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

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.     

Stimulation of the lysogenization of Pseudomonas aeruginosa cells by phage transposon B39 induced by plasmid Rms163

The influence of Rms163 plasmid on lysogenization of Pseudomonas aeruginosa cells by B39 phage was studied. Plasmid Rms163 was shown to increase the frequency of lysogenization of PAO1 cells 7-8 times. C-mutants of B39 phage were isolated. According to complementation test, c-mutants were distributed into two groups--cI and cII/III. The product of cI is essential for establishment and maintenance of lysogenic state, cII/cIII product being only necessary for establishment of lysogenization. The mutants with special characteristics were isolated: B39cx1 phage carries a mutation which seems to be located on a regulatory site essential for establishment of lysogenic state. The region of the B39 genome responsible for interaction with Rms163 plasmid was mapped. Possible mechanisms of Rms163 plasmid interference with transposable B39 phage are discussed.     

The effect of R-plasmids on the reproduction of Pseudomonas aeruginosa phage SM

The inheritance of plasmids Rms163 and R74 by Pseudomonas aeruginosa strain PAO hs been shown to effect the reproduction of a temperature bacteriophage SM. The decrease in plating efficiency of bacteriophage on Pseudomonas aeruginosa PAO (rms163) lawn is explained by the high degree of cell lysogenization by bacteriophage. Plasmid R74 inhibits bacteriophage SM propagation ultimately, evidently due to interruption of definite stages in vegetative development of bacteriophage by the products of plasmid specific genes.     

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.     

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.     

Properties of natural interspecific hybrids of transposable phages of Pseudomonas aeruginosa: specific characteristics of phage PL24 transposition

Properties of natural hybrid transposable phages (TP) of Pseudomonas aeruginosa, including phage PL24 and lysogens for this phage, were studied. PL24 possesses the properties of TP from two previously described groups, B3 and D3112. Its genome, unlike the genome of D3112, contains many sites susceptible to the SalGI restriction endonuclease and possesses no more than 100 nucleotides of bacterial origin located at the left genome end. However, unlike B3, phage PL24 failed to induce auxotrophic mutants upon integration in the bacterial genome. This phage differed from both B3 and D3112 in sensitivity to chloroform treatment. A more detailed examination of a group containing 25 randomly isolated lysogens for phage PL24 revealed previously unknown processes occurring at early stages of bacterial lysogenization. There are at least two different modes of cell lysogenization with phage PL24. In the first case, the emerging lysogens contained a single prophage genome located (in each lysogen) at individual sites. In the second case, polylysogenic bacteria appeared, and, after primary integration of a phage genome, replicative transposition occurred at new sites (often accompanied by the appearance of prophage clusters at these sites). The choice of the mode of lysogenization can be determined both by differences in the physiological state of bacteria and by specific features of phage PL24, which possibly affect the time of repressor accumulation to the concentration sufficient for blocking phage growth or the stability of the lysogenic state.     

Expression of Pseudomonas aeruginosa phage transposons in Pseudomonas putida PpG1 cells. II. Zygotic induction--a necessary condition for the formation of defective lysogens

The transfer of hybrid plasmid RP4::PT (where PT is the genome of a transposable phage specific for Pseudomonas aeruginosa) into recipient cells of P. putida strain PpG1 occurs with the same frequency as into P. aeruginosa, the homologous host for PT. Approximately 1/3 of all PpG1 exconjugants carrying RP4 markers lost the capability to produce viable PT phage. In contrast, in a cross with homologous recipient P. aeruginosa all exconjugant clones contained nondefective prophages in the hybrid plasmids. Zygotic induction is an obligatory condition for detection of PpG1 exconjugants with defective phages. The defective prophages in RP4::PT hybrid plasmids have deletions of different size; the other carry mutations indistinguishable from point mutations in an essential phage gene. Some of deletions also cover plasmid genes. At least some of the defective prophages, including deleted ones, have arisen in the recipient cells of P. putida after transfer of the hybrid plasmid.     

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.     

Phage-transposon interaction: the cip locus of prophage D3112 responsible for the inhibition of integration and transposition of related phage B39 of Pseudomonas aeruginosa

Bacterial cells lysogenic for D3112, a transposable Pseudomonas aeruginosa phage restrict the growth of a related heteroimmune B39 phage. The lysogens are divided into two different types PAO(D3112). In the lysogens of the type I the efficiency of B39 growth only decreases slightly, the lysogens of the type II restricting completely the growth of this phage (e.o.p. is less than 10(-7). As shown by the results of Southern hybridization experiments, lysogens of the type I are monolysogens, while those of the type II are double or polylysogens. Restriction of B39 in PAO(D3112) is caused by expression of a locus in the D3112 genome. The locus has been termed as cip (control of interaction of phages). The cip locus was mapped at the interval 1.3-2.45 kb of the D3112 physical map using different deletion derivatives of D3112. Expression of cip only takes place in the prophage state and not during the phage lytic development. When expressed, cip affects the early steps in the growth of B39 lowering the level of integration and transposition processes; the effect is not dependent on the way of initiation of the lytic cycle (through prophage induction or infection).     

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.     

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.     

Pseudomonas aeruginosa transposable bacteriophages D3112 and B3 require pili and surface growth for adsorption.

