Use of deletion mutants of plasmid RP4::D3112 for the genetic analysis of Pseudomonas aeruginosa bacteriophage D3112

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

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.     

Compatibility of transposable phages of Escherichia coli and Pseudomonas aeruginosa. I. Co-development of phages Mu and D3112 and integration of phage D3112 into RP4::Mu plasmid in Pseudomonas aeruginosa cells

The possibility of using a model system (which included RP4::Mu plasmid and D3112 phage in Pseudomonas aeruginosa cells) for analysis of compatibility of transposable Escherichia coli phage Mu and P. aeruginosa phage D3112, as phages and transposons, was studied. No interaction was observed during the vegetative growth of phages. The majority of the hybrid RP4::Mu plasmids lost the Mu DNA after insertion of D3112 into RP4::Mu. The phenomenon was not a result of transposition immunity. We consider the loss of the Mu DNA as a consequence either of plasmid RP4::Mu instability in P. aeruginosa cells, because of the lack of functional Mu repressor, or of some D3112-encoded activity involved in its transposition. For the inambiguous conclusion on compatibility of two phages as transposons, it is necessary to modify the model system, eliminating the possibility of Mu phage replication--transposition.     

Features of genome expression of phage transposon D3112 of Pseudomonas aeruginosa in Escherichia coli bacteria: dependence of bacterial phenotype on copy number of D3112 genome]

Escherichia coli (RP4 :: D3112) bacteria manifest Tcs phenotype (thirty centigrade sensitivity), i.e. the cells do not divide and form colonies under conditions of lowered temperature (30 degrees C and lower), while cells grow normally at 42 degrees C. In this work it is demonstrated that replication-transposition of D3112 and the Tcs phenotype depend on no recA system of E.coli. Following events lead to the loss of the Tcs phenotype (in E.coli (RP4 :: D3112) cells survived after growing at 30 degrees C): occurrence of mutations in bacterial, phage and plasmid genomes, elimination of DNA of hybrid plasmid or RP4 DNA (a portion of DNA) as well as integration of the hybrid plasmid into bacterial chromosome. In the latter case, the E.coli (D3112) cells acquired the properties shared by the initial bacteria and those with the Tcs phenotype. Such clones are designated tcl (thirty centigrade low sensitivity), they are able to form colonies at 30 degrees C but their growth is more slow, they maintain instability at lowered temperature and continue to produce D3112 phage. The tcl clones in which replication-transposition of D3112 DNA in less effective than in the tcs clones are a suitable object for the study of genetic rearrangements caused by D3112 phage transposon. It is shown that either complete RP4 genome or its portion are comprised between direct repeats of D3112 and are built into various chromosomal sites, i.e. cointegrates are being formed. Two types of deletions are revealed: eliminating sites of RP4 plasmid adjacent to the left end of D3112 genome as well as deletions of the D3112 genome. It is demonstrated that alteration in the growth nature of E.coli, carrying D3112 DNA, at 30 degrees C depends on the copy number of D3112 per bacterial cell.     

Effective, plasmid RP4-dependent replication-transposition of DNA of the transposable phage D3112 of Pseudomonas aeruginosa in a heterologous Escherichia coli system

The processes of replication and transposition of Pseudomonas aeruginosa transposable phage D3112 in cells of Escherichia coli (D3112) and E. coli (RP4::D3112) were studied. D3112 genome is a "silent cassette" ("conex-phage"--conditionally expressible) in E. coli cells incubated at 42 degrees C. Two compulsory conditions for D3112 genome expression are incubation at 30 degrees C and the presence in cells of RP4 plasmid. Processes of replication and transposition in E. coli are coupled. RP4 plasmid stimulates D3112 DNA synthesis in E. coli at least by two order of magnitude. In correspondence with this observation is the fact that when Mg2+ is present in high concentration (0.1 M) in a cultural medium, the production of mature phage is enhanced by two order of magnitude in E. coli (RP4::D3112) or in E. coli (D3112, RP4) cells, and is approx. 10(-1)-10(-2) phage per cell. No influence of Mg on phage production is observed in E. coli (D3112) cells.     

