Bacteriophage Ecology Group
Reference Abstracts (1999)
Dedicated to the ecology and evolutionary biology of the parasites of unicellular organisms (UOPs)
© Stephen T. Abedon
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© Phage et al. last updated on Wednesday, December 26, 2001
  1. Viruses help fight bacteria that resist antibiotics. ??? (1999). The Patriot Ledger Quincy, MA 13(183?), 183? (News Section)-same? Scientists have harnessed nature's way of tackling antibiotic- resistant bacteria. An injection of a virus that attacks bacteria only has saved the life of a patient after all other drugs proved useless. The technique -- the use of bacteriophages, or bacteria-eaters -- was pioneered in the former Soviet Union at around the time of the discovery of much more swiftly effective antibiotics. Although penicillin and other such drugs changed medicine, one team in Tbilisi, Georgia, kept research in phages going to the present day. [TOP OF PAGE]

  2. Genetic exchange between microorganisms. ??? (1999). p. ???-??? In Lengeler, J.W., Drews, G., and Shlegel, H.G. (eds.), Biology of Prokaryotes. Blackwell Science, Inc., [TOP OF PAGE]

  3. Bacteriophage T4 resistance to lysis-inhibition collapse. Abedon, S.T. (1999). Genet. Res. 74:1-11. Lysis-inhibition is a mechanism of latent-period extension and burst-size increase that is induced by the T4 bacteriophage adsorption of T4-infected cells. Mutants of T4 genes imm, sp, and 5 (specifically the ts1 mutant of the latter) display some lysis inhibition. However, these mutants experience lysis-inhibition collapse, the lysis of lysis-inhibited cells, earlier than wild type-infected cells (i.e., their collapse occurs prematurely). Lysis from without is a lysis induced by excessive T4 adsorption. Gp5 is an inducer of lysis from without while gpimm and gpsp effect resistance to lysis from without. This paper shows that interfering with the adsorption of phages to imm-, sp-, or 5ts1-mutant-infected cells, in a variety of contexts, inhibits premature lysis-inhibition collapse. From these data, it is inferred that wild-type T4-infected cells display resistance to lysis-inhibition collapse by a mechanism resembling resistance to lysis from without. [TOP OF PAGE]

  4. Longitudinal study on the susceptibility to bacteriophages of Staphylococcus aureus strains isolated from dairy farms in Trinidad. Adesiyun, A.A., Romain, H.T. (1999). Zentralblatt Fur Veterinarmedizin - Reihe B 46:567-581. A 6-month longitudinal study was conducted on 30 dairy cows in early lactation and their human handlers on six farms across Trinidad. Weekly samples of bulk milk, composite milk and anterior nares and hand swabs from human handlers were collected and cultured for Staphylococcus aureus on Baird-Parker agar (BPA). The susceptibility of S. aureus strains to bacteriophages and the relatedness of strains isolated over the study period were determined. Sixty-three (51.2%) of 123 strains of S. aureus from bulk milk were typable compared with 111 (57.3%) of 194 and 82 (61.7%) of 133 strains isolated from composite milk and human handlers, respectively. The differences were not statistically significant (P > 0.05; chi 2). Bovine phage 42D lysed 3.3% (4 of 123), 16.5% (32 of 194) and 12.0% (16 of 133) of S. aureus strains isolated from bulk milk, composite milk and human handlers, respectively. The differences were statistically significant (P < 0.001; chi 2). Amongst bulk milk isolates of S. aureus, 35 (31.8%) of 110 exhibited relatedness in 11 groups based on their phage patterns and groups. The mean maximum interval between the first and last detection of related S. aureus strains in a group was 11.5 +/- 7.3 weeks. Amongst composite milk strains of S. aureus, 23 (46.0%) of 50, 25 (62.5%) of 40 and 22 (53.7%) of 41 exhibited relatedness on farms IB 2, IB 27 and IC 23, respectively, but the differences were not statistically significant (P > 0.05; chi 2). On farm IB 2, five groups of related strains of S. aureus were detected with a mean maximum interval of detection of 18.2 +/- 8.5 weeks compared to farm IB 27 where five groups of related strains were also observed but with an interval of 13.8 +/- 8.2 weeks. On farm IC 23, a total of seven groups of related S. aureus strains were detected with a mean interval of 8.0 +/- 5.5 weeks. For human strains of S. aureus from farm IB 2, nine (56.3%) of 16 strains isolated from anterior nares exhibited relatedness in three groups with a mean maximum interval of 13.3 +/- 4.7 weeks compared to four (25.0%) of 16 hand swab isolates which exhibited relatedness in two groups with mean interval of detection of 11.0 +/- 1.4 weeks. The differences were not statistically significant (P > 0.05; chi 2). On farm IB 27, for anterior nares isolates, eight (72.7%) of 11 exhibited relatedness in two groups with a mean maximum interval of detection of 20.5 +/- 2.1 weeks compared to hand swab isolates, with six (50.0%) of 12 showing relatedness in two groups and a mean interval of 10.5 +/- 2.1 weeks. It was concluded that dairy cows and their human handlers carried particular strains of S. aureus at various sites for extended periods, which served as continuous sources of contamination of milk and may play a significant role in the occurrence of subclinical mastitis, with an obvious economic impact. [TOP OF PAGE]

  5. Iron modulates phenotypic variation and phosphorylation of P270 in double-stranded RNA virus-infected Trichomonas vaginalis. Alderete, J.F. (1999). Infect. Immun. 67:4298-4302. Trichomonas vaginalis infected with a double-stranded RNA virus undergoes phenotypic variation on the basis of surface versus cytoplasmic expression of the immunogenic protein P270. Examination of batch cultures by flow cytofluorometry with monoclonal antibody (MAb) to P270 yields both fluorescent and nonfluorescent trichomonads. Greater numbers and intensity of fluorescent organisms with surface P270 reactive with MAb were evident in parasites grown in medium depleted of iron. Placement of iron-limited organisms in medium supplemented with iron gave increased numbers of nonfluorescent trichomonads. Purified subpopulations of trichomonads with and without surface P270 obtained by fluorescence-activated cell sorting reverted to nonfluorescent and fluorescent phenotypes when placed in high- and low-iron media, respectively. No similar regulation by iron of P270 was evident among virus-negative T. vaginalis isolates or virus-negative progeny trichomonads derived from virus-infected isolates. Equal amounts of P270 were detectable by MAb on immunoblots of total proteins from identical numbers of parasites grown in low- and high-iron media. Finally, P270 was found to be highly phosphorylated in high-iron parasites. Iron, therefore, plays a role in modulating surface localization of P270 in virus-harboring parasites. [TOP OF PAGE]

  6. Primary structure and features of the genome of the Lactobacillus gasseri temperate bacteriophage phiadh. Altermann, E., Klein, J.R., Henrich, B. (1999). Gene (Amsterdam) 236:333-346. The complete DNA sequence of the Lactobacillus (Lb.) gasseri temperate phage phiadh was determined. The linear and double-stranded genome consists of 43.785 bp with a G+C content of 35.3% and 3' protruding cohesive ends of 12 nt. Sixty-two possible ORFs were identified. On the basis of homology comparisons, some of them could be assigned to possible functions, such as a helicase, a nucleic acid polymerase and a protease. In a non-coding area of the phiadh genome, structural features of a potential replication origin were detected. After subcloning, this region was functional as a replicon in Lb. gasseri and Lactococcus lactis. N-terminal aa sequencing and electron microscopic analysis of intact and defective phage particles enabled the identification of two capsid protein genes. One of their products, the major head protein, seems to be processed on the posttranslational level. [TOP OF PAGE]

  7. Stable expression of the Lactobacillus casei bacteriophage A2 repressor blocks phage propagation during milk fermentation. Alverez, M.A., Rodriguez, A., Suarez, J.E. (1999). Journal of Applied Microbiology 86:812-816. A general strategy was applied to implement resistance against temperate bacteriophages that infect food fermentation starters through cloning and expression of the phage repressor. Lactobacillus casei ATCC 393 and phage A2 were used to demonstrate its feasibility as milk fermentation is drastically inhibited when the strain is infected by this phage. The engineered strain Lact. casei EM40::cI, which has the A2 repressor gene (cI) integrated into the genome, was completely resistant and able to ferment milk whether phage was present or not. In addition, viable phages were eliminated from the milk, probably through adsorption to the cell wall. Finally, the integration of cI in the genome resulted in a stable resistance phenotype, being unnecessary selective pressure during milk fermentation. [TOP OF PAGE]

  8. Test tube evolution catches time in a bottle. Appenzeller, T. (1999). Science 284(June 25, 1999), 2108-2110. By running experiments on microbes for thousands of generations, researchers are exploring the roles of chance and history in evolution. [TOP OF PAGE]

  9. Application of bacteriophages as surrogates for mammalian viruses: a case for use in filter validation based on precedents and current practices in medical and environmental virology. Aranha-Creado, H., Brandwein, H. (1999). PDA Journal of Pharamaceutical Science and Technology 53:75-82. Infectivity-based assays are the assays of choice for the detection of pathogenic mammalian viruses. While it is intuitively appropriate to conduct testing and validation studies with the known viral burden or a closely related mammalian species, logistic considerations often dictate otherwise. Consequently, bacteriophages have served as suitable surrogates for mammalian viruses in both medical and environmental virology applications. The wide range of bacteriophages available offers a powerful analytical tool amenable to several different applications: filter validation studies (where removal is based on size exclusion), investigations into virus contamination control issues, evaluation of barrier materials, etc. There is a considerable body of evidence to suggest and support the use of bacteriophages as surrogates for mammalian viruses. Use of appropriately sized bacteriophages provides an innocuous, efficacious and expeditious method for economical testing and validation of viral clearance capabilities of virus removal filters, thus facilitating performance of filter validation studies in biopharmaceuticals under product- and process-specific conditions in an overall effort towards ensuring the virological safety of biologicals. This paper discusses the limitations associated with mammalian virus assays and provides a rationale for the use of bacteriophages as surrogates for mammalian viruses. Data from published literature documenting applicability of bacteriophages in filter validation studies, especially when removal is based on size exclusion, is reviewed along with examples of studies from the fields of medical and environmental virology. [TOP OF PAGE]

  10. The genetic element pSSVx of the extremely thermophilic crenarchaeon Sulfolobus is a hybrid between a plasmid and a virus. Arnold, H.P., She, Q., Phan, H., Stedman, K., Prangishvili, D., Holz, I., Kristjansson, J.K., Garrett, R., Zillig, W. (1999). Molecular Microbiology 34:217-226. A new Sulfolobus islandicus strain, REY15/4, harboured both a novel fusellovirus, SSV2, and a small plasmid, pSSVx. The plasmid spread in S. solfataricus P1 together with the virus after infection with either the supernatant of a culture of REY15/4 or purified virus. Spreading of the plasmid required co-transfection with either SSV2 or the related SSV1 as helpers. Virus purified from REY15/4 constituted a mixture of two sizes of particles, one with the dimensions of a normal fusellovirus and the other smaller. Cloned SSV2 produced only the larger particles and only SSV2 DNA, indicating that the smaller particles contained pSSVx packaged into capsids made up of SSV2 components. The 5.7 kb genome of pSSVx revealed regions of high sequence similarity to the cryptic Sulfolobales plasmids pRN1, pRN2 and pDL10. Thus, pSSVx belongs to the family of pRN plasmids that share a highly conserved region, which probably constitutes the minimal replicon. They also contain a variable region showing no sequence similarity. In pSSVx, this region contains three open reading frames (ORFs), two of which are juxtapositioned and show high sequence similarity to a tandem of ORFs in fusellovirus genomes. Neither pRN1 nor pRN2, which lack this tandem, spread in the presence of the fuselloviruses, which implies that the sequences of these ORFs enable pSSVx to use the packaging system of the viral helpers for spreading. [TOP OF PAGE]

  11. Characterization of six bacteriophages of Serratia liquefaciens CP6 isolated from the sugar beet phytosphere. Ashelford, K.E., Fry, J.C., Bailey, M.J., Jeffries, A.R., Day, M.J. (1999). Appl. Environ. Microbiol. 65:1959-1965. Six phages (Phi CP6-1 to (Phi CP6-6) that are commonly found in the phytosphere of sugar beet (Beta vulgaris var. Amethyst) were investigated, and their relative impacts on their host (Serratia liquefaciens CP6) were compared. There were fundamental differences between the two most abundant predators of CP6 Phi CP6-1 and Phi CP6-4), Like Phi CP6-2 and Phi CP6-5, Phi CP6-1 belonged to the family Siphoviridae, while Phi CP6-4 exhibited the morphology of the family Podoviridae, The other phages were members of the family Myoviridae, DNA-DNA crosshybridization revealed that Phi CP6-1 and Phi CP6-4 had little common DNA, although all of the other phages exhibited some genetic similarity. Like Phi CP6-2, Phi CP6-3, and Phi CP6-5, Phi CP6-1 was capable of forming a lysogenic association with its host, while Phi CP6-4 and Phi CP6-6 appeared to be entirely virulent. Single-step growth curve experiments revealed that Phi CP6-4 had a much shorter latent period and a smaller burst size than Phi CPC-1. Also, Phi CP6-1 could transduce a number of host chromosomal markers with transfer frequencies of 2.9 x 10(-9) to 3.9 x 10(-7), whereas Phi CP6-4 could not transduce S. liquefaciens CP6 genes. When viewed in the context of the strikingly different temporal niches of these phages, our data provide an insight into how bacteriophage interactions with their hosts might reflect the natural ecology of bacteriophages, Our data also illustrate how the potential for gene transfer changes over time in an environment that supports several different phages. [TOP OF PAGE]

