. Ecol. Prog. Ser. 145:269-277. [TOP OF PAGE]

  48. Phage susceptibility and plasmid profile analysis of Sinorhizobium fredii. Hashem, F.M., Kuykendall, L.D., Udell, S.E., Thomas, P.M. (1996). Plant and Soil 186:127-134. This is the first report identifying bacteriophages and documenting megaplasmids of Sinorhizobium fredii. Plasmid DNA content and bacteriophage typing of eighteen strains of S. fredii were determined. S. fredii strains fell into ten plasmid profile groups containing 1 to 6 plasmids, some evidently larger than 1000 MDa. Twenty-three S. fredii lytic phages were isolated from soil, and they lysed six different S. fredii strains. The host range and plaque morphology of these phages were studied. Susceptibility to S. fredii phages was examined for S. meliloti; Rhizobium leguminosarum bvs. viceae, trifolii and phaseoli; R. loti; Bradyrhizobium japonicum; B. elkanii and Bradyrhizobium sp. (Arachis). Several phages that originally lysed S. fredii strain USDA 206 also lysed strains of all three S. fredii serogroups described originally by Sadowsky et al. Phages that infected S. fredii strains USDA 191 and USDA 257 were highly specific and lysed only serogroup 193 strains. S. meliloti strains L5-30 and USDA 1005 were lysed by three of the phages that lysed S. fredii strain USDA 217. No other Rhizobium or Bradyrhizobium strain tested was susceptible to lysis by any of the S. fredii phages. The present investigation indicates that phage susceptibility in conjunction with plasmid profile analysis may provide a rapid method for identification and characterization of strains of S. fredii. [TOP OF PAGE]

  49. Comparative evaluation of four bacterial assays for the detection of genotoxic effects in the dissolved water phases of aqueous matrices. Helma, C., Mersch-Sundermann, V., Houk, V.S., Glasbrenner, U., Klein, C., Wenquing, L., Kassie, F., Knasmueller, R.S.-H. (1996). Environmental Science & Technology 30:897-907. The aim of this study was to evaluate the performance of four bacterial short-term genotoxicity assays (Salmonella/microsome assay, SOS Chromotest, Microscreen phage-induction assay, differential DNA repair test) that are widely used and/or have a promising potential for the genotoxicity testing of water samples. Twenty-three samples of different origins (drinking and bathing water, surface water, municipal and industrial wastewater, pulp mill effluents, groundwater, and landfill leachates) were tested in these assays. In total, 20 samples were genotoxic: 13 in the Salmonellalla/microsome assay, 13 in the SOS Chromotest, 8 in the Microscreen phage-induction assay, and 19 in the differential DNA repair test. Although the differential DNA repair test was the most sensitive system, positive results were obtained also with some of the negative control samples, and it had the least power to detect different genotoxic potencies. The Microscreen assay was the least sensitive system due to nonlinear results and sample toxicity. The Salmonella/microsome assay and the SOS Chromotest were of equal sensitivity, but the variance of the results was higher in the Salmonella/microsome assay. As the Salmonella/microsome assay also lacks toxicity correction for routine applications and ordinarily utilizes two strains, the SOS Chromotest appears to be the most promising test system for routine screening of water samples. Based on the present experiments, the investigated water samples were ranked according to their genotoxic potency as follows: landfill leachates gt effluents from pulp production gt wastewater gt surface water gt contaminated groundwater apprxeq drinking and bathing water gt control samples. The rankings obtained with the individual test systems were generally in good agreement. In addition, we present data on the impact of water treatment methods (activated sludge treatment, UV disinfection) and of alternative technologies (ozone vs ClO-2 pulp bleaching) on the genotoxicity of water samples. [TOP OF PAGE]

  50. Fracture aperture measurements and migration of solutes, viruses, and immiscible creosote in a column of Clay-Rich Till. Hinsby, K., McKay, L.D., Jorgensen, P., Lenczewski, M., Gerba, C.P. (1996). Ground Water 34:1065-1089. A series of ground-water flow and tracer experiments were performed on an undisturbed column of fractured clay-rich till, 0.5 m diameter by 0.5 m long, in a pressure-controlled cell. The measured hydraulic conductivity of the sample was 1.0 to 1.2 times 10-6 m/sec and the average hydraulic gradient during the tracer experiments ranged from 0.45 to 0.49. The experiments clearly show that ground-water flow and contaminant migration through the sample is primarily controlled by fractures and root holes. Tracer experiments using a solute (chloride), colloid-sized bacteriophage (PRD-1 and MS-2) and uncharged latex microspheres, indicated very fast transport rates of 4 to 360 m/day. These rates are similar to fracture flow velocities calculated on the basis of the measured bulk hydraulic conductivity of the column, and measured fracture spacing, using the cubic law for flow through parallel-walled fractures. Fracture aperture values calculated from the ground-water flow data (35 to 56 mu-m) are of the same magnitude as values calculated from the breakthrough of tracers (13 to 120 mu-m). Aperture values calculated for fractures (1 to 94 mu-m) and root holes (2 to 188 mu-m), on the basis of measured immiscible creosote entry pressures, are also comparable with these values. The injected creosote, a DNAPL, penetrated most of the visible and a few invisible fractures and root holes, indicating that, for this till, fractures and root holes are important conduits for the transport of DNAPL's. [TOP OF PAGE]

  51. Visualizing virus fitness gains [news; comment]. Holland, J.J. (1996). Nature Biotechnology 14:431-432. [TOP OF PAGE]

  52. Isolation and characterization of a virus infecting Phaeocystis pouchetii (Prymnesiophyceae). Jacobsen, A., Bratbak, G., Heldal, M. (1996). J. Phycol. 32:923-927. [TOP OF PAGE]

  53. Genotoxicity of photoilluminated riboflavin in the presence of Cu(II). Jazzar, M.M., Naseem, I. (1996). FREE RADICAL BIOLOGY AND MEDICINE 21:7-14. Riboflavin is known to generate superoxide anion (O2.-) and other reactive oxygen species in the presence of Cu(II) and light as well as cause fragmentation of DNA and protein in vitro. In the present study we examined the genotoxic effects of photoilluminated riboflavin in the presence of Cu(II). Using the phage inactivation assay, a significant decline in plaque-forming unit (PFU) is seen. Results of Ames testing have suggested that probably a frameshift mutation is caused by a riboflavin-Cu(II)-mediated reaction. Using neocuproine as a Cu(I) sequestering reagent, Cu(I) has been shown to be an essential intermediate generated in the reaction between Cu(II), photoilluminated riboflavin, and DNA. Results obtained with various scavengers of active oxygen species strongly suggest that the species predominantly responsible for DNA damage is oxygen (O2) in the singlet or triplet state, together with H2O2, hydroxyl radical, and hydroxyl ion, to a lesser extent. In the case of riboflavin, a ternary complex of DNA-drug-Cu(II) is presumably formed. A redox reaction, involving riboflavin and Cu(II) in the complex, may then occur with the formation of a DNA-oxidized riboflavin-Cu(I) complex. This probably acts as a catalyst for the oxidation of Cu (I) to Cu(II), during which molecular oxygen is reduced to generate a variety of active oxygen species. The probable mechanism for the generation of these reactive oxygen species has also been proposed. [TOP OF PAGE]

  54. Occurence of lysogenic bacteria in marine microbial communities as determined by prophage induction. Jiang, S.C., Paul, J.H. (1996). Mar. Ecol. Prog. Ser. 142:27-38. [TOP OF PAGE]

  55. Integration of the DNA of a novel filamentous bacteriophage VSK from Vibrio cholerae 0139 into the host chromosomal DNA. Kar, S., Ghosh, R.K., Ghosh, A.N., Ghosh, A. (1996). FEMS Microbiol. Let. 145:17-22. An unusual filamentous bacteriophage, VSK, containing single-stranded, circular DNA as its genome was isolated from Vibrio cholerae 0139 strains P07 and B04. Unlike other single-stranded DNA phages, VSK can integrate its genome into the chromosome of the host and enter into a lysogenic state. The double-stranded replicative form (RF) of the single-stranded phage DNA was isolated. A restriction map of the VSK RF DNA was constructed using HaeII, AvaII, ClaI and XbaI. By Southern blot analysis of the chromosomal DNA of the lysogen using labeled phage DNA as probe, the attachment site (attP) on the viral genome was also identified. [TOP OF PAGE]

