Characterization of pili determined by drug resistance plasmids R711b and R778b.

The bacterial drug resistance plasmids R711b and R778b, at present classified in the X incompatibility group, determine pili (designated 711) that resemble F pili morphologically. Like F pili, 711 pili adsorb F-specific filamentous bacteriophages to their tips, though more often in pairs, than singly. However, F-specific RNA-containing bacteriophages are not adsorbed to their sides, and strains carrying the plasmids are resistant to these phages. Pili determined by the only IncFV plasmid Folac are similar to 711 pili in their phage adsorption properties, but they are serologically different, as are F pili. It is concluded that F, Folac and 711 pili have basic differences in spite of a morphological resemblance.     

Bacteriophages F0lac h, SR, SF: phages which adsorb to pili encoded by plasmids of the S-complex

Phage F0lac is an RNA-containing phage which plates only on strains carrying the plasmid EDP208, a pilus derepressed derivative of the unique incompatibility plasmid F0lac. A host range mutant, phage F0lac h, was selected which plated on strains carrying the ungrouped plasmid pPLS::Tn5 and lysed strains carrying another ungrouped plasmid TP224::Tn10 or the Com9 plasmid R71. An RNA-containing phage, SR, was isolated from sewage on bacteria harbouring plasmid pPLS::Tn5. It was antigenically distinct from the above two phages but had the same host range as phage F0lac h. Phages F0lac h and SR adsorbed unevenly to the shafts of the conjugative pili. Another phage, SF, was filamentous and plated or propagated on strains carrying any of the above plasmids as well as on strains harbouring IncD or F-complex plasmids. Plasmids TP224::Tn10 and pPLS::Tn5 were compatible with representative plasmids of all Inc groups also encoding thick flexible pili. The four plasmids EDP208, R71, TP224::Tn10 and pPLS::Tn5 were compatible with one another except for the reaction of TP224::Tn10 in the presence of pPLS::Tn5 which was slightly ambiguous. The host ranges of the bacteriophages, together with the serological relatedness of the thick flexible pili determined by these four compatible plasmids, suggested that they constitute a new complex, here designated S.     

Filamentous Bacteriophages of Vibrio parahaemolyticus as a Possible Clue to Genetic Transmission

We have previously reported the isolation and characterization of two filamentous bacteriophages of Vibrio parahaemolyticus, designated Vf12 and Vf33. In this study, to understand the potential of these phages as tools for genetic transmission, we investigated the gene structures of replicative-form (RF) DNAs of their genomes and the distribution of these DNAs on chromosomal and extrachromosomal DNAs. The 7,965-bp nucleotide sequences of Vf12 and Vf33 were determined. An analysis of the overall gene structures revealed that Vf12 and Vf33 had conserved regions and distinctive regions. The gene organization of their conserved regions was similar to that of CTX phage of Vibrio cholerae and coliphage Ff of Escherichia coli, while their distinctive regions were characteristic of Vf12 and Vf33 phage genomes. Southern blot hybridization testing revealed that the filamentous phage genomes integrated into chromosomal DNA of V. parahaemolyticus at the distinctive region of the phage genome and were also distributed on some plasmids of V. parahaemolyticus and total cellular DNAs of one Vibrio damsela and one nonagglutinable Vibrio strain tested. These results strongly suggest the possibilities of genetic interaction among the bacteriophage Vf12 and Vf33 genomes and chromosomal and plasmid-borne DNAs of V. parahaemolyticus strains and of genetic transmission among strains through these filamentous phages.     

CTXphi infection of Vibrio cholerae requires the tolQRA gene products

CTXphi is a lysogenic filamentous bacteriophage that encodes cholera toxin. Filamentous phages that infect Escherichia coli require both a pilus and the products of tolQRA in order to enter host cells. We have previously shown that toxin-coregulated pilus (TCP), a type IV pilus that is an essential Vibrio cholerae intestinal colonization factor, serves as a receptor for CTXphi. To test whether CTXphi also depends upon tol gene products to infect V. cholerae, we identified and inactivated the V. cholerae tolQRAB orthologues. The predicted amino acid sequences of V. cholerae TolQ, TolR, TolA, and TolB showed significant similarity to the corresponding E. coli sequences. V. cholerae strains with insertion mutations in tolQ, tolR, or tolA were reduced in their efficiency of CTXphi uptake by 4 orders of magnitude, whereas a strain with an insertion mutation in tolB showed no reduction in CTXphi entry. We could detect CTXphi infection of TCP(-) V. cholerae, albeit at very low frequencies. However, strains with mutations in both tcpA and either tolQ, tolR, or tolA were completely resistant to CTXphi infection. Thus, CTXphi, like the E. coli filamentous phages, uses both a pilus and TolQRA to enter its host. This suggests that the pathway for filamentous phage entry into cells is conserved between host bacterial species.     