  12. In situ population dynamics of bacterial viruses in a terrestrial environment. Ashelford, K.E., Day, M.J., Bailey, M.J., Lilley, A.K., Fry, J.C. (1999). Appl. Environ. Microbiol. 65:169-174. Predation by bacteriophages is thought to control bacterial numbers and facilitate gene transfer among bacteria in the biosphere. A thorough understanding of phage population dynamics is therefore necessary if their significance in natural environments is to be fully appreciated. Here we describe the in situ population dynamics of three separate phage populations predating on separate bacterial species, living on the surface of field-grown sugar beet (Beta vulgaris var. Amethyst), as recorded over a 9-month period. The distributions of the three phage populations were different and fluctuated temporally in 1996 (peak density, approximately 10(3) PFU g-1). One of these populations, predating on the indigenous phytosphere bacterium Serratia liquefaciens CP6, consisted of six genetically distinct DNA phages that varied in relative abundance to the extent that an apparent temporal succession was observed between the two most abundant phages, phi CP6-1 and phi CP6-4. [TOP OF PAGE]

  13. Toward selection of internalizing antibodies from phage libraries. Becerril, B., Poul, M.A., Marks, J.D. (1999). Biochemical & Biophysical Research Communications 255:386-393. Antibodies which bind cell surface receptors in a manner whereby they are endocytosed are useful molecules for the delivery of drugs, toxins, or DNA into the cytosol of mammalian cells for therapeutic applications. Traditionally, internalizing antibodies have been identified by screening hybridomas. For this work, we studied a human scFv (C6.5) which binds ErbB2 to determine the feasibility of directly selecting internalizing antibodies from phage libraries and to identify the most efficient display format. Using wild-type C6.5 scFv displayed monovalently on a phagemid, we demonstrate that anti-ErbB2 phage antibodies can undergo receptor-mediated endocytosis. Using affinity mutants and dimeric diabodies of C6.5 displayed as either single copies on a phagemid or multiple copies on phage, we define the role of affinity, valency, and display format on phage endocytosis and identify the factors that lead to the greatest enrichment for internalization. Phage displaying bivalent diabodies or multiple copies of scFv were more efficiently endocytosed than phage displaying monomeric scFv and recovery of infectious phage was increased by preincubation of cells with chloroquine. Measurement of phage recovery from within the cytosol as a function of applied phage titer indicates that it is possible to select for endocytosable antibodies, even at the low concentrations that would exist for a single phage antibody member in a library of 10(9). [TOP OF PAGE]

  14. Viral evolution revealed by bacteriophage PRD1 and human adenovirus coat protein structures. Benson, S.D., Bamford, J.K., Bamford, D.H., Burnett, R.M. (1999). Cell 98:825-833. The unusual bacteriophage PRD1 features a membrane beneath its icosahedral protein coat. The crystal structure of the major coat protein, P3, at 1.85 A resolution reveals a molecule with three interlocking subunits, each with two eight-stranded viral jelly rolls normal to the viral capsid, and putative membrane-interacting regions. Surprisingly, the P3 molecule closely resembles hexon, the equivalent protein in human adenovirus. Both viruses also have similar overall architecture, with identical capsid lattices and attachment proteins at their vertices. Although these two dsDNA viruses infect hosts from very different kingdoms, their striking similarities, from major coat protein through capsid architecture, strongly suggest their evolutionary relationship. [TOP OF PAGE]

  15. Biological UV dosimeters in the assessment of the biological hazard from environmental radiation. Berces, A., Fekete, A., Gaspar, S., Grof, P., Rettberg, P., Horneck, G., Ronto, G. (1999). Journal of Photochemistry and Photobiology B, Biology 53:36-43. To determine the impact of environmental UV radiation, biological dosimeters that weight directly the incident UV components of sunlight have been developed, improved and evaluated in the frame of the BIODOS project. Four DNA-based biological dosimeters ((i) phage T7, (ii) uracil thin layer, (iii) spore dosimeter and (iv) DLR-biofilm) have been assessed from the viewpoint of their biological relevance, spectral response and quantification of their biological effectiveness. The biological dosimeters have been validated by comparing their readings with weighted spectroradiometer data, by comparison with other biological doses, as well as with the determined amounts of DNA UV photoproducts. The data presented here demonstrate that the biological dosimeters are potentially reliable field dosimeters for measuring the integrated biologically effective irradiance for DNA damage. [TOP OF PAGE]

  16. Isolation of Shigella sonnei lysogenic for a bacteriophage encoding gene for production of Shiga toxin. Beutin, L., Strauch, E., Fischer, I. (1999). Lancet 353:1498-1498. [TOP OF PAGE]

  17. Reconsidering the relationship between virally induced bacterial mortality and frequency of infected cells. Binder, B. (1999). Aquat. Microb. Ecol. 18:207-215. The relative contribution of viral lysis to overall mortality in aquatic bacterial populations is often estimated as twice the frequency of infected cells (FIC). The `factor-of-two rule' upon which this estimate is based assumes (1) steady-state conditions, (2) that latent period is equivalent to generation time, and (3) that infected cells are not grazed. FIC values for this calculation are themselves derived from measurements of the frequency of visibly infected cells (FVIC) by the use of a simple conversion factor. A steady-state model was developed to more rigorously define the relationships between FIC, FVIC, and the fraction of mortality from viral lysis (FMVL). This model shows that even under the restrictive assumptions listed above, the factor-of-two rule systematically overestimates FMVL for typically reported values of FVIC. The model also shows that although grazing on infected cells further reduces FMVL for a given estimate of FIC, at the same time such grazing increases FIC for a given measurement of FVIC. In combination, these 2 effects minimize the influence of grazing on the calculation of FMVL from FVIC. Overall, the relationship between FMVL and FVIC is well approximated as follows: FMVL epsilon FVIC/[ gamma ln(2) (1 - epsilon - FVIC)], where gamma = the ratio between the latent period and generation time, and \g?\ = the fraction of the latent period during which viral particles are not yet visible. Using typically observed values of FVIC, and assuming that gamma = 1 (per assumption 2, above) and \g?\ = 0.186 (per literature estimates), the model suggests that, on average, viral lysis accounts for approximately 22% (range: 4.5 to 45%) of total bacterial mortality in a range of aquatic environments, corresponding to a mean overestimate of 24% (range: 4 to 44%) by the factor-of-two rule. Perhaps most importantly, the model shows that calculations of FMVL from FIC or FVIC are very sensitive to changes in the relative length of the latent period ( gamma ) and in the assumed proportion of the latent period during which viral particles are not recognizable ( epsilon ). Constraining these 2 factors would greatly improve the reliability of FMVL calculations. [TOP OF PAGE]

  18. Vaginal bacterial phaginosis? Blackwell, A.L. (1999). Sexually Transmitted Infections 75:352-353. The hypothesis that a sexually transmitted lactobacillus phage may specifically destroy the endogenous healthy lactobacillus vaginal flora and secondarily permit overgrowth of endogenous aerobic bacteria and G. vaginalis may explain why anaerobic vaginosis behaves epidemiologically as a sexually transmitted agent but recurrence rate is unaffected by antibacterial treatment of male partners. Unfortunately this hypothesis raises as many questions as answers. Are phages capable of destroying lactobacilli carried on the penis or are the lactobacillus phages derived from the woman's own gut flora and merely transferred into the vagina by sexually activity. Are there any phage resistant strains of lactobacilli which could be of therapeutic use? Is t here any possibility that a vaccine could be developed that could be given to women with recurrent anaerobic vaginosis, particularly perhaps, those who smoke or who have cervical intraepithelial neoplasia. Until the pathogenesis of anaerobic vaginosis is more fully understood, argument will undoubtedly remain concerning the best name for the conditions (?vaginal bacterial phaginosis) and treatment will be unsatisfactory. As a result some women will be burdened not only by the social consequences of recurrent genital malodour but may also be at risk of a plethora of complications. [see: http://sti.bmjjournals.com/cgi/reprint/75/5/352.pdf for full-text pdf]. [TOP OF PAGE]

  19. Rezervoary, interakcie a stabilita genov rezistencie na antibiotika. Syndrom "easy to get--hard to lose". [Reservoirs, interactions and stability of genetic resistance to antibiotics. The "easy to get--hard to lose" syndrome]. Blahova, J., Kralikova, K., Krcmery, V. (1999). CASOPIS LEKARU CESKYCH 138:424-428. Enthusiasm after discovery of antibiotics and their use in clinical practice led to presumption that problems of bacterial infections will be soon resolved and forgotten and attention will be turned to other serious problems, such as viral infections or neoplastic diseases. However, instead of disappearance of bacterial infections, bacterial pathogens become more resistant to many antibiotics. The ability of bacterial strains to acquire resistance genes from other bacteria, even of different species, causes increasing stability of resistance of bacteria. Transferable elements--resistance genes--often interact and create changed structures; this enables to preserve, stabilize, or under special conditions, transfer resistance genes. Transferable elements include plasmids, transposons, integrins and gene cassettes. Conjugation of bacteria, transduction by bacteriophages and transformation are the mechanisms by which these elements are transferred. A very significant property of transferable, mobilizable and transposable genetic systems of resistance is their stability and ability to adapt to new hosts. They do not lose it in the absence of antibiotics. The generally pessimistic view on future antibacterial chemotherapy should be a challenge to prevent the existence and spread of resistant strains of bacteria. It is much simpler and more convenient than "quench the fire" later. Best scheme is to stop resistance before it starts. [TOP OF PAGE]

  20. Transduction of antibiotic resistance in Pseudomomas aeruginosa: relationship between lytic and transducing activity of phage isolate AP-423. Blahova, J., Kralikova, K., Krcmery, V.S., Jezek, P. (1999). Acta Virol. 43:395-398. Isolation and propagation of a wild type phage, isolate AP-423, from an apparently lysogenic strain of Pseudomonas aeruginosa, resistant to a series of anti-pseudomonadal antibiotics, and its use for transduction of resistance determinants is described. The phage isolate AP-423 showed a phenomenon of host restriction, i.e. it was lysogenic only for some of the recipient strains tested. Its transduction capacity, both in sets of genes transduced and frequency of transduction, was different in two recipient strains of P. aeruginosa. This phage showed also some restriction in titers, to which it could be propagated, only in certain recipient strains. [TOP OF PAGE]

  21. High-frequency transduction (HFT) of resistance to ceftazidime and other antibiotics by a wild-type Pseudomonas aeruginosa phage. Blahová, J., Králiková, K., Krcméry, V., Sr., Bartoníková, N., Mikovicova, A. (1999). Zentralblatt Fuer Bakteriologie 289:179-183. [TOP OF PAGE]

  22. High-frequency transduction of antibiotic resistance in Pseudomonas aeruginosa by a wild-type bacteriophage with restricted specificity for recipient strains. Blahová, J., Králiková, K., Krcméry, V., Sr., Bartoníková, N. (1999). European Journal of Clinical Microbiology & Infectious Diseases 18:152-154. [TOP OF PAGE]

  23. Epistatic interactions can lower the cost of resistance to multiple consumers. Bohannan, B.J.M., Travisano, M., Lenski, R.E. (1999). Evolution 53:292-295. It is widely assumed that resistance to consumers (e.g., predators or pathogens) comes at a "cost"; i.e., that when the consumer is absent the resident organisms are less fit than their susceptible counterparts. It is unclear what factors determine this cost. We demonstrate that epistasis between genes that confer resistance to two different consumers can alter the cost of resistance. We used as a model system the bacterium Escherichia coli and two different viruses (bacteriophage), T4 and l, that prey upon E. coli. Epistasis tended to reduce the costs of multiple resistance in this system. However, the extent of cost savings and its statistical significance depended on the environment in which fitness was measured, whether the null hypothesis for gene interaction was additive or multiplicative, and subtle differences among mutations that conferred the same resistance phenotype. [TOP OF PAGE]

  24. Effect of prey heterogeneity on the response of a food chain to resource enrichment. Bohannan, B.J.M., Lenski, R.E. (1999). Am. Nat. 153:73-82. We demonstrated that the presence of invulnerable prey can result in a shift in the balance between top-down and bottom-up control of a model food chain. Our model food chain consisted of the bacterium Escherichia coli and the bacteriophage T4 (a virus that feeds on E. coli) in chemostats supplied with different concentrations of glucose. The E. coli population consisted of individuals that were susceptible to predation by T4 ("edible" E. coli) and individuals that were resistant to predation by T4 ("inedible" E. coli). The equilibrium density of a hetergeneous prey population (consisting of edible and inedible E. coli) increased strongly in response to an enrichment of its resources. This response consisted of an increase in the inedible fraction of the prey population but no change in the edible fraction. In contrast, a homogeneous prey population (edible E. coli only) increased only marginally. The equilibrium density of the predator population (bacteriophage T4) did not significantly increase in response to enrichment when its prey were heterogeneous, but it increased when its prey were homogeneous. [TOP OF PAGE]