  56. Variability of the biological characteristics of Vibrio cholerae isolated from environmental objects. [Russian]. Khaitovich, A.B. (1996). Zhurnal Mikrobiologii, Epidemiologii i Immunobiologii 0:23-26. The study of the biological characteristics used for the identification of the species, biovar and serovar of V. cholerae O1 isolated from environmental objects on the territory of 8 regions of Ukraine for the period of 1974-1993 revealed the tendency towards an increase in the number of altered cultures. Phage sensitivity (60,7%) and capacity for agglutination with cholera species- and type-specific sera (24,6%) proved to be the most variable properties. Resistance to polymyxin (4,8%), the absence of hemagglutination with chick red blood cells (8,7%), the absence of diastatic activity (28,9%) were also detected. The study established that the temporal, ecosystemic and territorial geographical dependence could be observed in the character of variability of V. cholerae O1. The data thus obtained indicate the expediency of monitoring the dynamics of the properties of V. cholerae O1 in order to establish the relationship between its variability and the epidemic process. [TOP OF PAGE]

  57. Evidence that the hanA gene coding for HU protein is essential for heterocyst differentiation in, and cyanophage A-4(L) sensitivity of, Anabaena sp. strain PCC 7120. Khudyakov, I., Wolk, C.P. (1996). Journal of Bacteriology [J. BACTERIOL. ] 178:3572-3577. The highly pleiotropic, transposon-generated mutant Anabaena sp. strain PCC7120 exhibits slow growth, altered pigmentation, cellular fragility, resistance to phage A-4(L), and the inability to differentiate heterocysts. Reconstruction of the transposon mutation in the wild-type strain reproduced the phenotype of the original mutant. Sequencing of the flanking DNA showed that the transposon had inserted at the beginning of a gene, which we call hanA, that encodes Anabaena HU protein. Mapping of the transposon insertion by pulsed-field gel electrophoresis showed that hanA is located at ca. 4.76 Mb on the physical map of the chromosome and is transcribed clockwise. Repeated subculturing of of filament fragmentation, presumably because of one or more compensatory mutations; however, the mutant retained its A-4(L) super(r) Het super(-) phenotype. The mutation in strain be complemented by a fragment of wild-type DNA bearing hanA as its only open reading frame. [TOP OF PAGE]

  58. Rapid transport of virus, bromide, rhodamine-wt in a floodplain aquifer. Kiley, Q.T., Ball, P., Bushur, J.M., DeBorde, D.C., Woessner, W.W. (1996). Abstracts with Programs - Geological Society of America 28:352 Proposed legislation restricts virus concentrations in drinking water to no more that 1 per 10,000,000 liters. Such limits require prediction of virus concentrations as current detection limits for enteric virus are about 1 per 2,000 l. Virus typically survive for 10's of days in cold groundwater, and low numbers cause infection. Developing needed models requires extensive field investigations in widely different hydrogeologic settings. To investigate the transport of virus in a highly conductive aquifer, a 64 well monitoring and tracer well network was established in the floodplain of the Clark Fork River, near Missoula, Montana. The shallow, coarse-grained aquifer is 20 ft thick and depth to water ranges between 1 and 7 ft below land surface. Estimates for the hydrogeologic properties of the shallow aquifer include: a gradient of 0.0005, conductivites ranging from 26,000 to 37,000 ft/d, and transport velocities of 65 to 94 ft/d. Three bromide and rhodamine-wt tracer experiments and one virus seeding experiment, using the coliphage MS2, were conducted. Peak concentrations of each constituent traveled at approximately the same velocity, tending to act conservatively. However, over 95% of the virus attached to the aquifer material and break through plots exhibited long tailing, indicating a delayed release of viruses for months. This process, and hydrodynamic dispersion, indicates a predicted reduction in peak concentration of 10 orders of magnitude over a distance of 175 ft. Die off was determined to be insignificant over a one week time period. The next phase of this research will attempt to develop numerical tools that will allow simulation of primary processes controlling observed virus transport. [TOP OF PAGE]

  59. Analysis of Lactobacillus phages and bacteriocins in American dairy products and characterization of a phage isolated from yogurt. Kilic, A.O., Pavlova, S.I., Ma, W.G., Tao, L. (1996). Appl. Environ. Microbiol. 62:2111-2116. Yogurt and acidophilus milk that contain Lactobacillus acidophilus could promote human health because L. acidophilus can inhibit enteric and food-borne microbial pathogens. To evaluate the stability of diary L. acidophilus cultures, we studied whether some diary lactobacilli could be inhibited by phages or bacteriocins released by other dairy lactobacilli. From 20 yogurts and two acidophilus milks purchased at local food markets, 38 Lactobacillus strains were isolated. Eight Lactobacillus type strains were used as controls. With mitomycin induction and agar spot assay, phages and bacteriocins were isolated from these strains and their activities were analyzed. Lactobacillus strains from 11 yogurts released phages, while the strains from most of the remaining products released bacteriocins. One phage, designated phi y8, was characterized. It was spontaneously released from its host strain L. acidophilus Y8, at a rate of about 10(4)/ml. This phage lysed nine other dairy Lactobacillus strains tested. It had a burst size of 100, an elongated prolate head of 39 by 130 nm, a long, flexible but noncontractile tail of 300 nm, and a 54.3-kb linear double-stranded DNA. DNA fingerprinting analysis indicated that L. acidophilus phages of nine yogurts in this study belonged to the same type as phi y8. Although they may be sensitive to bacteriocins, all lysogens resisted further phage attacks, whereas most nonlysogens were sensitive to both phages and bacteriocins. Therefore, Lacotbacillus cultures of some American yogurts and acidophilus milks may be unstable or unsafe because they can either be inhibited by phages or bacteriocins or release them to inhibit lactobacilli or other diary products. [TOP OF PAGE]

  60. Isolation of a mutant bacteriophage T7 deleted in nonessential genetic elements, gene 19.5 and m. Kim, S.H., Chung, Y.B. (1996). Virology 216:20-25. Half of the 55 potential genes of bacteriophage T7 appear to be dispensable. One of the major obstacles in the study of these nonessential genes is the difficulty in obtaining mutants. During a study of genes involved in the packaging of bacteriophage T7, we hypothesized that some nonessential genes may be required for optimal growth. Mutant phages lacking such nonessential genes may form plaques but grow slowly. One gene located at the extreme right end of the linear T7 genome, gene 19.5 with no known mutants, and a genetic element m responsible for a unique hairpin end, were studied. Mutant T7 phages deleted in gene 19.5 and m (T7 delta 19.5-M) were generated in vivo by homologous recombination with a recombinant plasmid. This phage produces small plaques and the production of progeny phage particles per infected cell was reduced fourfold. Investigation of the intracellular DNA after infection with T7 delta 19.5-M showed the persistence of Escherichia coli DNA as well as delayed conversion of concatemers to unit-length T7 DNA. The inefficiency of concatemer processing confirmed the proposed function of the M-hairpin in duplication of the concatemer junction. Since it is not likely that the M-hairpin influences the degradation of host DNA, we propose that the gene 19.5 product is partly responsible for the degradation of E. coli chromosomal DNA. [TOP OF PAGE]

  61. Inducible gene expression and environmentally regulated genes in lactic acid bacteria. Kok, J. (1996). Antonie van Leeuwenhoek 70:129-145. Relatively recently, a number of genes and operons have been identified in lactic acid bacteria that are inducible and respond to environmental factors. Some of these genes/operons had been isolated and analysed because of their importance in the fermentation industry and, consequently, their transcription was studied and found to be regulatable. Examples are the lactose operon, the operon for nisin production, and genes in the proteolytic pathway of Lactococcus lactis, as well as xylose metabolism in Lactobacillus pentosus. Some other operons were specifically targetted with the aim to compare their mode of regulation with known regulatory mechanisms in other well-studied bacteria. These studies, dealing with the biosynthesis of histidine, tryptophan, and of the branched chain amino acids in L. lactis, have given new insights in gene regulation and in the occurrence of auxotrophy in these bacteria. Also, nucleotide sequence analyses of a number of lactococcal bacteriophages was recently initiated to, among other things, specifically learn more about regulation of the phage life cycle. Yet another approach in the analysis of regulated genes is the 'random' selection of genetic elements that respond to environmental stimuli and the first of such sequences from lactic acid bacteria have been identified and characterized. The potential of these regulatory elements in fundamental research and practical (industrial) applications will be discussed. [References: 77]. [TOP OF PAGE]

