Two loci in the genome of the transposable phage B39 of Pseudomonas aeruginosa affecting the process of integration. I. Study of the properties of phages with the Pde- phenotype and mapping of the rms site

Mutants and recombinants of transposable Pseudomonas aeruginosa bacteriophage B39 with a specific phenotype Pde- (pleiotropic developmental effect) were studied. Pde- phages produce clear minute plaques on lawns of P. aeruginosa PAO1 and fail to grow in cells of PAO1 harbouring Rms 163 (Inc P5) plasmid. Pde+ character is under control of the two loci in phage genome which were designated pdeX and pdeY. In hybrid phages the pdeX and pdeY loci originating from different transposable phages (pdeX from B39 and pdeY from PH132) do not accomplish their function and, as a result, the hybrid phages have the Pde- phenotype. The frequency of integration (f.o.i.) of Pde- phages into bacterial chromosome is lower than f.o.i. for Pde+ phages, as well as the frequency of stable lysogenization of infected bacteria; lytic development of the Pde- phages is also limited. The great difference among the transposable phages in their reaction to the presence of Rms163 plasmid is caused by some differences in the specific rms site in the phage genome. The site is located inside the interval 1.1-3.9 kb of the physical genome map, being closely linked to cI gene of phage B39. The growth of Pde- phages in cells with Rms163 can be restored, due to additional mutations in phage genes affecting lysogenization.     

Effect of the Prophage and Penicillinase Plasmid of the Recipient Strain Upon the Transduction and the Stability of Methicillin Resistance in Staphylococcus aureus

Transduction of a methicillin-resistance determinant (mec) in Staphylococcus aureus RN450 was dependent on its prior lysogenization with an appropriate temperate phage. In addition, an appropriate transduced penicillinase plasmid was usually required. Some phage 80-resistant variants of RN450 or of its parental lysogenic strain, NCTC 8325, were also effective recipients for transduction of mec. Elimination of prophage from RN450 abrogated its effectiveness as a transductional recipient of mec. Elimination of prophage from a methicillin-resistant transductant of RN450 reduced resistance to undetectable levels in six of seven phage-eliminated strains. In four of these a variable number of clones again became phenotypically resistant after lysogenization alone or lysogenization combined with reintroduction of a penicillinase plasmid. In two prophage-eliminated strains, no evidence of residual mec could be adduced. The establishment, expression, or stability of the transduced mec in strain RN450 appeared to depend on some function determined by a prophage or a prophage and a penicillinase plasmid.     

Control of lysogeny and a genetic map of the temperate phage SM of Pseudomonas aeruginosa

76 mutants with impaired ability to lysogenize host cells were isolated in SM phage after mutagenesis using several chemical mutagens. By means of complementation test, these mutants were distributed into two groups, cI and cII. The mutants of the cI group were similar phenotypically to the cI mutants of phage lambda defective in synthesis of repressor. The mutants of the cII group establish and support the lysogenic state in infected cells with very low frequency. Temperature-sensitive mutants belonging to 13 complementation groups and nonlysogenizing mutants of the cI and cII groups were used in genetic mapping of SM phage. Mutual positions of markers and relative distances between them were determined by the method of two-factorial crosses. The greatest distance equal to 20 units of recombination was determined between ts 88 marker and one of early genes marked with ts 105 mutation. The genes cI and cII are closely linked to each other and also to ts 105 marker and are situated at one end of the genetic map.     

Lysogenic conversion caused by phage phi 80. III. The mapping of the conversion gene and additional characterization of the phenomenon

The cor gene specifies lysogenic conversion caused by the lambdoid phage phi 80. The cor gene product inhibits tonA function in infected and lysogenic cells. The cells harboring pBR322 plasmid with the cloned cor gene of phi 80 became resistant to the phages T1 and phi 80 (TonA phenotype). The cor gene was mapped between 24 and 13 genes on the phi 80 phage genetic map. It is not essential for phage lytic growth. Its presence in lysogens leads up to accumulation of tonA mutants in a cell population.     

Genetic and physical characterization of lysogeny by bacteriophage MX8 in Myxococcus xanthus

Myxophage MX8 can initiate a lysogenic cycle in Myxococcus xanthus. The lysogenic phage was gentically stable in vegetative cells and persisted in the latent state through many cell generations in the absence of extracellular phage reinfection. The latent state also was stable during the host developmental cycle, since myxospores transmitted latent MX8 genetic information to future progeny cells. DNA hybridization experiments to probe the structure of the lysogenic phage provided physical evidence that MX8 formed a prophage. During lysogenization, MX8 DNA was cut at a specific site (attP) on phage DNA, and we have concluded that genetic recombination between attP and a bacterial DNA site (attB) leads to integration of MX8 DNA and formation of stable MX8 prophage. The genetic and physical properties of MX8 that we describe should make MX8 useful in the analysis of development of M. xanthus by genetic methods.     

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.     

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.     