  25. Effect of resource supply rate on host-pathogen dynamics. Bohannan, B.J.M. (1999). AnonymousProceedings of the 8th International Symposium on Microbial Ecology. The dynamics of model host cell (E. coli) and model pathogen (bacteriophage) populations were studied in chemostats with different resource supply rates. Resource supply rate was manipulated by altering the concentration of the limiting resource (glucose) in the incoming media. Population responses to increased resourse supply rate were influenced by the vulnerability of the host cells to infection. When the host cell population consisted entirely of cells equally vulnerable to infection, both pathogen and host cells responded to increased resource supply rate with an increase in their average densities. In contrast, when the host cell contained some cells that were less vulnerable to infection (i.e., partially phage-resistant E. coli), only the pathogen population responded to increased supply rate with a signficant increase in average density. Furthermore, when the host cell population contained some cells completely invulnerable to infection (i.e., phage-resistant E. coli) only the host cell population responded to increased supply rate with an increase in average density. These responses were in general agreement with the predictions of mechanistic models of resource-consumer interactions. [TOP OF PAGE]

  26. Antimicrobial properties and morphological characteristics of two Photorhabdus luminescens strains. Bondi, M., Messi, P., Sabia, C., Baccarani Contri, M., Manicardi, G. (1999). New Microbiologica 22:117-127. The biological properties of two Photorhabdus luminescens isolates (MU1 and MU2) of environmental source and the activity of antimicrobial agar diffusible agents (AADA) produced by the same are reported. With regard to cultural features, two variant forms for P. luminescens MU1 and three for P. luminescens MU2 (including an intermediate phase I-like form) have been found. These three forms differ in biological and biochemical properties: beta-lactamase, urease, bioluminescence and antimicrobial agar diffusible substance production associated with the phase I form, were less evident in the intermediate phase I-like MU2 and were absent in phase II form. Antimicrobial activity was present in both strains, with the production of a large amount of a diffusible compound with a wide spectrum of action against bacteria of other genera; a reduced activity against correlated species was also observed. Examination by electron microscopy of MU1 and MU2 purified broth cultures revealed the presence of particles belonging to the class of the phage tail-like bacteriocins, described in recent studies as responsible for antibacterial activity against correlated bacteria, a result never confirmed "in vitro". A plasmid of 21 Mdal was observed in all the form variants of P. luminescens MU2, suggesting that plasmids are not involved in the transition from primary to secondary phase; no plasmid was detected in P. luminescens MU1. [TOP OF PAGE]

  27. Alternative mechanism of cholera toxin acquisition by Vibrio cholerae: Generalized transduction of CTXPHI by bacteriophage CP-T1. Boyd, E.F., Waldor, M.K. (1999). Infect. Immun. 67:5898-5905. Horizontal transfer of genes encoding virulence factors has played a central role in the evolution of many pathogenic bacteria. The unexpected discovery that the genes encoding cholera toxin (ctxAB), the main cause of the profuse secretory diarrhea characteristic of cholera, are encoded on a novel filamentous phage named CTXPHI, has resulted in a renewed interest in the potential mechanisms of transfer of virulence genes among Vibrio cholerae. We describe here an alternative mechanism of cholera toxin gene transfer into nontoxigenic V. cholerae isolates, including strains that lack both the CTXPHI receptor, the toxin coregulated pilus (TCP), and attRS, the chromosomal attachment site for CTXPHI integration. A temperature-sensitive mutant of the V. cholerae generalized transducing bacteriophage CP-T1 (CP-T1ts) was used to transfer a genetically marked derivative of the CTX prophage into four nontoxigenic V. cholerae strains, including two V. cholerae vaccine strains. We demonstrate that CTXPHI transduced by CP-T1ts can replicate and integrate into these nontoxigenic V. cholerae strains with high efficiency. In fact, CP-T1ts transduces the CTX prophage preferentially when compared with other chromosomal markers. These results reveal a potential mechanism by which CTXPHI+ V. cholerae strains that lack the TCP receptor may have arisen. Finally, these findings indicate an additional pathway for reversion of live-attenuated V. cholerae vaccine strains. [TOP OF PAGE]

  28. Use of plaque assay to detect enteric viruses in a rural watershed. Brenner, F.J., Brenner, E.K., Schwartz, T.E. (1999). Journal of Environmental Quality 28:845-849. Water samples were collected from four locations within the Munnell Run Watershed in Mercer County, Pennsylvania, and analyzed for fecal coliforms by MPN and enteric phages by plaque assay using Salmonella typhimurium WG 49 and Bacteroides fragilis HSP 40 as hosts. Fecal coliform concentrations and the number of phages varied seasonally (P < 0.001), as well as among the different sampling stations (P < 0.001). At all sampling stations positive for phages, S. typhimurium WG 49 PFUs outnumbered B. fragilis HSP 40 PFUs. Phages also were isolated from a septic discharge pipe and a wetland receiving septic drainage, but not from three avian species, 12 mammalian species, two streams without human wastes or from natural wetland, indicating that these viruses were of human origin. MPNs of fecal coliforms and S. typhimurium and B. fragilis PFUs were correlated with stream temperature (P < 0.001) and rainfall (P < 0.01). Only fecal coliforms and S. typhimurium WG 49 phages were correlated with suspended solids concentrations (P < 0.01). Likewise, there was a significant interaction among these parameters and MPNs of fecal coliform and HSP 40 and WG 49 PFUs (P < 0.001). The presence of host specific phages indicate the existence of septic discharges in the watershed, but both fecal coliforms and enteric viruses persist in stream systems, especially during the summer months. [TOP OF PAGE]

  29. Iodine disinfection of a model bacteriophage, MS2, demonstrating apparent rebound. Brion, G.M., Silverstein, J. (1999). Water Res. 33:169-179. MS2 coliphage viruses suspended in buffered distilled water were rapidly inactivated by < 5 mg/L iodine doses, losing 6 logs (99.9999%) of infectivity within less than 3 min contact time. The effect of pH on MS2 inactivation within the range of 6 to 8 was not statistically significant. However, in the presence of dissolved organic substances, such as detergents and proteins, the inactivation of MS2 viruses decreased significantly to less than 4 logs (99.99%). Of special interest was that in the presence of beef extract proteins, an apparent reversal of MS2 inactivation, dubbed rebound, was observed. It was observed that after an initial 5 to 6 log reduction in infectivity, a consistent and statistically significant increase in the number of plaque forming units (PFU), as much as 2 logs, was measured. MS2 rebound occurred only when the oxidized iodine residual had been quickly consumed by beef extract proteins in solution. Neither virus particle aggregation nor water salinity were found to account for the increase in PFU values. Based on other investigators' suggestions that iodine disinfection caused changes to viral protein coats, it was hypothesized that conformational changes in MS2's protein coat caused by iodine would result in a change in the isoelectric focusing point of whole MS2 virions. A shift in isoelectric focusing point from an acidic pH value of 3.9 to more basic values, and a dispersion of the virus band after exposure to high levels of iodine was observed, supporting the hypothesis that iodine caused changes in the charge distribution characteristics of the protein coat. [TOP OF PAGE]

  30. Unexplored reservoirs of pathogenic bacteria: protozoa and biofilms. Brown, M., Barker, J. (1999). Trends in Microbiology 7:???-??? [TOP OF PAGE]

  31. Flow cytometric analyses of virus infection in two marine phytoplankton species, Micromonas pusilla (Prasinophyceae) and Phaeocystis pouchetii (Prymnesiophyceae). Brussaard, C.P.D., Thyrhaug, R., Marie, D., Bratbak, G. (1999). Journal of Phycology 35:941-948. Cell characteristics of two axenic marine phytoplankton species, Micromonas pusilla (Butscher) Manton et Parke and Phaeocystis pouchetii (Hariot) Lagerheim, were followed during viral infection using flow cytometry, Distinct differences between noninfected and infected cultures were detected in the forward scatter intensities for both algal species. Changes in side scatter signals on viral infection were found only for P, pouchetii. Chlorophyll red fluorescence intensity per cell decreased gradually over time in the infected cultures, DNA analyses were performed using the nucleic acid-specific fluorescent dye SYBR Green I. Shortly after infection the fraction of algal cells with more than one genome equivalent increased for both species because of the replication of viral DNA in the infected cells. Over time, a population of algal cells with low red autofluorescence and low DNA fluorescence developed, likely representing algal cells just prior to viral lysis, The present study provides insight into basic virus-algal host cell interactions. It shows that flow cytometry can be a useful tool to discriminate between virus infected and noninfected phytoplankton cells. [TOP OF PAGE]

  32. Evolution by small steps and rugged landscapes in the RNA virus phi6. Burch, C.L., Chao, L. (1999). Genetics 151:921-927. Fisher's geometric model of adaptive evolution argues that adaptive evolution should generally result from the substitution of many mutations of small effect because advantageous mutations of small effect should be more common than those of large effect. However, evidence for both evolution by small steps and for Fisher's model has been mixed. Here we report supporting results from a new experimental test of the model. We subjected the bacteriophage phi6 to intensified genetic drift in small populations and caused viral fitness to decline through the accumulation of a deleterious mutation. We then propagated the mutated virus at a range of larger population sizes and allowed fitness to recover by natural selection. Although fitness declined in one large step, it was usually recovered in smaller steps. More importantly, step size during recovery was smaller with decreasing size of the recovery population. These results confirm Fisher's main prediction that advantageous mutations of small effect should be more common. We also show that the advantageous mutations of small effect are compensatory mutations whose advantage is conditional (epistatic) on the presence of the deleterious mutation, in which case the adaptive landscape of phi6 is likely to be very rugged. [TOP OF PAGE]

  33. Induction and characterization of Pediococcus acidilactici temperate bacteriophage. Caldwell, S.L., McMahon, D.J., Oberg, C.J., Broadbent, J.R. (1999). Systematic and Applied Microbiology. 22:514-519. Mitomycin C was used to induce temperate bacteriophage from three strains of Pediococcus acidilactici. The new bacteriophage, designated pa97, pa40, and pa42, were characterized based on morphology, DNA homology, and major protein profiles. Morphological attributes (small isometric heads with non-contractile tails) place these bacteriophages within the B1 group of the family Siphovirdae. Restriction endonuclease digests suggested that the bacteriophage genomes were linear molecules without cohesive ends, and between 33 and 37 kilobases in length. All three bacteriophages possessed one major protein with an estimated mass of 30 to 35 kilodaltons. Bacteriophage pa42 also contained a second major protein of approximately 47 kilodaltons. DNA-DNA hybridization showed bacteriophages pa40 and pa42 were homologous to each other, but not to pa97, suggesting that Pediococcus acidilactici bacteriophage fall into at least two different species. [TOP OF PAGE]

  34. Phage therapy: past history and future prospects. Carlton, R.M. (1999). Archivum Immunologii et Therapiae Experimentalis 47:267-274. Bacterial viruses (bacteriophages, also called "phages") can be robust antibacterial agents in vitro. However, their use as therapeutic agents, during a number of trials from the 1920s to the 1950s, was greatly handicapped by a number of factors. In part, there were certain limitations inherent in phage physiology (e. g. narrow host range, and rapid clearance from the body); in part there were technological limitations in the era (e.g. lysogeny not yet discovered); but the greatest limitation was the highly inadequate scientific methodologies used by practitioners at the time (e.g., their failure to conduct placebo-controlled studies, to remove endotoxins from the preparations, and to re-confirm phage viability after adding sterilizing agents to the preparations). In recent years, well-controlled animal models have demonstrated that phages can rescue animals from a variety of fatal infections, while non-controlled clinical reports published in Eastern Europe have shown that phages can be effective in treating drug-resistant infections in humans. This encouraging data, combined with the fact that drug-resistant bacteria have become a global crisis, have created a window of opportunity for phage therapy to be tested anew, this time using modem technologies and placebo-controlled designs. If successful, it can be used as a stand-alone therapy when bacteria are fully resistant to antibiotics, and as a valuable adjunct to antibiotics when the bacteria are still susceptible. [TOP OF PAGE]

  35. Adsorption of bacteriophages on clay minerals. Chattopadhyay, S., Puls, R.W. (1999). Environmental Science & Technology 33:3609-3614. The ability to predict the fate of microorganisms in soil is dependent on an understanding of the process of their sorption on soil and subsurface materials. Presently, we have focused on studying the thermodynamics of sorption of bacteriophages (T-2, MS-2, and variant phiX-174) on clays (hectorite, saponite, kaolinite, and clay fraction of samples collected from a landfill site). The thermodynamic study not only determines the feasibility of the process but also provides information on the relative magnitudes of the different forces under a particular set of conditions. The total free energy of interaction during sorption of bacteriophages on clays (DELTAG) has been assumed to be the summation of DELTAGH (DELTAG due to hydrophobic interactions) and DELTAGEL (DELTAG due to electrostatic interactions). The magnitude of DELTAGH was determined from the different interfacial tensions (gamma) present in the system, while DELTAGEL was calculated from zeta-potentials of the colloidal particles. Calculated results show that surface hydrophobicities of the selected sorbents and sorbates dictate sorption. Among the selected bacteriophages, maximum sorption was observed with T-2, while hectorite has the maximum sorption capacity. Experimental results obtained from the batch adsorption studies also corroborated those obtained from the theoretical study. [TOP OF PAGE]