  62. Selection and properties of totally phage-resistant mutant Pseudomonas putida PpG1. Krylov, V.N., Rasskazchikova, S.A., Al'nikin, A.F. (1996). Genetika 32:348-353. The efficiency of using bacteria in open systems to degrade different anthropogenic toxic pollutants can depend strongly on the interaction between these bacteria and natural bacteriophages. The possibility of selecting bacterial Pseudomonas putida mutants resistant to all bacteriophages of this species known so far was tested (in our work, these mutants were designated totally phage-resistant mutants). In a model experiment, changes in the composition of a population upon prolonged growth of bacteria in the presence of one of the virulent phages were examined. On the basis of the results obtained, it is postulated that: (1) Mutants differing in resistance to various phages accumulate in a population; relative numbers of different mutants can undergo alterations over the course of time; mutants selected in the presence of a given virulent phage do not often manifest complete resistance to this phage. (2) It is possible to isolate totally phage-resistant mutants of P. putida PpG1. These mutants carry up to three different mutations simultaneously; however, these mutants regain sensitivity to many phages upon pseudoreversion occurrence. [TOP OF PAGE]

  63. Evolution of T4-related phages. Kutter, E., Gachechiladze, K., Poglazov, A., Marusich, E., Shneider, M., Aronsson, P., Napuli, A., Porter, D., Mesyanzhinov, V. (1996). Virus Genes 11:285-297. Much progress has been made in understanding T-even phage biology in the last 50 years. We now know the entire sequence of T4, encoding nearly 300 genes, only 69 of which have been shown to be essential under standard laboratory conditions; no specific function is yet known for about 140 of them. The origin of most phage genes is unclear, and only 42 genes in T4 have significant similarity to anything currently included in GenBank. Comparative analysis of related phages is now being used to gain insight into both the evolutionary origins and interrelationships of these phage genes, and the functions of their protein products. The genomes of phages isolated from Tbilisi hospitals, Long Island sewage plants, the Denver zoo, and Khabarovsk show basic similarity. However, these phages show substantial insertions and deletions in a number of regions relative to each other, and closer investigation of specific sequences often reveals much more complex relationships. There are only a few cases in T4- related phages in which there is evidence for evolution through DNA duplication. These include the fibrous products of genes 12, 34, and 37; head proteins gp23 and gp24; and the Alt enzyme and its downstream neighbors. T4 also contains 13 apparent relatives of group I and group II intron homing endonucleases. Distal portions of the tail fibers of various T-even phages contain segments closely related total-fiber regions of other DNA coliphages, such as Mu, P1, P2, and lambda. Horizontal gene transfer clearly emerges as a major factor in the evolution of at least the tail-fiber regions, where site-specific recombination probably is involved in the exchange of host-range determinants. [TOP OF PAGE]

  64. Effects of dilution rate on bacteriophage development in an immobilized cell system used for continuous inoculation of lactococci in milk. Lapointe, M., Champagne, C.P., Vuillemard, J.C., Lacroix, C. (1996). Journal of Dairy Science 79:767-774. The response to bacteriophage contamination of a free cell and an immobilized cell bioreactor was studied during continuous pH-controlled fermentation of milk with Lactococcus lactis ssp. lactis. After phage infection (1 times 10-5 pfu/ml), the phage population reached 1010 pfu/ml in a free cell bioreactor operated at a dilution rate of 0.5/h and then declined to about 10-7 pfu/ml as a phage-resistant cell population became established in the system. In the immobilized cell bioreactor operated at dilution rates of 0.5 and 3/h, the phage population continuously increased until reaching 10-10 pfu/ml where it remained throughout the 48 h of continuous culture. Conversely, phage populations decreased during the first 30 min following contamination at dilution rates of 10 and 15/h but subsequently increased. For all tested conditions in the immobilized cell bioreactor, the phage-resistant population increased to 10-2 to 10-4 cfu/ml, but the effluent milk contained mostly phage-sensitive cells. Analysis of bead populations showed the implantation of the phage as well as a limited population of phage-resistant cells. The effluent biomass from the immobilized cell bioreactor sharply reduced acidifying activity because this biomass was composed mainly of phage-sensitive cells and contained high phage populations. [TOP OF PAGE]

  65. Virus occurrence and transport in an unconfined, sand and gravel aquifer, western Montana. Lauerman, B.C., Woessner, W.W., DeBorde, D.C., Ball, P.N. (1996). Abstracts with Programs - Geological Society of America. 1-1-1996.[TOP OF PAGE]

  66. Smaller fleas ... ad infinitum: therapeutic bacteriophage redux. Lederberg, J. (1996). Proc. Natl. Acad. Sci. USA 93:3167-3168. [TOP OF PAGE]

  67. Imaging the propagation of viruses. Lee, Y., Yin, J. (1996). Biotechnology and Bioengineering 52:438-442. The propagation of viruses in a growing plaque has been measured using a digital image acquisition and analysis system. Plaques of phage T7 incubated at 37°C and illuminated against a dark field emerged as dark growing spots against a background of host bacteria. Images of the growth were acquired using a charge-coupled device (CCD) camera at 1-h intervals over 24 h. The first 10 h of plaque development coincided with rapid growth of the agar-immobilized Escherichia coli host, measured as a reduction in gray value. Following this period, the average radial velocity of plaque growth remained constant at 0.059 mm/h while the standard deviation about this velocity increased. These results suggest the suitability of the system for spatially resolving the dynamics of viral evolution during plaque growth. [TOP OF PAGE]

  68. Detection of evolving viruses. Lee, Y., Yin, J. (1996). Nature Biotechnology 14:491-493. The spread of viruses on a homogeneous lawn of receptive hosts provides an opportunity to detect the dynamics of their evolution. We have previously found that when repeated virus passages are confined to the expanding perimeter of a growing plaque, the appearance and outgrowth of genetically diverse strains (all descended from the same parent strain) can be traced along different radii of the plaque. As a plaque grows, the random mutation and selection of new fast-growing strains reduce the roundness or circularity of the growing plaque. Here we have quantified such changes in growing plaques of bacteriophage T7 using a digital imaging system. We find that T7 populations not adapted for fast growth exhibit a broader diversity of growth rates than populations adapted for fast growth. These results provide a foundation for understanding how viruses exploit mutation and selection processes to persist in nature. [TOP OF PAGE]

  69. The meaning of soil characteristics and temperature for the survival of bacteriophages in naturally contaminated soil samples. Leonardopoulos, J., Papaconstantinou, A., Georgakopoulou-Papandreou, E. (1996). Deltion Ellenikes Mikrobiologikes Etaireias 41:309-316. In this work we tested the survival of bacteriophage 1 of the old and new S. typhimurium phage typing scheme in four types of naturally contaminated soil and three different temperatures. The agar layer technique was employed in order to obtain a phage stock of high titre. Each soil sample was inoculated with 5ml of the bacteriophage suspension. The number of phage particles at inoculation was 1,3 times 10-9 per g of soil. After inoculation each type of soil was kept at 4 degree C, 20 degree C and 37 degree C. The survival period of the bacteriophage in the different types of soil was followed by estimating at 3 days intervals, the PFU per g of soil by removing after mixing 1g of soil and suspending it in 10ml saline. All phage suspensions were titrated by the spot technique, using ten fold dilutions of the phage suspensions. From the results of this work and their statistic analysis it can be concluded that the survival of bacteriophage 1 of the S. typhimurium phage typing scheme in four different types of naturally contaminated soil (clayloam, clayish, sandy clayloam, clayloam clayish) and three different temperatures (37 degree C, 20 degree C and 4 degree C) varied from 5 to 30 days. The temperature of incubation is a very important factor for the survival (increase of the temperature causes shortening of the survival time), while the type of soil does not seem to have an independent relation to the survival. [TOP OF PAGE]

  70. Survival of S. typhimurium in the soil in the presence of bacteriophages. Leonardopoulos, J., Achladi-Aliferi, P., Papaconstantinou, A. (1996). Deltion Ellenikes Mikrobiologikes Etaireias 41:586-597. It is well known that S. typhimurium can survive for a long period in the soil. In this work we studied the survival of a S. typhimurium strain (STM 36) when inoculated together with the bacteriophage 1 of the S. typhimurium phage typing scheme, in four types of sterile soil (clay loam, clayish, sandy clay loam, clay loam clayish) and three different temperatures (4, 20 and 37 degree C). The survival of S. typhimurium in this experiment varied from 18 to 30 days. It was found that under these conditions neither temperature of incubation nor the type of soil affected in a statistically considerable way the survival of S. typhimurium. The long survival period of S. typhimurium in the soil shortens dramatically in the presence of the proper bacteriophages in all kinds of soil and the different temperatures. Failing to isolate the microorganism from the soil does not always mean its absence, because its fast destruction by co-existing bacteriophages is possible. [TOP OF PAGE]

