Isolation and characterization of a temperature-sensitive generalized transducing bacteriophage for Vibrio cholerae.

CP-T1 is the only described generalized transducing bacteriophage for the intestinal pathogen Vibrio cholerae, yet many of its basic biological parameters remain unknown. Due to low frequencies of transduction and pseudolysogen formation, CP-T1 has not been widely used as a genetic tool. To overcome these limitations, we have isolated a conditional mutant of CP-T1 that exhibits temperature-sensitive plaque formation. Several biological properties of CP-T1ts were determined, including its restrictive temperature, adsorbance profile to host cells, burst time, and burst size. Based on these properties, an optimized transduction protocol was designed which resulted in several fold higher transduction frequencies for a variety of genetic markers from a number of chromosomal loci. Generalized transduction was also demonstrated between classical and E1 Tor biotype strains of V. cholerae.     

Physical characterization of bacteriophage MX4, a generalized transducing phage for Myxococcus xanthus.

A generalized transducing bacteriophage of Myxococcus xanthus has been examined. The phage particle consists of an isometric head and a contractile tail. The genome of the phage is a linear DNA molecule of molecular weight 39 ± 2.1 × 10⁶, which contains the normal DNA bases 70% of which are guanosine + cytosine. No overall heterogeneity of base composition is present. The DNA does not carry easily detectable cohesive ends nor is it cyclically permuted. It does contain a large and somewhat variable terminal redundancy. Heating phage particles in the presence of EDTA causes tail sheath contraction and ejection of DNA, some of which remains attached to the tail. Digestion of tail-bound DNA with restriction enzymes shows that the phage tail can be attached to either end of the DNA. Thus the DNA probably contains recognition sites for the packaging of its DNA at both ends. These results suggest possible mechanisms for the genesis of transducing particles by phage MX4.     

Molecular evidence for the lysogenic state of microorganisms belonging to the genus Bordetella and characterization of Bordetella parapertussis temperate bacteriophage 662-2

A new bacteriophage ?K 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 17 903 cells by B. pertussis bacteriophage ?134. Bacteriophage ?K is identical to previously described Bordetella bacteriophages ?T, ?134, and ?214 in morphology and some biological properties but has a permuted genome different from all other phages. DNA of bacteriophage ?K is not integrated in the chromosome of B. parapertussis 17 903, similar to DNA of bacteriophages ?T, ?134, and ?214 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 ?K 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 ?K phage and of B. pertussis and B. bronchiseptica for closely related phages ?T, ?134, and ?214 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.     

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.     

Purification and characterization of VSH-1, a generalized transducing bacteriophage of Serpulina hyodysenteriae.

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

Isolation and characterization of a temperate bacteriophage from Streptomyces galilaeus

A new temperate actinophage from Streptomyces galilaeus ATCC 31133 was purified after that strain was crossed with S. peucetius ATCC 29050. Sensitive hosts became lysogenized and yielded turbid plaques of 2 to 3 mm in diameter. Host-range analysis indicated that 16 of 27 Streptomyces strains tested were sensitive to infection on solid medium. S. lividans and S. coelicolor A3(2) were among those not infected by this new actinophage. The new actinophage, designated phi SPK1, belongs to the Bradley group B morphological type, the pH optimum for infection is 6.75 to 7.0, it is not efficiently induced by mitomycin C or UV irradiation, it has a circular chromosome of 35.8 +/- 0.5 kilobase pairs in length containing overlapping (cohesive) ends, and the G+C content of its DNA was calculated from the buoyant density of 1.7240 to be 69 mol%. The DNA of phage phi SPK1 was cleaved by the restriction endonucleases ApaI, AluII, EcoRI, PvuII, and SalI, but, in all cases except that with EcoRI, treatment yielded greater than 20 restriction fragments. No sites were detected for BamHI, BclI, BglII, ClaI, HindIII, MluI, PstI, SmaI, SphI, SstI, XbaI, or XhoI.     