  36. The primary immunity determinant in modulating the lysogenic immunity of the filamentous bacteriophage cf [published erratum appears in J Mol Biol 1999 Nov 5;293(4):987]. Cheng, C.M., Wang, H.J., Bau, H.J., Kuo, T.T. (1999). J. Mol. Biol. 287:867-876. Bacteriophage cf is the first single-stranded DNA phage that has been shown to set up a stable lysogenic state with its genome integrated into the host chromosome. From the isolation and characterization of a virulent mutant, cf-tv2, we report the first investigation into the mechanisms of the immunity established by the filamentous bacteriophage. The mutation in cf-tv2 enables the phage to produce plaques on lawns of cf lysogenic cells. The mutation was defined as a 49-nucleotide deletion located in a 0.59 kb NcoI/KpnI fragment of cf replicative form DNA. Two messages, cM1 and cM2, transcribed from the immunity region of wild-type cf but in opposite directions, were detected. In cf-tv2, the 49-nucleotide deletion abolishes cM2 transcription. The primer extension assay suggests a possible RNA-RNA interaction directed by base-pairing of the cM1 and cM2 RNAs. A frameshift mutation of the open reading frame ORF 165, encoded by cM2, resulted in a 10(5) plating efficiency on the cf lysogen. These observations suggest that both RNA-RNA interaction and repressor protein inhibition are involved in the mechanism of cf immunity. A model is proposed for the regulation of cf immunity. [TOP OF PAGE]

  37. Procaryotic infections in the mussel Mytilus galloprovinciallis and in its parasite the turbellarian Urastoma cyprinae. Comps, M., Tige, G. (1999). Diseases of Aquatic Organisms 38:211-217. Mussels Mytilus galloprovincialis from the Thau lagoon (Mediterranean coast of France) were regularly sampled to determine the prevalence and intensity of parasitic infections. Microscopically, hepatopancreatic tubules of the mussel appeared infected by a rickettsia-like organism (RLO). Each RLO were surrounded by 2 unit membranes, and colonies composed of several bacteria were enclosed within a vacuolar membrane of the host cell. In addition, examination by transmission electron microscopy revealed that the RLO was infected by phage particles. Histological investigations of the turbellarian Urastoma cyprinae parasitizing the mussels have shown that this ectoparasite was also infected by 2 types of procaryotes, a chlamydia-like organism (CLO) and a mollicute-like organism (MLO). The CLO displayed characteristic developmental stages of the Chlamydiales and was secondarily infected by electron-dense particles presumed to be phage particles. The MLO exhibited some morphological characteristics of the mollicutes, in that the microorganisms were bounded by a single membrane sharing a trilaminar structure. Neither of these microorganisms have previously been reported in Platyhelminthes. [TOP OF PAGE]

  38. Inactivation of faecal indicator microorganisms in waste stabilisation ponds: Interactions of environmental factors with sunlight. Davies-Colley, R.J., Donnison, A.M., Speed, D.J., Ross, C.M., Nagels, J.W. (1999). Water Res. 33:1220-1230. Sunlight exposure is considered to be the most important cause of "natural" disinfection in waste stabilisation ponds (WSPs). We examined the influence of dissolved oxygen (DO), pH, and particulate and dissolved constituents in WSP effluent, on sunlight inactivation of faecal micro-organisms, using small reactors operated under controlled physico-chemical conditions. Inactivation of both enterococci and F-RNA phages increased strongly as DO was increased, and also depended on light-absorbing pondwater constituents, but pH was not influential over the range investigated (7.5 to 10). Inactivation of E. coli increased strongly when pH increased above 8.5, as well as being strongly dependent on DO. Inactivation of F-DNA phage was independent of the factors investigated. These results are consistent with the F-DNA phages being inactivated as a result of direct DNA damage by UVB in sunlight, whereas the other three microbiological indicators are inactivated as a result of photo-oxidative damage, although the target of damage is apparently different. Our findings of diverse influences of physico-chemical conditions suggest difficulties in interpreting data for a single micro-organism to indicate WSP effluent quality. However, sunlight remains the factor of over-riding importance, and disinfection in WSPs may be enhanced by increasing sunlight exposure. [TOP OF PAGE]

  39. The Vibrio cholerae O139 Calcutta bacteriophage CTXphi is infectious and encodes a novel repressor. Davis, B.M., Kimsey, H.H., Chang, W., Waldor, M.K. (1999). J. Bacteriol. 181:6779-6787. CTXphi is a lysogenic, filamentous bacteriophage. Its genome includes the genes encoding cholera toxin (ctxAB), one of the principal virulence factors of Vibrio cholerae; consequently, nonpathogenic strains of V. cholerae can be converted into toxigenic strains by CTXphi infection. O139 Calcutta strains of V. cholerae, which were linked to cholera outbreaks in Calcutta, India, in 1996, are novel pathogenic strains that carry two distinct CTX prophages integrated in tandem: CTX(ET), the prophage previously characterized within El Tor strains, and a new CTX Calcutta prophage (CTX(calc)). We found that the CTX(calc) prophage gives rise to infectious virions; thus, CTX(ET)phi is no longer the only known vector for transmission of ctxAB. The most functionally significant differences between the nucleotide sequences of CTX(calc)phi and CTX(ET)phi are located within the phages' repressor genes (rstR(calc) and rstR(ET), respectively) and their RstR operators. RstR(calc) is a novel, allele-specific repressor that regulates replication of CTX(calc)phi by inhibiting the activity of the rstA(calc) promoter. RstR(calc) has no inhibitory effect upon the classical and El Tor rstA promoters, which are instead regulated by their cognate RstRs. Consequently, production of RstR(calc) renders a CTX(calc) lysogen immune to superinfection by CTX(calc)phi but susceptible (heteroimmune) to infection by CTX(ET)phi. Analysis of the prophage arrays generated by sequentially integrated CTX phages revealed that pathogenic V. cholerae O139 Calcutta probably arose via infection of an O139 CTX(ET)phi lysogen by CTX(calc)phi. [TOP OF PAGE]

  40. Rapid transport of viruses in a floodplain aquifer. DeBorde, D.C., Woessner, W.W., Kiley, Q.T., Ball, P. (1999). Water Res. 33:2229-2238. An unconfined floodplain aquifer near Missoula, MT, was instrumented with 89 monitoring wells and 20 four-port multilevel samplers. Bromide, bacteriophages MS2, PRD1 and phiX174 and the attenuated enterovirus, polio virus (type-1 CHAT strain), were seeded into the aquifer as slug injections. Bromide transport rates ranged between 22-29 m/d. Input concentrations of the tracers and the placement of monitoring wells limited detection of bromide and polio virus to 19.4 m and the detection of three bacteriophage to 40.5 m downgradient from the injection point. After 7.5 m of transport, the calculated relative attenuations (Harvey R. W and Garabedian S. P. (1991) Env. Sci. Tech. 25, 178-185) for MS2, PRD-1, phiX174 and attenuated polio virus were 49, 71, 65 and 99%, respectively. During the 72-h experiment, die-off was negligible (less than 1%) and attachment of virus to sediment surfaces resulted in the overall differences in bromide and virus behavior. Although relative attenuations at downgradient monitoring wells indicated that the virus tracers were attaching to aquifer material along the flowpath, virus peaks arrived at observation wells at rates similar to the bromide peak. The high collision efficiency of the attenuated polio virus resulted in breakthrough curve truncation. Natural attenuation of slug input virus over a "typical" source-supply set-back distance of 30.5 m would most likely not reduce virus concentrations to proposed acceptable risk levels in this or a similar cold-water high-velocity groundwater system. [TOP OF PAGE]

  41. An estimator of the mutant frequency in assays using transgenic animals. Delongchamp, R.R., Malling, H.V., Chen, J.B., Heflich, R.H. (1999). Mutation Research 440:101-108. The Poisson distribution is a fundamental probability model for count data, and is a natural model for the observed plaque counts in mutation assays using animals with lambda or PhiX174 transgenes. The Poisson likelihood for observed counts is a function of the mutant fraction, and it is straightforward to derive the associated maximum likelihood estimate of the mutant fraction and its variance. The estimate is easy to calculate, and if not the same, very similar to ad hoc estimates in current use. The model indicates the proper way to combine data from a number of plates, possibly prepared with different sample dilutions. The estimator of the mutant fraction is biased as a consequence of dividing by a random variable, the plaque count used to calculate the total recovered plaque-forming units. Fortunately, the bias becomes negligible as this count becomes large. On the other hand, increasing this count can increase the variance by decreasing the amount of sample assayed for mutant phages. Concurrent heed to the bias and the variance provides some guidance as to the optimum allocation of a sample into portions assayed for mutant phages and total recovered phages. The distribution of the estimate of the mutant fraction is related to the binomial distribution. This relationship implies a binomial distribution for the mutant count conditional on an overall count (either the sum of mutant and counted total plaques or the sum of counted mutant and non-mutant plaques). A special but important case occurs when each plate can be evaluated for mutant plaques and non-mutant plaques. Then, the observed proportion of mutants estimates the mutant fraction. More generally, the relationship to a binomial distribution provides a procedure for calculating a confidence interval. [TOP OF PAGE]

  42. Genetic organization and functional analysis of a novel phage abortive infection system, AbiL, from Lactococcus lactis. Deng, Y.M., Liu, C.Q., Dunn, N.W. (1999). Journal of Biotechnology 67:135-149. A plasmid-encoded phage abortive infection mechanism (AbiL) was identified from Lactococcus lactis biovar. diacetylactis LD10-1. AbiL conferred complete resistance to the small isometric-headed phage phi 712 (936 species) and partial resistance to the prolate-headed phage phi c2 (c2 species) when introduced into L. lactis LM0230. However, AbiL was not effective against the small isometric-headed phage ul36 (P335 species). The AbiL determinant was sequenced and it consists of two open reading frames, abiLi and abiLii. Their encoded proteins did not share significant homology with any known proteins in the protein databases. Transcriptional analysis indicated that abiLi and abiLii are organized as a single operon. Deletion within abiLii abolished the phage resistance. The levels of four phi c2-specific transcripts, three within the early transcribed region and one within the late transcribed region, were examined by RT-PCR, no effect of AbiL on synthesis of these transcripts was detected, suggesting that AbiL may act at a point after the transcription of phi c2 in L. lactis. [TOP OF PAGE]

  43. Comparative sequence analysis of the DNA packaging, head, and tail morphogenesis modules in the temperate cos-site Streptococcus thermophilus bacteriophage Sfi21. Desiere, F., Lucchini S, Brussow, H. (1999). Virology 260:244-253. The temperate Streptococcus thermophilus bacteriophage Sfi21 possesses 15-nucleotide-long cohesive ends with a 3' overhang that reconstitutes a cos-site with twofold hyphenated rotational symmetry, Over the DNA packaging, head and tail morphogenesis modules, the Sfi21 sequence predicts a gene map that is strikingly similar to that of lambdoid coliphages in the absence of any sequence similarity. A nearly one to one gene correlation was found with the phage lambda genes Nul to H, except for gene B-to-E complex, where the Sfi21 map resembled that of coliphage HK97. The similarity between Sfi21 and HK97 was striking: both major head proteins showed an N-terminal coiled-coil structure, the mature major head proteins started at amino acid positions 105 and 104, respectively, and both major head genes were preceded by genes encoding a possible protease and portal protein. The purported Sfi21 protease is the first viral member of the CipP protease family The prediction of Sfi21 gene functions by reference to the gene map of intensively investigated coliphages was experimentally confirmed for the major head and tail gene. Phage Sfi21 shows nucleotide sequence similarity with Lactococcus phage BK5-T and a lactococcal prophage and amino acid sequence similarity with the Lactobacillus phage A2 and the Staphylococcus phage PVL. PVL is a missing link that connects the portal proteins from Sfi21 and HK97 with respect to sequence similarity. These observations and database searches, which demonstrate sequence similarity between proteins of phage from gram-positive bacteria, proteobacteria, and Archaea, constrain models of phage evolution. [TOP OF PAGE]

  44. [An accelerated method for detecting coliphages in the drinking water]. Dmitrieva, R.A., Doskina, T.V., Nedachin, A.E., Sidorenko, S.G. (1999). Gigiena i Sanitariia 71-72. [TOP OF PAGE]

  45. Presence of bacteriophages in different stages of wastewater treatment. Donia, D., Divizia, M., Pana, A., Gabrieli, R., Gasbarro, M., Capuani, L., Morelli, A.L. (1999). Igiene Moderna 111:239-251. The presence and correlation among somatic coliphages, F-specific phages and the phage of Bacteroides fragilis in a wastewater treatment plant was evaluated. The efficiency of the treatment for bacteriological parameters was confirmed with a reduction of total coliforms of 99,9%, fecal coliforms of 96,0% and fecal streptococci of 97,9%. A similar abatement was present for F-specific phages and the phage of Bacteroides fragilis (2.0 log) whereas the somatic coliphages appeared stable. Cytopathogenic enteric viruses were also recovered in the different withdrawal points with different efficiency according to the methods used. A positive correlation was found in the inlet samples among coliphages, phage of Bacteroides fragilis and enteric viruses. [TOP OF PAGE]

  46. Horizontal gene transfer among bacteria in terrestrial and aquatic habitats as assessed by microcosm and field studies. Droege, M., Puehler, A., Selbitschka, W. (1999). Biology and Fertility of Soils 29:221-245. Genetic interactions among bacteria are mediated by one of the three distinct gene-exchange mechanisms: conjugation, transformation or transduction. Conjugative gene exchange relies on mobile elements, such as plasmids, which transfer between donor and recipient cells. In natural transformation, competent cells take up DNA and incorporate it into their genome. Gene transfer via transduction is mediated by bacteriophages which accidentally package donor DNA in their phage head and transfer it to recipient cells. Driven mainly by biosafety research and research into the rapid dissemination of antibiotic resistance, the evaluation of gene flux among bacteria in their natural habitats has become a focus of scientific interest in recent years. Accordingly, gene transfer has been assessed in laboratory-based studies employing model ecosystems, as well as in field experiments. Conjugative gene exchange has been shown to occur under a wide range of environmental conditions. Factors identified as conducive for conjugation include the presence of nutrients provided by the rhizosphere of plants. Studies addressing gene transfer via transformation have demonstrated that naturally transformable bacteria develop competence and take up DNA under in situ conditions. Moreover, DNA has been shown to persist to some extent in the environment, and thus be available for uptake by naturally competent cells. Gene exchange via transduction has been demonstrated under conditions of nutrient depletion and low densities of host cells. Whereas gene transfer is readily observed in the laboratory, more importantly, field studies have provided direct evidence that all three gene transfer mechanisms also occur in nature. DNA transfer frequencies observed in the environment in some cases differed considerably from those obtained under laboratory conditions. Transfers of low frequency observed in laboratory-based experiments have been readily detected in the environment in the presence of selective forces. [TOP OF PAGE]