Pseudomonas aeruginosa transposable bacteriophages D3112 and B3 were found to require pili for infection. Seventy mutants of P. aeruginosa PAO selected by resistance to D3112 or B3 were also resistant to the phage not used in the selection and suggested that the receptors of these two phages are identical. Of five resistant mutants examined, all were defective in the production of pili and did not adsorb either phage. P. aeruginosa PAK strains altered in pilus expression, such as hyperpiliated or nonpiliated mutants, adsorbed the phage but were not productively infected, implying that an additional host function was required for infection. The cell-associated lipopolysaccharide was not required for D3112 or B3 infection, since mutants deficient in O side-chain and core biosynthesis were still capable of adsorption and productive infection. This is in contrast to Escherichia coli mutator phages Mu and D108, which are dependent on lipopolysaccharide for adsorption. The P. aeruginosa phages adsorbed only to cells grown on solid media or in liquid media supplemented with agents that increase the macroviscosity, such as polyvinylpyrrolidone. Adsorption time course studies of D3112 and B3 using cells grown in solid media revealed similar but not identical adsorption patterns. These studies suggested that expression of the D3112 and B3 cell receptor is induced by growth on solid media.     

Multiplicity of the integration sites of Mu-like bacteriophages into Pseudomonas aeruginosa chromosome and plasmids

It has been shown that D3112 prophage can be integrated into different chromosomal sites of Pseudomonas aeruginosa. The other Mu-like phages (B3, B39, PM69) are capable to insert their genomes during infection process into the plasmids RPL11, RMS148, RMS163. Their integration is occasionally accompanied by formation of mutations in plasmid genes. The certain types of auxotrophic and morphological mutants (thi, met, pigmented, met - pigmented) can be found at a frequency about 10% among survivors after a long (48 h) incubation at 42 degrees C of PAO (D3112cts15) or PAO (B39cts1) lysogens. The spectrum of mutants might depend on the time of heat induction. After a short exposure (10-20 min), arg and pigmented mutants can be found. Accumulation of certain kinds of mutants after heat induction is quite a specific phenomenon for Mu-like phages; heat induction of PAO (F116ts245) does not lead to selection of these specific bacterial mutants (F116 is unrelated to Mu-like phages and has extrachromosomal location).     

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.     

Study of the diversity in a group of phages of Pseudomonas aeruginosa species PB1 (Myoviridae) and their behavior in adsorbtion-resistant bacterial mutants

A group of 12 Pseudomonas aeruginosa virulent bacteriophages of different origin scored with regard to the plaque phenotype are assigned to PB1-like species based on the similarity in respect to morphology of particles and high DNA homology. Phages differ in restriction profile and the set of capsid major proteins. For the purpose of studying adsorption properties of these phages, 20 random spontaneous mutants of P. aeruginosa PAO1 with the disturbed adsorption placed in two groups were isolated. Mutants of the first group completely lost the ability to adsorb all phages of this species. It is assumed that their adsorption receptors are functionally inactive or lost at all, because the attempt to isolate phage mutants or detect natural phages of PB1 species capable of overcoming resistance of these bacteria failed. The second group includes five bacterial mutants resistant to the majority of phages belonging to species PB1, These mutants maintain the vigorous growth of phage SN and poor growth of phage 9/3, which forms turbid plaques with low efficiency of plating. In the background of weak growth, phage 9/3 yields plaques that grew well. The examination of the progeny of phage 9/3, which can grow on these bacteria, showed that its DNA differed from DNA of the original phage 9/3 by restriction profile and is identical to DNA of phage PB1 with regard to this trait. Data supported a suggestion that this phage variant resulted from recombination of phage 9/3 DNA with the locus of P. aeruginosa PAO1 genome encoding the bacteriocinogenic factor R. However, this variant of phage 9/3 did not manifest the ability to grow on phage-resistant mutants of the first group. Possible reasons for the difference between phages 9/3 or SN and the remaining phages of PB1 species are discussed. A preliminary formal scheme of the modular structure for adsorption receptors on the surface of P. aeruginosa PAO1 bacteria was constructed based on the analysis of growth of some other phage species on adsorption mutants of the first type.     

Modular structure of the genes of phages-transposons of Pseudomonas aeruginosa

The basic criterion to confirm the recombinational origin of bacteriophages belonging to the same phage family is revealing several different combinations of differentiated segments in phage genomes which determine specific functions (modules). The results of phage-to-phage comparison of several regions in genomes of closely related transposable phages of Pseudomonas aeruginosa D3112, B39, PH2, PH51, PH93, PH132 have supported the modular hypothesis for this group of phages.     

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.     

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

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.     

Differences in the allele state of genes controlling the specificity of adsorption in transposable phages of Pseudomonas aeruginosa

To reveal possible differences in adsorptional specificities of transposable phages of Pseudomonas aeruginosa and to study the genetical control of this character, we isolated a group of phage-resistant P. aeruginosa mutants using some temperate and virulent phages. The study of resistance of the mutants to all the phages permitted us to find some types of mutants and to build a formal scheme of distribution of adsorptional receptors on the surface of P. aeurginosa cell. According to the results obtained, there are two main "receptor chains", where the receptors for all phages under study are grouped. For the majority of phages, just a single adsorptional receptor is obligatory, and at least two essential receptors are needed for adsorption of virulent phage E79. Two receptors were found also for another virulent phage, phi 11, one of them only being essential. Transposable phages can be grouped into three types, according to their adsorptional specificities. No correlations of adsorptional specificity types and all other characteristics of transposable phages studied (including the sub-groups of transposable phages belonging to different DNA homology types) were found. Genes of natural transposable phages controlling the differences in adsorptional specificities revealed can recombine in phage crosses.