[Role of phage LØ7 lysogeny in genetic variability of Escherichia coli].

AIM: Determine the possibility of Iysogenization of Escherichia coli single strain DNA (ssDNA) by 1ø7 bacteriophage from the Microviridae family and determine the role of phage lø7 lysogeny in genetic variability of these bacteria. MATERIALS AND METHODS: A method of E. coli K12 lysogenization by phage lø7 was developed. A spot-test for the control of resistance of the obtained lysogens against phage lø7 and determination of lysogen lø7 spontaneous production was worked out. Criteria for phage lø7 identification, that is spontaneously produced by E. coli K12 lysogens, were proposed. A kit of isogenic E. coli strains, that vary by mutations in ptsI, ptsH and fruA genes, that code phosphoenolpyruvate (PEP): carbohydrate phosphotransferase system (PTS) proteins, was constructed. RESULTS: The ability of highly virulent bacteriophage lø7 to lysogenize E. coli was shown. A reduction of lø7 titers in ptsI, ptsH and fruA E. coli K12 mutants was demonstrated compared with titers in wild-type bacteria. Lytic bacteriophage lø7 was also able to lysogenize ptsI, ptsH and fruA mutants at a high frequency. Lysogens are resistant to phages lø7, phiX174 of Microvirus genus and spontaneously produce lø7. CONCLUSION: Bacteriophage lø7 of the Microviridae family is able to lysogenize E. coli K12 and vertically transfer genome of this lytic phage. As a result, lytic phage lø7 takes part in bacterial variability as a factor of lysogen selection in bacteria population corresponding to PTS mutants by phenotype.

[The influence of Bordetella pertussis bvgAS operon on formation and resolution of plasmid-chromosome cointegrates in Escherichia coli K12 mutants with damages in common components of the phosphoenolpyruvate:sugar phosphotransferase system].

The plasmids containing the genetically marked variants of Bordetela pertussi transposon TnBP were synthesized on the base of the plasmid with thermosensitive replication. The integration frequency of these plasmids into the E.coli K12 chromosome at non-permissive temperature (42 degrees C) was determined. It was found that the frequency of forming of RSBP-induced plasmid-chromosome cointegrated in bacteria E.coli K12 deficient in HPr or Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system was decreased. The bvgAS operon from B.pertussis in trans-position restores the ability of mutant E.coli K12 to form and resolve.

[Precise excision of TN5 in Escherichia coli K12 mutants with alterations in common component of phosphotransferase system and in subunits of DNA-gyrase].

The effect of ptsH and gyrA mutations on precise excision (PE) of transposon Tn5 was studied in Escherichia coli K12. The conjugative plasmid with Tn5 integrated in the tet gene of Tn10 was used as a model in experiments. It was shown that mutational damage of HPr, a common component of the bacterial PEP-dependent phosphotransferase system (PTS), increased the frequency of PE. The alteration of the subunit A of DNA-gyrase (gyrA mutation) was found to enhance the frequency of PE. The ptsH mutation had the same effect. Mutational damage of the DNA-gyrase subunit B (gyrB mutation) had no effect on the frequency of PE. The data reported in this work are evidence of the necessity of the intact PTS for the balanced supercoiled DNA state maintained by DNA-gyrase.

[Functional activity of transposase of Bordetella pertussis IS element in Escherichia coli cells].

An Escherichia coli strain producing transposase of a repeated sequence of Bordetella pertussis chromosome (RSBP) was constructed. A defective MGE-helper plasmid method, which allowed the determination of transposase functional activity was developed. It was shown that transposase synthesized in E. coli cells ensures transposition of "defective" RSBP into the host chromosome. Overexpression of transposase was shown to markedly decrease the vital activity of E. coli cells under selective cultivation conditions. Reasons for a decrease in viability transposase-producing cells are discussed. Results showing the impact of transposase on replication of recombinant plasmids and E. coli cell division were obtained.

[Identification of the open reading frame coding transposase of Bordetella pertussis RS-element]. / Identifikatsiia otkrytoi ramki schityvaniia, kodiruiushchei transpozazu RS-élementa Bordetella pertussis.