  47. Survival, physiology, and lysis of Lactococcus lactis in the digestive tract. Drouault, S. (1999). Appl. Environ. Microbiol. 65:4881-4886. The survival and the physiology of lactococcal cells in the different compartments of the digestive tracts of rats were studied in order to know better the fate of ingested lactic acid bacteria after oral administration. For this purpose, we used strains marked with reporter genes, the luxA-luxB gene of Vibrio harveyi and the gfp gene of Aequora victoria, that allowed us to differentiate the inoculated bacteria from food and the other intestinal bacteria. Luciferase was chosen to measure the metabolic activity of Lactococcus lactis in the digestive tract because it requires NADH, which is available only in metabolically active cells. The green fluorescent protein was used to assess the bacterial lysis independently of death. We report not only that specific factors affect the cell viability and integrity in some digestive tract compartments but also that the way bacteria are administrated has a dramatic impact. Lactococci which transit with the diet are quite resistant to gastric acidity (90 to 98% survival). In contrast, only 10 to 30% of bacteria survive in the duodenum. Viable cells are metabolically active in each compartment of the digestive tract, whereas most dead cells appear to be subject to rapid lysis. This property suggests that lactococci could be used as a vector to deliver specifically into the duodenum the proteins produced in the cytoplasm. This type of delivery vector would be particularly appropriate for targeting digestive enzymes such as lipase to treat pancreatic deficiencies. [TOP OF PAGE]

  48. Integrity of powder-free examination gloves to bacteriophage penetration. Edlich, R.F. (1999). J. Biomed. Mater. Res. 48:755-758. The purpose of this study was to compare the resistance to viral penetration of powder-free synthetic examination gloves with powder-free latex examination gloves commonly used in hospitals. Because these gloves had no holes, this study examined viral penetration through a membrane. Using a standard bacteriophage penetration model, no bacteriophage penetration was detected through the membrane for any of the gloves tested. The new powder-free nitrile and polyvinyl chloride synthetic examination gloves provided comparable resistance to viral penetration as did the powder-free latex examination gloves. [TOP OF PAGE]

  49. Molecular evidence for a new bacteriophage of Borrelia burgdorferi. Eggers, C.H., Samuels, D.S. (1999). J. Bacteriol. 181:7308-7313. We have recovered a DNase-protected, chloroform-resistant molecule of DNA from the cell-free supernatant of a Borrelia burgdorferi culture. The DNA is a 32-kb double-stranded linear molecule that is derived from the 32-kb circular plasmids (cp32s) of the B. burgdorferi genome. Electron microscopy of samples from which the 32-kb DNA molecule was purified revealed bacteriophage particles. The bacteriophage has a polyhedral head with a diameter of 55 nm and appears to have a simple 100-nm-long tail. The phage is produced constitutively at low levels from growing cultures of some B. burgdorferi strains and is inducible to higher levels with 10 microg of 1-methyl-3-nitroso-nitroguanidine (MNNG) ml(-1). In addition, the prophage can be induced with MNNG from some Borrelia isolates that do not naturally produce phage. We have isolated and partially characterized the phage associated with B. burgdorferi CA-11.2A. To our knowledge, this is the first molecular characterization of a bacteriophage of B. burgdorferi. [TOP OF PAGE]

  50. Bacteriophage-like particles associated with the gene transfer agent of Methanococcus voltae PS. Eiserling, F., Pushkin, A., Gingery, M., Bertani, G. (1999). J. Gen. Virol. 80:3305-3308. The methanogenic archaeobacterium Methanococcus voltae (strain PS) is known to produce a filterable, DNase-resistant agent (called VTA, for voltae transfer agent), which carries very small fragments (4400 bp) of bacterial DNA and is able to transduce bacterial genes between derivatives of the strain. Examination by electron microscopy of two preparations of VTA that were concentrated and partially purified by different methods showed virus-like particles with isometric heads, about 40 nm in diameter, and with 61 nm long tails. These particles co-sedimented with the minute bacteriophage fX174 in a sucrose density gradient. [TOP OF PAGE]

  51. Development of reduced acridines as antiprophage agents. El-Bermawy, M.A., Kadry, A., El-Didamony, G., Amin, M. (1999). Chinese Pharmaceutical Journal (Taipei) 51:191-200. A set of 9,10-diphenyldecahydroacridine-1, 8-diones 3-7 with different electronic characteristics were synthesized and tested as antiprophages, curing and as antimicrobial agents. All compounds except the 4-nitrophenyl 7 showed inhibition of prophage lambda (lambda) induction. These compounds appeared to have different levels of antiprophage activity. The 4-methyl derivative 5 has the highest inhibition of the prophage lambda induction. The prepared acridines were tested against standard strains of microorganisms. The minimum inhibitory concentrations (MICs) were 0.5, 0.6, 3.0, 3.5 and 4.0 mg/mL for compounds 7, 5, 6, 3 and 4 respectively. The mutagenic activity of the prophage inducing agent 7 was also investigated. Compound 7 has a curing activity on the plasmids of clinical isolates of E. coli. [TOP OF PAGE]

  52. Little evidence for synergism among deleterious mutations in a nonsegmented RNA virus. Elena, S.F. (1999). J. Mol. Evol. 49:703-707. Several models have been proposed to account for the segmentation of RNA viruses. One of the best known models suggests that segmentation, and mixing of segments during coinfections, is a way to eliminate deleterious mutations from the genome. However, for validity, this model requires that deleterious mutations interact in a synergistic way. That is, two mutations together should have a more deleterious effect than the result of adding their individual effects. Here I present evidence that deleterious mutations in foot-and-mouth disease virus produce a decline in fitness but that the relationship between the number of mutations fixed and the magnitude of fitness decline is compatible mainly with a nonsynergistic model. However, the statistical uncertainties associated with the data still give some room for the existence of very weak synergistic epistasis. [TOP OF PAGE]

  53. Rate of deleterious mutation and the distribution of its effects on fitness in vesicular stomatitis virus. Elena, S.F., Moya, A. (1999). Journal of Evolutionary Biology 12:1078-1088. Despite their importance, the parameters describing the spontaneous deleterious mutation process have not been well described in many organisms. If mutations are important for the evolution of every living organism, their importance becomes critical in the case of RNA-based viruses, in which the frequency of mutation is orders of magnitude larger than in DNA-based organisms. The present work reports minimum estimates of the deleterious mutation rate, as well as the characterization of the distribution of deleterious mutational effects on the total fitness of the vesicular stomatitis virus (VSV). The estimates are based on mutation-accumulation experiments in which selection against deleterious mutations was minimized by recurrently imposing genetic bottlenecks of size one. The estimated deleterious mutation rate was 1.2 mutations per genome and generation, with a mean fitness effect of -0.39% per generation. At the end of the mutation-accumulation experiment, the average reduction in fitness was 38% and the distribution of accumulated deleterious effects was, on average, left-skewed. The magnitude of the skewness depends on the initial fitness of the clone analysed. The implications of our findings for the evolutionary biology of RNA viruses are discussed. [TOP OF PAGE]

  54. Evaluation of a bacteriophage-based assay (Phage amplified biologically assay) as a rapid screen for resistance to isoniazid, ethambutol, streptomycin, pyrazinamide, and ciprofloxacin among clinical isolates of Mycobacterium tuberculosis. Eltringham, I.J. (1999). J. Clin. Microbiol. 37:3528-3532. Rapid molecular assays for the detection of mutations associated with rifampin resistance in Mycobacterium tuberculosis are commercially available. However, they are complex and expensive and have predictive values of 90 to 95%. Molecular assays for other drugs are less predictive of resistance. Ideally, assays based on phenotypic markers should be used for susceptibility testing, but these can take weeks to complete. We previously described a rapid phenotypic assay, the phage amplified biologically (PhaB) assay, for the rapid determination of rifampin and isoniazid susceptibility in clinical isolates of M. tuberculosis. In this study, we extended the assay to the study of ethambutol, pyrazinamide, streptomycin, and ciprofloxacin. After the optimization of antibiotic concentrations and incubation conditions, the assay was applied to each drug for a total of 157 isolates. The correlations between the results of the PhaB assay and the resistance ratio method were 94% for isoniazid, 96% for streptomycin, 100% for ciprofloxacin, 88% for ethambutol, and 87% for pyrazinamide. For ciprofloxacin, ethambutol, and pyrazinamide, significantly better correlations were found when a 90% reduction in plaque count was used as the cutoff. Turnaround times for the PhaB assay were 2 to 3 days, compared with 10 days for the resistance ratio method. We believe that this low-cost assay may have widespread applicability for the rapid screening of drug resistance in M. tuberculosis isolates, especially in developing countries. [TOP OF PAGE]

  55. Evaluation of reverse transcription-PCR and a bacteriophage-based assay for rapid phenotypic detection of rifampin resistance in clinical isolates of Mycobacterium tuberculosis. Eltringham, I.J. (1999). J. Clin. Microbiol. 37:3524-3527. New rapid phenotypic assays for the detection of rifampin resistance in Mycobacterium tuberculosis have recently been described, but most of these require liquid cultures, which reduces the utility of many tests in terms of turnaround times. In the United Kingdom, over 90% of rifampin-resistant isolates are also resistant to isoniazid, so rifampin resistance can be used as a sensitive marker for multidrug-resistant tuberculosis. In this study, two new rapid phenotypic assays were compared to the standard resistance ratio method on 91 clinical isolates of M. tuberculosis. One, the phage amplified biologically (PhaB) assay, has been described previously and is based on the inability of susceptible isolates of M. tuberculosis to support the replication of bacteriophage D29 in the presence of inhibitory doses of rifampin. The other employed reverse transcription (RT)-PCR to demonstrate a reduction in inducible dnaK mRNA levels in susceptible isolates treated with rifampin. After incubation for 18 h with 4 g of rifampin per ml, the PhaB assay showed concordance with the resistance ratio method for 46 of 46 (100%) susceptible and 31 of 31 (100%) resistant isolates, while RT-PCR showed concordance for 46 of 48 (96%) susceptible and 35 of 36 (97%) resistant isolates. We believe these assays provide a reliable rapid means of susceptibility testing with a total turnaround time of only 48 h, although the PhaB assay is better in terms of its lower technical demand and cost and its applicability to tuberculosis susceptibility testing in developing countries. [TOP OF PAGE]

  56. Lysogenic Conversion of Environmental Vibrio mimicus Strains by CTXF. Faruque, S.M., Rahman, M.M., Asadulghani, K.M., Islam, N., Mekalanos, J.J. (1999). Infect. Immun. 67:5723-5729. The filamentous bacteriophage CTXF, which encodes cholera toxin (CT) in toxigenic Vibrio cholerae, is known to propagate by infecting susceptible strains of V. cholerae by using the toxin coregulated pilus (TCP) as its receptor and thereby causing the origination of new strains of toxigenic V. cholerae from nontoxigenic progenitors. Besides V. cholerae, Vibrio mimicus strains which are normally TCP negative have also been shown to occasionally produce CT and cause diarrhea in humans. We analyzed nontoxigenic V. mimicus strains isolated from surface waters in Bangladesh for susceptibility and lysogenic conversion by CTXF and studied the expression of CT in the lysogens by using genetically marked derivatives of the phage. Of 27 V. mimicus strains analyzed, which were all negative for genes encoding TCP but positive for the regulatory gene toxR, 2 strains (7.4%) were infected by CTX-KmF, derived from strain SM44(P27459 ctx::km), and the phage genome integrated into the host chromosome, forming stable lysogens. The lysogens spontaneously produced infectious phage particles in the supernatant fluids of the culture, and high titers of the phage could be achieved when the lysogens were induced with mitomycin C. This is the first demonstration of lysogenic conversion of V. mimicus strains by CTXF. When a genetically marked derivative of the replicative form of the CTXF genome carrying a functional ctxAB operon, pMSF9.2, was introduced into nontoxigenic V. mimicus strains, the plasmid integrated into the host genome and the strains produced CT both in vitro and inside the intestines of adult rabbits and caused mild-to-severe diarrhea in rabbits. This suggested that in the natural habitat infection of nontoxigenic V. mimicus strains by wild-type CTXF may lead to the origination of toxigenic V. mimicus strains which are capable of producing biologically active CT. The results of this study also supported the existence of a TCP-independent mechanism for infection by CTXF and showed that at least one species of Vibrio other than V. cholerae may contribute to the propagation of the phage. [TOP OF PAGE]