Isolation and characterization of a temperate bacteriophage from a ruminal acetogen

Nine acetogenic bacterial cultures recently isolated from the bovine rumen were tested for phage susceptibility by plaque formation. Both clear plaques and plaques with turbid centers were occasionally seen, but could not be used repeatedly to lyse pure cultures of acetogens, suggesting the possibility of a temperate phage. Five of the nine acetogenic isolates showed a response to mitomycin C induction. Acetogenic isolate H3HH was chosen for further study because it produced the greatest lysogenic response to mitomycin C. The bacteriophage was induced with mitomycin C, examined by transmission electron microscopy, and shown to have a hexagonal head (diameter, 59 m), a long flexible tail (192 m), and a flat collar (diameter, 31 m). The bacteriophage was classified within Bradley's group B. Bacteriophage DNA was determined to contain 36.2 kilobases of linear double-stranded DNA.Journal Paper #14665 of the Agricultural Research Programs of Purdue University.     

MP13, a generalized transducing bacteriophage for Bacillus megaterium.

The first generalized transducing bacteriophage reported for Bacillus megaterium has been characterized. Optimum conditions for lysate production and transduction procedures were established so that transducing frequencies of 8 x 10(-6) and higher are now possible. The phage, MP13, has a head diameter of 97 nm and a contractile tail (202 by 17 nm) and adsorbs to the periphery of the cell. MP13 was inactivated rapidly at 60 degrees C, but not at 55 degrees C, and was sensitive to toluene, ether, and chloroform. When centrifuged in a neutral CsCl gradient, two bands were observed, a major band of 1.490 g cm-3 and a minor band of 1.482 g cm-3 buoyant density. The major band contained only infective particles, whereas the minor band contained both infective and transducing particles. Phage DNA was resistant to several restriction endonucleases, but yielded 9 fragments with MboI, more than 34 with HindIII, and 7 with BstEII. The molecular weights for the fragments from MboI-BstEII double digests total 97 x 10(9).     

The isolation and characterization of plaque-forming arabinose transducing bacteriophage lambda.

Non-defective arabinose transducing phage, λpara, were isolated in two steps: first, Escherichia coli strains containing rare insertions of λ DNA into the arabinose C or B genes were selected; and second, these lysogens were induced and transducing phage were selected from the resulting lysates. The approximate location of the bacterial substitution on the phage and the ara gene content of the substitution were determined genetically. The precise location of the substitution was determined by electron microscopy of DNA heteroduplexes.Transducing phage, derived from the strain possessing λ inserted into the araC gene, carried part of the araC gene, the ara regulatory region, and all of the araB gene. Transducing phage, derived from eight independent strains possessing λ inserted in the same orientation and in the same position in the araB gene, carried a portion of the araB gene, the ara regulatory region and all of the araC gene. In these nine cases the ara DNA on the phage was immediately adjacent to the normal phage attachment site, indicating that the transducing phage were formed by the same type of abnormal excision which produces gal or bio transducing λ phage. The relative orientations of ara and phage genes were deduced from the topology of such excisions. One anomalous transducing phage was also characterized.     

Induction and characterization of Pediococcus acidilactici temperate bacteriophage

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.     

Isolation and characterization of a temperate bacteriophage from Streptomyces galilaeus.

A new temperate actinophage from Streptomyces galilaeus ATCC 31133 was purified after that strain was crossed with S. peucetius ATCC 29050. Sensitive hosts became lysogenized and yielded turbid plaques of 2 to 3 mm in diameter. Host-range analysis indicated that 16 of 27 Streptomyces strains tested were sensitive to infection on solid medium. S. lividans and S. coelicolor A3(2) were among those not infected by this new actinophage. The new actinophage, designated phi SPK1, belongs to the Bradley group B morphological type, the pH optimum for infection is 6.75 to 7.0, it is not efficiently induced by mitomycin C or UV irradiation, it has a circular chromosome of 35.8 +/- 0.5 kilobase pairs in length containing overlapping (cohesive) ends, and the G+C content of its DNA was calculated from the buoyant density of 1.7240 to be 69 mol%. The DNA of phage phi SPK1 was cleaved by the restriction endonucleases ApaI, AluII, EcoRI, PvuII, and SalI, but, in all cases except that with EcoRI, treatment yielded greater than 20 restriction fragments. No sites were detected for BamHI, BclI, BglII, ClaI, HindIII, MluI, PstI, SmaI, SphI, SstI, XbaI, or XhoI.     

