[Study of intraspecific variations of the bacterium Lactococcus lactis in adaptation to high acidity of the medium].

This paper reports on the study of acid tolerance of lactic acid bacteria as a property of cells, determining their ability to divide efficiently and retain viability under conditions of increased nutrient medium acidity during bacterial growth. The bacteria of the strain TV2, isolated from a self-soured curd, similar to the bacteria of the strain STE05 (Russian National Collection of Industrial Microorganisms), were assigned to the species Lactococcus lactis according to their G+C composition (36.7-36.5 mol %) and the high level of DNA-DNA hybridization (93%). However, these strains were essentially different in the number and size of the plasmids and the chromosomal DNA restriction fragments, as well as in the sensitivity to phages of lactic acid bacteria. It was found that bacteria of the strain TV2 were stable (i.e., they divided efficiently at a pH as low as 5.3) and tolerant to the lactic acid that they produced while growing (i.e., they retained viability at pH 4.4). Bacteria of the strain STE05 lacked acid tolerance (at pH below 6.5, growth was retarded, and pH 5.0 was the lowest value at which the cells remained viable). The acid tolerance and phage resistance of TV2 bacteria are likely to characterize their higher adaptive capacity in comparison with STE05 bacteria. Acid tolerance is inherited in a stable manner and retained by the segregants of TV2 strain obtained in the course of long-term storage of the bacteria. Specifically, the strains TV29, TV13, and TV 229, which displayed this property, had altered physiological and biochemical characteristics (accumulation of biomass and fermentation of lactose) in spite of their genetic identity to the original strain (pulsed-field gel electrophoresis of chromosomal DNA restriction fragments).

[Transcriptional mapping and study of transcription regulation of the Pseudomonas aeruginosa phage-transposon D3112]. / Transkriptsionnoe kartirovanie i izuchenie reguliatsii transkriptsii faga-transpozona D3112 Pseudomonas aeruginosa.

Regulation of transcription was studied in a wild-type transposable phage (TP) D3112 of Pseudomonas aeruginosa and its mutants for different genes. For this purpose, [3H]RNA-DNA hybridization with denatured fragments from various regions of the D3112 genome bound to nitrocellulose filters was used. A transcriptional map of TP D3112 was constructed based on the data obtained. The map comprised six independent transcriptional units corresponding to the modular organization of the phage genome. Only the repressor gene cI was transcribed in the lysogenic state. After repressor thermoinactivation, the cI transcription ceased, and transcription occurred in the same order as the genes (modules) were located on the D3112 phage genetic map (from left to right): cip1 (a negative regulator) and early genes A and B (controlling the replication-transposition of the phage), nonessential genome region, genes C and the locus marked with the ts47 mutation (positive regulators of late gene transcription), the genes of the head morphogenesis, gene c91 (a positive regulator responsible for the lysogenic state), and the genes of the tail morphogenesis. Similarities between nonhomologous TPs D3112 of P. aeruginosa and Mu of Escherichia coli with respect to genetic organization and transcription regulation are discussed.

[Phenotypic effect of mutation ts47 in the Pseudomonas aeruginosa phage transposon D3112 on expression of late phage genes]. / Izuchenie fenotipicheskogo éffekta mutatsii ts47 faga-transpozona D3112 Pseudomonas eruginosa na vyrazhenie pozdnikh genov faga.

At nonpermissive temperature (42 degrees C) the ts47 mutation causes substantial abnormalities in the late phase of the phage intracellular development. In these conditions DNA of the D3112 phage is detected both in a free form and integrated into bacterial chromosome. The transcription kinetics in the ts47 mutant at 42 degrees C was indistinguishable from that typical to other early gene mutants (A, B, and C): specifically, the preservation of the first transcription peak along with low activity of late transcription were observed. Similarly to the C gene, the ts47 mutation-carrying locus is involved in regulating the transcription of the D3112 transposable phage late genes. It is suggested that the mechanism underlying the action of the ts47 mutation differs from that of the C gene product. One of the possible explanations is based on the fact that the product of the ts47 locus affects the activity of cellular RNA polymerase via providing more effective recognition of the phage promoters by the RNA polymerase modified with the phage protein C.

[Recombinational origin of natural transposable phages of related species belonging to group B3, active in Pseudomonas aeruginosa species]. / Rekominatsionnoe proiskhozhdenie prirodnykh fagov-transpozonov rodstvennykh vidov gruppy B3, aktivnykh na bakteriiakh vida Pseudomonas aeruginosa.

A heteroduplex analysis of four related transposable phages--B3, PM57, PM62, and Hw12--of the Pseudomonas aeruginosa B3 group was performed. Heteroduplex structures, restriction maps, and data on DNA-DNA hybridization obtained upon hybridization of phage DNA restriction fragments with labeled probes representing different regions of the phage genomes are in good agreement. The data obtained strongly confirmed the recombinational origin of the analyzed phages. Thus, all natural transposable phages of P. aeruginosa, including phages from both group B3 and species D3112, were shown to have a recombinational origin.