IncI2 plasmids specify sensitivity to filamentous bacteriophage IKe.

Bacterial strains carrying the derepressed incompatibility group IncI2 plasmids TP114drp-l or R721pilc were lysed by the filamentous bacteriophages IKe, I2-2, and X. Phage I2-2 was serologically related to IKe, but phage X was not. Phage IKe adsorbed to the tips of thick pili determined by the IncI2 plasmids, but not to the well-known thin I2 pili.     

Exposure of conjugative plasmid carrying Escherichia coli biofilms to male-specific bacteriophages

Escherichia coli carrying a natural conjugative F-plasmid generates F-pili mating pairs, which is important for early biofilm formation. In this study, we investigated the effect of male-specific filamentous single stranded DNA bacteriophage (f1) and RNA bacteriophage (MS2) on the formation of biofilms by E. coli carrying a natural conjugative F-plasmid. We showed that the early biofilm formation was completely inhibited by addition of the f1 phage, but not the MS2 phage. This suggests that the tip of F-pili is the specific attachment site for mating pairs formation and the side of F-pili has a non-obligatory role during biofilm formation. The inhibitory effect of the f1 phage was dependent on the time of addition during the biofilm formation. No inhibitory effect was observed when the f1 phages were added to the mature biofilms. This resistant mechanism of the mature biofilms could be attributed to the biofilm-specific phenotypes representing that the F-pili mating pairs were already formed and then the curli production commenced during the biofilm maturation. The pre-formed mating pairs seemed to resist the f1 phages. Altogether, our results indicate a close relationship between the presence of conjugative plasmid and male-specific bacteriophages within sessile biofilm communities, as well as the possibility of using the male-specific bacteriophages to control biofilm formation.     

Inhibition of Formation of Escherichia coli Mating Pairs by f1 and MS2 Bacteriophages as Determined with a Coulter Counter

The effect of male-specific filamentous deoxyribonucleic acid (f1) and isometric ribonucleic acid (MS2) bacteriophages on the formation of mating pairs in Escherichia coli conjugation was examined directly in the Coulter counter. When a sufficient multiplicity of infection (MOI) was used, the f1 phage immediately and completely inhibited the formation of mating pairs. On the other hand, the MS2 phage at a relatively high MOI also inhibited the formation of mating pairs significantly although not completey. The inhibitory effect of MS2 phage was dependent on the time of addition and the MOI used. At relatively low MOI (<20), the MS2 phage showed some inhibitory effect when added to a male culture prior to mixing with females, whereas no effect was observed when phages were added after mating pair formation had already commenced. At a high MOI (>400) MS2 phage disrupted the mating pairs already formed. Some preformed mating pairs were resistant to the high MOI of MS2 phages, however, and the "sensitive" (to high MOI) mating pairs seem to mature into "resistant" mating pairs as a function of time. We conclude that the tip of an F pilus is the specific attachment site for mating. The following process of mating pair formation has been formulated by deduction. (i) The sides of F pili weakly contact female cells, (ii) then the tips of F pili attach to the specific receptor sites to form initial mating pairs, and (iii) those pairs mature into mating pairs that are resistant to the high MOI of MS2 phages. The high MOI of MS2 prevents the first step, whereas f1 phages affect the second step-the binding between the tips of F pili and the receptor sites.     

An efficient and reproducible method for transformation of genetically recalcitrant bifidobacteria

The epidemic community-associated methicillin-resistant clone Staphylococcus aureus USA300 is a major source of skin and soft tissue infections and involves strains with a diverse set of resistance genes. In this study, we report efficient transduction of penicillinase and tetracycline resistance plasmids by bacteriophages φ80α and φJB between clinical isolates belonging to the USA300 clone. High transduction frequencies (10(-5) - 10(-6) CFU/PFU) were observed using phages propagated on donor strains as well as prophages induced from donors by ultraviolet light. Quantitative real-time PCR was employed to detect penicillinase plasmids in transducing phage particles and determine the ratio of transducing particles in phage lysates to infectious phage particles (determined as approximately 1 : 1700). Successful transfer of plasmids between strains in USA300 clone proves transduction is an effective mechanism for spreading plasmids within the clone. Such events contribute to its evolution and to emergence of new multiple drug-resistant strains of this successful clone.     