Isolation and characterization of bacteriophage BCJA1, a novel temperate bacteriophage active against the alkaliphilic bacterium, Bacillus clarkii

The isolation and characterization of a novel bacteriophage active against the obligately alkaliphilic bacterium Bacillus clarkii is described. The bacteriophage, designated BCJA1, is a member of the Siphoviridae family with a B1 morphology. It possesses an isometric head, which measures 65 nm between opposite apices, and a noncontractile tail of 195 nm length. It had a buoyant density of 1.518 g/ml and an estimated particle mass of 37 × 10⁷ daltons. BCJA1 was stable over the pH range of 6–11. A one-step growth experiment conducted at pH 10 demonstrated a latent period of about 40 min and a burst size of approximately 40. The purified bacteriophage appeared to consist of 10 proteins with the major head and tail proteins likely to be of molecular weight 36 500 and 28 000, respectively. The genome size was estimated to be between 32.1 and 34.8 kb. The percent G + C content of purified bacteriophage DNA was 45.6. The wildtype bacteriophage is temperate but a clear plaque mutant was isolated. Received: May 25, 1997 / Accepted: August 5, 1997     

Marine transducing bacteriophage attacking a luminous bacterium

The isolation and partial characterization of a marine bacteriophage attacking a strain of luminous bacteria is described, including some physical, biological, and genetic properties. It is a DNA phage of density of 1.52 with a long flexible tail and an apparently icosohedral head. With respect to stability in suspension, it has a rather specific requirement for the sodium ion in high concentration; it is further stabilized by the addition of calcium and magnesium ions. These same ions are likewise all required for both good plating efficiency and plaque uniformity. Although it goes through a typical lytic growth cycle (about 45 min), with a burst size of 100, and no stable lysogens have been isolated, it is nevertheless a transducing phage specifically for the tryptophan region, transducing several, but not all, independently isolated Trp⁻ auxotrophs to protrophy. No other auxotrophs of a variety of amino acids were transduced by this phage to prototrophy. Phage infection does not change the normal expression of the luminescent system, and light remains at near normal levels until cell lysis occurs.     

Isolation and sequencing of a temperate transducing phage for Pasteurella multocida

A temperate bacteriophage (F108) has been isolated through mitomycin C induction of a Pasteurella multocida serogroup A strain. F108 has a typical morphology of the family Myoviridae, presenting a hexagonal head and a long contractile tail. F108 is able to infect all P. multocida serogroup A strains tested but not those belonging to other serotypes. Bacteriophage F108, the first P. multocida phage sequenced so far, presents a 30,505-bp double-stranded DNA genome with cohesive ends (CTTCCTCCCC cos site). The F108 genome shows the highest homology with those of Haemophilus influenzae HP1 and HP2 phages. Furthermore, an F108 prophage attachment site in the P. multocida chromosome has been established to be inside a gene encoding tRNA(Leu). By using several chromosomal markers that are spread along the P. multocida chromosome, it has been demonstrated that F108 is able to perform generalized transduction. This fact, together with the absence of pathogenic genes in the F108 genome, makes this bacteriophage a valuable tool for P. multocida genetic manipulation.     

Isolation and characterization of a Lactobacillus fermentum temperate bacteriophage from Chinese yogurt

AIMS: The aim of this study was to investigate the properties of temperate bacteriophage of Lactobacillus fermentum, based on its morphology, restriction patterns, protein profile and the impact on the growth of host strain. METHODS AND RESULTS: With Mitomycin C, seven temperate phages were induced from Lactobacilli derived from Chinese yogurt. The temperate phages induced belong to the most common Bradley's group B, having hexagonal head and long, noncontractile tail. They were furthermore confirmed to be the same bacteriophage by identical restriction patterns. SDS-PAGE profile showed that the phage studied had one major structure protein about 31.9 kDa. The presence of the prophage influenced the cell shape and colony size of its lysogenic strain. CONCLUSIONS: The phage obtained had similar, but not complete identical properties with other L. fermentum phages reported. It influenced the growth behaviour of its lysogenic strain. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides some information about bacteriophages occurring in the Chinese yoghurt manufacture and contributes to our knowledge on the bacteriophage diversity in the dairy industry.     

