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.     

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.     

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.     

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.     

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.     

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).     

Genetic map of the transposable phage D3112 of Pseudomonas aeruginosa

Using a large group of newly isolated deletion mutants of prophage D3112 the location of all known mutations of D3112 phage was more precisely defined. The mutations affecting establishment of lysogenic state were mapped in two regions of the genome- 0-1.3 and 29-30 kb. The replicative A gene is mapped between 1.3 and 4.9 kb, the second replicative B gene being situated on the right of the A gene, between 4.9 and 9.4 kb. The C gene which is responsible for positive regulation of phage late genes' expression is mapped within the 9-12 kb region. It is suggested that promoter of the gene C is situated within the same interval. Mutations were isolated in the Lys gene which is responsible for host cell lysis. The gene is located within the interval 14-22 kb of the physical map. The order of morphogenetic genes in the late genome region was also established.     

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.     

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.     

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.     

Mini-D3112 bacteriophage transposable elements for genetic analysis of Pseudomonas aeruginosa.

Small bacteriophage D3112 transposable elements deleted for most of the phage-lytic functions while retaining the sites required for transposition and packaging were constructed to facilitate genetic studies in Pseudomonas aeruginosa. These mini-D derivatives were constructed with the terminal 1.85 kilobases (kb) of the phage left end and 1.4 kb of the phage right end and either the Tn5 kanamycin resistance or the pSC101 (pBR322) tetracycline resistance determinant. Thermally induced lysates of strains lysogenic for both a mini-D element and D3112 cts (temperature-sensitive repressor) transduced P. aeruginosa PAO recipients to drug resistance at frequencies of between 10(-4) and 10(-5)/PFU of the helper phage. As for the parent plaque-forming D3112 phage, the mini-D171 element could insert itself into many different sites in the chromosome but the frequency of insertion into particular genes varied widely. Among 1,000 insertions, none resulted in auxotrophy but 10 resulted in pigment production. Insertions were also selected in a cloning plasmid with a transduction scheme. At least eight different insertion sites were found to have been used among 10 individual insertions. Transductants harboring these mini-D elements were immune to infection by D3112, since they contained the D3112 repressor gene in the left 1.85-kb terminal fragment. Chromosomal genes were transduced in a generalized fashion 100 to 1,000 times more frequently by the mini-D-D3112 cts lysates than by the D3112 cts phage alone. Mini-D171-D3112 cts lysates also yielded some transductants that retained the drug resistance marker of the mini-D element and which were unstable for the chromosomal transduced marker. This is consistent with the miniduction properties of Mu whereby transduced genes are flanked by two mini-D elements in the same orientation.     

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.     

Complete genome sequence of Pseudomonas aeruginosa lytic bacteriophage PA1O which resembles temperate bacteriophage D3112

A novel Pseudomonas aeruginosa lytic bacteriophage (phage), PA1Ø, was isolated, and its genome was sequenced completely. This phage is able to lyse not only P. aeruginosa but also Staphylococcus aureus. Genome analysis of PA1Ø showed that it is similar to a P. aeruginosa temperate phage, D3112, with the exception of the absence of a c repressor-encoding gene, which is known to play a critical role in the maintenance of the lysogenic state of D3112 in P. aeruginosa.     

Insertion and replication of the Pseudomonas aeruginosa mutator phage D3112

D3112 is a temperate bacteriophage of P. aeruginosa with heterogeneous sequences at one extremity of the virion DNA molecule. Infection of strain PAOl with phage D3112 results in a 40- to 65-fold increase in the frequency of ami mutants resistant to fluoroacetamide. Nine ami::D3112 prophages have been mapped to distinct sites within the ami locus by Southern blotting experiments with a cloned ami+ probe. All prophages have the same restriction map as the D3112 genome extracted from phage particles. The position of D3112 insertions correlates with the phenotype and reversion behavior of the ami mutants. Induction of D3112cts prophages results in amplification of internal prophage segments as discrete restriction fragments before the terminal viral fragments are visible as sharp hybridizing species. This indicates that D3112 replication is accompanied by recombination of prophage termini to numerous sites in the bacterial genome. Chromosomal junction fragments of an ami::D3112cts prophage are maintained through most of the replication cycle but are cleaved shortly before cell lysis, apparently by the viral encapsidation system.     

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.