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.     

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.     

2 types of molecular structure (composition) of a genome in one species of transposable bacteriophages of Pseudomonas aeruginosa

Comparison of heteroduplexes (HD) between DNAs of different transposable phages of Pseudomonas aeruginosa belonging to two previously described subgroups (D3112 and B3) revealed two types of structure (composition) of the bacteriophages, designated "type A" and "type B". The properties of genome structure of type A (phages of D3112 subgroup) are as follows: high level of conservation (up to 70% of genomes of different phages are represented as blocks of homologous DNA sequences); substitutions in genomes revealed as nonhomology regions in HD are, as a rule, small and located in certain sites; the distribution of the nonhomologous regions in HD of these phages is highly reproducible in independent experiments. Bacteriophages of subgroup B3 have genomes of type B: only a small part (approx. 30%) of genomes retain homology general for all of the phages; the nonhomologous regions are distributed in a large number of sites in HD; the sizes of nonhomologous regions are substantially larger than for the phages of subgroup D3112; distribution of the regions in HD is highly variable, which is characteristic of DNAs with partial homology. There is no difference between genomes of types A and B in G + C content (approx. 61-63%). Viable recombinants can be formed in crosses between phages of different genome types not only in regions with earlier revealed large DNA/DNA homology (right ends of genomes), but also in central portions of the genomes. Nevertheless, functional incompatibility of some regions of phage genomes of types A and B was demonstrated.     

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.     

Complete genomic sequence of bacteriophage B3, a Mu-like phage of Pseudomonas aeruginosa

Bacteriophage B3 is a transposable phage of Pseudomonas aeruginosa. In this report, we present the complete DNA sequence and annotation of the B3 genome. DNA sequence analysis revealed that the B3 genome is 38,439 bp long with a G+C content of 63.3%. The genome contains 59 proposed open reading frames (ORFs) organized into at least three operons. Of these ORFs, the predicted proteins from 41 ORFs (68%) display significant similarity to other phage or bacterial proteins. Many of the predicted B3 proteins are homologous to those encoded by the early genes and head genes of Mu and Mu-like prophages found in sequenced bacterial genomes. Only two of the predicted B3 tail proteins are homologous to other well-characterized phage tail proteins; however, several Mu-like prophages and transposable phage D3112 encode approximately 10 highly similar proteins in their predicted tail gene regions. Comparison of the B3 genomic organization with that of Mu revealed evidence of multiple genetic rearrangements, the most notable being the inversion of the proposed B3 immunity/early gene region, the loss of Mu-like tail genes, and an extreme leftward shift of the B3 DNA modification gene cluster. These differences illustrate and support the widely held view that tailed phages are genetic mosaics arising by the exchange of functional modules within a diverse genetic pool.     

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.     

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).     

Superinfection exclusion reveals heteroimmunity between Pseudomonas aeruginosa temperate phages

Temperate siphophages (MP29, MP42, and MP48) were isolated from the culture supernatant of clinical Pseudomonas aeruginosa isolates. The complete nucleotide sequences and annotation of the phage genomes revealed the overall synteny to the known temperate P. aeruginosa phages such as MP22, D3112, and DMS3. Genome-level sequence analysis showed the conservation of both ends of the linear genome and the divergence at the previously identified dissimilarity regions (R1 to R9). Protein sequence alignment of the c repressor (ORF1) of each phage enabled us to divide the six phages into two groups: D3112 group (D3112, MP29, MP42, and MP48) and MP22 group (MP22 and DMS3). Superinfection exclusion was observed between the phages belonging to the same group, which was mediated by the specific interaction between the c repressor and the cognate operator. Based on these, we suggest that the temperate siphophages prevalent in the clinical strains of P. aeruginosa represent at least two distinct heteroimmunity groups.     

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).     

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.     

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.     

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.     

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.     

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.     

Genetic control of morphogenesis of Pseudomonas aeruginosa transposable phage D3112

The influence of ts mutations in the early and late genes of transposable phage D3112 on phage morphogenesis was studied. The mutations in the early genes A, B and C were shown to suppress morphogenesis of D3112. Six genes (D, E, F, G, H and I), located from 14 to 29 kbp of the phage physical map, control morphogenesis of phage head. Five genes (J, K, L, M and N), clustered in the 29-36 kbp region of the map, control morphogenesis of tail. The similarity of genetic organization of the Escherichia coli transposable phage Mu and the Pseudomonas aeruginosa phage D3112 is discussed.