Isolation and characterization of temperate bacteriophages of Clostridium difficile

The lack of information on bacteriophages of Clostridium difficile prompted this study. Three of 56 clinical C. difficile isolates yielded double-stranded DNA phages phiC2, phiC5, phiC6, and phiC8 upon induction. Superinfection and DNA analyses revealed relatedness between the phages, while partial sequencing of phiC2 showed nucleotide homology to the sequenced C. difficile strain CD630.     

Resistance of a Bacillus subtilis mutant to a group of temperate bacteriophages

A mutant of Bacillus subtilis 168 was isolated which resists infection by all the group III temperate bacteriophages except SPR, while allowing full infection by phages of the other groups (I, II and IV). The mutation conferring this phenotype, pha-3, shows 52-54% PBS1-mediated cotransduction with the hisAl marker, mapping therefore in the gtaA and gtaB region of the B. subtilis chromosome. Nevertheless, it does not affect the infection by phages sensitive to gta mutations.     

Interaction of heterologous bacteriophages: growth suppression of temperate PP56 phage of Pseudomonas putida by the transposable phage D3112 of Pseudomonas aeruginosa

We have found an inhibiting effect of hybrid RP4::D3112 plasmid (where D3112 is represented as genome of a transposable phage specific for Pseudomonas aeruginosa) on the development of temperate P. putida phage PP56. The study of the effect has revealed a previously unknown locus (in the region 12-14.2 kb of the D3112 genome) which functions in the prophage state. The locus affects PP56 decreasing phage yield. Mutants of PP56 insensitive to inhibition were found.     

Comparison of new giant bacteriophages OBP and Lu11 of soil pseudomonads with bacteriophages of phiKZ-supergroup of Pseudomonas aeruginosa

Study of two recently isolated giant bacteriophages Lu11 and OBP that are active on Pseudomonas putida var. Manila and Pseudomonas fluorescens, respectively, demonstrated their similarity in morphology, genome size, and size of phage particles, with giant bacteriophages of Pseudomonas aeruginosa assigned to the supergroup of phiKZ-like phages of the family Myoviridae designated in this manner according to the best studied phage phiKZ that belongs to the species of this group widely distributed in nature. Comparison of major polypeptide sizes of mature particles suggests the similarity of certain proteins in the phages examined. In OBP particles visualized with an electron microscope, an "inner body" was detected, which points to the specific DNA package intrinsic to phages of phiKZ group. In the meantime, phages Lul11 and OBP do not exhibit resemblance among themselves or with any of earlier described phiKZ-like phages in respect to other traits; particularly, they have no detectable DNA homology. Note that phage Lu11 of P. putida var. Manila exhibits very slight homology with phage Lin68 of the family of P. aeruginosa phiKZ-like phages detected only in blot hybridization. This suggests the possible involvement of these phages in interspecies recombination ("gene shuffling") between phages of various bacterial species. Results of partial sequencing of phage genomes confirmed the phylogenetic relatedness of phage OBP to phages of the phiKZ-supergroup, whereas phage Lu11 most probably belongs to a novel species that is not a member of supergroup phiKZ composition. The results of the study are discussed in terms of the evolution of these phages.     

Isolation and characterization of Pseudomonas putida R-prime plasmids

A number of enhanced chromosome mobilizing (ECM) plasmids derived from the wide host range plasmid R68 have been used to construct R-prime plasmids carrying a maximum of two map minutes of the Pseudomonas putida PPN chromosome, using Pseudomonas aeruginosa PAO as the recipient. For one ECM plasmid, pMO61, the ability to form R-primes did not correlate with the ability to mobilize chromosomes in intrastrain crosses, suggesting that different mechanisms are involved. Physical analysis of one R-prime showed that 3.5 kb of chromosomal DNA had been inserted between the tandem IS21 sequences carried by the parent ECM plasmid.     

Isolation of alginate-producing mutants of Pseudomonas fluorescens, Pseudomonas putida and Pseudomonas mendocina

Spontaneous alginate-producing (muc) variants were isolated from strains of Pseudomonas fluorescens, P. putida and P. mendocina at a frequency of 1 in 10(8) by selecting for carbenicillin resistance. The infrared spectrum of the bacterial exopolysaccharide was typical of an acetylated alginate similar to that previously described in Azotobacter vinelandii and in mucoid variants of P. aeruginosa. Mucoid variants were not isolated from P. stutzeri, P. pseudoalcaligenes, P. testosteroni, P. diminuta, P. acidovorans, P. cepacia or P. maltophilia.     

Isolation of a third lipoamide dehydrogenase from Pseudomonas putida.