A computer-aided analysis of the repeating sequence of Bordetella pertussis chromosome (RSBP3) revealed 3 open reading frames, one of whose (ORF1) can code a protein whose structure and properties are similar to those of transposasas, i.e. enzymes in charges for the traveling of migrating genetic elements of pro- and eukaryote. Mutants of the RSBP3 insertion sequence with the affected and unaffected ORF1 sequence were constructed in order to substantiate the above assumption. Two independent experimental models (formation of inter-plasmid co-integrates and of co-integrates between plasmid and E. coli chromosome) were used to show that the RSBP3-stimulated formation of co-integrates is only true for plasmids containing RSBP3 with the unaffected ORF1 sequence. An activity of the Hpr protein (a component of the phosphoenolpyruvate-dependent phosphotransferase) was proven to influence the formation process of inter-plasmid co-integrates.

[The multifunctional fructose-specific component of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli K12--fruA gene product]. / O mul'tifunktsional'nosti fruktozospetsifichenogo komponenta fosfoenolpiruvatzavisimoi fosfotransferaznoi sistemy Escherichia coli K12--produkta gena fruA.

Mutational damage of the ptsH gene leads to pleiotropic disturbance of sugar utilization in Escherichia coli K12. A fruS mutation suppresses the defect because of a constitutional expression of the fruB and fruA genes. FruB protein possessing a pseudo-HPr activity replaces the HPr. It was shown that wild type allele fruS+ dominates over the fruS1156 mutation in heterozygous merodiploid. The existence of thermosensitive mutations (fruS4 and fruS12) which repair the ptsH damage was also demonstrated. The fruS mutations were located in the fru operon. Fructose utilization was not disturbed in fruS1156 mutant, but fruS2 and fruS12 mutants were unable to utilize fructose. Spontaneous mutations (fruS6 and fruS13) possessing the same phenotype at any temperature similar to the thermosensitive ones under nonpermissive conditions were isolated. They were mapped using the P1vir transduction. The fruS mutations were found in the structural gene of the fructose operon. Presumably it is the fruA gene that cods for the fructose-specific multidomain protein IIB'Bc of the phosphoenolpyruvate-dependent phosphotransferase system.

[Isolation and properties of mutants devoid of pseudo-HPr activity of the fructose transfer system in Escherichia coli K12]. / Vydelenie i svoistva mutantov, lishennykh psevdo-HPr aktivnosti sistemy perenosa fruktozy u Escherichia coli K12.

Mutants without pseudo-HPr activity intrinsic to the H domain of the FruB protein were obtained in Escherichia coli K12 through insertional mutagenesis by means of the MudIlac phage and TnPhoA transposon. For isolating these mutants, double mutants of enteric bacterium (ptsH fruR of ptsH fruS) were used as original strains. These double mutants were inactive with respect to the total HPr protein of the phosphoenolpyruvate-carbohydrate phosphotransferase system and could not provide constitutive synthesis of fructose-specific proteins of this system. The mutants obtained were designated fruH, mapped, and genetically characterized. Insertions were shown to be located in exactly the portion of the fruB gene that specifies synthesis of the H domain of the FruB protein. The fruH mutations decreased motility of bacterial both in a medium free of carbohydrates and in a medium with glucose, fructose, or maltose. In addition, fruH mutants with the MudIlac genome insertion had sharply decreased activity of beta-galactosidase.

[Determination of the direction of transcription of Escherichia coli K-12 structural genes of the fructose regulon in vivo]. / Opredelenie in vivo napravleniia transkriptsii strukturnykh genov fruktoznogo regulona Escherichia coli K-12.

The direction of transcription of specific components of the fructose phosphotransferase system, the fruA, fruK, and fruB genes, was determined in vivo by plasmid F'ts1141ac. Transcription from each of these genes was shown to run in the same direction, counterclockwise with respect to the E. coli chromosomal map.

[Shigella flexneri mutation giving rise to the appearance of fosfomycin-resistant avirulent forms with disordered carbohydrate utilization]. / Mutatsiia u Shigella flexneri, vyzyvaiushchaia poiavlenie fosfomitsinrezistentnykh avirulentnykh form s narusheniiami v utilizatsii uglevodov.

The present work analyzes pt44 mutation in Sh. flexneri resulting in the appearance of the following phenotypical properties: resistance to phosphomycin, avirulence, pleiotropic disturbances in carbohydrate utilization. The data provided by the biochemical and genetic analysis have indicated that pts44 mutation occupies the region between purC and ptsI loci on the chromosome of Sh. flexneri. Merodiploids containing the mutant allele pts44 and the plasmid including purC-ptsI-ptsH genes from E. coli K12 acquired the capacity for fermenting carbohydrates, but at the same time retained resistance to phosphomycin and avirulent properties. The presence of phosphoenol pyruvate-dependent carbohydrate phosphotransferase system in Sh. flexneri has been proved.