Expression of the genome of Mu-like phage D3112 specific for Pseudomonas aeruginosa in Escherichia coli and Pseudomonas putida cells

The behavior of Escherichia coli cells carrying RP4 plasmid which contains the genome of a Mu-like D3112 phage specific for Pseudomonas aeruginosa was studied. Two different types of D3112 genome expression were revealed in E. coli. The first is BP4-dependent expression. In this case, expression of certain D3112 genes designated as "kil" only takes place when RP4 is present. As a result, cell division stops at 30 degrees C and cells form filaments. Cell division is not blocked at 42 degrees C. The second type of D3112 genome expression is RP4-independent. A small number of phage is produced independently of RP4 plasmid but this does not take place at 42 degrees C. No detectable quantity of the functionally active repressor of the phage was determined in E. coli (D3112). It is possible that the only cause for cell stability of E. coli (D3112) or E. coli (RP4::D3112) at 42 degrees C in the absence of the repressor is the fact of an extremely poor expression of D3112. In another heterologous system, P. putida both ways of phage development (lytic and lysogenic) are observed. This special state of D3112 genome in E. coli cells is proposed to be named "conditionally expressible prophage" or, in short, "conex-phage", to distinguish it from a classical lysogenic state when stability is determined by repressor activity. Specific blockade of cell division, due to D3112 expression, was also found in P. putida cells. It is evident that the kil function of D3112 is not specific to recognize the difference between division machinery of bacteria belonging to distinct species or genera. Protein synthesis is needed to stop cell division and during a short time period this process could be reversible. Isolation of E. coli (D3112) which lost RP4 plasmid may be regarded as an evidence for D3112 transposition in E. coli. Some possibilities for using the system to look for E. coli mutants with modified expression of foreign genes are considered.     

Introduction of the hybrid plasmid RP4::D3112 into Pseudomonas putida cells requires the presence of specific mutation in the phage genome

The wild type of D3112, a transposable phage of Pseudomonas aeruginosa can not be introduced as a portion of the hybrid plasmid RP4::D3112 into Pseudomonas putida cells. It is only possible when phage D3112 carries mutations designated lpc (lethal for P. putida and Escherichia coli). Analysis of heteroduplex molecules between DNAs of phages D3112w+ and D3112lpc demonstrated the absence of nonhomology regions, which suggests that lpc is a point mutation. The lpc2 mutation was located within the interval 20-29.9 kb of the phage genome.     

Plasmid transduction using the D3112DeltaH miniphage in Pseudomonas aeruginosa cells]

Plasmid DNA transduction with mini-D3112 delta H, deletion derivative of phage D3112, which lost the genes essential for phage growth but retained the sites required for transposition and packaging was studied. Unlike D3112, mini-D3112 delta H element can transduce plasmids and plasmid markers at frequencies of 10(-5)-10(-8) in rec+ cells of Pseudomonas aeruginosa. Plasmids R1162 and R388 of the size smaller than phage genome were transduced intact. Large plasmids, like RP4 and R151, were deleted under transduction. By this way, we isolated deletion derivatives of RP4. The smallest derivative pN2 contained a 4.5 kb fragment of RP4. Unlike the latter, pN2 plasmid had narrow host range and did not maintain in Escherichia coli cells.     

Coupling and rec-independence of the processes of replication and transposition of Pseudomonas aeruginosa phage D3112. The effect of the phage genes controlling the replication of DNA of D3112

D3112 phage was shown to replicate via the process of coupled replication--transposition: the phage DNA is not excised from the chromosome after prophage induction and new phage copies insert into many different sites. The transposition is controlled by two D3112 early genes--A (mapped in the 1.5-3 kbp region) and B (3-4.5 kbp), and requires intact attL site (involvement of the phage right end attR not studied). D3112 is capable to transpose RP4 plasmid into the chromosome; both the D3112 and RP4 transpositions are rec-independent. The product of the early C gene which is not required for D3112 transposition has pleiotropic effect on the development of D3112 and is necessary for the process of D3112 DNA excision from the chromosome, for cell lysis as well as for mature phage production. We suggest that this gene is responsible for positive regulation of D3112 late genes expression, similar to the C gene of Mu phage or Q gene of lambda. Mutations in four D3112 late genes ts25, ts35, ts73 and ts110 do not affect transposition or excision processes. No detectable (less than 0.02 copies per cell) amount of linear or circular D3112 DNA is formed during the replication--transposition. Hence, in the course of replication and transposition processes D3112 genome has its ends permanently bound covalently to the chromosome. The excision of the D3112 DNA takes place at late stages.     

