[The use of Miniphage D3112 deltaHcts as an integrative vectorin Pseudomonas aeruginosa cells]. / Ispol'zovanie minifaga D3112 deltaHcts v kachestve integrativnogo vektora v kletkakh Pseudomonas aeruginosa.

Catechol 2,3-dioxygenase gene (xylE) was cloned into the Pseudomonas aeruginosa miniphage D3112 delta Hcts. Miniphage provides an effective transduction of gene xylE and its transposition into different replicons. It can be used as an integrative vector in the P. aeruginosa cells. Besides, gene xylE is rather convenient genetic marker for identification of miniphage both in Excherichia coli and Pseudomonas species.

[Minigenomes of the transposable phage D3112 of Pseudomonas aeruginosa and their properties]. / Mini-genomy faga-transpozona D3112 Pseudomonas aeruginosa i ikh svoistva.

Two derivatives of D3112 prophage with large internal deletions (mini-D3112) have been constructed. Mini-D3112 delta H is about 3.5 kb long, containing the repressor c1 gene. Mini-D3112 delta E is about 12 kb long, contains the c1 gene, several structural genes and replication gene A. These mini-D3112 are unable to replicate. However, they could replicate and maturated in the presence of the helper D3112cts. Mini-D3112 mediate translocation of the gene argH from the chromosome into the R' plasmids, the translocated fragment being sandwiched between two mini-D3112 genomes.

[Two loci in the genome of the Pseudomonas aeruginosa transposable phage B39 which affect the integration process. II. Mapping of the pdeX and pdeY loci by restriction and heteroduplex analysis]. / Dva lokusa v genome faga-transpozona B39 Pseudomonas aeruginosa, vliiaiushchie na protsess integratsii. Soobshchenie II. Opredelenie polozheniia lokusov pdeX i pdeY metodami restriktsionnogo i geterodupleksnogo analizov.

The coordinate function of two loci - pdeX and pdeY - in the genome of a transposable phage (TP) provides the phage function pde+ (good growth on bacteria with Rms163 plasmid). When these two loci in hybrid phages originate from different TP, some of the hybrid phages have Pde- phenotype. To localize pdeX and pdeY, the structure of hybrid TP genomes with Pde+ and Pde- phenotype obtained in crosses between B39ts+ and PH132 were studied using restriction and heteroduplex analysis. On the basis of data obtained, pdeX and pdeY were mapped in 2.85-6.4 and 6.4-16 kbp regions, respectively.

[Wide distribution of transposable phages in natural Pseudomonas aeruginosa populations]. / Shirokaia rasprostranennost' transpozonnykh fagov v prirodnykh populiatsiiakh Pseudomonas aeruginosa.

Five phages (PH2, PH51, PH59, PH93 and PH132) which have some characteristics common with D3112, the transposable phage of Pseudomonas aeruginosa, were isolated from clinical P. aeruginosa isolates. The phages were distributed into 4 different immunity groups. The basic criteria used for selection of transposable phages have been: 1) Morphology of a phage particle, host range, similar inactivation with antiserum; 2) Similar sizes of phage genomes; 3) The presence of a variable non-phage nucleotide sequences covalently linked to phage genome DNA, which could be identified using restriction endonucleases or by heteroduplex analyses. The DNAs of the new phages are resistant to treatment with BamH1 endonuclease, like the DNAs of phages D3112, B39 and B3 described earlier. The restriction maps of the phage genomes are constructed.

[Use of deletion mutants of plasmid RP4::D3112 for the genetic analysis of Pseudomonas aeruginosa bacteriophage D3112]. / Ispol'zovanie deletsionnykh mutantov plazmidy RP::4D3112 dlia geneticheskogo analiza bakteriofaga D3112 Pseudomonas aeruginosa.

The hybrid plasmid RP4::D3112 becomes unstable in Escherichia coli K-12 cells under certain growth conditions. The deletion mutants of this plasmid are formed at a high frequency. All the deletions selected have a specific feature: they start in the left end, at the point of joining of plasmid and phage DNA, and remove different portions of the phage genome. The deletion mutants have been used for genetic mapping of D3112. We have localized the repressor gene cI (0-1.3 kb), 3 early genes (1.3-14.2 kb) and two groups of late genes (14.2-29.9 and 29.9-38 kb). Electron microscope studies of RP4::D3112 DNA and its deletion derivatives have shown that integration of D3112 genome in RP4 occurs through the ends of the genome, without permutations. It appears that bacterial nucleotide sequences joined to DNA from mature D3112 particles, to the right end of D3112 genome, are lost. Thus, transposable phages D3112 of Pseudomonas aeruginosa and E. coli Mu phage have some similarities in the genome organization and in the way of their integration into the host DNA.

[Transposition of the phage D3112 genome in Escherichia coli cells]. / Transpozitsiia genoma faga D3112 v kletkakh Escherichia coli.

It has been demonstrated that the genome of phage D3112 of Preudomonas aeruginosa can be transposed into Escherichia coli chromosome as a component of the hybrid plasmid RP4 TcrKms::D3112. Also, transposition of D3112 from E. coli (D3112) chromosome into RP4 plasmid occurs. The phage stimulates the chromosome mobilizing activity of RP4 plasmid, similar to other transposons. E. coli (RP4::D3112) cells were previously shown to form no colonies at 30 degrees C. Auxotrophic mutants and mutants incapable of utilizing different carbohydrates were found among E. coli clones survived after a long incubation at 30 degrees C (at frequencies approximately 10(-3) - 10(-4). These mutants inherited stably the capability to produce D3112 phage. E. coli auxotrophic mutants have arisen indeed as a consequence of phage integration into the E. coli chromosome, since prototrophic transductants derived from these mutants after their treatment with generalized transducing P1 phage have lost the ability to produce D3112 phage. Clones with mutations in Km or Tc genes of RP4 plasmid, occurring at high frequencies (about 3%) were found after introduction of RP4 into E. coli (D3112). These mutant RP4 plasmids carry insertions of D3112 genomes. Clones of E. coli which lost mutant plasmids still produce D3112 and retain their initial auxotrophic mutations.