[Characteristics of attenuated immunogenic mutants of shigella flexneri selected from phosphomycin-resistant clones]. / Kharakteristika attenuirovannykh, immunogennykh mutantov shigell Flexnera, otobrannykh sredi klonov, rezistentnykh k fosfomitsinu.

Sh. flexneri mutants No. 2a, resistant to phosphomycin (25 microgram/ml), differ in their fermentability in respect to glucose, mannitol, mannose and fructose. The mutants fall into 7 groups by this characteristic. All the resistant mutants, irrespective of the type of hydrocarbon fermentation, have lost their ability of causing keratoconjunctivitis. The decreased virulence of the phosphomycin-resistant mutants is linked with the loss of their penetration properties, although their adhesiveness and proliferative capacity on the surface HeLa cells is retained. A change in the virulence of the mutants under study is not connected with disturbances in the synthesis of O-antigen LPS. Most of these mutants are highly immunogenic.

[Effect of the dose of ptsI- and ptsH-genes on carbohydrate transport and regulation of lac-operon activity in Escherichia coli K-12]. / Vliianie dozy ptsI- i ptsH-genov na transport uglevodov i reguliatsiiu aktivnosti lac-operona u Escherichia coli K12.

Phage Mu-1 cts61 was used for transposition of pts1 and ptsH genes. The received F'-factors AUF2 and AUF3 carry short fragments of the bacterial chromosome. Merodiploid strains with double pts genes were selected in sexduction crosses with the appropriate recA recipients. Effect of the gene dose was not registered in pts+/pts+ strains in the case of accumulation of the substrates of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) and in the case of bacterial growth in the presence of these carbohydrates. This indicates that the enzyme (enzymes) II of the PTS is the limiting step in the transpost process. Induction of beta-galactosidase and the growth on carbohydrates not transported via the PTS (maltose, lactose) were greatly reduced in pts mutant. Introduction of the pts+ allele with episome lead to the restoration of the two above processes. These data show that the phospho approximately HPr generating system of the PTS is directly (or in indirect manner) involved in the regulation of catabolite-sensitive operons. Glucose repression was markedly increased in pts+/pts+ merodiploids as compared with pts+/pts- ones and with pts+ bacteria. Possible mechanisms of this effect are discussed.

[Glucose transport system and regulation of gene expression in Escherichia coli]. / Sistema transporta gliukozy i reguliatsiia gennoi aktivnosti u Escherichia coli.

The object of this work was to study the effect of mutations damaging protein components of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) of E. coli on the regulation of the activity of catabolite-sensitive operons. Mutations ptsI and ptsH affecting the activity of the enzyme I and HPr protein made the synthesis of catabolite-sensitive enzymes resistant to the action of glucose, and at the same time decreased the rate of transport of this compound. Mutation tgl affecting the activity of the glucose enzyme II lead to the same effect on the enzyme syntheses, though utilization was not altered in this case. The disturbance of beta-galactosidase synthesis in ptsI and ptsH mutants is due to interference of pts mutations into transcription of the lac operon at the lac promoter level. It is concluded that the proteins of the Escherichia coli PTS take part not only in glucose transport, but are also involved in the regulation of transcription of the catabolite sensitive operons.

[Isolation and properties of merodiploid strains with F-factor including the pts-region of the Escherichia coli K-12 chromosome]. / Vydelenie i svoistva marodiploidnykh shtammov s F'-faktorom, vkliuchaiushchim pts-oblast' khromosomy Escherichia coli K-12.

A technique of hybridization of haploid methanol-utilizing yeast Pichia pinus MH4 is worked out using UV- and N-nitrosoguanidine-induced auxotrophic mutants. Vegetative diploid cultures are isolated. Tetrad analysis and random spore analysis have revealed a meiotic nature of spores, recombination of genetic material in the process of sporulation and the chromosomal nature of some mutations. A possibility to construct a genetic map of the yeast Pichia pinus MH4 is demonstrated on the basis of tetrad analysis. Three linkage groups are revealed. The life cycle in a homothalic haploid yeast, Pichia pinus, was demonstrated. They are capable to form zygotes and meiotic spores under conditions preventing vegetative growth.