  57. Inducible prophages contribute to Salmonella virulence in mice. Figueroa-Bossi, N., Bossi, L. (1999). Molecular Microbiology 33:167-176. We show that Salmonella typhimurium harbours two fully functional prophages, Gifsy-1 and Gifsy-2, that can be induced by standard treatments or, more effectively, by exposing bacteria to hydrogen peroxide. Curing bacteria for the Gifsy-2 prophage significantly reduces Salmonella's ability to establish a systemic infection in mice. Cured strains recover their virulence properties upon relysogenization. Phage Gifsy-2 carries the sodC gene for a periplasmic [Cu,Zn]-superoxide dismutase previously implicated in the bacterial defences against killing by macrophages. The contribution of the Gifsy-1 prophage to virulence - undetectable in the presence of Gifsy-2 as prophage - becomes significant in cells that lack Gifsy-2 but carry the sodC gene integrated in the chromosome. This confirms the involvement of Gifsy-2-encoded SodC protein in Salmonella pathogenicity and suggests that the Gifsy-1 prophage carries one or more additional virulence genes that have a functional equivalent on the Gifsy-2 genome. [TOP OF PAGE]

  58. Identification of four phage resistance plasmids from Lactococcus lactis subsp. cremoris HO2. Forde, A., Daly, C., Fitzgerald, G.F. (1999). Appl. Environ. Microbiol. 65:1540-1547. The bacteriophage-host sensitivity patterns of 16 strains of Lactococcus lactis originally isolated from a mixed strain Cheddar cheese starter culture were determined. Using phages obtained from cheese factory whey, four of the strains were found to be highly phage resistant. One of these isolates, Lactococcus lactis subsp. cremoris HO2, was studied in detail to determine the mechanisms responsible for the phage insensitivity phenotypes. Conjugal transfer of plasmid DNA from strain HO2 allowed a function to be assigned to four of its six plasmids. A 46-kb molecule, designated pCI646, was found to harbor the lactose utilization genes, while this and plasmids of 58 kb (pCI658), 42 kb (pCI642), and 4.5 kb (pCI605) were shown to be responsible for the phage resistance phenotypes observed against the small isometric-headed phage phi712 (936 phage species) and the prolate-headed phage phic2 (c2 species). pCI658 was found to mediate an adsorption-blocking mechanism and was also responsible for the fluffy pellet phenotype of cells containing the molecule. pCI642 and pCI605 were both shown to be required for the operation of a restriction-modification system. [TOP OF PAGE]

  59. Bacteriophage defence systems in lactic acid bacteria. Forde, A. (1999). Antonie van Leeuwenhoek 76:89-113. The study of the interactions between lactic acid bacteria and their bacteriophages has been a vibrant and rewarding research activity for a considerable number of years. In the more recent past, the application of molecular genetics for the analysis of phage-host relationships has contributed enormously to the unravelling of specific events which dictate insensitivity to bacteriophage infection and has revealed that while they are complex and intricate in nature, they are also extremely effective. In addition, the strategy has laid solid foundations for the construction of phage resistant strains for use in commercial applications and has provided a sound basis for continued investigations into existing, naturally-derived and novel, genetically-engineered defence systems. Of course, it has also become clear that phage particles are highly dynamic in their response to those defence systems which they do encounter and that they can readily adapt to them as a consequence of their genetic flexibility and plasticity. This paper reviews the exciting developments that have been described in the literature regarding the study of phage-host interactions in lactic acid bacteria and the innovative approaches that can be taken to exploit this basic information for curtailing phage infection. [TOP OF PAGE]

  60. Phage typing of Campylobacter jejuni and Campylobacter coli and its use as an adjunct to serotyping. Frost, J.A., Kramer, J.M., Gillanders, S.A. (1999). Epidemiology and Infection 123:47-55. Campylobacter is the most commonly reported cause of gastro-intestinal infection in England and Wales, with over 50000 reported cases in 1997. The majority of human campylobacter isolates in England and Wales are C. jejuni (c. 90%) with most of the remainder being C. coli. We describe the use of phage typing as an extension to serotyping for more detailed characterization within these two species. The scheme was piloted during a study of 2407 C. jejuni and 182 C. coli strains isolated in Wales between April 1996 and March 1997. Fifty-seven C. jejuni phage types were identified, with the ten most prevalent phage types accounting for 60% of isolates tested; 16% of isolates were untypable. The most common phage type was PT 1 which represented c. 20% of isolates. A further 7% of isolates reacted with the phages but did not conform to a designated type (RDNC). Only 12 phage types were identified among C. coli, with the two most common types, PT 2 and PT 7 accounting for 75.2% ofisolates. When used in conjunction with serotyping, the ability of phage typing to identify between 6 and 29 subtypes within each of the predominant HS types has enabled a further level of discrimination to be achieved that enhances the epidemiological typing of C. jejuni and C. coli. [TOP OF PAGE]

  61. Causative agents of bacterial mortality and the consequences to marine food webs. Fuhrman, J.A., Noble, R.T. (1999). p. ???-??? In Bell, C.R., Brylinsky, M., and Johnson-Green, P. (eds.), Microbial Biosystems: New Frontiers. Proc 8th Int Symp Microb. Ecol. Atlantic Canada Society for Microbial Ecology, Halifax, Canada. [TOP OF PAGE]

  62. Marine viruses and their biogeochemical and ecological effects. Fuhrman, J.A. (1999). Nature 399:541-548. Viruses are the most common biological agents in the sea, typically numbering ten billion per litre. They probably infect all organisms, can undergo rapid decay and replenishment, and influence many biogeochemical and ecological processes, including nutrient cycling, system respiration, particle size-distributions and sinking rates, bacterial and algal biodiversity and species distributions, algal bloom control, dimethyl sulphide formation and genetic transfer. Newly developed fluorescence and molecular techniques leave the field poised to make significant advances towards evaluating and quantifying such efforts. [TOP OF PAGE]

  63. Impact of viruses on planktonic bacteria. Fuhrman, J.A. (1999). p. ???-??? In Kirchman, D.L. (ed.), Microbial Ecology of the Oceans. Wiley & Sons, ??? [TOP OF PAGE]

  64. The microbial quality of a Wetland Reclamation Facility used to produce an effluent for unrestricted non-potable reuse. Fujioka, R.S., Bonilla, A.J., Rijal, G.K. (1999). Water Science and Technology 40:369-374. An auxiliary Wetland Reclamation Facility (WRF) was constructed to receive stabilization pond treated sewage and further treat it with water hyacinth ponds, chemical flocculation, filtration and ultraviolet light disinfection. This was the first facility in Hawaii which was approved to produce the highest quality reclaimed water using alternative treatment schemes. We assessed the effectiveness of the WRF by monitoring water samples after each of the WRF treatment schemes for five genetically different groups of sewage borne microorganisms (fecal coliform, enterococci, C. perfringens, FRNA phage, total heterotrophic bacteria). The concentrations of all fecal indicator microorganisms, especially FRNA phage were low in the influent water to the WRF indicating that extended pond treatment may be especially effective in removing human viruses from sewage. The WRF treatment scheme was calculated to be able to reduce >99.99% of fecal coliform and therefore was able to produce an effluent meeting the non-potable, unrestricted reuse standard of a geometric mean of <1 fecal coliform/100 ml. [TOP OF PAGE]

  65. Horizontal gene transfer in glycosyl hydrolases inferred from codon usage in Escherichia coli and Bacillus subtilis. Garcia-Vallve, S., Palau, J., Romeu, A. (1999). Molecular Biology and Evolution 16:1125-1134. Glycosyl hydrolase (GH) genes from Escherichia coli and Bacillus subtilis were used to search for cases of horizontal gene transfer. Such an event was inferred by G + C content, codon usage analysis, and a phylogenetic congruency test. The codon usage analysis used is a procedure based on a distance derived from a Pearson linear correlation coefficient determined from a pairwise codon usage comparison. The distances are then used to generate a distance-based tree with which we can define clusters and rapidly compare codon usage. Three genes (yagH from E. coli and xynA and xynB from B. subtilis) were determined to have arrived by horizontal gene transfer and were located in E. coli CP4-6 prophage, and B. subtilis prophages 6 and 5, respectively. In this study, we demonstrate that with codon usage analysis, the proposed horizontally transferred genes can be distinguished from highly expressed genes. [TOP OF PAGE]

  66. Bacteriophages of dairy propionibacteria. Gautier, M., Rouault, A., Herve, C., Sommer, P., eret, V., Jan, G., Fraslin, J.M., Prevot, F., Coste, A. (1999). Lait- 79:93-104. Characteristics of 2 types of phages infecting propionic acid bacteria that were isolated from Swiss-type cheeses were examined. One type belonged to group B1 of Bradley's classification and the other was a filamentous phage, the 1st of its type identified in Gram positive bacteria. Diversity, host spectrum and origin of the phages were studied. The phages were detected in raw milk and cheeses, but not in curd or cooked curd. Group 1 phages showed high homology and were probably derived from a common ancestor. All phages showed a very narrow host spectrum. Information on phages infecting Propionibacterium freudenreichii was used in the development of a cloning system for this bacterium. [This paper was presented at the 2nd Symposium on Propionibacteria, which was held in Cork, Republic of Ireland on 25-27 June 1998.]. [TOP OF PAGE]

  67. Optimization of artificial wetland design for removal of indicator microorganisms and pathogenic protozoa. Gerba, C.P., Thurston, J.A., Falabi, J.A., Watt, P.M., Karpiscak, M.M. (1999). Water Science and Technology 40:363-368. The enhancement of water quality by artificial wetland systems is increasingly being employed throughout the world. Three wetlands were studied in Tucson, AZ to evaluate their individual performance in the removal of indicator bacteria (coliforms), coliphage, and enteric pathogens (Giardia and Cryptosporidium). A duckweed-covered pond, a multi-species subsurface flow (SSF) and a multi-species surface flow (SF) wetland were studied. Removal of the larger microorganisms, Giardia and Cryptosporidium, was the greatest in the duckweed pond at 98 and 89 percent, respectively. The lowest removal occurred in the SF wetland, 73 percent for Giardia and 58 percent removal for Cryptosporidium. In contrast, the greatest removal of coliphage, total and fecal coliforms occurred in the SSF wetland, 95, 99, and 98 percent respectively, whereas the pond had the lowest removals (40, 62, and 61 percent, respectively). Sedimentation may be the primary removal mechanism within the duckweed pond since the removal was related to size, removal of the largest organisms being the greatest. However, the smaller microorganisms were removed more efficiently in the SSF wetland, which may be related to the large surface area available for adsorption and filtration. This study suggests that in order to achieve the highest treatment level of secondary unchlorinated wastewater, a combination of aquatic ponds and subsurface flow wetlands may be necessary. [TOP OF PAGE]

  68. Biocontrol of Erwinia amylovora using bacteriophage. Gill, J.J., Svircev, A.M., Myers, A.L., Castle, A.J. (1999). Phytopathology 89:S27 [TOP OF PAGE]

  69. Roles of Pseudomonas aeruginosa las and rhl quorum-sensing systems in control of twitching motility. Glessner, A., Smith, R.S., Iglewski, B.H., Robinson, J.B. (1999). J. Bacteriol. 181:1623-1629. Pseudomonas aeruginosa is a ubiquitous environmental bacterium and an important human pathogen. The production of several virulence factors by P. aeruginosa is controlled through two quorum-sensing systems, las and rhl. We have obtained evidence that both the las and rhl quorum-sensing systems are also required for type 4 pilus-dependent twitching motility and infection by the pilus-specific phage D3112cts. Mutants which lack the ability to synthesize PAI-1, PAI-2, or both autoinducers were significantly or greatly impaired in twitching motility and in susceptibility to D3112cts. Twitching motility and phage susceptibility in the autoinducer-deficient mutants were partially restored by exposure to exogenous PAI-1 and PAI-2. Both twitching motility and infection by pilus-specific phage are believed to be dependent on the extension and retraction of polar type 4 pili. Western blot analysis of whole-cell lysates and enzyme-linked immunosorbent assays of intact cells were used to measure the amounts of pilin on the cell surfaces of las and rhl mutants relative to that of the wild type. It appears that PAI-2 plays a crucial role in twitching motility and phage infection by affecting the export and assembly of surface type 4 pili. The ability of P. aeruginosa cells to adhere to human bronchial epithelial cells was also found to be dependent on the rhl quorum-sensing system. Microscopic analysis of twitching motility indicated that mutants which were unable to synthesize PAI-1 were defective in the maintenance of cellular monolayers and migrating packs of cells. Thus, PAI-1 appears to have an essential role in maintaining cell-cell spacing and associations required for effective twitching motility. [TOP OF PAGE]

  70. Development and characterization of a fluorescent-bacteriophage assay for detection of Escherichia coli O157:H7. Goodridge, L., Chen, J., Griffiths, M. (1999). Appl. Environ. Microbiol. 65:1397-1404. In this paper we describe evaluation and characterization of a novel assay that combines immunomagnetic separation and a fluorescently stained bacteriophage for detection of Escherichia coli O157:H7 in broth. When it was combined with flow cytometry, the fluorescent-bacteriophage assay (FBA) was capable of detecting 10(4) cells/ml. A modified direct epifluorescent-filter technique (DEFT) was employed in an attempt to estimate bacterial concentrations. Using regression analysis, we calculated that the lower detection limit was between 10(2) and 10(3) cells/ml; however, the modified DEFT was found to be an unreliable method for determining bacterial concentrations. The results of this study show that the FBA, when combined with flow cytometry, is a sensitive technique for presumptive detection of E. coli O157:H7 in broth cultures. [TOP OF PAGE]