  71. Evaluation of a phage-inhibitory medium for lactococcus starters in pilot cheese manufacture. Lerayer, A.L.S., Van Dender, A.G.F., Moreno, I., Mori, E.E.M., Parada, J.L. (1996). International Dairy Journal 6:529-535. Antiphage medium MAB18 was designed to inhibit phage multiplication in Lactococcus cells during propagation steps for cheesemaking. This medium has the essential nutrients that allow bacterial growth, and polyphosphates as internal pH control and as Ca-2+ sequestrant. In the pilot experiments it was used in a mixed starter L. lactis subsp. cremoris and L. lactis subsp. lactis and its specific phage vphi-IL25. Lactic acid production was higher and the pH lower when cells were grown in MAB18 than in milk, while the number of initial added phages did not increase. Using starter prepared in the artificial medium, the yield of "Minas Frescal" cheese with and without phages was higher than that obtained with starters grown in milk. The number of phages remained low during propagation in the medium and also in whey and cheese. According to physico-chemical characteristics and sensory evaluation, the cheeses produced using MAB18 were well accepted and within the standards of this typical cheese. [TOP OF PAGE]

  72. Phage therapy revisited: the population biology of a bacterial infection and its treatment with bacteriophage and antibiotics. Levin, B.R., Bull, J.J. (1996). Am. Nat. 147:881-898. Phage therapy is the use of bacterial viruses (bacteriophage) to treat bacterial infections. It has been practiced sporadically on humans and domestic animals for nearly 75 yr. Nevertheless, phage therapy has remained outside the mainstream of modern medicine, presumably because of doubts about its efficacy, and possibly because it was eclipsed by antibiotics and other chemotherapeutic agents. In this report, we develop the study of phage therapy and antibiotic therapy as a population biological phenomenon-the dynamic interaction of bacteria with a predator (phage) or a toxic chemical (antibiotic) inside a host whose immune and other defenses also affect the interaction. Our goal is to identify the conditions under which phage and antibiotics can successfully control a bacterial infection and when they cannot. We review data published in the 1980s by H. Williams Smith and J. B. Huggins on the use of phage and antibiotics to control lethal, systemic infections of Escherichia coli in experimentally inoculated mice. We show that some of their observations can be accommodated by a quantitative model that invokes known or plausible assumptions about host defenses and the interactions of bacteria with phage and antibiotics; some observations remain unexplained by the model. Our analysis identifies several hypotheses about the population dynamics of phage and antibiotic therapy that can be tested experimentally. Included among these are hypotheses that account for variation in the efficacy of the different phages employed by Smith and Huggins and why, in their study, phages were more effective than antibiotics. [TOP OF PAGE]

  73. The evolution and maintenance of virulence in microparasites. Levin, B.R. (1996). Emerging Infectious Diseases 2:93-102. In recent years, population and evolutionary biologists have questioned the traditional view that parasite-mediated morbidity and mortality_virulence_is a primitive character and an artifact of recent associations between parasites and their hosts. A number of hypotheses have been proposed that favor virulence and suggest that it will be maintained by natural selection. According to some of these hypotheses, the pathogenicity of HIV, Vibrio cholerae, Mycobacterium tuberculosis, the Shigella, as well as Plasmodium falciparum, and many other microparasites, are not only maintained by natural selection, but their virulence increases or decreases as an evolutionary response to changes in environmental conditions or the density and/or behavior of the human population. Other hypotheses propose that the virulence of microparasites is not directly favored by natural selection; rather, microparasite-mediated morbidity and mortality are either coincidental to parasite-expressed characters (virulence determinants that evolved for other functions) or the product of short-sighted evolution in infected hosts. These hypotheses for the evolution and maintenance of microparasite virulence are critically reviewed, and suggestions are made for testing them experimentally. [TOP OF PAGE]

  74. Cholera: Nice bacteria and bad viruses. Levin, B.R., Tauxe, V. (1996). Current Biology 6:1389-1391. The genes coding for cholera toxin are borne on, and can be infectiously transmitted by, a filamentous bacteriophage, raising intriguing questions about the mechanisms and evolution of bacterial pathogenesis, and the taxonomy, epidemiology and control of cholera and other bacterial diseases. [TOP OF PAGE]

  75. Genetic control of the resistance to phage C1 of Escherichia coli K-12. Likhacheva, N.A., Samsonov, V.V., Samsonov, V.V., Sineoky, S.P. (1996). J. Bacteriol. 178:5309-5315. Escherichia coli K-12 lytic phage C1 was earlier isolated in our laboratory. Its adsorption is controlled by at least three bacterial genes: dcrA, dcrB, and btuB. Our results provide evidence that the dcrA gene located at 60 min on the E. coli genetic map is identical to the sdaC gene. This gene product is an inner membrane protein recently identified as a putative specific serine transporter. The dcrB gene, located at 76.5 min, encodes a 20-kDa processed periplasmic protein, as determined by maxicell analysis, and corresponds to a recently determined open reading frame with a previously unknown function. The btuB gene product is known to be an outer membrane receptor protein responsible for adsorption of BF23 phage and vitamin B-12 uptake. According to our data the DcrA and DcrB proteins are not involved in these processes. However, the DcrA protein probably participates in some cell division steps. [TOP OF PAGE]

  76. Construction of luciferase reporter bacteriophage A511::luxAB for rapid and sensitive detection of viable Listeria cells. Loessner, M.J., Rees, C.E., Stewart, G.S., Scherer, S. (1996). Appl. Environ. Microbiol. 62:1133-1140. Specific transfer and expression of bacterial luciferase genes via bacteriophages provides an efficient way to detect and assay viable host cells. Listeria bacteriophage A511 is a genus-specific, virulent myovirus which infects 95% of Listeria monocytogenes serovar 1/2 and 4 cells. We constructed recombinant derivative A511::luxAB, which carries the gene for a fused Vibrio harveyi LuxAB protein inserted immediately downstream of the major capsid protein gene (cps). Efficient transcription is initiated by the powerful cps promoter at 15 to 20 min postinfection. Site-specific introduction of the luciferase gene into the phage genome was achieved by homologous recombination in infected cells between a plasmid carrying A511 DNA flanking luxAB and phage DNA. Recombinants occurred in the lysate at a frequency of 5 x 10(-4) and were readily identified by the bioluminescent phenotype conferred on newly infected host cells. A511::luxAB can be used to directly detect Listeria cells. Following infection and a 2-h incubation period, numbers as low as 5 x 10(2) to 10(3) cells per ml were detected by using a single-tube luminometer. Extreme sensitivity was achieved by including an enrichment step prior to the lux phage assay; under these conditions less than 1 cell of L. monocytogenes Scott A per g of artificially contaminated salad was clearly identified. The assay is simple, rapid, inexpensive, and easy to perform. Our findings indicate that A511::luxAB is useful for routine screening of foods and environmental samples for Listeria cells. [TOP OF PAGE]

  77. A super lytic actinophage system as a pre-treatment in the isolation of non-streptomycete actinomycetes from soil. Long, P.F., Amphlett, G.E. (1996). Letters in Applied Microbiology 22:62-65. An increase in the abundance and diversity of nonstreptomycete actinomycetes isolated from soils was achieved after the number of streptomycetes was reduced by pre-incubating the soil with streptomycete-specific actinophage. This method is described as a super lyric actinophage system. [TOP OF PAGE]

  78. Wastewater treatment and elimination of pathogens: new prospects for an old problem. Lopez-Pila, J.M., Dizer, H., Dorau, W. (1996). Microbiologia 12:525-536. Although the development of wastewater treatment technology is more than one hundred years old, most wastewater treatment plants existing today do not eliminate pathogens satisfactorily. Even in highly developed nations, receiving waters, serving in many cases as drinking water resources, are contaminated with pathogens. Surface waters also contain large concentration of phosphate due to long lasting wastewater discharges. Cyanobacteria and algal overgrowth is the consequence. Present drinking water technology only partially overcomes the pollution; it can not be ruled out that drinking water originating from polluted resources contains pathogens. This situation frequently goes on unnoticed because current indicator organisms are not representative for all pathogens. As studies have shown that small concentrations of pathogens also pose a risk for the consumer health, this state of affairs is a matter of concern. Microfiltration technology is able to significantly eliminate bacteria and protists from wastewater. Viruses, although smaller than the pore size of the filters, are reduced too because, in wastewater, they are frequently bound to larger particles. If the microfiltration of wastewater is preceded by the addition of coagulants for the precipitation of phosphate, the precipitate will be retained by the filter. The effluent obtained contains very low concentrations of phosphate. As viruses also adsorb to the precipitate, the amount of viruses eliminated increases and with increasing amounts of coagulant they become undetectable. [TOP OF PAGE]