Transfer of chromosomal genes and formation of R'-derivatives using PR4::D312cts15 plasmids in Pseudomonas aeruginosa cells

Several hybrid RP4 plasmids containing the genome of heat-inducible D3112cts15 phage integrated into 2 different sites of RP4 were selected. It was shown that the plasmids RP4::D3112cts15 mobilized the chromosome of Pseudomonas aeruginosa from many sites located in different chromosome regions. Chromosomal recombinants are, formed at frequencies of about 10(-4) per recipient cell. Analysis of coinheritance of unselected markers showed that the majority of recombinants inherited short donor chromosome fragments (about 5 min). R' plasmids can be easily selected by mating with a rec- recipient. For instance, the frequency of selection of R' plasmids containing argH+ locus was about 10(-5) per donor cell. Conjugative transfer of RP4::D3112cts15 into nonlysogenic strains PAO P. aeruginosa results in partial or complete loss of prophage from a hybrid plasmid. The RP4::D3112cts15 plasmids appear to have retained the broad host range of the original RP4 (they are maintained in P. putida and Escherichia coli).     

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.     

Erwinia carotovora as a recipient system for the natural hybrid plasmid RP4::Mu cts62

The plasmid RP4::Mu cts62 is transferred from Escherichia coli cells into a recipient strain Erwinia carotovora 268 by conjugation with the frequency 1.5-5 x 10(-7) per donor cell. The maximal frequencies of transfer are obtained by cultivation of donor and recipient cells for 3-5 h on the filters. Structural and functional validity of the plasmid in transconjugants is expressed in preservation of all antibiotic-resistant markers of RP4, thermosensitivity to growth at 42 degrees C as well as spontaneous and thermally-induced production and zygotic induction of bacteriophage determined by the genome of Mu cts62, total length of the plasmid restricts. Location and orientation of Mu cts62 genome in the plasmid restricts. Location and orientation of Mu cts62 genome in the plasmid RP4::Mu cts62 in Erwinia carotovora transconjugant cells has been determined. A single bacteriophage genome has been shown to transpose into the chromosome of the cell with the elimination of RP4 fragment under the conditions of thermal induction.     

Expression of Pseudomonas aeruginosa transposable phages in Pseudomonas putida cells. I. The establishment of a lysogenic state and the effectiveness of lytic development

Expression of transposable phages (TP) of Pseudomonas aeruginosa in the cells of P. putida was studied. The high efficiency of phage lytic development was shown both as a consequence of zygotic induction after transfer of the RP4::TPc+ plasmid into nonlysogenic recipients, and as a result of heat induction of lysogens PpG1 (D3112cts15). The high phage yield (20-25 particles of D3112cts phage per one cell of P. putida) is an evidence for a high level of transposition in the cells of this bacterial species. Plasmids RP4::TP are transferred into cells of PpG1 and PAO1 with similar frequency. However, the efficiency of establishment of the lysogenic state is lower in PpG1. Transposable phages of P. aeruginosa can integrate into the chromosome of PpG1 producing stable inducible lysogens. The presence of RP4 in the P. putida cells is not necessary for expression of transposable phages. The transposable phage D3112cts15 can be used in experiments of interspecies transduction of plasmids and chromosomal genes.     

Characteristics of phages-transposons of Pseudomonas aeruginosa belonging to 2 groups distinguished by DNA-DNA homology

14 new transposable phages (TP) were isolated from approx. 200 clinical isolates of Pseudomonas aeruginosa. The frequent occurrence of TP of P. aeruginosa has been confirmed. There are at least two different groups of TP, namely, the group of D3112 and that of B3. The distinctive features of phages belonging to the groups are as follows: 1) low level of DNA-DNA homology (less than 10%), the whole region of homology in phage genomes of different groups being located on right genome end (29-38 kb); only one of phages of the B3 group shows an additional homology with D3112 DNA outside the above mentioned region; 2) a variable DNA is observed on the left end of the B3 group phage genomes and no such DNA is revealed on the left end of genomes of the D3112 group phages; 3) all phages of the B3 group have specific type of interaction with RPL11 plasmid, which distinguish them from phages of the D3112 group; 4) phages belonging to the two groups differ greatly in their growth in cells harbouring pMG7 plasmid which mediates production of PaeR7 endonuclease and in the number of DNA sites sensitive to SalGI, PstI, BglII endonucleases. Since some of the B3 group phage genomes possess BamH1 sites, resistance to this enzyme cannot be regarded as a general characteristics for all TP of P. aeruginosa, as it was earlier proposed. Some aspects of modular hypothesis of bacteriophage evolution concerning, in particular, the ways of module formation are discussed.     