Molecular evidence for the lysogenic state of microorganisms belonging to the genus Bordetella and characterization of Bordetella parapertussis temperate bacteriophage 66(2.2)

A new bacteriophage phiK of microorganisms belonging to the genus Bordetella was isolated from cells of the earlier characterized strains 66(2-2) (1 and 2) obtained upon phage conversion of B. parapertussis 17903 cells by B. pertussis bacteriophage phi134. Bacteriophage phiK is identical to previously described Bordetella bacteriophages phiT, phi134, and phi214 in morphology and some biological properties but has a permuted genome different from all other phages. DNA of bacteriophage phiK is not integrated in the chromosome of B. parapertussis 17903, similar to DNA of bacteriophages phiT, phi134, and phi214 that are not integrated into B. pertussis and B. bronchiseptica chromosomes, but may be present in a small part of the bacterial population as linear plasmids. Sequences homologous to DNA of bacteriophage phiK were detected in the chromosome of strain 66(2-2) (1 and 2) and in chromosomes of all tested strains B. pertussis and B. bronchiseptica. Prophage integration in chromosomes of microorganisms of the genus Bordetella may vary in different bacterial strains and species. An assumption about abortive lysogeny of B. parapertussis bacteria for phiK phage and of B. bronchiseptica for closely related phages phiT, phi134, and phi214 has been advanced. The possibility of involvement of B. pertussis insertion sequences in the formation of the chromosomal structure in 66(2-2) convertants and in phage genomes is considered.     

Consensus structure of Pf1 filamentous bacteriophage from X-ray fibre diffraction and solid-state NMR

Filamentous bacteriophages (filamentous bacterial viruses or Inovirus) are simple and well-characterised macromolecular assemblies that are widely used in molecular biology and biophysics, both as paradigms for studying basic biological questions and as practical tools in areas as diverse as immunology and solid-state physics. The strains fd, M13 and f1 are virtually identical filamentous phages that infect bacteria expressing F-pili, and are sometimes grouped as the Ff phages. For historical reasons fd has often been used for structural studies, but M13 and f1 are more often used for biological experiments. Many other strains have been identified that are genetically quite distinct from Ff and yet have a similar molecular structure and life cycle. One of these, Pf1, gives the highest resolution X-ray fibre diffraction patterns known for filamentous bacteriophage. These diffraction patterns have been used in the past to derive a molecular model for the structure of the phage. Solid-state NMR experiments have been used in separate studies to derive a significantly different model of Pf1. Here we combine previously published X-ray fibre diffraction data and solid-state NMR data to give a consensus structure model for Pf1 filamentous bacteriophage, and we discuss the implications of this model for assembly of the phage at the bacterial membrane.     

The effect of the IncP-2-group plasmid on the growth of Pseudomonas aeruginosa bacteriophages

The influence of plasmids of the IncP-2 group on development of bacteriophages of Pseudomonas aeruginosa was studied. Six different types of phage growth inhibition conferred by natural plasmids of the IncP-2 group were found. All these plasmids were shown to have no effect on adsorption and injection of phage DNA into cells, only blocking intracellular phage development. The differences between phage inhibition mechanisms were shown by comparison of efficiency of colony formation by cells containing different plasmids, in the presence of different phages. The presence of the RpL11 plasmid reduces the frequency of lysogenization with G101 phage but not with B3 phage. The mutants of pMG53 plasmid having modified phage inhibition spectrum were obtained. It was inferred that inhibition of different phages is under control of different loci of this plasmid. The mutants of phage B3 overcoming inhibition by plasmids were obtained. It was supposed that the plasmids act at least at three different sites of the phage B3 genome.     

Bacteriophage selection against a plasmid-encoded sex apparatus leads to the loss of antibiotic-resistance plasmids

Antibiotic-resistance genes are often carried by conjugative plasmids, which spread within and between bacterial species. It has long been recognized that some viruses of bacteria (bacteriophage; phage) have evolved to infect and kill plasmid-harbouring cells. This raises a question: can phages cause the loss of plasmid-associated antibiotic resistance by selecting for plasmid-free bacteria, or can bacteria or plasmids evolve resistance to phages in other ways? Here, we show that multiple antibiotic-resistance genes containing plasmids are stably maintained in both Escherichia coli and Salmonella enterica in the absence of phages, while plasmid-dependent phage PRD1 causes a dramatic reduction in the frequency of antibiotic-resistant bacteria. The loss of antibiotic resistance in cells initially harbouring RP4 plasmid was shown to result from evolution of phage resistance where bacterial cells expelled their plasmid (and hence the suitable receptor for phages). Phages also selected for a low frequency of plasmid-containing, phage-resistant bacteria, presumably as a result of modification of the plasmid-encoded receptor. However, these double-resistant mutants had a growth cost compared with phage-resistant but antibiotic-susceptible mutants and were unable to conjugate. These results suggest that bacteriophages could play a significant role in restricting the spread of plasmid-encoded antibiotic resistance.     