  79. A field experiment on competition of Rhizobium leguminosarum bv. viciae. Lorkiewicz, Z., Kowalczuk, E. (1996). Bulletin of the Polish Academy of Sciences Biological Sciences 44:221-224. Transconjugants of Rhizobium leguminosarum bv. viciae crossed with Azotobacter vinelandii were tested for their symbiotic effectiveness determined by seed crops. The most effective appeared to be the strains N24 and R6. The competitiveness of the strains was evaluated by identification of rhizobial antibiotic resistance and phage sensitivity patterns. Seven strains examined presented an elevated competitiveness reaching from 62 to 92%. [TOP OF PAGE]

  80. Active surveillance for Vibrio cholerae O1 and vibriophages in sewage water as a potential tool to predict cholera outbreaks. Madico, G., Checkley, W., Gilman, R.H., Bravo, N., Cabrera, L., Calderon, M., Ceballos, A. (1996). Journal of Clinical Microbiology 34:2968-2972. The 1991 Peruvian cholera epidemic has thus far been responsible for 600,000 cholera cases in Peru. In an attempt to design a cholera surveillance program in the capital city of Lima, weekly sewage samples were collected between August 1993 and May 1996 and examined for the presence of Vibrio cholerae O1 bacteria and V. cholerae O1 bacteriophages (i.e., vibriophages). During the 144 weeks of surveillance, 6,323 cases of clinically defined cholera were recorded in Lima. We arbitrarily defined an outbreak as five or more reported cases of cholera in a week. The odds of having an outbreak were 7.6 times greater when V. cholerae O1 was present in sewage water during the four previous weeks compared with when it was not (P < 0.001). Furthermore, the odds of having an outbreak increased as the number of V. cholerae O1 isolations during the previous 4 weeks increased (P < 0.001). The odds of having an outbreak were 2.4 times greater when vibriophages were present in sewage water during the four previous weeks compared with when they were not, but this increase was not statistically significant (P = 0.15). The odds of having an outbreak increased as the number of vibriophage isolations during the previous 4 weeks increased (P < 0.05). The signaling of a potential cholera outbreak 1 month in advance may be a valuable tool for implementation of preventive measures. In Peru, active surveillance for V. cholerae O1 and possibly vibriophages in sewage water appears to be a feasible and effective means of predicting and outbreak of cholera. [TOP OF PAGE]

  81. The use of an automated assay to assess phage survival after a biocidal treatment. Maillard, J.Y., Beggs, T.S., Day, M.J., Hudson, R.A., Russell, A.D. (1996). J. Appl. Bacteriol. 80:605-610. The coliphage K-1-5 has been used in an automated assay to monitor the viricidal activity of various disinfectants. This indirect assay based on the spectrophotometric reading of the lysis of the host cell (Escherichia coli D837) produced encouraging results and was faster than the overlay counting method (previously studied) which relies on plaque formation. However, differences in sensitivity towards some disinfectants were observed between the two methods. [TOP OF PAGE]

  82. Bacteriophages: a model system for human viruses f. Maillard, J.Y. (1996). Letters in Applied Microbiology 23:273-274. It is important to assess and control the presence of viruses and their inactivation from surfaces (e.g. inanimate surfaces, body tissues, nosocomial equipment) and water (e.g. drinking, sewage and sea water). Since the detection and use of mammalian viruses can be fastidious, bacteriophages (bacterial viruses) offer potential alternatives in the following areas: 1. Bacteriophages as a model system 2. Bacteriophages as an index system for enteric pathogens 3. Bacteriophages as an indicator system for enteric viral pathogens 4. Bacteriophages as tools for studying mechanisms of viral disinfection. [TOP OF PAGE]

  83. Opinion. Maillard, J.Y. (1996). Letters in Applied Microbiology 23:273-274. [TOP OF PAGE]

  84. Rapid detection of mutagens by induction of luciferase-bearing prophage in Escherichia coli. Maillard, K.I., Benedik, M.J., Willson, R.C. (1996). Environmental Science and Technology [ENVIRON. SCI. TECHNOL. ] 30:2478-2483. Mutagenicity in bacteria is highly correlated with carcinogenicity in humans, and many industrial wastes and manufacturing sites contain mutagenic components. This has stimulated interest in rapid screening assays for genotoxic agents. We describe a rapid and sensitive bacterial test for detecting DNA-damaging agents, based on bioluminescence reporting of prophage lambda induction. A promoterless cassette bearing genes encoding bacterial luciferase was transposed randomly into the lambda chromosome from the suicide plasmid pUTmini-Tn5lux lysogenized with the resulting phages. In the lysogens, the luciferase genes are expressed from phage promoters, which allows rapid detection of DNA-damage-triggered prophage induction with a simple whole-cell luciferase assay. One lysogen was selected among several for its high ratio of induced to spontaneous luciferase activity upon exposure to mitomycin C. Within 4 h, the assay detects ethyl methanesulfonate, methyl methanesulfonate, and mitomycin C with sensitivity comparable to those of assays requiring more time, although not all tested mutagens were detected. The test is also automatable and generates very little waste, which makes it a potentially valuable tool for rapid screening and process monitoring. [TOP OF PAGE]

  85. Sentinel school. Manuel, J. (1996). Environmental Health Perspectives 104:934-936. [TOP OF PAGE]

  86. High concentrations of viruses in the sediments of Lac Gilbert, Quebec. Maranger, R., Bird, D.E. (1996). Microb. Ecol. 31:141-151. [TOP OF PAGE]

  87. The effect of environmental pH on the physiology and surface structures of Salmonella serotype enteritidis phage type 4. McDermid, A.S., McKee, A.S., Dowsett, A.B., Marsh, P.D. (1996). J. Med. Microbiol. 45:452-458. The incidence of food-poisoning caused by Salmonella serotype Enteritidis PT4 has increased. Implicated food products display pH levels between 4 and 9. Accordingly, the effect of growth at extremes of pH on the presence of surface structures and the carriage of a 38-MDa plasmid was determined by growing a clinical isolate of Enteritidis PT4 in a chemostat. Steady-state growth was possible over the pH range 4.35-9.45, corresponding to the pH extremes associated with key reservoirs implicated in outbreaks. Without pH control, cultures stabilised at pH 7.10. Growth at extremes of pH had significant effects on the distribution of cell surface structures; at pH 9.45, only 3% of cells were fimbriate compared with 52% at pH 7.10 and 20% at pH 4.35. The proportion of motile cells and the presence of flagella was also reduced at extremes of pH. A 38-MDa plasmid was present in cells grown in the chemostat at pH 7.10, but not in cells grown at pH 4.35 or pH 9.45. Thus, environmental pH may have a significant impact on the virulence potential of Enteritidis PT4. [TOP OF PAGE]

  88. WHO study on subtyping Listeria monocytogenes: results of phage-typing. McLauchlin, J., Audurier, A., Frommelt, A., Gerner-Smidt, P., Jacquet, C., Loessner, M.J., van der Mee-Marquet, N., Rocourt, J., Shah, S., Wilhelms, D. (1996). Int. J. Food Microbiol. 32:289-299. A multi-centered study on phage typing of Listeria monocytogenes was carried out using 80 cultures sent under code and tested in six different laboratories. Phage typing was performed using an international phage set in five laboratories and phage sets unique to two laboratories. Testing of cultures sent in duplicate showed similar levels of reproducibility to that previously reported. Analysis of results from groups of epidemiologically related cultures showed a high level of agreement in all laboratories. Patterns of phage susceptibility were relatively stable on retesting strains in the same laboratory after long periods of time. However, there was limited comparability between results obtained from testing the same cultures using the same phages in different laboratories. It is recommended that the phages in the international set be reviewed, and that better inter-laboratory reproducibility may be achieved by standardisation of phage suspensions, propagation strains and methodology, together with the use of centrally propagated phages. [TOP OF PAGE]

  89. Use of bacteriophages as selective factors in bacteriological diagnosis of mixed infections. [Russian]. Men'Shikov, D.D., Yanisker, G.Y., Orlova, N.Y., Men'Shikova, E.D. (1996). Klinicheskaya Laboratornaya Diagnostika 0(2). A new method is proposed for detecting aerobic and anaerobic bacteria during diagnostic culturing by exposure of mixed microorganism populations to bacteriophages. By lysing homologous bacteria, the phages facilitate the detection and isolation of associants resistant to them in pure cultures. The proposed method is compared with the selective media techniques and a conclusion is made on the advantages of selective decontamination of biological samples by bacteriophages in experiments and diagnostic culturing of material from patients with pyoinflammatory processes. [TOP OF PAGE]