Pseudomonads are the only organisms so far known to produce two lipoamide dehydrogenases (LPDs), LPD-Val and LPD-Glc. LPD-Val is the specific E3 component of branched-chain oxoacid dehydrogenase, and LPD-Glc is the E3 component of 2-ketoglutarate and possibly pyruvate dehydrogenases and the L-factor of the glycine oxidation system. Three mutants of Pseudomonas putida, JS348, JS350, and JS351, affected in lpdG, the gene encoding LPD-Glc, have been isolated; all lacked 2-ketoglutarate dehydrogenase, but two, JS348 and JS351, had normal pyruvate dehydrogenase activity. The pyruvate and 2-ketoglutarate dehydrogenases of the wild-type strain of P. putida were both inhibited by anti-LPD-Glc, but the pyruvate dehydrogenase of the lpdG mutants was not inhibited, suggesting that the mutant pyruvate dehydrogenase E3 component was different from that of the wild type. The lipoamide dehydrogenase present in one of the lpdG mutants, JS348, was isolated and characterized. This lipoamide dehydrogenase, provisionally named LPD-3, differed in molecular weight, amino acid composition, and N-terminal amino acid sequence from LPD-Glc and LPD-Val. LPD-3 was clearly a lipoamide dehydrogenase as opposed to a mercuric reductase or glutathione reductase. LPD-3 was about 60% as effective as LPD-Glc in restoring 2-ketoglutarate dehydrogenase activity and completely restored pyruvate dehydrogenase activity in JS350. These results suggest that LPD-3 is a lipoamide dehydrogenase associated with an unknown multienzyme complex which can replace LPD-Glc as the E3 component of pyruvate and 2-ketoglutarate dehydrogenases in lpdG mutants.     

A comparative study of the NAH and TOL catabolic plasmids in Pseudomonas putida

A comparative study of the NAH and TOL catabolic plasmids was carried out to provide information for future genetic manipulation experiments involving these two plasmids. The plasmids were studied in a strain of P. putida and its mutant derivatives. The NAH and TOL plasmids were found to be incompatible. Under the conditions used in these experiments the TOL plasmid transferred into some strains into which NAH was unable to transfer. The use of mutants to remove certain catabolic activities encoded by the bacterial host cell facilitated the allocation of growth genotypes to the NAH and TOL plasmids. TOL encoded the degradation of benzoate, m-toluate and p-toluate, whereas NAH encoded the degradation of naphthalene and salicylate. The other plasmid-associated growth phenotypes were partly plasmid-specified and partly specified by the host cell. The pH optimum of the catechol 2,3-dioxygenase specified by the TOL plasmid was approximately 6.7, whereas that of the NAH-encoded enzyme was approximately 8.3.     

Study of morphology and genome structure of Pseudomonas putida bacteriophages for their classification

A group of 27 bacteriophages specific for Pseudomonas putida strains PpG1 and PpN has been isolated. The phages were characterized and compared with the previously described virulent (pf 16, af, tf and PMW) and temperate (PP56 and PP71) phages. The new phages belong to B1 and C1 morphotypes, according to Ackerman's classification. Phage DNAs were digested with several endonucleases; the molecular weights and homology of the DNAs were determined. All phages of P. putida isolated up to now were distributed into 10 species (groups), on the basis of particle morphology, genome size and the results of homology studies. Recombination processes are believed to participate in formation of phages belonging to certain species.     

Comparison of new giant bacteriophages OBP and Lu11 of soil pseudomonads with bacteriophages of the ϕKZ-supergroup of Pseudomonas aeruginosa

Study of two recently isolated giant bacteriophages Lu11 and OBP that are active on Pseudomonas putida var. Manila and Pseudomonas fluorescens, respectively, demonstrated their similarity in morphotype, genome size, and size of phage particles, with giant bacteriophages of Pseudomonas aeruginosa assigned to the supergroup of ?KZ-like phages of the family Myoviridae. This supergroup was designated in this manner according to the best studied phage ?KZ that belongs to the species of this group widely distributed in nature. Comparison of major polypeptide sizes of mature particles suggests similarity of certain proteins in the phages examined. In OBP particles visualized with an electron microscope, an “inner body” was detected, which points to specific DNA package intrinsic to phages of ?KZ group. In the meantime, phages Lu11 and OBP do not exhibit resemblance among themselves or with any of earlier described ?KZ-like phages in respect to detectable DNA homology. Note that phage Lu11 of P. putida var. Manila exhibits very slight homology with phage Lin68 of the family of P. aeruginosa ?KZ-like phages detected only in blot hybridization. This suggests the possible involvement of these phages in interspecies recombination (“gene shuffling”) between phages of various bacterial species. Results of partial sequencing of phage genomes confirmed the phylogenetic relatedness of phage OBP to phages of the ?KZ supergroup, whereas phage Lu11 most probably belongs to a novel species that is not a member of supergroup ?KZ composition. The results of the study are discussed in terms of the evolution of these phages.     