[Role of divergence in evolution of group B3 Pseudomonas aeruginosa transposable phage evolution]. / Vyiavlenie roli divergentsii v évoliutsii fagov-transpozonov gruppy B3 Pseudomonas aeruginosa.

A heteroduplex analysis was performed to identify and map divergent DNA sequences in the genomes of the P. aeruginosa transposable phages (TPs) of group B3 using different formamide concentrations (30, 50, and 70%). Six PTs were classified into three related species--B3, PM681, and PM57. The role of DNA divergence in the evolution of TPs within one species is insignificant: the genomes of phages pM105 and PM681 (species PM681) and phages Hw12 and pM57 (species pM57) were shown to contain either homologous (98%) or nonhomologous DNA (2%). Homologous, divergent, and nonhomologous DNA regions (modules) were identified in the genomes of the TP of different species. Homologous modules with a level of DNA homology higher than 86% constitute approximately 30% of the phage genome; they are located at the left (1-5 kb) and right (29-38 kb) ends of the phage genome. Divergent modules with a DNA homology level between 50 and 67% and nonhomologous modules represent 30 to 35% and 25 to 30% of the phage genome, respectively. These regions form a mosaic structure in a 5-29-kb region. Thus, the key role of DNA divergence in the evolution of the natural TPs of three related species of group B3 was shown. A single region containing a 5-11-kb divergent DNA sequence was detected in the pM62 phage genome (species pM57). As shown by our previous data, this region was integrated into phage pM62 via interspecific recombination with a phage of species B3.

[Interaction between Pseudomonas aeruginosa phages-transposons: genetic analysis of the trait of inhibition by prophage D3112 of phage B39 development]. / Vzaimodeistvie fagov-transpozonov Pseudomonas aeruginosa: geneticheskii analiz priznaka podavleniia profagom D3112 razvitiia faga B39.

A novel, previously unknown cip gene of Pseudomonas aeruginosa phage D3112 was discovered. The cip gene is responsible for inhibiting the intracellular growth of the related heteroimmune phage B39 of P. aeruginosa. It was shown that the cip gene product inhibits replication of the B39 genome, interacting, apparently, with the definite s+ site in the phage B39 genome. Mutants cip- of phage D3112, incapable of inhibiting the growth of phage B39, and B39 mutants, insensitive to the action of the cip gene product of phage D3112, were isolated.

[Cloning and study of the expression of Pseudomonas aeruginosa cip1 gene by phage-transposon D3112 in a homologous host and in Escherichia coli]. / Klonirovanie i izuchenie ékspressii gena cip1 faga-transpozona D3112 Pseudomonas aeruginosa v gomologichnom khoziaine i v Escherichia coli.

Regulatory gene cipl of Pseudomonas aeruginosa transposable phage D3112 was cloned, and its expression was studied in P. aeruginosa and Escherichia coli. Overexpression of the cipl gene prevents transcription and replication of phage D3112 DNA and also lysogenization of bacteria P. aeruginosa PAO1 by phage D3112. The direction of cipl gene transcription within the vector was determined in the study of cipl gene expression, dependent on its orientation toward the gene lacZ promoter. The expression of the cloned cipl gene inhibited the specific TCS phenotype of E. coli (RP4 :: D3112) cells. The functional homology of the cipl gene of phage D3112 and the negative regulator ner of E. coli Mul phage was discussed.

[The phenotype of rpi mutant of Pseudomonas aeruginosa phage-transposon D3112 expressed in a heterologous Escherichia coli host]. / Fenotip mutanta rpi faga-transpozona D3112 Pseudomonas aeruginosa, proiavliaemyi v geterologichnom khozaine Escherchia coli.

A clone of Escherichia coli II-16 with unique properties was isolated upon incorporation of hybrid plasmid RP4::D3112 with an integrated genome of phage-transposon D3112 Pseudomonas aeruginosa into E. coli C600 cells. The cells of this clone produce viable phage and are not sensitive to growth under low temperatures, which is characteristic of the majority of E. coli (RP4::D3112) clones with the genome of wild type phage. The clone E. coli II-16 contains phage genome both in an integrated state within the chromosome and in plasmid RP4. The properties of phage D3112 in the clone II-16 demonstrated that the phage carried a mutation. The mutation was designated RP4-phage interaction (rpi). The phenotypic effect of this mutation is expressed as phage inability to replicate in response to the presence of plasmid RP4 at 30 C (Tcs phenotype). The mutant rpi differs in its characters from the previously described mutants in the early regulator gene cip, the analog of the ner gene of E. coli phage Mu1, and from the known mutations in the A gene. Plasmid RP4::D3112 rpi exerts an inhibitory effect on the burst size of RP4::D3112 in E. coli.