  90. Long-circulating bacteriophage as antibacterial agents. Merril, C.R., Biswas, B., Carlton, R., Jensen, N.C., Creed, G.J., Zullo, S., Adhya, S. (1996). Proc. Natl. Acad. Sci. USA 93:3188-3192. The increased prevalence of multidrug-resistant bacterial pathogens motivated us to attempt to enhance the therapeutic efficacy of bacteriophages. The therapeutic application of phages as antibacterial agents was impeded by several factors: (i) the failure to recognize the relatively narrow host range of phages; (ii) the presence of toxins in crude phage lysates; and (iii) a lack of appreciation for the capacity of mammalian host defense systems, particularly the organs of the reticuloendothelial system, to remove phage particles from the circulatory system. In our studies involving bacteremic mice, the problem of the narrow host range of phage was dealt with by using selected bacterial strains and virulent phage specific for them. Toxin levels were diminished by purifying phage preparations. To reduce phage elimination by the host defense system, we developed a serial- passage technique in mice to select for phage mutants able to remain in the circulatory system for longer periods of time. By this approach we isolated long-circulating mutants of Escherichia coli phage lambda and of Salmonella typhimurium phage P22. We demonstrated that the long- circulating lambda mutants also have greater capability as antibacterial agents than the corresponding parental strain in animals infected with lethal doses of bacteria. Comparison of the parental and mutant lambda capsid proteins revealed that the relevant mutation altered the major phage head protein E. The use of toxin-free, bacteria-specific phage strains, combined with the serial-passage technique, may provide insights for developing phage into therapeutically effective antibacterial agents. [TOP OF PAGE]

  91. Effects of viruses on nutrient turnover and growth efficiency of noninfected marine bacterioplankton. Middelboe, M., Jorgensen, N.O.G., Kroer, N. (1996). Appl. Environ. Microbiol. 62:1991-1997. The effects of virus infection and lysis of a marine Vibrio sp. on C, N, and P turnover and the growth efficiency of noninfected bacterioplankton were studied in a series of dilution cultures. The cultures were enriched with various sources of organic matter and N and P. The growth of the Vibrio host and the growth of the natural bacterioplankton were measured by immunofluorescence and 4',6-diamidino-2-phenylindole staining methods, respectively. Lysis products resulting from infection of the Vibrio sp. caused an increase in metabolic activity and cell production by the noninfected bacterioplankton. In P-limited cultures, the addition of viruses increased the uptake of dissolved organic carbon by 72% and the potential alkaline phosphatase activity by 89% compared with control cultures without viruses. Our data suggest that input of available phosphorus through virus-induced Vibrio lysates occurred, which caused an increase in the bacterial nutrient uptake. The growth efficiency of noninfected bacteria was reduced in the presence of viruses compared with the control without viruses (growth efficiencies, 0.08 plus or minus 0.03 and 0.24 plus or minus 0.02, respectively). We suggest that the decrease in growth efficiency may be explained by an increase in bacterial energy demand associated with extracellular degradation of polymeric organic nitrogen and phosphorus in cell lysates. [TOP OF PAGE]

  92. Evolution of the genetic switch in temperate bacteriophage. I. Basic theory. Mittler, J.E. (1996). J. Theor. Biol. 179:161-172. While the molecular mechanisms underlying lysogeny and induction in bacteriophage have been intensely studied, relatively little has been done to relate these findings to their presumed selective functions. To explore the ecological basis for these traits, I have used a resource-based model for competition between bacteriophage with different probabilities of lysogeny and different spontaneous induction rates. In any given habitat the fitness of a phage will depend on the inputs of sensitive cells and nutrient resources. In equable environments (modeled here using chemostats with constant inputs of nutrients and sensitive cells), bacteriophage with low probabilities of lysogeny and low induction rates can always invade when rare and will generally be good competitors. In variable environments (chemostats with seasonal inputs), bacteriophage with higher probabilities of lysogeny and higher induction rates are favored. In both equable and variable environments, the ability of a phage to invade when rare will depend on the properties of the resident phage, and it is possible for phages with divergent parameter values to coexist. The modeling suggests that bacteriophage that have evolved moderately low induction and lysogeny rates will be able to "hedge their bets" against environmental change without sacrificing the ability to compete well in a constant environment. Implications of this theory for understanding the molecular basis of gene regulation in temperate bacteriophage and other viruses are discussed. [TOP OF PAGE]

  93. Marine bacteriophage reproduction under nutrient-limited growth of host bacteria. I. Investigations with six phage-host systems. Moebus, K. (1996). Mar. Ecol. Prog. Ser. 144:1-12. Bacteriophage reproduction was investigated with 6 phage-host systems (PHS) isolated from the North Sea near Helgoland, Germany, with the hosts adapted to growth at 6 or 0.6 mg organic nutrients 1-1. For 5 of the PHS, similarities in production were observed to depend upon the time of infection during a transition period which included the last hours of the logarithmic growth phase and the first 1 to 2 d of the stationary growth phase of the host bacteria. Over this period the extent and/or the rate of phage production d-1 decreased greatly. After longer incubation before addition of phage, the relative ability of host cells to propagate phage either ceased (3 PHS) or was regained (1 PHS) or remained the same as during the transition period in regard to final phage concentrations (1 PHS). The remaining PHS showed no phage reproduction at the low nutrient concentration. With phage-resistant mutant bacteria serving as competitors for nutrients, phage production was drastically reduced. The present findings are in agreement with observations concerning concentrations of infective virions in fresh seawater samples. They failed, however, to provide evidence for the hypothesis that release of mature phage in starving marine bacteria is delayed until sufficient nutrients become available. [TOP OF PAGE]

  94. Marine bacteriophage reproduction under nutrient-limited growth of host bacteria. II. Investigations with phage-host system [H3: H3/1]. Moebus, K. (1996). Mar. Ecol. Prog. Ser. 144:13-22. When host bacterium H3 is cultivated in artificial seawater at a concentration of 0.6 mg organic nutrients ml-1, its reaction to infection by phage H3/1 changes dramatically with the duration of incubation before phage attack. Cells infected when still in their logarithmic growth phase rapidly produce progeny phage until breakdown of the phage-sensitive population. For cells infected after entering the stationary phase, rate and extent of phage propagation by resting cells decreases for some time, but both parameters rather suddenly increase again for cells infected after prolonged incubation. Phage production may then reach the same level attained by cells infected during logarithmic growth phase; although, with increasing phage concentration, resting cells very effectively become protected from phage attack by pseudolysogeny. This course of events is mainly influenced by the method of incubation of the host (shaken vs still), by the age of the cells before infection, and presumably by changes in physiological traits of the bacteria when serially subcultured for extended periods of time. The reduction in volume of bacterial culture by repeated withdrawal of aliquots was found to be of minor importance; however, phage production was measurably affected by the transfer of cells to fresh receptacles. With cells which entered their stationary phase up to 36 h before infection, an initial phage concentration of between 10 and 10-3 PFU (plaque forming units) ml-1 was found to determine the extent, but not the rate, of phage production. The aforementioned observations were also made with cells starved for 3, 8, and 22 additional days before inoculation, except with initial PFU concentrations of 10 and 10-2 PFU ml-1, when an appreciable increase in phage production was found. In cell suspensions seeded with phage 48 d after set up of the experiment, the highest phage production was found with the lowest initial phage concentration and vice versa. This finding, i.e. the inverse relationship between production and initial concentration, is not in agreement with any of the current hypotheses concerning bacteriophage ecology. [TOP OF PAGE]

  95. Isolation and characterization of lactococcal bacteriophages from cultured buttermilk plants in the United States. Moineau, S., Borkaev, M., Holler, B.J., Walker, S.A., Kondo, J.K., Vedamuthu, E.R., Vandenbergh, P.A. (1996). Journal of Dairy Science 79:2104-2111. From July 1993 and June 1994, 27 different lactococcal bacteriophages were isolated from 27 US cultured buttermilk plants located in 23 states. Phages were characterized by DNA homology, electron microscopy, restriction patterns, genome size, host range, and serology. Over 80% (22 of 27) of the phages were classified into the 936 species, and the remaining phages were divided almost equally between the P335 species (3 of 27) and the c2 species (2 of 27). The 936 and c2-type phages had the same basic morphological and genetic characteristics as other phages from the same species isolated in other countries. Very closely related 936 phages were isolated from widely separated areas in the US. The P335 phages had a very narrow host range and showed noticeable genetic and immunological diversity. None of the phages could propagate on the two exopolysaccharide-producing Lactococcus lactis strains tested. Novel mechanisms for phage resistance should be tested for efficiency against members of the lactococcal phage species 936, c2, and P335. To our knowledge, this study is the first thorough examination of industrial lactococcal phages isolated from buttermilk plants. [TOP OF PAGE]