  71. The use of a fluorescent bacteriophage assay for detection of Escherichia coli O157:H7 in inoculated ground beef and raw milk. Goodridge, L., Chen, J., Griffiths, M. (1999). Int. J. Food Microbiol. 47:43-50. The objective of this study was to develop a fluorescent bacteriophage assay (FBA) for the detection of E. coli O157:H7 in ground beef and raw milk. The FBA is a two step assay that combines immunomagnetic separation, to separate the target organism from mixed culture, with a highly specific fluorescently stained bacteriophage to label the E. coli O157:H7 cells. When used in conjunction with flow cytometry, the FBA was able to detect 2.2 CFU/g of artificially contaminated ground beef following a 6 h enrichment. Between 10(1) and 10(2) CFU/ml of artificially contaminated raw milk were detectable after a 10 h enrichment step. The results show that the FBA is potentially useful as a rapid technique for the preliminary detection of E. coli O157:H7 in food. [TOP OF PAGE]

  72. Bacteriophage lambda: The untold story. Gottesman, M. (1999). J. Mol. Biol. 293:177-180. The study of bacteriophage lambda has provided key insights into fundamental biological processes. This review recalls some highlights in the history of lambda research, and relates how simple (but elegant) experiments yielded major scientific breakthroughs. What we know about recombination, gene regulation, and protein folding, for example, derives in large part from bacteriophage lambda genetics. Lambda not only represents a model system of scientific logic in a technology-driven age, but continues to reveal new principles of molecular biology. [TOP OF PAGE]

  73. Removal of MS-2 and PRD-1 bacteriophages from an ultrapure water system. Governal, R.A., Gerba, C.P. (1999). Journal of Industrial Microbiology & Biotechnology 23:166-172. Viruses must be removed from the ultrapure water environment, as they have the potential to deposit on microelectronic devices and generate killer defects. Controlled and well-defined challenges by MS-2 and PRD-1 bacteriophages were treated in a pilot-scale ultrapure water system using ultraviolet radiation (UV), ozone, mixed bed ion exchange adsorption, and reverse osmosis filtration technologies typical of those used in industrial systems. Applying a first order kinetic model to the data generated rate constants for MS-2 removal by UV-185, 50 mg L-1 ozone, mixed bed ion exchange or reverse osmosis filtration of 15.5, 12.9, 3.9, and 10.4 min-1, respectively, and PRD-1 removal of 13.8, 15.5, 8.2, and 11.9 min-1, respectively. In all cases, removal of viruses by oxidative mechanisms such as ozone and UV were far superior to adsorption and filtration mechanisms. A theoretical viral population balance was generated to model the removal of the bacteriophages by these unit operations.This model relates the inlet time-dependent profile of viruses to the output, destruction, and accumulation profiles; it also relates these profiles to the unit operation's treatment mechanisms including oxidation, adsorption, and filtration. This model is the first step in generating a site-independent theoretical model to project the persistence of viruses in ultrapure water systems. [TOP OF PAGE]

  74. Elimination of viruses, phages, bacteria and Cryptosporidium by a new generation Aquaguard point-of-use water treatment unit. Grabow, W.O., Clay, C.G., Dhaliwal, W., Vrey, M.A., Muller, E.E. (1999). Zentralblatt fur Hygiene und Unweltmedizin 202:399-410. The elimination of human viruses, phages, bacteria and Cryptosporidium oocysts by a new generation commercial Aquaguard purifier for the domestic treatment of drinking water, has been evaluated. The unit basically consists of a candle prefilter, activated carbon filter and ultraviolet irradiation compartment. Drinking water seeded with selected laboratory test strains of resistant micro-organisms was passed through the unit. Similar tests were carried out with sewage-contaminated river water and secondary treated waste water containing naturally occurring organisms. Test procedures were based on internationally accepted principles for the evaluation of point-of-use water treatment units, including a standard test protocol of the United States Environmental Protection Agency. Reduction in numbers of seeded test organisms at several log levels higher than those expected in water for which the unit is intended, was determined by the cultivation of viable organisms. In the case of seeded viruses and Cryptosporidium parvum oocysts the qualitative absence of nucleic acid was determined by the reverse transcriptase polymerase chain reaction (RT-PCR). At the design flow rate of one litre per minute, numbers of polio, hepatitis A, adeno types 2 and 41, rota SA11, human rota and astro viruses, as well as somatic and MS2 coliphages, and Escherichia coli, Streptococcus faecalis, Clostridium perfringens, total coliform bacteria, enterococci, heterotrophic bacteria and C. parvum oocysts, were reduced by more than 99.999% in all waters tested. This efficiency conforms to specifications for such units. The quality of the treated water was well within microbiological limits of international specifications for drinking water. [TOP OF PAGE]

  75. Virus removal by filtration. Graf, E.G., Jander, E., West, A., Pora, H., Aranha-Creado, H. (1999). Dev. Biol. Standard. 99:89-94. Advances in membrane technology have allowed the expansion of the size-exclusion removal principle to viruses of concern in the processing of pharmaceutical drug products derived from biological fluids and cell-culture techniques. Direct flow- and cross-flow filters are complementary techniques for virus removal and may be used either independently or as an adjunct to other virus clearance methods. Representative virus titre reduction data for microfiltration and ultrafiltration membranes are presented along with a validation model using bacteriophages as challenge viruses. Non-destructive filter integrity tests before and after filtration and a stringent process validation regime are applied to enhance product safety. [TOP OF PAGE]

  76. Viral lysis and bacterivory during a phytoplankton bloom in a coastal water microcosm. Guixa-Boixareu, N., Lysnes, K., Pedros-Alio, C. (1999). Appl. Environ. Microbiol. 65:1949-1958. The relative importance of viral lysis and bacterivory as causes of bacterial mortality were estimated. A laboratory experiment was carried out to check the kind of control that viruses could exert over the bacterial assemblage in a non-steady-state situation. Virus-like particles (VLP) were determined by using three methods of counting (DAPI [4',6-diamidino-2-phenylindole] staining, YOPRO staining, and transmission electron microscopy). Virus counts increased from the beginning until the end of the experiment. However, different methods produced significantly different results. DAPI-stained VLP yielded the lowest numbers, while YOPRO-stained VLP yielded the highest numbers. Bacteria reached the maximal abundance at 122 h (3 x 10 super(7) bacteria ml super(-1)), after the peak of chlorophyll a (80 mu g liter super(-1)). Phototrophic nanoflagellates followed the same pattern as for chlorophyll a. Heterotrophic nanoflagellates showed oscillations in abundance throughout the experiment. The specific bacterial growth rate increased until 168 h (2.6 day super(-1)). The bacterivory rate reached the maximal value at 96 hours (0.9 day super(-1)). Bacterial mortality due to viral infection was measured by using two approaches: measuring the percentage of visibly infected bacteria (%VIB) and measuring the viral decay rates (VDR), which were estimated with cyanide. The %VIB was always lower than 1% during the experiment. VDR were used to estimate viral production. Viral production increased 1 order of magnitude during the experiment (from 10 super(6) to 10 super(7) VLP ml super(-1) h super(-1)). The percentage of heterotrophic bacterial production consumed by bacterivores was higher than 60% during the first 4 days of the experiment; afterwards, this percentage was lower than 10%. The percentage of heterotrophic bacterial production lysed by viruses as assessed by the VDR reached the highest values at the beginning (100%) and at the end (50%) of the experiment. Comparing both sources of mortality at each stage of the bloom, bacterivory was found to be higher than viral lysis at days 2 and 4, and viral lysis was higher than bacterivory at days 7 and 9. A balance between bacterial losses and bacterial production was calculated for each sampling interval. At intervals of 0 to 2 and 2 to 4 days, viral lysis and bacterivory accounted for all the bacterial losses. At intervals of 4 to 7 and 7 to 9 days, bacterial losses were not balanced by the sources of mortality measured. At these time points, bacterial abundance was about 20 times higher than the expected value if viral lysis and bacterivory had been the only factors causing bacterial mortality. In conclusion, mortality caused by viruses can be more important than bacterivory under non-steady-state conditions. [TOP OF PAGE]

  77. Evolutionary relationships among diverse bacteriophages and prophages: All the world's a phage. Hendrix, R.W., Smith, M.C.M., Burns, R.N., Ford, M.E., Hatfull, G.F. (1999). Proc. Natl. Acad. Sci. USA 96:2192-2197. We report DNA and predicted protein sequence similarities, implying homology, among genes of dsDNA bacteriophages and prophages spanning broad phylogenetic range of host bacteria. The sequence matches reported here establish genetic connections not always direct, among the lambdoid phages of Escherichia coli, phage fC31 of Streptomyces, phages of Mycobacterium, a previously unrecognized cryptic prophage, fFlu, in the Haemophilus influenzae genome, and two small prophage-like elements fRv1 and fRV2, in the genome of M. tuberculosis. The results imply that these phage genomes, and very possibly all of the dsDNA tailed phages, share common ancestry. We propose a model for the genetic structure and dynamics of the global phage population in which all dsDNA phage genomes are mosaics with access by horizontal exchange, to a large common genetic pool, but in which access to the gene pool is not uniform for all phage. [TOP OF PAGE]

  78. Evolution: the long evolutionary reach of viruses. Hendrix, R.W. (1999). Current Biology 9:R914-R917 The structure of a phage capsid protein provides good evidence this phage shares ancestry with an animal virus. In this and similar cases, either the viral lineages predate the emergence of the three contemporary domains of life, or viruses have been leaping the phylogenetic chasms that separate the domains. [TOP OF PAGE]

  79. Removal of viruses by microfiltration membranes at different solution environments. Herath, G., Yamamoto, K., Urase, T. (1999). Water Science and Technology 40:331-338. Rejection change by nuclepore microfiltration membranes with solution environment was investigated by using the RNA coliphages Qbeta, MS2, fr and DNA coliphage T4. The obtained rejection results showed a higher rejection at lower pH than at higher pH for all viruses. The highest rejection for all viruses were obtained at pH closer to their isoelectric points and also the rejection variation indicates a similar pattern of behavior. This phenomenon of higher virus rejection at lower pH is explained with a possible viruses aggregation with each other due to their own electrostatic charge and isoelectric points. Also it was observed that the virus rejection was enhanced when they are in mixed environment. Finally the effect of protein was studied where, the virus rejection below pH 5.0 showed protein influence. [TOP OF PAGE]

  80. Preparation of phage-insensitive strains of Tetragenococcus halophila and its application for soy sauce fermentation. Higuchi, T., Uchida, K., Abe, K. (1999). Biosci. Biotech. Biochem. 63:415-417. Production of _D-10 phage-insensitive mutants of Tetragenococcus halophila D-10, used in industrial fermentation of soy sauce, is reported. Phage contact during selection initially resulted in lysogeny. Subsequently, phage-insensitive mutants were screened by replica plating so that mutant cells did not touch the phage during selection. 2 strains were selected from approx. 150 000. They grew normally in soy sauce mash (moromi) in the presence of phage _D-10, although had a similar extent of adsorption of _D-10, as did the parent strain. Industrial application of these strains in moromi fermentation is discussed. [TOP OF PAGE]

  81. The catalytic group-I introns of the psbA gene of Chlamydomonas reinhardtii : core structures, ORFs and evolutionary implications. Holloway, S.P., Deshpande, N.N., Herrin, D.L. (1999). Current Genetics 36:69-78. The sequences and predicted secondary structures of the four catalytic group-I introns in the psbA gene of Chlamydomonas reinhardtii, Cr.psbA-1-Cr.psbA-4, have been determined. Cr.psbA-1 and Cr.psbA-4 are subgroup-IA1 introns and have similar secondary structures, except at the 3 end where Cr.psbA-1 contains a large inverted-repeat domain. Cr.psbA-4 is closely related to intron 1 of the Chlamydomonas moewusii psbA gene, with which it shares the same location, high nucleotide identity in the core, and an identically placed ORF that shows 58% amino-acid identity. Cr.psbA-2 is a subgroup-IA3 intron, and shows similarities to the Chlamydomonas eugametos rRNA intron, Ce.LSU-1. Cr.psbA-3 is a subgroup-IA2 intron, and is remarkably similar to the T4 phage intron, sunY. Interestingly, a degenerate version of Cr.psbA-3 is located in the intergenic region between the chloroplast petA and petD genes. All four introns contain ORFs, which potentially code for basic proteins of 11-38 kDa. The ORFs in introns 2 and 3 contain variants of the GIY-YIG motif; however, the Cr.psbA-2 ORF is free-standing, whereas the Cr.psbA-3 ORF is contiguous and in-frame with the upstream exon. The Cr.psbA-4 ORF contains an H-N-H motif, and possibly a GIY-YIG motif. These data indicate that the C. reinhardtii psbA introns have multiple origins, and illustrate some of the evolutionary DNA dynamics associated with group-I introns in Chlamydomonas. [TOP OF PAGE]

  82. Uptake and intracellular fate of phage display vectors in mammalian cells. Ivanenkov, V.V., Felici, F., Menon, A.G. (1999). Biochim. Biophys. Acta 1448:450-462. Receptor-mediated endocytosis is exploited in experimental systems for selective delivery of genes and drugs into specific cells. To improve targeting efficiency of delivery vectors, we have used phage display technology to isolate novel ligands for endocytosed receptors. We show here that phage vectors internalized by mammalian cells via integrin-mediated endocytosis can be rescued by cell lysis and quantitated by infection of bacteria. Immediately following uptake, phage enter an intracellular compartment where they remain intact, with phage titer unaffected by the addition of chloroquine. Phage are then translocated to a second intracellular compartment in which they are inactivated and their titer affected by chloroquine. Immunofluorescence microscopy showed an association of the second compartment with supranuclear organelles. The ability to recover internalized phage in an infectious form from two distinctive intracellular compartments provides a means to select novel ligands from phage libraries for targeted delivery of macromolecules into mammalian cells. [TOP OF PAGE]