[The effect of certain Escherichia coli genes on the appearance of the TCS phenotype, conferred by plasmid RP4 with an integrated genome of the D3112 Pseudomonas aeruginosa phage]. / Vliianie nekotorykh genov Escherichia coli na proiavlenenie fenotipa TCS, soobshchaemogo kletke plazmidoi RP4 s integrirovannym genomom faga D3112 Pseudomonas aeruginosa.

The possibility of the SOS system activation caused by introduction of a hybrid plasmid RP4::D3112 (where D3112 is a genome of the transposable phage of Pseudomonas aeruginosa) into Escherichia coli was examined. It has been shown previously that the presence of this plasmid confers to E. coli a so called TCS phenotype: E. coli (RP4::D3112) forms normal colonies and grows at 42 degrees C but does not divide and becomes filamentous at 30 degrees C, probably because of E. coli DNA damages generated in the course of D3112 replication-transposition. It was shown that the level of prophage lambda induction is not elevated during the growth of E. coli (lambda) (RP4::D3112) cells at 30 degrees C. The character of the TCS phenotype was not affected by lex A3 or lex A51 mutations (which cause correspondingly non-inducibility or constitutive expression of the SOS regulon). It was concluded therefore that the TCS phenotype is not related to induction of the SOS response. It was found also that the mutation to heat shock gene dnaJ (dnaJ259) is a cause for significant decrease in phage D3112 production in E. coli dnaJ259 (RP4::D3112) cells. The DNA hybridization data of labelled RP4 and D3112 DNA with total E. coli dnaJ259 (RP4::D3112) DNA suggest that the dnaJ259 probably affected the late D3112 functions.

[Some developmental features of Escherichia coli Mu phage in Pseudomonas aeruginosa cells]. / Nekotorye osobennosti razvitiia faga Mu Escherichia coli v kletkakh Pseudomonas aeruginosa.

In order to determine the replication-transposition (RT) efficiency of Escherichia coli phage Mu in Pseudomonas aeruginosa cells, the change of Mu DNA copy number after transfer of P. aeruginosa (RP4::Mu) from 42 (the condition of RP4::Mu plasmid stability and low phage production level in P. aeruginosa) to 30 degrees C (the condition of RP4::Mu plasmid instability and higher phage production level in P. aeruginosa) was analysed. It was shown that the temperature shift causes no increase in Mu DNA copy number, although free phage DNA is revealed after transfer of the cells at 30 degrees C. Considering that the studied cells contained also a linear RP4 DNA and the free Mu DNA hybridized with the RP4 DNA, we proposed that the mature Mu DNA arises as a result of Mu genome packaging from the original plasmid. So, the Mu genome RT is uneffective in P. aeruginosa and all of the phage particles released from P. aeruginosa (RP4::Mu) cells contain Mu DNA apparently originated from the DNA of hybrid plasmid RP4::Mu. Moreover, these results suggest that the Mu DNA packaging is not effective in P. aeruginosa (taking into account that the P. aeruginosa (RP4::Mu) cells release about 10(-2) p.f.u./cell and that originally the copy number of RP4::Mu > or = 1).

DNA homology in species of bacteriophages active on Pseudomonas aeruginosa.

Using electron microscopy and DNA-DNA-hybridization, 113 virulent and temperate bacteriophages specific for P. aeruginosa have been assigned to 23 species. In most cases, especially in virulent phages, both particle morphology and DNA homology types were in good correlation and their use was sufficient for clear-cut definition of phage species. No virulent phages of different species had any DNA homology. DNA homology was detected between temperate phages of several species. Temperate phages formed two large groups of two and seven species, respectively. The first group included all transposable bacteriophages. The extent of interspecies DNA homology of phages belonging to each group was not more than 10-15% (except for 25% for phages D 3 and KF 1). No DNA homology was between phages of different groups. The possible origin and function of homologous sequences (genetic modules, linkers, occasional insertional sequences) are discussed. One of the phages (phi C 15) may be considered as the result of recombination between phages belonging to two different species, 295 and SM.

[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]. / Osobennosti vyrazheniia genoma faga-transpozona D3112 Pseudomonas aeruginosa v bakteriiakh Escherichia coli: zavisimost' fenotipa bakterii ot chisla kopii genoma D3112.

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.

[Phage-stable mutants of Pseudomonas putida: new types of stability]. / Fagoustoichivnye mutanty Pseudomonas putida: novye tipy ustoichivosti.