Plaque forming specialized transducing phage P1: Isolation of P1CmSmSu, a precursor of P1Cm

Summary E. coli strains lysogenic for various types of P1-R hybrids were isolated. These carry all the essential genes for vegetative phage production and lysogenization including P1 immunity and P1 incompatibility, together with drug resistance genes derived from the R plasmid NR1. In particular, P1Cm and P1CmSmSu derivatives were studied. When strains lysogenic for these phages were induced in the absence of helper phage, yields of phage particles as high as with wild type P1 were obtained. All P1Cm phages isolated were of plaque forming type and usually every plaque contained Cm^r lysogens. Lysates of P1CmSmSu lysogens transduced Cm^rSm^rSu^r at high frequency and they formed plaques with an efficiency of 10^-4 to 10^-2 per phage particle. Only a minority of these plaques contained drug resistant bacteria. Cm^rSm^rSu^r transductants isolated from bacteria infected at a high multiplicity with phage P1CmSmSu were lysogens for the original P1CmSmSu. In contrast, Cm^rSm^rSu^r transductants isolated after infection at low multiplicity appeared to carry the Cm^rSm^rSu^r markers integrated into the host chromosome. The results described suggest that P1CmSmSu prophages carry the resistance genes transposed into the P1 genome without in principle causing a loss of essential gene functions. However, since these prophages are longer than the wild type P1 genome, the DNA packaged into phage particles has a reduced redundancy which seriously affects the reproduction and lysogenization abilities.Plaque forming P1Cm can be obtained from P1CmSmSu. Thus, P1CmSmSu is a precursor of P1Cm. P1Cm is also obtainable from P1 and NR1 under the recA ^- condition. The mechanism of formation of plaque forming P1Cm is discussed.     

Generalized transduction between Salmonella typhi and Salmonella typhimurium by phage j2 and characterization of the j2 plasmid in Escherichia coli

Phage j2, a P1-like phage in Salmonella typhi, was heteroimmune to phage P1 and existed in the lysogenic state as a plasmid of molecular size 58.6 MDal. The phage j2 plasmid was incompatible with the P1 plasmid (IncY group). A j2-sensitive mutant of Salmonella typhimurium LT2 was isolated by transduction of j2Ap phage into LT2 followed by curing of the prophage. The mutant was used to demonstrate transduction between S. typhi and S. typhimurium by phage j2.     

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.     

Characteristics of lysogenization of Escherichia coli K-12 strains by phage MupAp1 during zygotic induction

Genetic proofs of the fact that lysogenization of recipient cells by Mu phage in the course of zygotic induction is normally a slowed process were obtained. Stabilization of lysogenic state seems to occur after the stage of induced excision of Mu DNA during the process of conjugational crossing followed by integration as such of Mu DNA-containing recombinant structure into the recipient chromosome.     

Lysogenization by bacteriophage lambda

Summary An easy and sensitive way of measuring the proportion of E. coli cells which are lysogenized by lambda phage or lambda mutants has been devised. With this assay it was possible to analyse the lysogenic response as a function of the average phage input per cell. The results indicate that lysogenization of exponentially growing cells requires that the cells are infected by at least two phages able to replicate, or three or four phages unable to replicate.     

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.     

Genetic mapping of the regionc of the bacteriophage G101 (Pseudomonas aeruginosa)

Morphological mutants of the c type of the bacteriophage G101 (Pseudomonas aeruginosa) were isolated after mutagenesis with hydroxylamine. Complementation analysis of 27 c mutants showed that the c region is formed by at least two genes. Two types of c mutants were obtained. One of them (cI26) behaves analogously to a mutant in the gene controlling the synthesis of the repressor of phage lambda. The second type of the c mutants (cII1, cII18) specifies a gene having probably an auxiliary function in the "c" region. According to the low frequency of recombination between the genes cI26 and c II18 (1.37 recombination units), these genes responsible for lysogenization are localized in a short region of the chromosome.     

Der Einfluß der Infektionsmultiplizität auf die Lysogenisierung im System Salmonella typhimurium — Phage P 22

Summary The increase of lysogenization in phage infected cells has been investigated with increasing multiplicities of infection in the system Salmonella thyphimurium-phage P 22. The increase of infection resp. lysis and lysogenization with multiplicity follows first order reaction kinetics as concluded from multiplicities<0.3. Under the experimental conditions employed, the probability per phage is 0.57 for lysogenization and 0.43 for lysis. If multiplicity is>0.3 and cells are infected with more than one phage, the lysogenizations increase according to one hit kinetics, whereas the lysis of cells decreases. It is concluded, that lytic reactions in multicomplexes, which can be initiated independently by every one of the infecting phage particles will be suppressed by lysogenic reactions initiated by other independently infecting phages of the complex. Our experiments suggest, that immunity of the prelysogenic condition is the process responsible for the suppression of the lytic reaction. Therefore, in multicomplexes the immunity induced by one of the infecting phages is superimposed upon the one hit lytic infection causing the percentage of lysogenization increasing with multiplicity.     

Mapping of the c-region of phage phi80

A set of c-mutants of the phage phi80 is isolated. These mutants fit into three genes. According to plaque morphology and frequency of lysogenization of mutants, the genes were named cI, cII and cIII as it was previously done for phage lambda. Their order, determinated by mutant phage crosses, is cIII-sus326-cI-cII-sus250. Sus326 is a mutation in the gene 15, so it is probably an analogue of the N gene of the phage lambda. Thermoinducible mutants of the phage phi80 cts11 and cts12 correspond to the mutant types cItsB and cItsA of the phage lambda and they complement each other. Thus, it is supposed that phi80 phage repressor molecules consist of few protein subunits.     

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.