Expression of benzene dioxygenase from Pseudomonas putida ML2 in cis-1,2-cyclohexanediol-degrading pseudomonads

Benzene dioxygenase (BDO; EC 1.14.12.3) from Pseudomonas putida ML2 dihydroxylates benzene to produce cis-1,2-dihydroxy-cyclohexa-3,5-diene. As well as oxidising benzene and toluene, cell-free extracts of Escherichia coli JM109 expressing recombinant BDO oxidised cyclohexene, 1-methylcyclohexene and 3-methylcyclohexene. In an attempt to construct a novel metabolic pathway for the degradation of cyclohexene (via an initial BDO-mediated dihydroxylation of cyclohexene), cis-1,2-cyclohexanediol-degrading bacteria were isolated by enrichment culture. The bedC1C2BA genes encoding BDO (under the control of the tac promoter) were sub-cloned into pLAFR5, successfully conjugated into seven of the Gram-negative cis-1,2-cyclo-hexanediol-degrading isolates and stably maintained and expressed in three of them. However, despite their ability to grow on cis-1,2-cyclohexanediol as sole carbon source, express an active BDO and oxidise cyclohexene, none of the three strains was able to grow on cyclohexene as sole carbon source. Analysis revealed that BDO oxidised cyclohexene to a mixture of two products, a monohydroxylated (2-cyclohexen-1-ol) product and a dihydroxylated (cis-1,2-cyclohexanediol) product; and failure to grow on cyclohexene was attributed to the toxicity of metabolic intermediates accumulating from the 2-cyclohexen-1-ol metabolism.     

Isolation and characteristics of new nonlysogenic bacteriophages of Pseudomonas aeruginosa

A large group of nonlysogenic bacteriophages specific for Pseudomonas aeruginosa was studied. According to their absorption characteristics and serological properties, the phages were subdivided into four groups: luminal diameter k, luminal diameter m, luminal diameter mnP78 and luminal diameter mnF82. Within each of the groups, the phages were similar in the morphology of their particles and certain physiological characteristics. The luminal diameter m phages were similar to the P. aeruginosa bacteriophage E79 in their adsorption properties and antigenic specificity. The phages of the other groups differed in the above characteristics from the known P. aeruginosa bacteriophages. The effect of some plasmids on the growth of bacteriophages luminal diameter k, luminal diameter m, luminal diameter mnP78 and luminal diameter mnF82 was studied. The growth of new bacteriophages on certain plasmid-containing strains was inhibited in some cases.     

Comparative study of temperate bacteriophages isolated from Yersinia

170 Yersinia strains belonging to various species were investigated for the presence of temperate bacteriophages. By induction with mitomycin C seven phages were isolated from Y. enterocolitica strains and one phage from a Y. frederiksenii strain. The phages were characterized on the basis of their morphology, host range, genome size, DNA homology, and protein composition. They belong to different phage families and reveal narrow to moderate wide host ranges. Some of the isolated phages were able to infect pathogenic as well as nonpathogenic strains of Y. enterocolitica. The genomes of all isolated phages were found to be composed of double stranded DNA ranging from about 40 to 60 kb. In addition to the analysed phages, a number of putative phages were induced in strains of Y. frederiksenii, Y. kristensenii, Y. intermedia, and Y. mollaretii. The putative phages were identified by isolation of phage DNA from cell free lysates but could not be propagated on indicator strains. Southern hybridization experiments revealed relationships between phages belonging to different families. Moreover, DNA homologies were observed between phages isolated from nonpathogenic Yersinia strains and a phage which was isolated from a pathogenic Y. enterocolitica serogroup O:3 strain.     

Search for destruction factors of bacterial biofilms: Comparison of phage properties in a group of Pseudomonas putida bacteriophages and specificity of their halo-formation products

Comparison of Pseudomonas putida group of phages attributed to five species (af, ?15, ?27, ?2F, and pf16) with their common property of halo-formation (formation of lightening zones) around phage plaques was conducted. The halo around phage plaques appears as a result of reduction or disappearance of bacterial polysaccharide capsules. The concentration of viable bacteria remains unchanged within the halo. A comparison of specificities of halo-formation products from various phages was conducted by a simple method. These products were shown to be highly specific and inactive on other species of pseudomonads. Phage-resistant P. putida mutants scored with respect to various phages, which lost phage adsorption ability, were tolerant to the effect of halo-formation products in most cases. Apparently, the capsular polysaccharides, which serve as a substrate for depolymerases and are the primary phage receptors, may be often lost. Results of partial sequencing of the af phage genome revealed an open reading frame that encodes the enzyme transglycosylase similar rather to transglycosylases of oligotrophic bacteria belonging to different species than to lysozymes of other phages. Possibly, it is a polyfunctional enzyme combining functions of lysozyme and an enzyme that executes the penetration of phage particle across extracellular slime and capsule.     

