Biomedical subjects

I A Khmel'

Publications and source records attributed to I A Khmel'.

At least 19 recordsLinked to original sources

PubMed2005

[Activation of the expression of the microcin C51 operon upon glucose starvation of cells at the exponential growth phase].

It was earlier shown that expression of the microcin C51 operon in Escherichia coli cells is activated upon decelerated growth of cells during their transition to the stationary growth phase and depends on the sigmaS subunit of RNA polymerase. Using a single-copy construct containing the cloned promoter region of the microcin C51 operon and a promoterless lac operon (P(mcc)-lac), it was shown that the promoter of the microcin operon was also induced by stress caused by the transition of cells at the exponential growth phase into the medium without glucose as a sole carbon source. Activation of P(mcc)-lac expression upon severe glucose starvation occurred in rpoS+ and rpoS- strains. In cells carrying the rpoD800 mutation that renders the sigma70 subunit of RNA polymerase temperature-sensitive, an activation of P(mcc)-lac expression was observed at nonpermissive temperature, in contrast to its complete inhibition in E. coli cells at the phase of delayed growth. Other stressors-nitrogen starvation, high temperatures, osmotic shock, tetracycline and chloramphenicol-did not activate P(mcc)-lac expression in cells at the exponential growth phase.

Bacteriocins↗

PubMed2005

[Regulation of expression of bacterial genes in the absence of active cell growth].

Global changes in cell physiology and metabolism take place in bacterial cells entering the stationary phase. The processes that ensure cell survival under adverse environmental conditions are activated, and cells with an improved resistance to many stress factors are formed as a result of the general stress response. The review considers the molecular mechanisms of cell differentiation in Gram-negative bacteria (exemplified by Escherichia coli) upon deceleration or complete suppression of cell growth, including changes in all steps of gene expression. The transition to the stationary phase is accompanied by DNA compaction, which is due to qualitative and quantitative changes in the protein composition of the nucleoid. Changes in the translation machinery under these conditions include the formation of functionally inactive 100S ribosome dimers with participation of RMF. Dramatic changes are observed in the transcription machinery: while the general level of gene expression decreases, activation or induction is characteristic of many genes and operons that are silent in actively growing cells. The key role in stationary stage-associated regulation of gene transcription is played by the sigmaS subunit of RNA polymerase. Expression of the rpoS gene, which codes for sigmaS, is regulated by an intricate multicomponent regulatory network, which functions at the levels of transcription of the rpoS gene, translation of the rpoS mRNA, and proteolysis of sigmaS. In addition, some issues of selection of sigmaS for transcription initiation and the roles of other factors in regulating gene expression in cells with decelerated or completely suppressed growth are discussed. Data are presented on the role of quorum-sensing systems in regulating the processes that occur at a high density of a bacterial population.

DNA, Bacterial↗

PubMed2004

[Phytase activity in some groups of bacteria. Search for and cloning of genes for bacterial phytases].

A search for phytase genes in 9 Bacillus strains from the collection of IMGAN was implemented. The growth optimum of strains IX-22, IX-12B, K17-2, K18, IMG I, IMG II, M4 and M8 was 50-60 degrees C; the optimal growth temperature for Bacillus sp. 790 was 45-47 degrees C. According to the sequence data of 16S RNA genes, Bacillus sp. 790 belongs to the B. subtilis/amyloliquefaciens group. The other 8 strains were identified as B. licheniformis. Selection of Bacillus strains, potentially containing the phytase genes, was performed via PCR with primers designed on the basis of the conserved sequence regions of the phyA gene from B. amyloliquefaciens FZB45 with chromosomal DNA being used as the template. The nucleotide sequences of all PCR fragments showed a high level of homology to the known Bacillus phytase genes. The gene libraries of B. licheniformis M8 and B. amyloliquefaciens 790 in E. coli were constructed and phytase-containing clones were selected from them. Twenty-four Pseudomonas strains of different species, 5 Xanthomonas maltophilia strains and 1 Xanthomonas malvacearum (all from the mentioned collection) were tested for phytase activity. Such activity was found in 13 Pseudomonas strains and in 6 Xanthomonas strains. The accumulation of phytase in Pseudomonas was shown to take place at later (over 2 days') growth stages. The optimum pH for phytase from 3 Pseudomonas strains were established. The enzymes were found to be most active at pH 5.5.

6-Phytase↗

PubMed2004

[Involvement of sigma S and sigma 70 subunits of RNA polymerase and the CRP protein in the regulation of microcin C51 operon expression].