  83. Changing consumer water-use patterns and their effect on microbiological water quality as a result of an engineering intervention. Jagals, P., Bokako, T.C., Grabow, W.O.K. (1999). Water S A (Pretoria) 25:297-326. A previous study done during 1994-1995 in a section of a large, low socio-economic urban development with limited sanitary facilities and drinking-water provision indicated that the community was exposed to water-related health risks when consuming the water supplied. The study indicated that, although the public supplied water was of a good quality, the stored water, once fetched from the standpipes, deteriorated to a quality often not safe for human consumption. Based on the findings of this previous study, the local authority decided to install standpipes for each individual family in the area concerned and these were placed in the house yards. The closer proximity of the standpipes immediately altered the water-fetching and storing patterns of the community. The consequent study, on which this abstract is based, assessed the potential risk of infection posed to health by the altered water-use pattern. Weekly water samples were collected from standpipes outside as well as from containers kept inside houses of selected families. Total coliforms, faecal coliforms, heterotrophic plate counts, Clostridium perfringens and somatic coliphages were used as microbiological indicators. Although the improvement of water accessibility enhanced the microbiological quality of stored water, the results indicated that hygienic quality still deteriorated. This situation indicated that a suitable education and information programme to enhance the quality gains of such engineering interventions should accompany engineering improvement of water accessibility. [TOP OF PAGE]

  84. Development of lytic Lactococcus lactis bacteriophages in a Cheddar cheese plant. Josephsen, J., Petersen, A., Neve, H., Nielsen, E.W. (1999). Int. J. Food Microbiol. 50:163-171. The mixed TK5 starter culture was used in a Danish factory as the only starter for production of Cheddar cheese for more than 11 years before the factory experienced serious bacteriophage attacks with inhibition of the acid production in the curd. The cheese whey contained some phages from the beginning, and gradually new phages appeared able to infect an increasing number of isolates. Three bacteriophages jw30, jw31 and jw32 were isolated from the factory whey collected in 1989-94 and compared with lytic bacteriophages isolated in the period 1982-86 (Josephsen et al., 1994) DNA hybridisation showed that the type phage P008 had high homology to the new phages jw30, jw31 and jw32 as had the phages isolated in 1982-84. The new phages had broader host ranges and higher burst sizes than the previously isolated phages, showing that the lytic phages had become more virulent with time. [TOP OF PAGE]

  85. [Cryostabilization of biological properties of plague phages]. Kadetov, V.V., Kudriakova, T.A., Terent'ev, A.N., Kachkina, G.V., Borodina, T.N., Saiamov, S.R. (1999). Vopr. Virusol. 44:136-139. Conditions of cryostabilization of Yersinia pestis phages preserving their biological properties at very low temperature are studied. [TOP OF PAGE]

  86. Bacteriocin-like inhibitory activities among various species of Listeria. Kalmokoff, M.L., Daley, E., Austin, J.W., Farber, J.M. (1999). Int. J. Food Microbiol. 50:191-201. Three hundred Listeria isolates were examined for inhibitory activities using a deferred antagonism plating assay. Approximately 75% of the surveyed isolates produced inhibitory activity, the majority of which (71%) resulted from the production of bacteriophage or defective bacteriophage particles. Twenty-three isolates (8%) produced inhibitory activities distinct from those resulting from bacteriophage. Four of these isolates (Listeria innocua 743, L. innocua 755, L. innocua 228, L. monocytogenes 538) produced heat-stable, protease sensitive peptides, which demonstrated broad-spectrum inhibitory activities against all L. monocytogenes serotypes tested. [TOP OF PAGE]

  87. Characterization of wild lambdoid bacteriophages: detection of a wide distribution of phage immunity groups and identification of a nus-dependent, nonlambdoid phage group. Kameyama, L., Fernandez, L., Calderon, J., Ortiz-Rojas, A., Patterson, T.A. (1999). Virology 263:100-111. Temperate phages were isolated from fresh human fecal samples. Lambdoid phages were screened for growth on Nus+ but not Nus- bacteria. Approximately 100 independent lysogens of Nus-dependent phages were constructed and tested for immunity to superinfection by the same Nus-dependent phages. This identified 20 different phage immunity groups, 18 of which belonged to the lambdoid phage family. The DNA from the majority of these phages hybridized with a lambda DNA probe, and approximately 50% were recognized by anti-lambda antibodies. Furthermore most were inducible by UV light. Eleven phage recombinants with different immunity were obtained when a phage from each group was coinfected with lambda or its derivative lambdaBLK20. We also identified another immunity group with 48 members. None of these hybridized with either lambda or phi80 DNA probes nor were they recognized by anti-lambda serum. Most were not induced by UV light treatment, and no recombinants were obtained when crossed with either lambda or lambdaBLK20. Consequently, this group of Nus-dependent phages represent a new nonlambdoid phage family. [TOP OF PAGE]

  88. An extrachromosomal prophage naturally associated with Bacillus thuringiensis serovar israelensis. Kanda, K., Ohderaotoshi, T., Shimojyo, A., Kato, F., Murata, A. (1999). Letters in Applied Microbiology 28:305-308. Bacillus thuringiensis serovar israelensis, an entomopathogen for mosquito larvae, was demonstrated to be lysogenized by temperate phage SU-11 whose genome was located extrachromosomally in the cell. The prophage SU-11 was cured at high frequency from the parental strain by continuous sub-culture at high temperature, but the ability to produce delta-endotoxin remained in the prophage cured strain. Moreover, phage induction was found to occur after mating of serovar israelensis with its prophage cured strain, as well as with B. thuringiensis serovar thuringiensis, B. cereus and B. subtilis. [TOP OF PAGE]

  89. A bacteriophage encoding a pathogenicity island, a type-IV pilus and a phage receptor in cholera bacteria [see comments]. Karaolis, D.K., Somara, S., Maneval, D.R.Jr., Johnson, J.A., Kaper, J.B. (1999). Nature 399:375-379. The virulence properties of many pathogenic bacteria are due to proteins encoded by large gene clusters called pathogenicity islands, which are found in a variety of human pathogens including Escherichia coli, Salmonella, Shigella, Yersinia, Helicobacter pylori, Vibrio cholerae, and animal and plant pathogens such as Dichelobacter nodosus and Pseudomonas syringae. Although the presence of pathogenicity islands is a prerequisite for many bacterial diseases, little is known about their origins or mechanism of transfer into the bacterium. The bacterial agent of epidemic cholera, Vibrio cholerae, contains a bacteriophage known as cholera-toxin phage (CTXphi), which encodes the cholera toxin, and a large pathogenicity island called the VPI (for V. cholerae pathogenicity island) which itself encodes a toxin-coregulated pilus that functions as a colonization factor and as a CTXphi receptor. We have now identified the VPI pathogenicity island as the genome of another filamentous bacteriophage, VPIphi. We show that VPIphi is transferred between V. cholerae strains and provide evidence that the TcpA subunit of the toxin-coregulated type IV pilus is in fact a coat protein of VPIphi. Our results are the first description of a phage that encodes a receptor for another phage and of a virus-virus interaction that is necessary for bacterial pathogenicity. [TOP OF PAGE]

  90. Shiga toxins even when different are encoded at identical positions in the genomes of related temperate bacteriophages. Karch, H., Schmidt, H., Janetzki-Mittmann, C., Scheef, J., Kroger, M. (1999). Molecular and General Genetics 262:600-607. The nucleotide sequence of an 11,142-bp region including the stx2 operon in the genome of the temperate bacteriophage 933W in the EDL933 strain of Escherichia coli 0157 was determined and compared to the respective regions derived from other lambdoid bacteriophages. In phage 933W, a region of ORFs interlinked by overlapping start-stop codons (ATGA) was detected preceding the toxin gene. These ORFs show a high degree of sequence identity to genes of the nin region of phage lambda. Immediately downstream of these nin genes we identified an ORF that may code for an anti-terminator similar to the lambda Q protein. It is concluded that toxin expression is directly associated with the initiation of cell lysis. Downstream of the stx2 operon we identified an ORF that is homologous to the holin gene S of bacteriophage PA-2. PCR primers were designed, which, based on a comparison of the phage sequences, appeared to be common to both stx1- and stx2-harbouring phages. However, only seven of the 22 STEC strains investigated from serogroups 0157, 026, 0103 and 0111 yielded the expected PCR amplification product. The data reported here may be useful in developing new strategies for inhibiting the expression of Stx and for developing universal diagnostic primers for use in tracking the origin and evolution of Shiga toxins and the phages that carry them. [TOP OF PAGE]

  91. Genetic selection of phage engineered for receptor-mediated gene transfer to mammalian cells. Kassner, P.D., Burg, M.A., Baird, A., Larocca, D. (1999). Biochemical & Biophysical Research Communications 264:921-928. Although phage display is a powerful way of selecting ligands against purified target proteins, it is less effective for selecting functional ligands for complex targets like living cells. Accordingly, phage display has had limited utility in the development of targeting agents for gene therapy vectors. By adapting a filamentous bacteriophage for gene delivery to mammalian cells, however, we show here that it is possible to screen phage libraries for functional ligands capable of delivering DNA to cells. For example, when targeted with epidermal growth factor (EGF), M13 bacteriophage were capable of delivering a green fluorescent protein (GFP) gene to EGF receptor bearing cells in a ligand-, time-, and phage concentration-dependent manner. The EGF-targeted phage transduced COS-1 cells in a highly specific manner as demonstrated by competition with excess free EGF or alternatively with anti-EGF receptor antibodies. We further demonstrate that EGF-phage can be selected, by their ability to transduce EGF receptor bearing cells from libraries of peptide display phage. When phage were incubated with COS-1 cells, EGF ligand-encoding sequences were recovered by PCR from FACsorted, GFP-positive cells and the EGF-displaying phage were enriched 1 million-fold by four rounds of selection. These data suggest the feasibility of applying molecular evolution to phage gene delivery to select novel cell-specific DNA-targeting ligands. The same approach could be used to select genetically altered phage that are specifically designed and evolved as gene therapy vectors. [TOP OF PAGE]

  92. Anticodon nuclease: a bacterial RNA restriction enzyme. Kaufmann, G. (1999). TIBS ???:???-??? The tRNALys-specific anticodon nuclease exists in an E. coli isolate in latent form, complexed with a DNA restriction enzyme. A phage T4-encoded alleviator of DNA restriction disrupts this masking interaction but other phage proteins repair the damaged tRNALys. Detection of a homologous system in Neisseria and a different anticodon nuclease in Colicin E5 suggest widespread occurrence of versatile tRNA restriction endonucleases. Studying them may provide new insights into the evolution of RNA recognition and cleavage mechanisms. [TOP OF PAGE]

  93. Biological projectiles (phage, yeast, bacteria) for genetic transformation of plants. Kikkert, J.R., Humiston, G.A., Roy, M.K., Sanford, J.C. (1999). In Vitro Cellular & Developmental Biology Plant 35:43-50. Bacteriophage lambda particles, yeast cells, and bacterial cells were tested as projectiles to deliver marker/reporter genes into plant cells via the biolistic process. When phage particles were complexed to tungsten or gold particles and used to bombard tobacco cells, fewer than 15 cell clusters per plate transiently expressed beta-glucuronidase (GUS). Cells of wild-type Saccharomyces cerevisiae were too large to be effective projectiles, but use of a reduced-size mutant resulted in a small number of transformants. Escherichia coli cells complexed with tungsten were the most effective projectile for plant transformation. Various methods to prepare E. coli were tested to reduce particle size, improve binding of bacteria to metal particles, and/or minimize particle clumping. In maize, the number of transformants was highest when bacteria/tungsten particles were air-dried onto macrocarriers from an aqueous solution. When maize cells were bombarded with bacteria/tungsten projectiles, rates of transient gene expression (2000 per plate) and stable transformation (50 per plate) were only two- to threefold lower than when purified DNA was used. Transformation of tobacco with E. coli projectiles was improved when the bacteria were treated with a series of ethanol and ether washes, then dried into a powder. Nevertheless, tobacco transformation was still 24- (transient) and 200-fold (stable) less than when purified DNA was used. Biological projectiles can be effective for plant transformation and are advantageous because once a DNA construct is made and put into the appropriate microorganism, the need to isolate and purify DNA for the biolistic process is eliminated, which saves time and lessens DNA shear. Such projectiles may be especially well suited where high molecular weight DNA constructs are needed. [TOP OF PAGE]

  94. Alternative origins for nannobacteria-like objects in calcite. Kirkland, B.L., Folk, R.L., Lynch, F.L., McLean, R.J.C., Molineux, I.J., Rahnis, M.A. (1999). Geology (Boulder) 27:347-350. More than 40 calcite-precipitation experiments were performed under sterile conditions in order to investigate the origins of 25-300 nm spherical-, rod-, and ovoid-shaped objects that have been widely interpreted as evidence of nanometer-scale life (i.e., nannobacteria). Individual experiments included the addition of soluble organic compounds, common species of eubacteria, or phage-induced eubacterial lysates. These experiments indicate that many of the nanometer-scale objects have inorganic or nonnannobacterial origins. In the precipitation experiments, calcite formed euhedral crystals 50-800 nm in diameter and smaller (<50 nm) anhedral or rounded particles or protocrystals. The small anhedral or rounded solids resembled nannobacteria. The relative amount of anhedral or rounded calcite was greatest in experiments with a dissolved organic component. These controlled experiments are in accord with observations