Genomic organization and molecular characterization of SM1, a temperate bacteriophage of Streptococcus mitis

The direct binding of Streptococcus mitis to human platelets is mediated in part by two proteins (PblA and PblB) encoded by a lysogenic bacteriophage (SM1). Since SM1 is the first prophage of S. mitis that has been identified and because of the possible role of these phage-encoded proteins in virulence, we sought to characterize SM1 in greater detail. Sequencing of the SM1 genome revealed that it consisted of 34,692 bp, with an overall G+C content of 39 mol%. Fifty-six genes encoding proteins of 40 or more amino acids were identified. The genes of SM1 appear to be arranged in a modular, life cycle-specific organization. BLAST analysis also revealed that the proteins of SM1 have homologies to proteins from a wide variety of lambdoid phages. Bioinformatic analyses, in addition to N-terminal sequencing of the proteins, led to the assignment of possible functions to a number of proteins, including the integrase, the terminase, and two major structural proteins. Examination of the phage structural components indicates that the phage head may assemble using stable multimers of the major capsid protein, in a process similar to that of phage r1t. These findings indicate that SM1 may be part of a discrete subfamily of the Siphoviridae that includes at least phages r1t of Lactococcus lactis and SF370.3 of Streptococcus pyogenes.     

Isolation and characterization of a temperate bacteriophage from the ruminal anaerobe Selenomonas ruminantium

A temperate bacteriophage was obtained from an isolate of the ruminal anaerobe Selenomonas ruminantium. Clear plaques that became turbid on further incubation occurred on a lawn of host bacteria. Cells picked from a turbid plaque produced healthy liquid cultures, but these often lysed on storage. Mid-log-phase liquid cultures incubated with the bacteriophage lysed and released infectious particles with a titer of up to 3 X 10(7) PFU/ml. A laboratory strain of S. ruminantium, HD-4, was also sensitive to this bacteriophage, which had an icosohedral head (diameter, 50 nm) and a flexible tail (length, 140 nm). The bacteriophage contained 30 kilobases of linear, double-stranded DNA, and a detailed restriction map was constructed. The lysogenic nature of infection was demonstrated by hybridization of bacteriophage DNA to specific restriction fragments of infected host genomic DNA and by identification of a bacteriophage genomic domain which may participate in integration of the bacteriophage DNA. Infection of S. ruminantium in vitro was demonstrated by two different methods of cell transformation with purified bacteriophage DNA.     

Isolation of bacteriophage MX4, a generalized transducing phage for Myxococcus xanthus.

The structure of α-chitin has been determined by X-ray diffraction, based on the intensity data from deproteinized lobster tendon. Least-squares refinement shows that adjacent chains have alternating sense (i.e. are antiparallel). In addition, there is a statistical distribution of side-chain orientations, such that all the hydroxyl groups form hydrogen bonds. The unit cell is orthorhombic with dimensions a = 0.474 ± 0.001 nm, b = 1.886 ± 0.002 nm and c = 1.032 ± 0.002 nm (fiber axis); the space group is P2₁2₁2₁ and the cell contains disaccharide sections of the two chains passing through the center and corner of the ab projection. The chains form hydrogen-bonded sheets linked by CO…HN bonds approximately parallel to the a axis, and each chain has an O-3′H…O.5 intramolecular hydrogen bond, similar to that in cellulose. Adjacent chains along the ab diagonal have different conformations for the CH₂OH groups: on one chain these groups form O.6H…O.6′ intermolecular hydrogen bonds to the CH₂OH group on the adjacent chain along the ab diagonal. The latter group is oriented to form an intramolecular O.6′H…O.7 bond to the carboxyl oxygen on the next residue. The results indicate that a statistical mixture of CH₂OH orientations is present, equivalent to half oxygens on each residue, each forming inter- and intramolecular hydrogen bonds. As a result the structure contains two types of amide groups, which differ in their hydrogen bonding, and account for the splitting of the amide I band in the infrared spectrum. The Inability of this chitin polymorph to swell on soaking in water is explained by the extensive intermolecular hydrogen bonding.     

Improved generalized transducing bacteriophage for Caulobacter crescentus.

Caulobacter phage phi Cr30T, a temperature-sensitive derivative of the lytic, generalized transducing phage phi Cr30, was isolated as a double temperature-sensitive recombinant in a cross between phi Cr30ts1 and phi Cr30ts2, phi Cr30T mediated generalized transduction of Caulobacter crescentus at frequencies comparable to those of phi Cr30 and eliminated the requirement for irradiation of transducing lysates to prevent killing of transductants on the plate.