Expression of the microcin C51 operon in Escherichia coli cells is activated during cell entry into the stationary growth phase and depends on the sigmaS subunit of RNA polymerase (RpoS). The null rpoS mutations retained the residual expression level of the transcriptional P(mcc)-lac fusion, which indicates that other sigma subunit can participate in the regulation of transcription of the microcin C51 operon. Data presented in this work show that the overproduction of sigma70 in rpoS- cells diminished the level of P(mcc)-lac expression, as in wild-type cells, which seems to be the consequence of competition between sigma factors for a limited number of core RNA polymerase molecules. In the presence of the rpoD800 mutation that renders sigma70 temperature-sensitive, expression of P(mcc)-lac was not induced in the phase of delayed culture growth at nonpermissive temperature, which indicates that sigma70 is indispensable for microcin operon expression. Point substitutions in the -10 P(mcc) region, leading to the formation of 5'-TGaTATAAT-3' site, enhanced promoter activity but did not affect the relationship between P(mcc)-lac transcription and growth phase, sigmaS, and the activator protein CRP. The activator protein CRP was shown to bind a DNA fragment containing the TGTGA(AATGAA)TCTAT site in the -59.5 bp position relative to the start site of transcription. Mutation in the ssrI gene encoding 6S RNA did not disturb P9mcc)-lac expression; these results indicate that 6S RNA does not participate in the regulation of microcin C51 operon expression.

Bacteriocins↗

PubMed2003

[Clinical strains of Burkholderia cepacia: characteristic and detection of the components in the quorum sensing regulatory system].

Described in the paper are characteristics of B. cepacia clinical strains isolated from patients at Moscow hospitals. The strains were investigated for the presence of proteolytic, chitinolytic, hemolytic and lipase activities as well as for presence of components of the "Quorum sensing" gene activity regulatory system by using biological test-systems and in the polymerase chain reaction with primers to genes cepI and cepR.

Anti-Bacterial Agents↗

PubMed2002

[Synthesis of signaling N-acyl-homoserine-lactones participating in quorum sensing in rhizosphere and soil bacteria Pseudomonas and Xanthomonas].

Signaling molecules assigned to N-acyl-homoserine-lactones (AHL) serve as autoinducers for the genes controlling the quorum sensing regulatory system. In many gram-negative bacteria, AHL are the key factors responsible for density-dependent regulation of exoenzyme and secondary metabolite production; they also participate in interaction between bacteria and higher organisms. The soil and rhisosphere bacteria Pseudomonas and Xanthomonas from different geographical zones of Russia and the former USSR were analyzed for the presence of the AHL producers. Screening was conducted by using a test system based on the mutant strain Chromobacterium violaceum, which was unable to synthesize AHL but produced a pigment violacein in the presence of exogenous AHL. The AHL-like compounds proved to be formed by 9.7% of the studied bacteria. Various Pseudomonas species differed in the capacity to synthesize this compounds. In at least a half of the isolated P. aureofaciens and P. aeruginosa, an intense AHL production was observed, whereas the AHL-producers were far less frequent among the P. fluorescens, P. chlororaphis, P. lemonnieri, P. geniculata, and P. putida. None of the 41 Xanthomonas maltophilia strains examined synthesized AHL.

Homoserine↗

PubMed2001

[Features of functioning of the promoter of microcin C51 promoter under various conditions of Escherichia coli cell growth].

The level of transcription from the promoter of the microcin C51 operon (Pmcc) depends on the growth phase of Escherichia coli cells: transcription proceeds with low efficiency at the exponential phase of growth and with higher efficiency when growth of cells is delayed during entry into the stationary phase. The functioning of Pmcc was studied in cells grown in different media by a single-copy construct, which contained the cloned promoter region of the microcin C51 operon and the promoterless lac operon. A decrease in the rate of cell growth caused by changes in the sole carbon source in minimal medium correlated with an increase in the level of transcription from the Pmcc promoter at the exponential phase of growth; the expression of Pmcc-lac during cell entry into the stationary phase was higher under unfavorable medium conditions. The use of composite rich media impaired this feature. The addition of l-leucine (100 micrograms/ml) to the medium decreased the expression of Pmcc-lac in wild-type cells carrying the delta lrp mutation. A further increase in leucine concentration and the presence of other amino acids in the medium enhanced transcription that started from Pmcc during cell entry into the stationary growth phase. The capacity of the Pmcc promoter and of the wild-type lacZ gene promoter was virtually the same upon IPTG induction. A mutation in the ompR gene did not markedly influence transcription started from Pmcc.

Amino Acid Sequence↗

PubMed1999

[Cloning and expression of a serine proteinase gene from Thermoactinomyces sp. in Bacillus subtilis cells].

A gene coding for thermostable serine protease from Thermoactinomyces sp. K50 is cloned and expressed in Bacillus subtilis cells. Restriction map of cloned DNA fragment is determined. Thermostability and temperature optimum of proteolytic activity of the cloned gene product are lower than those of the natural proteinase of Thermoactinomyces sp. K50. Serine protease, a product of cloned gene, is highly sensitive to proteolysis and its degradation can be prevented by Ca2+ ions.

Bacillus subtilis↗

PubMed1999

[Microcins--peptide antibiotics of enterobacteria: genetic control of the synthesis, structure, and mechanism of action].

