[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.

[Nitrogen assimilation enzymes in Bacillus subtilis mutants with hyperproduction of riboflavin].

Three groups of the nitrogen assimilation cycle enzymes (glutamate synthases (GTS), glutamine synthases (GS), and glutamate dehydrogenases (GD)) were studied in Bacillus subtilis strains with hyperproduction of riboflavin (vitamin B2). It was found that in all strains tested activity of GS was virtually the same, activity of GD was absent, and activity of GTS was reduced. In strains 41 and 24, riboflavin producers, activity of GTS was 30-60% the enzyme activity in the original strain (wild-type RosR). The most pronounced decrease in the activity of GTS (0-12% relative to RosR) was observed in the strain AS5, which had the highest level of biosynthetic activity relative to the other strains. According to the results of determination of the sensitivity of induction of beta-xylosidase to glucose- and fructose-induced catabolic repression, none of the strains studied was characterized by disorders in the protein CcpA, a global regulator of the catabolic repression in gram-positive bacteria, which is required for reducing amination and resulting activation of biosynthesis of glutamic acid in cell. It was suggested that mutations responsible for partial or complete inhibition of GTS biosynthesis caused an increase in the intracellular pool of glutamine. The intracellular pool of glutamine is a nitrogen source for riboflavin in cell. It follows from the results of this work that there is a trend toward an increase in the rate of biosynthesis of vitamin B2 in mutants with inhibited GTS activity. However, the complexity of the processes of regulation of nitrogen assimilation enzymes makes it difficult to find a distinct correlation between GTS activity and riboflavin biosynthesis in these strains.

[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.

[Properties of mutants of bacteria belonging to the genus Erwinia devoid of common components of the phosphoenolpyruvate-dependent phosphotransferase system]. / Svoistva mutantov bakterii roda Erwinia, lishennykh obshchikh komponentov fosfoenolpiruvatzavisimoi fosfotransferaznoi sistemy.

Biochemical consequences of mutational damage to common components of the Erwinia phosphoenolpyruvate-dependent phosphotransferase system (the HPr protein and enzyme I) were studied. The transport of glucose, mannose, fructose, and mannitol in Erwinia was shown to require a preliminary induction of proteins of the phosphotransferase system. A drastic decrease in the rate of the transport of these carbohydrates was observed in ptsI and ptsH mutants. A disturbance in the common components suppresses the synthesis of inducible enzymes (beta-galactosidase, complexes of pectolyases and cellulases) and renders it resistant to catabolite repression by glucose, but mutants were shown to retain intracellular cAMP content. Erwinia mutants devoid of common components of the system lack phytopathogenic features. The appearance of an intact ptsI allele in the cell completely corrected pleiotropic disturbances in these mutants.

[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.

[Mutation fruB in the fructose regulon affecting beta-galactosidase synthesis and adenylate cyclase activity of E. coli K12]. / Mutatsiia fruB vo fruktoznom regulone, vliiaiushchaia na sintez beta-galaktozidazy i aktivnost' adenilattsiklazy u E. coli K12.

Escherichia coli mutant devoid of fructosespecific factor III (factor IIIfru) of phosphoenolpyruvate (PEP) carbohydrate phosphotransferase system was isolated. The mutation fruB was localized on 46 min of chromosomal map of Escherichia coli in the fru-operon region. The mutant bacteria are unable to accumulate fructose. PEP-dependent phosphorylation of this carbohydrate in cellular extracts was considerably decreased. The mutational damage of factor IIIfru results in the suppression of beta-galactosidase synthesis. The impaired synthesis of the enzyme was partially resistant to glucose catabolite repression. However, the deficiency was suppressed by addition of exogenous cyclic AMP. The adenylatecyclase activity in fruB mutant was found to be 50% lower as compared with the one in the parent strain.

[Isolation and mapping of a mutation leading to damage in the cytoplasmic-specific component of the fructose transport system in Escherichia coli K-12 bacteria]. / Vydelenie i kartirovanie mutatsii, privodiashchei k povrezhdeniiu tsitoplazmaticheskogo spetsificheskogo komponenta sistemy transporta fruktozy u bakterii Escherichia coli K-12.

A mutant delta ptsH of Escherichia coli was used to obtain a mutation which damages a function of cytoplasmic specific component of the fructose phosphotransferase system--FPr protein. This mutation was mapped using a number of genetical methods. In conjugational crosses the mutation was localized at 47 min of the E. coli chromosomal map. The gene responsible for this defect was designated fpr. In P1 mediated transduction and in conjugational three-factorial crosses the order of the markers in this region was established as: gyrA-ompC-fpr-ptsF-...-his. The frequency of cotransduction of the fpr gene was 4.5 and 35% with gyrA and ompC markers, respectively. In fructose containing minimal medium the doubling time of the ptsH+fpr mutant was lower than that of the delta ptsHfpr+ mutant. Also, the doubling time of the fpr mutant depends on concentration of fructose in growth medium but is independent of the amount of this sugar in the case of the delta ptsH mutant.