Genomic sequence and receptor for the Vibrio cholerae phage KSF-1phi: evolutionary divergence among filamentous vibriophages mediating lateral gene transfer

KSF-1phi, a novel filamentous phage of Vibrio cholerae, supports morphogenesis of the RS1 satellite phage by heterologous DNA packaging and facilitates horizontal gene transfer. We analyzed the genomic sequence, morphology, and receptor for KSF-1phi infection, as well as its phylogenetic relationships with other filamentous vibriophages. While strains carrying the mshA gene encoding mannose-sensitive hemagglutinin (MSHA) type IV pilus were susceptible to KSF-1phi infection, naturally occurring MSHA-negative strains and an mshA deletion mutant were resistant. Furthermore, d-mannose as well as a monoclonal antibody against MSHA inhibited infection of MSHA-positive strains by the phage, suggesting that MSHA is the receptor for KSF-1phi. The phage genome comprises 7,107 nucleotides, containing 14 open reading frames, 4 of which have predicted protein products homologous to those of other filamentous phages. Although the overall genetic organization of filamentous phages appears to be preserved in KSF-1phi, the genomic sequence of the phage does not have a high level of identity with that of other filamentous phages and reveals a highly mosaic structure. Separate phylogenetic analysis of genomic sequences encoding putative replication proteins, receptor-binding proteins, and Zot-like proteins of 10 different filamentous vibriophages showed different results, suggesting that the evolution of these phages involved extensive horizontal exchange of genetic material. Filamentous phages which use type IV pili as receptors were found to belong to different branches. While one of these branches is represented by CTXphi, which uses the toxin-coregulated pilus as its receptor, at least four evolutionarily diverged phages share a common receptor MSHA, and most of these phages mediate horizontal gene transfer. Since MSHA is present in a wide variety of V. cholerae strains and is presumed to express in the environment, diverse filamentous phages using this receptor are likely to contribute significantly to V. cholerae evolution.     

Bacteriophage D: an IncD group plasmid-specific phage

The existence of the plasmid incompatibility group D was reaffirmed as a result of compatibility experiments done on plasmids R687, R711b, R778b and R840 which were previously tentatively accepted as constituting the group. The group was further delineated by the isolation of a phage, phage D, which adsorbed specifically to IncD plasmid-encoded pili produced by Escherichia coli K12 strains and strains of Salmonella typhimurium, Proteus morganii and Klebsiella oxytoca harbouring one of these plasmids. Plaque formation, like that of phage pilH alpha, was temperature sensitive in that plaques formed at 26 degrees C but not at 37 degrees C. Plaques were fairly clear, regular in outline and varied from pinpoint to about 1.5 mm in diameter on E. coli hosts where plaques were detected, but on the other hosts the plaques were more turbid and often irregular in outline. The phage did not plate (or propagate) on IncD plasmid-carrying strains of Providencia alcalifaciens, Providencia stuartii or Serratia marcescens. The phage had an isometric hexagonal outline with a diameter of about 27 nm. It contained RNA and resembled two other RNA-containing phages, M and pilH alpha, by being sensitive to chloroform. It adsorbed to the sides of the very distal ends of the shafts of IncD plasmid-coded pili.     

Ten new temperate bacteriophages of<Emphasis Type="Italic">Citrobacter youngae</Emphasis>

In a cross-test, we examined 55 strains of Citrobacter youngae against each other as potential producers of temperate bacteriophages and as potential sensitive indicators for them. Ten strains (18.2 %) showed the production of phages. Seven different strain-specific spectra of activity (from 1 to 11 strains each) were found. Phage production by 6 strains was inducible with mitomycin C, in 4 strains it was not inducible. The plaques of the phages were more or less turbid, without a lytic halo, tiny to small, 0.2-1.3 mm in diameter. Using a polyclonal, specific anti-lambda serum, all 10 phages were found to be clearly distinct from E. coli lambda phage, the phage 31/47 showing the highest neutralization titre of all. Interspecific tests with 15 strains of 8 species of Enterobacteriaceae revealed not a single case of activity of Citrobacter phages towards any of them. Five phage-immune clones lysogenized with 5 of the phages kept their remaining phage sensitivity spectra, though extended by sensitivity to 1-3 phages; 2 of these strains acquired also sensitivity to phage lambda. The phages belong to the morphotypes of Myoviridae (6 phages) and Siphoviridae (4 phages), with head diameters of 51-58 nm and tail length of 97-173 nm. Three strains produced corpuscular bacteriocins.     