Introduction of bacteriophage Mu into bacteria of various genera and intergeneric gene transfer by RP4::Mu.

The host range of coliphage Mu was greatly expanded to various genera of gram-negative bacteria by using the hybrid plasmic RP4::Mu cts, which is temperature sensitive and which confers resistance to ampicillin, kanamycin, and tetracycline. These drug resistance genes were transferred from Escherichia coli to members of the general Klebsiella, Enterobacter, Citrobacter, Salmonella, Proteus, Erwinia, Serratia, Alcaligenes, Agrobacterium, Rhizobium, Pseudomonas, Acetobacter, and Bacillus. Mu phage was produced by thermal induction from the lysogens of all these drug-resistant bacteria except Bacillus. Mu phage and RP4 or the RP4::Mu plasmid were used to create intergeneric recombinant strains by transfer of some genes, including the arylsulfatase gene, between Klebsiella aerogenes and E. coli. Thus, genetic analysis and intergeneric gene transfer are possible in these RP4::Mu-sensitive bacteria.     

The infection of bacilli by Mu cts phage integrated into the plasmid

The infection of Bacillus thuringiensis, B. cereus, B. mesentericus and B. polymyxa strains with temperate E. coli bacteriophage Mu cts62 integrated into plasmid RP4 under conditions of conjugative transfer is shown possible. The investigated strains of bacilli are not able to produce intact phage particles but they acquire the thermosensitive property determined by the phage genome. Gel electrophoresis and blot hybridization of DNA have confirmed the transfer of Mu cts62 genome or a part of it in the investigated strains of bacilli.     

Internal EcoRI-generated deletion in prophage Mu DNA

The hybrid plasmid consisting of the plasmid pRP1.2 (derivative of RP4) genome and deleted prophage Mucts 62 genome which lost the central EcoRI fragment of DNA was constructed. The ability of deleted Mu phage to carry out E. coli chromosomal genes transposition was still retained.     

Conjugation in Rhodopseudomonas sphaeroides mediated by R plasmids

Plasmids R68.45, RP4, RP4::Mu cts62, RP1ts::Tn10, RP1ts::Tn9, Rts1 and RP41 were transferred into cells of photosynthetic nitrogen-fixation bacterium Rhodopseudomonas sphaeroides from Escherichia coli and Pseudomonas aeruginosa. The transfer of plasmids occurred with high frequency of 10(-1) to 10(-2) per donor cell in all cases. Mobilization of R. sphaeroides 2R chromosome was obtained by RP4 and Rts1 plasmids at a frequency of 10(-7) to 10(-8) per donor cell in all cases. Mobilization of R. sphaeroides 2R chromosome was obtained by RP4 and Rts1 plasmids at a frequency of 10(-7) to 10(-8) per donor cell. Bacteriophage Mu cts62 could be induced from the plasmid DNA in R. sphaeroides 2R cells and was capable of the lytic growth and producing phage progeny. It was demonstrated that an increase in the efficiency of donor chromosomal genes transfer into recipient cells could be achieved in crosses with the donor carrying RP4::Mcts62 plasmid.     

Effect of the polymerase activity of DNA polymerase I on the development of moderate bacteriophages Mu, lambda red- and lambda red-gam-. II. The effect of the polymerase activity of DNA polymerase I on the development of bacteriophage Mu

The paper reports on the influence of polymerizing activity of DNA-polymerase I on different developmental stages of temperate bacteriophage Mu in Escherichia coli K-12 cells. This activity is shown to be necessary for optimization of phage Mu primary integration into cell chromosomes. The relative frequency of Mu integration into bacterial chromosomes is 5-6 times lower in polA cells than in isogenic polA+ control strains, the phage yield from cells being delayed during the phage infectious development, but not in the course of induction from the prophage state. Data have been obtained that show the process of phage Mu DNA integration into the plasmid pRP1 .2 and the process of Mu transposition from the cell chromosome into the plasmid to be independent of the polymerizing activity of DNA-polymerase I.