[Interaction of Pseudomonas aeruginosa plasmids and mu-like phages: the suppression of the early stages of cell infection by phage D3112 in the presence of plasmid RPL11]. / Vzaiodeistvie nekotorykh plazmid i Mu-podobnykh fagov Pseudomonas aeruginosa: podavlenie rannikh étapov infektsii kletok fagom D3112 v prisutstvii plazmidy RPL11.

The growth of Mu-like, D3112, B39 and B3 bacteriophages of Pseudomonas aeruginosa on bacterial strains containing R plasmids was studied. Plasmids RPL11, Rms148 and Rms163 were shown to interfere with phage growth: 1) D3112 and B39 phages do not produce plaques on a lawn of PAO1 (Rms148) giving e.o.p. less than 10(-9); 2) RPL11 plasmid restricts phage D3112 growth (e.o.p. less than 10(-9), the growth of phage B3 being also restricted by this plasmid, though in considerably less extent; 3) phage B39 makes small and very turbid plaques on a lawn of PAO1 (Rms163) with e.o.p. 0,13, while c mutants form clear plaques on this lawn and grow with e.o.p. 1,0. The interference of plasmid RPL11 with phage D3112 growth was examined in detail. The plasmid did not affect phage D3112 adsorption and no restriction of phage DNA in R+ cells was found. However, phage genes controlling establishment of lysogeny and the lytic cycle were not expressed after infection. It was observed though, that if a cell contains both prophage D3112 and plasmid RPL11, no interference with repressor synthesis or phage development takes place after induction of prophage. The results obtained allow to conclude that: 1) RPL11 plasmid interference with phage D3112 growth is caused by the plasmid effect on one of the early stages in the development preceding phage DNA integration; 2) the process of primary integration after infection and that of reintegration of DNA after prophage induction are likely to differ.

[Isolation and analysis of phage-resistant Pseudomonas putida mutants using new bacteriophages]. / Vydelenie i analiz fagoustoichivykh mutantov Pseudomonas putida s pomoshch'iu novykh bakteriofagov.

Three new bacteriophages of Pseudomonas putida PpG1 have been isolated. Bacteriophages differ in many characteristics and are unrelated. Phage-resistant mutants of Pseudomonas putida PpG1 are selected using these phages and pf16 bacteriophage. No lysogenic variants were detected among these mutants. They have been distributed in seven groups according to their resistance to bacteriophages. The resistance to PMW phage was caused by at least two different mutations. Some of the phage-resistant mutants seem to cause the killing effect on Pseudomonas putida PpG1 cells.

[Pseudomonas aeruginosa phages whose DNA structure is similar to the DNA structure of phage Mu 1. III. The isolation and analysis of the hybrid phages D3112 and B39: localization of the immunity region and of certain genetic factors]. / Bakteriofagi Pseudomonas aeruginosa, struktura DNK kotorykh skhodna so strukturoi DNK faga Mu 1. Soobshchenie III. Vydelenie i analiz gibridov fagov D3112 i B39: lokalizatsiia oblasti immuniteta i nekotorykh geneticheskikh faktorov.

Several different hybrids have been isolated in a cross between heteroimmune Mu-like Pseudomonas aeruginosa bacteriophages D3112 and B39. Analysis of hybrid phage DNAs treated with specific endonucleases and heteroduplex studies have permitted to map the immunity region of phages in the left part of their genomes. Also, a ts-mutation in the wild-type B39 and some genetic factors influencing the growth of D3112 in P. aeruginosa strain PA0 (B39) and in bacteria harbouring RPL11 plasmid, have been localized. All recombinants studied have arisen by double crossingover and can be considered as substitutions of different continuous parts of the D3112 genome by fragments of the B39 genome. A comparison of distribution of non-homologous regions in B39 and D3112 genomes as well as locations of endonuclease-sensitive sites have given some unexpected results. In contrast to the B39-D3112 pair, the other pair of related Mu-like phages, B3-D3112, have no visible homology within the most part of their genomes.

[Pseudomonas aeruginosa bacteriophages with DNA structure similar to the DNA structure of Mu1 phage. II. Evidence for similarity between D3112, B3, and B39 bacteriophages: analysis of DNA splits by restriction endonucleases, isolation of D3112 and B3 recombinant phages]. / Bakteriofagi Pseudomonas aeruginosa, struktura DNA kotorykh skhodna so strukturoi DNK faga Mu1. Soobshchenie II. Dokazatel'stvo rodstva bakteriofagov D3112, B3 i B39: analiz rasshchepleniia DNK éndonukleazami restriktsii, vydelenie rekombinanta fagov D3112 i B3.

It is found that bacteriophages B3 and B39 specific for Pseudomonas aeruginosa have the same genome structure as previously described phage D3112. On the right (S) end of their genomes a variable non-phage DNA is located (approximately 0.9-2.5 kilobases for different phages). It is probable that this variable DNa has its origin from different regions of bacterial chromosome. In genome of one of the phages, B3 phage, such variable DNA (not more than 150 base pairs) was found on the left end of DNA molecule. Isolation of a viable B3XD3112 recombinant phage and analysis of its genome with restriction technique and with studies of homo- and heteroduplex molecules had confirmed genetical relationship of B3 and D3112. Some essential non-homology of B3 and D3112 DNAs have been found on the right ends of genomes of the phages.