  96. Nitrogen metabolism by ruminal microorganisms: Current understanding and future perspectives. Morrison, M., Mackie, R.I. (1996). Australian Journal of Agricultural Research 47:227-246. This review presents an outline of our current understanding of ruminal nitrogen metabolism from three perspectives: proteolytic microorganisms and their enzymes, intraruminal recycling of microbial protein, and enzymes of ammonia assimilation. Some of the pending advances and future research opportunities in these areas are also discussed. The 'smugglin' concept appears to offer the potential to inhibit peptide-utilizing bacteria selectively in the rumen, as demonstrated by initial studies with Prevotella ruminicola. The relative contributions of protozoa-, bacteriophage-, and self-mediated lysis of bacteria to intraruminal recycling of microbial protein are not yet quantified, and further efforts to understand the biology and dynamics of ruminal bacteriophage and protozoa populations are warranted. In Ruminococcus flavefaciens and Prevotella ruminicola, glutamate dehydrogenase (GDH) appears to be the predominant route of ammonia assimilation irrespective of ammonia concentration, and peptides modulate GDH activity in P. ruminicola. The physiological basis behind the difference between optimal ammonia concentrations for ruminal fibre digestion and microbial protein synthesis remains unclear. Molecular biology techniques extend beyond their application in pursuit of the 'superbug' concept, by offering new and exciting opportunities to understand better microbial physiology, diversity, and ecology. Fundamental research in these areas must be continued if further advances in feed utilization and nutrient retention are to be realized. [TOP OF PAGE]

  97. The disintigration process of a Heterosigma akashiwo (Raphidophyceae) red tide in norther Hiroshima Bay, Japan, during the summer of 1994. Nagasaki, K., Itakura, S., Imai, I., Nakagiri, S., Yamaguchi, M. (1996). pp. 251-254. In In Yasumoto, T., Oshima, Y., and Fukuyo, Y. (eds.), Harmful and Toxic Algal Blooms. UNESCO, Paris. [TOP OF PAGE]

  98. The role of ciliate protozoa in the lysis of methanogenic archaea in rumen fluid. Newbold, C.J., Ushida, K., Morvan, B., Fonty, G., Jouany, J.P. (1996). Letters in Applied Microbiology 23:421-425. Predation by ciliate protozoa can account for 90% of the eubacterial protein turnover in the rumen. However, little is known about the factors affecting the lysis of archaea in rumen fluid. Bacterial lysis was followed from the release of acid-soluble 14C from 14C leucine-labelled bacteria. The rumen methanogen Methanobrevibacter MF1 was broken down more rapidly than other non-ruminal archaea in rumen fluid withdrawn from sheep harbouring either a mixed protozoal population or monofaunated with Polyplastron multivesiculatum or Entodinium spp. The removal of protozoa from the rumen fluid had little effect on the breakdown of Methanobrevibacter, while lysis of the non-methanogenic ruminal bacterium Selenomonas ruminantium decreased by over 70%. Substantial lysis of Methanobrevibacter occurred in cell-free rumen fluid and this effect could be abolished by autoclaving. In view of the high number of bacteriophages in rumen fluid and susceptibility of ruminal bacteria to phage-induced lysis it is tempting to suggest that phages have a role in the lysis of archaea in rumen fluid. [TOP OF PAGE]

  99. A model on the production of temperate phages in continuous culture. Noack, D. (1996). pp. 233-241. In AnonymousContinuous Cultivation of Microorganisms, Proceedings of the Fourth Symposium. ???, ??? [TOP OF PAGE]

  100. Repeated transfer of small RNA virus populations leading to balanced fitness with infrequent stochastic drift. Novella, I.S., Elena, S.F., Moya, A., Domingo, E., Holland, J.J. (1996). Molecular and General Genetics 252:733-738. The population dynamics of RNA viruses have an important influence on fitness variation and, in consequence, on the adaptative potential and virulence of this ubiquitous group of pathogens. Earlier work with vesicular stomatitis virus showed that large population transfers were reproducibly associated with fitness increases, whereas repeated transfers from plaque to plaque (genetic bottlenecks) lead to losses in fitness. We demonstrate here that repeated five-plaque to five-plaque passage series yield long-term fitness stability, except for occasional stochastic fitness jumps. Repeated five-plaque passages regularly alternating with two consecutive large population transmissions did not cause fitness losses, but did limit the size of fitness gains that would otherwise have occurred. These results underscore the profound effects of bottleneck transmissions in virus evolution. [TOP OF PAGE]

  101. Large-population passages of vesicular stomatitis virus in interferon-treated cells select variants of only limited resistance. Novella, I.S., Elena, S.F., Kohn, J., Moya, A., Domingo, E., Holland, J.J. (1996). J. Virol. 70:6414-6417. Vesicular stomatitis virus (VSV) populations were repeatedly passaged in L-929 cells treated with alpha interferon (IFN-alpha) at levels of 25 U/ml. This IFN-alpha concentration induced a 99.9% inhibition of viral yield in standard infections. Analysis of viral fitness (overall replicative ability measured in direct competition with a reference wild-type VSV) after 21 passages in IFN-treated cells showed only a limited increase or no increase in fitness, compared with the greater increase upon parallel passage in cells not treated with IFN-alpha. However, this limited increase in fitness was more pronounced when competition assays were carried out with IFN-alpha-treated cells, suggesting the selection of VSV populations with a low level of resistance to IFN-alpha. Thus, despite the extensively documented capacity of VSV to adapt to changing environments, the antiviral state induced by IFN-alpha imposes adaptive constraints on VSV which are not readily overcome. [TOP OF PAGE]

  102. AbiG, a genotypically novel abortive infection mechanism encoded by plasmid pCI750 of Lactococcus lactis subsp. cremoris UC653. O'Connor, L., Coffey, A., Daly, C., Fitzgerald, G. (1996). Appl. Environ. Microbiol. 62:3075-3082. AbiG is an abortive infection (Abi) mechanism encoded by the conjugative plasmid pCI750 originally isolated from Lactococcus lactis subsp. cremoris UC653. Insensitivity conferred by this Abi manifested itself as complete resistance to vphi-712 (936 phage species) with only partial resistance to vphi-c2 (c2 species). The mechanism did not inhibit phage DNA replication. The smallest subclone of pCI750 which expressed the Abi phenotype contained a 3.5-kb insert which encoded two potential open reading frames. abiGi (750 bp) and abiGii (1,194 bp) were separated by 2 bp and appeared to share a single promoter upstream of abiGi. These open reading frames showed no significant homology to sequences of either the DNA or protein databases; however, they did exhibit the typical low G+C content (29 and 27%, respectively) characteristic of lactococcal abi genes. In fact, the G+C content of a 7.0-kb fragment incorporating the abiG locus was 30%, which may suggest horizontal gene transfer from a species of low G+C content. In this context, it is notable that remnants of IS elements were observed throughout this 7.0-kb region. [TOP OF PAGE]

  103. Occurrence of a temperate cyanophage lysogenizing the marine cyanophyte Phormidium persicinum. Ohki, K., Fujita, Y. (1996). Journal of Phycology 32:365-370. A temperate cyanophage was found to lysogenize the marine cyanophyte Phormidium persicinum (Reinke) Gom. (Provasoli strain). The lytic cycle was induced by the addition of mitomycin C or by brief illumination with ultraviolet light. The lytic process observed under the electron microscope showed that phage particles appeared in a nucleoplasm region 15 to 24 h after the addition of mitomycin C. The induction of the lytic process occurred simultaneously in almost all cells of every trichome. Matured phage particles were released to the medium 30 to 50 h after the addition of mitomycin C Phage particles isolated from algal lysates had a polyhedral head (about 40 nm in diameter) with a long (about 300 nm) and noncontractile tail. The most abundant protein, presumably a structural protein, had an apparent molecular mass of about 38 kDa. The genome size estimated from restriction analysis was about 50 kbp. Phage DNA was digested with several restriction endonucleases including Sau3AI and DpnI. However, MboI failed to digest the pha DVA, suggesting that the phage DNA is highly methylated. Southern blot analysis suggested that some part of the phage was in the lytic cycle in algal cells growing under normal conditions. A possible role of temperate cyanophages in the regulation of cyanophyte populations in the marine environment is discussed. [TOP OF PAGE]