Isolation of plasmid deoxyribonucleic acid from Pseudomonas putida.

Conditions suitable for reproducible recovery of covalently closed circular deoxyribonucleic acid from strains of Pseudomonas putida containing degradative plasmids (CAM, SAL, OCT, etc.) have been defined. These degradative plasmids could not be isolated by the usual procedure, whereas RP1, an R factor of the P group, present in the isogenic strain of P. putida, was isolated equally well by either the usual procedure or the modified procedure. Characterization by electron microscopy of RP1 deoxyribonucleic acid confirmed the molecular weight (about 40 X 10(6)) previously determined by sucrose gradient centrifugation.     

Effect of plasmids from various incompatibility groups on the development of bacteriophages specific to Pseudomonas aeruginosa and Pseudomonas putida

The aim of this work was to study the effect of plasmids belonging to different incompatibility groups on the growth of bacteriophages in Pseudomonas aeruginosa and Pseudomonas putida strains. The growth of bacteriophages was shown to be limited most often due to the presence in cells of plasmids belonging to the P-2 incompatibility group. Plasmids of the Inc P-2 group differed from one another in the spectrum of bacteriophages whose growth they limited. Phages whose growth was suppressed in strains containing plasmids of the P-5, P-9 or P-10 incompatibility groups were found. Some plasmids showed no specific interaction with bacteriophages. The plasmids investigated differed in the studied trait in P. aeruginosa and P. putida cells. In contrast to P. aeruginosa PAO, P. putida PpGI plasmid containing cells did not maintain the growth of donor-specific bacteriophages and, to a lesser degree, limited the growth of phages specific for P. putida PpGI.     

Phenogenetic characterization of a group of giant Phi KZ-like bacteriophages of Pseudomonas aeruginosa

A comparative study was made of a group of Pseudomonas aeruginosa virulent giant DNA bacteriophages similar to phage phi KZ in several genetic and phenotypic properties (particle size, particle morphology, genome size, appearance of negative colonies, high productivity, broad spectrum of lytic activity, ability to overcome the suppressing effect of plasmids, absence of several DNA restriction sites, capability of general transduction, pseudolysogeny). We have recently sequenced the phage phi KZ genome (288,334 bp) [J. Mol. Biol., 2002, vol. 317, pp. 1-19]. By DNA homology, the phages were assigned to three species (represented by phage phi KZ, Lin68, and EL, respectively) and two new genera (phi KZ and EL). Restriction enzyme analysis revealed the mosaic genome structure in four phages of the phi KZ species (phi KZ, Lin21, NN, and PTB80) and two phages of the EL species (EL and RU). Comparisons with respect to phage particle size, number of structural proteins, and the N-terminal sequences of the major capsid protein confirmed the phylogenetic relatedness of the phages belonging to the phi KZ genus. The origin and evolution of the phi KZ-like phages are discussed. Analysis of protein sequences encoded by the phage phi KZ genome made it possible to assume wide migration of the phi KZ-like phages (wandering phages) among various prokaryotes and possibly eukaryotes. Since the phage phi KZ genome codes for potentially toxic proteins, caution must be exercised in the employment of large bacteriophages in phage therapy.     

Isolation and characterization of toluene-sensitive mutants from Pseudomonas putida IH-2000

Two toluene-sensitive mutants were generated from Pseudomonas putida IH-2000, the first known toluene-tolerant isolate, by Tn5 transposon mutagenesis. These mutants were unable to grow in the presence of toluene (log P_ow 2.8) but they could grow in medium overlaid with organic solvents having a log P_ow value higher than that of toluene such as p-xylene (log P_ow 3.1), cyclohexane (log P_ow 3.4) and n-hexane (log P_ow 3.9). The Tn5 transposable element knocked out a cyoB-like gene in one mutant and a cyoC-like gene in the other mutant. Seven open reading frames were found in a 5.5-kb region containing the cyoB- and cyoC-like genes of strain IH-2000. ORFs 3–7 showed significant identity to the cyoABCDE gene products of Escherichia coli, but ORFs 1 and 2 showed no significant homology to any protein reported so far. The growth patterns of the Tn5 mutants with the inactivated cyo-like gene were similar to that of the wild-type strain in the absence of organic solvents, although the doubling times were slightly longer than that of the wild-type strain. Our findings indicate that cyo is an important gene for toluene tolerance, although its role is still unclear.