Site-specific recombination systems in filamentous phages

Since the discovery of the integration mechanism of the filamentous phage CTX? of Vibrio cholerae, integrating filamentous phages have been discovered to be more abundant and diverse than previously recognized. However, the integration systems of filamentous phages have not been fully investigated. The present review provides a short overview on the different strategies employed by filamentous bacteriophages for integration into the host chromosome. This is the first review to describe the diversity of site-specific recombination in filamentous phages.     

Selection of scFv phages on intact cells under low pH conditions leads to a significant loss of insert-free phages

Display of functional antibody fragments on the surface of filamentous bacteriophages allows fast selection of specific phage antibodies against a variety of target antigens. However, enrichment of single chain variable fragment (scFv)-displaying phages is often hampered by the abundance of bacteriophages lacking antibody fragments. Moderate adhesive binding activities and production advantages of these "empty" phages results in their subsequent enrichment during selection on target cells. To date, very limited effort has been made to develop strategies removing nonspecific binding phages during the selection processes. To efficiently reduce insert-free phages when panning on intact cells, we increased the washing stringency by lowering the pH of the buffer with citric acid. Under standard washing procedures (pH 7.4), only approximately 73% of recovered phages were insert-free after three rounds of selection. Using stringent washing procedures (pH 5.0), approximately 12% of recovered phages contained no scFv. Using this protocol, we have cloned an antibody fragment from a mouse/human hybridoma cell line directed against the disialoganglioside GD2. This study confirms that selection of phage antibodies on cells is efficiently enhanced by assays augmenting the stringency to remove nonspecific binding phages.     

Serological characteristics of pili determined by the plasmids R711b and F0lac.

The plasmids R711b (at present IncX) and F0lac (IncFV) both determine pili morphologically like those of F (IncFI), and confer sensitivity to the F-specific filamentous bacteriophages, but not to the F-specific isometric RNA phages. Detailed serological studies show that the two pilus types are unrelated, and that neither is related to any of the previously defined F pilus serotypes. Adsorption of the isometric RNA phage MS2 to R711b pili occurs in the presence but not in the absence of formalin, which presumably prevents elution of reversibly adsorbed virions. No adsorption occurs with F0lac pili. MS2 multiplication, as measured by titre increase tests in liquid medium, is found with neither plasmid. The two plasmids are not incompatible. These observations indicate that R744b and F0lac are different both from one another and from the plasmids belonging to the incompatibility groups IncFI--IV.     

A novel strategy for the functional cloning of enzymes using filamentous phage display: the case of nucleotidyl transferases

In vitro selections for catalytic activity have been designed for the isolation of genes encoding enzymes from libraries of proteins displayed on filamentous phages. The proteins are generally expressed as C-terminal fusions with the N-terminus of the minor coat protein p3 for display on phages. As full-length cDNAs generally contain several stop codons near their 3′ end, this approach cannot be used for their expression on the surface of phages. Here we show that in vitro selection for catalytic activity is compatible with a system for expression of proteins as N-terminal fusions on the surface of bacteriophages. It is highlighted for the Stoffel fragment of Taq DNA polymerase I and makes use of (p3–Jun/Fos–Stoffel fragment) fusions. The efficiency of the selection is measured by an enrichment factor found to be about 55 for a phage polymerase versus a phage not expressing a polymerase. This approach could provide a method for the functional cloning of nucleotidyl transferases from cDNA libraries using filamentous phage display.     

New waterborne bacteriophages active on Yersinia enterocolitica.

Seven bacteriophages active on Yersinia enterocolitica (YE) were isolated from surface water samples collected in Granada, Spain. A comparison of the respective host ranges of these new phages and of reference phages used for YE phage typing showed that YE strains belonging to various phage types, grown at either 37 or 25 degrees C, expressed susceptibility to reference sewage water phages whereas susceptibility to new waterborne phages, as well as to reference phages from lysogenic YE, was only demonstrated in YE strains grown at 25 degrees C. A YE strain isolated by stool culture from a pig was lysogenic for a bacteriophage which behaved like waterborne phages and reference phages from lysogenic YE strains. The possibility that the isolation of waterborne bacteriophages might, in certain circumstances, reflect the presence of lysogenic YE was raised.