  104. USE OF HYDROLYZED WHEY PEPTIDE BLOCKERS TO NHIBIT CULTURE AGGLUTINATION AND PHAGE PROLIFERATION IN LACTIC BULK STARTER CULTURE. ONUORAH, C.E. (1996). University of Kentucky. Peptides fractionated from papain hydrolyzed whey, collected through a 10,000 MW cut off hollow fiber Romicon ultrafiltration membrane were used to prepare a culture agglutination resistant and phage inhibitory medium. Permeate solids were used to replace the whey solids that are part of a commercially prepared Insure$/sp/circler$ medium. The freeze dried and liquid permeates containing the 10,000; 3,000; and 1,000 MW cut off peptides (calculated on a solids basis as 60% of the solids) were added to an internal pH control buffer salt mixture (Insure$/sp/circler$, salts, as 40% of the solids). A control medium was prepared from commercially prepared Insure$/sp/circler$ medium using 75.7 g/L. All media were heat treated at 85$/sp/circ$C for 45 min, cooled to 31$/sp/circ$C. Graduated cylinders (25 ml) containing each medium (10 ml) were inoculated with 4% of culture (B62, and E72 cultures) and incubated with continuous agitation at room temperature (23-25$/sp/circ$C) for 16-18 h or until pH of at least 5.30 was achieved. These starters were used to inoculate 240-ml aliquots of pasteurized (62$/sp/circ$C for 30 min) skim milk which were incubated at 31$/sp/circ$C for 5 h in 250 ml graduated cylinders. A pH measurement was determined at the top and bottom of the cylinders initially and after each h of the incubation. All cultures grown in the presence of peptides were observed to be less agglutinatinated (P $<$.0011) than the control (inoculated with starter culture without peptides). Of the three peptides sizes tested, 1,000 MW cut off was the most effective in inhibiting agglutination. Bulk starter was prepared from M17 media inoculated with WWA4 single strain Lactococcus lactis ssp cremoris. M17 media (heat treated at 85$/sp/circ$C for 45 min and cooled to 24$/sp/circ$C) containing whey peptide blockers (0,.025,.05,.075, and.1 g/ml) was inoculated with WWA4 bulk starter and incubated for 6 h. WWA4-phage (10$/sp5$ pfu) was added to the growing culture after 90 min of incubation. Cell growth was monitored spectrophotometrically (Ag$/rm/sb[600nm]$) at 10 min intervals. As peptide blocker concentration increased, time to bacterial cell lyse increased and rate of bacterial lyse decreased. Time from phage introduction to maximum absorbance increased linearly from 85 min (0 g/ml blocker) to 147.5 min (.1 g/ml blocker). Slope of bacterial lysis did not change until blocker concentration exceed.05 g/ml. A linear decrease in lysis rate was observed for blocker concentration of.05 to.1 g/ml, suggestion that antigenic sites on the surface of the lactic cell must be saturated before the attachment of phage could be competitively inhibited. In experiments where phage concentration varied from 10$/sp[-3]$ to 10$/sp[-5]$ pfu, time to maximum absorbance decreased as phage concentration increased. However, the time differential between media with and without blockers increased linearly with phage concentration suggesting that blocker concentration was most effective when lactic cell numbers were lowest. [TOP OF PAGE]

  105. Viruses and DNA in marine environments. Paul, J.H., Kellogg, C.A., Jiang, S.C. (1996). pp. 115-124. In In Colwell, R.R., Simidu, U., and Ohwada, K. (eds.), Microbial Diversity in Time and Space. Plenum Press, New York. [TOP OF PAGE]

  106. Construction of D29 shuttle phasmids and luciferase reporter phages for detection of mycobacteria. Pearson, R.E., Jurgensen, S., Sarkis, G.J., Hatfull, G.F., Jacobs, W.J. (1996). Gene 183:129-136. Diseases caused by Mycobacterium tuberculosis, M. leprae and M. avium, cause significant morbidity and mortality worldwide. Effective treatments require that the organisms be speciated and that drug susceptibilities for the causative organisms be characterized. Reporter phage technology has been developed as a rapid and convenient method for identifying mycobacterial species and evaluating drug resistance. In this report we describe the construction of luciferase reporter phages from mycobacteriophage D29 DNA. Shuttle phasmids were first constructed with D29 in order to identify non-essential regions of the D29 genomes and to introduce unique cloning sites within that region. Using this approach, we observed that all of the D29 shuttle phasmids had the cosmid vector localized to one area of the phage genome near one cohesive end. These shuttle phasmids had been constructed with a cosmid that could be readily excised from the D29 genome with different sets of restriction enzymes. Luciferase reporter phages were made by substituting the luciferase cassette for the cosmid vector. Recombinant phages with the luciferase cassette fall into two groups. One group produced light and had the expression cassette oriented with the promoter directing transcription away from the cohesive end. In contrast, the other group had the expression cassette in the opposite orientation and failed to produce light during lytic infection, but did produce light in L5 lysogens which are known to repress D29 promoters. These results suggest that a phage promoter of the D29 phage can occlude the expression of a promoter introduced into this region. D29 luciferase reporter phages are capable of detecting low numbers of L5 lysogens like L5 luciferase phages. However, unlike L5 luciferase phages, D29 luciferase phages can readily infect M. tuberculosis and M. bovis BCG, demonstrating that these phages can be used to evaluate drug susceptibilities of many types of mycobacteria. [TOP OF PAGE]

  107. Exclusion of T4 phage by the hok/sok killer locus from plasmid R1. Pecota, D., Wood, T.K. (1996). J. Bacteriol. 178:2044-2050. The hok (host killing) and sok (suppressor of killing) genes (hok/sok) efficiently maintain the low-copy-number plasmid R1. To investigate whether the hok/sok locus evolved as a phage- exclusion mechanism, Escherichia coli cells that contain hok/sok on a pBR322-based plasmid were challenged with T1, T4, T5, T7, and lambda phage. Upon infection with T4, the optical density of cells containing hok/sok on a high-copy-number plasmid continued to increase whereas the optical density for those lacking hok/sok rapidly declined. The presence of hok/sok reduced the efficiency of plating of T4 by 42% and decreased the plaque size by apprx 85%. Single-step growth experiments demonstrated that hok/sok decreased the T4 burst size by 40%, increased the time to form mature phage (eclipse time) from 22 to 30 min, and increased the time to cell lysis (latent period) from 30 to 60 min. These results further suggest that single cells exhibit altruistic behavior. [TOP OF PAGE]

  108. Occurrence of airborne bacteria and pathogen indicators during land application of sewage sludge. Pillai, S.D., Widmer, K.W., Dowd, S.E., Ricke, S.C. (1996). Appl. Environ. Microbiol. 62:296-299. Glass impingers (AGI-30) were used at a commercial sludge application site to determine the levels of airborne bacteria and pathogen indicators. Even though heterotrophic bacteria averaged 10-5 CFU/m-3, none of the sites showed the presence of Salmonella spp. or indicators such as fecal coliforms or coliphages. Indicators such as H-2S producers and pathogenic clostridia were present in locations having significant physical agitation of the sludge material. PCR-based ribotyping using the 16S-23S interspacer region is a promising method to identify the genetic relatedness and origins of airborne clostridia. [TOP OF PAGE]

  109. Reproducible nonlinear population dynamics and critical points during replicative competitions of RNA virus quasispecies. Quer, J., Novella, I.S., Tsimring, L., Domingo, E., Holland, J.J. (1996). J. Mol. Biol. 264:465-471. RNA virus evolution is generally considered to be highly unpredictable, but tests of determinism in the evolution of competing populations during viral infections have not been performed. Here we study the fate of two closely related evolving quasispecies: of vesicular stomatitis virus, by determining the relative concentration of a wild-type clone and a surrogate marked virus subclone (MARM-C) upon extensive competitive replication in a constant cell culture environment. A highly predictable nonlinear behaviour of the two competing populations was found. In addition, the presence of critical points, which are defined as points from which viral competitions may follow different trajectories, has been documented. Critical points were reached after nearly constant periods of time. The dynamics of relative fitness values for both competing populations were calculated during the replication passages. Concomitant with expected fitness gain of both competing viral popu