More than 170 phage-resistant mutants (PRM) of the first order of Pseudomonas putida strain PpG1 were obtained using newly isolated and previously described bacteriophages specific for this strain. According to the results of analysis of resistance of the mutants to each of 31 phages of PpG1 strain and 8 phages of the PpN strain, the PRM strains were distributed into 20 groups. In most cases, the reason for resistance is loss of absorption capacity of bacteria. However, no direct relation between the level of absorption and efficiency of phage plating was detected. It was shown that some of the PRM of P. putida PpG1 strains acquired the ability to maintain the growth of phages specific for the other P. putida strain, PpN. Frequencies of isolating mutants of various resistance types depend on the concrete phage used. In accordance with their absorption specificity, all phages were distributed into 23 groups, and a tridimensional formal scheme of receptor sites for these phages on the PpG1 strain was drawn. In the process of selection of the PpG1 clones resistant to non-lysogenizing mutant of temperate PP71 phage, a variant of this strain manifesting the phenomenon of "auto-plaquing" was found. These results support the mutational origin of this phenomenon in some cases.

[Genetic control of morphogenesis of Pseudomonas aeruginosa transposable phage D3112]. / Geneticheskii kontrol' morfogeneza faga-transpozona D3112 Pseudomonas aeruginosa]

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.

[Study of morphology and genome structure of Pseudomonas putida bacteriophages for their classification]. / Izuchenie morfologii chastits i struktury genomov bakteriofagov Pseudomonas putida s tsel'iu ikh klassifikatsii.

A group of 27 bacteriophages specific for Pseudomonas putida strains PpG1 and PpN has been isolated. The phages were characterized and compared with the previously described virulent (pf 16, af, tf and PMW) and temperate (PP56 and PP71) phages. The new phages belong to B1 and C1 morphotypes, according to Ackerman's classification. Phage DNAs were digested with several endonucleases; the molecular weights and homology of the DNAs were determined. All phages of P. putida isolated up to now were distributed into 10 species (groups), on the basis of particle morphology, genome size and the results of homology studies. Recombination processes are believed to participate in formation of phages belonging to certain species.

[Integration of phage Mu into the RP4::D3112A-plasmid in Escherichia coli cells]. / Integratsiia faga mu v plazmidu RP4::D3112A v kletkakh Escherichia coli.

Hybrid plasmids obtained as a result of Mu phage insertions into the RP4::D3112 plasmid in Escherichia coli cells were studied. Stable maintenance of RP4::D3112 plasmid in E. coli cells was provided by using the D3112 phage genome with a point polar mutation in the A gene which prevented early genes' expression. The presence of D3112A- in the RP4 plasmid has been shown to have no effect on efficiency of phage Mu transposition into this plasmid. Moreover, RP4 and D3112 genomes were equivalent targets for Mu integration. The integration of transposable phage into genome of nonrelated phage can be used as one of the approaches to construct recombinant phage genomes in vivo in the absence of DNA homology.

[Transcription of the genome of Pseudomonas aeruginosa phage-transposon D3112 in an homologous host]. / Izuchenie transkriptsii genoma faga-transpozona D3112 Pseudomonas aeruginosa v gomologichnom khoziaine.

In vivo transcription of Pseudomonas aeruginosa transposable phage D3112 was studied. The 3H-labelled RNA isolated from the lysogenic cells PAO1 (D3112cts) after heat induction was hybridized with D3112 DNA to estimate phage-specific RNA. Two main stages of D3112 transcription, including transcription of the early (first 6-8 min) and the late (after 8th min) genes, were revealed. The transcription rate of D3112 early genes achieves the maximum at 3-5 min, being reduced to the minimum at 6-8 min after heat induction. These data point to the existence of negative regulation of D3112 early genes' transcription. The influence of ts mutations in early A, B and C genes and in late ts25 gene on transcription of D3112 phage was studied. It is shown that the genes A and B have no effect on the late transcription, while gene C regulates transcription of the D3112 late genes.

[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]. / Izuchenie sovmestimosti fagov-transpozonov Escherichia coli i Pseudomonas aeruginosa. Soobshchenie I. Sovmestnoe razvitie fagov Mu i D3112 i integratsiia faga D3112 v plazmidu RP4::Mu v kletkakh Pseudomonas aeruginosa.

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.

[Transfer of chromosomal genes and formation of R'-derivatives using PR4::D312cts15 plasmids in Pseudomonas aeruginosa cells]. / Perenos khromosomnykh genov i obrazovanie R'-proizvodnykh s pomoshch'iu plazmid PR4::D3112cts15 v kletkakh Pseudomonas aeruginosa.

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).

[2 types of molecular structure (composition) of a genome in one species of transposable bacteriophages of Pseudomonas aeruginosa]. / Dva tipa molekuliarnoi struktury (sostava) genoma v predelakh odnogo vida bakteropfagov-transpozonov 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.