A general survey of the poorly studied peptide antibiotics, microcins, which are produced by enterobacteria and effect gram-negative bacteria is conducted. In most cases, plasmid genes are responsible for microcin synthesis and the host cell immunity to the produced microcin. A complex system of plasmid and chromosomal genes is involved in the production and immunity to microcins. The chemical structure of three studied microcins is unusual. It was shown that peptides of the microcin molecules are synthesized on ribosomes and often modified posttranslationally. New types of posttranslational modifications were revealed in the biosynthesis of the microcin B17. These modifications are related to the formation of the thiazole and oxazole rings in its molecule; plasmid genes are responsible for these modifications. It was demonstrated that the smallest gene (21 bp) of all known genes controls the synthesis of the heptapeptide contained in the microcins of the C type.

Anti-Bacterial Agents↗

PubMed1997

[Role of the rpoS gene in the regulation of expression of plasmid genes determining the synthesis of C51 microcin].

In cells mutant for the rpoS gene coding the sigma s subunit of RNA polymerase, synthesis of antibiotic microcin C51, which is produced by Escherichia coli, is absent or extremely reduced. In experiments with a cloned promoter of the microcin operon, the sigma s subunit was shown to participate in regulation of transcription of plasmid genes that determine microcin synthesis.

Anti-Bacterial Agents↗

PubMed1996

[Identification and expression of plasmid genes participating in the synthesis of microcin C51].

A structural gene of heptapeptide, which is a component of the microcin C51 molecule, was identified by hybridization of plasmid DNA fragments and a mixture of synthesized oligonucleotides with the sequence corresponding to that of amino acids in the peptide. Sequence analysis of the structural gene of microcin peptide and its promoter region was performed. The data obtained indicate that the peptide of microcin C51 is synthesized on ribosomes. Four polypeptides of 67, 39, 16, and 14 kDa were identified using the system of minicells. These polypeptides are specified by a DNA fragment responsible for microcin synthesis and immunity in a producer cell. Apparently, three of these polypeptides with molecular masses of 67, 39, and 16 kDa are responsible for microcin production and immunity. The 67 kDa polypeptide is involved in the expression of immunity to microcin and, probably, in microcin production.

Anti-Bacterial Agents↗

PubMed1994

[Cloning and analysis of genetic determinants determining the production of microcin B2 and B27].

Cloning of plasmid genes for the synthesis of two peptide broad-spectrum antibiotics-microcins B2 and B27- and for host cell immunity to their action was performed. Recombinant plasmids containing these genes were designated pBE108 and pVB27, respectively. Deletional derivatives of plasmid pBE108 and mutant plasmids were obtained via transposon Tn5 insertions, which did not determine production of microcin B2 and immunity to it. Phenotypic study and physical mapping of these plasmids demonstrated that a 4.2-kb DNA fragment is responsible for B2 microcin production; immunity is provided by a 1.4-kb DNA fragment. A 5-kb DNA fragment is necessary for microcin B27 synthesis and expression of immunity to its action. Homology between these fragments and with plasmid DNA for the synthesis of microcin B17 and immunity to it was found. Homology between plasmid genes determining synthesis of type B and C microcins and host cell immunity to them was not observed. The production of B27 microcin is controlled by the product of the ompR gene; B2 microcin synthesis does not depend on this product. The mutations recA and lexA increase the susceptibility of Escherichia coli cells to the action of microcins B2 and B27 but not microcin C51.

Anti-Bacterial Agents↗

PubMed1994

[Plasmid genes participating in the synthesis of microcin C51].

Microcin C51 is an antibiotic with a wide application range produced by Escherichia coli cells. Using insertions of transposon Tn5 a set of mutations was induced in the recombinant plasmid pAST, which determines synthesis of microcin C51. The mutations were physically mapped. Complementation analysis was performed for insertions and deletions in the Mic+ plasmids pAST and pUHAB that lead to the absence of microcin or immunity to it. The analysis showed that at least three plasmid genes take part in microcin production and two genes determine immunity of producer cells to microcin. Functioning of the ompR gene product was necessary for synthesis of microcin C51.

Anti-Bacterial Agents↗

PubMed1993

[Characterization of enterobacteria producing the broad-spectrum antibiotics microcins].

As a result of screening among 11956 enterobacteria strains isolated from feces of normal children, grown-ups and lambs, seven active microcin-producing strains were obtained. The microcins were shown to be peptides or their derivatives with a low molecular weight (less than 10,000) and a broad spectrum of activity, mainly against gram-negative bacteria. According to cross immunity criteria the microcins studied belonged to two different types. Those of type I could be further classified into two subtypes on the account of difference in the spectrum of antibacterial activity. In 5 cases out of 7 the microcin-producing ability and immunity to microcins have been attributed to plasmids that the strains harboured. The effect of microcins on sensitive cells depended on ompR and ompF gene products.

Animals↗

PubMed1992

[Mapping of plasmid genes responsible for microcin R51 synthesis and immunity to it].

Microcin R51 is plasmid-determined low-molecular-weight peptide antibiotic produced by Escherichia coli. The spectrum of its action includes many different species of gram negative and some gram positive bacteria. Microcinogenic strains are immune to the action of the microcin they synthesize. As shown earlier, genes responsible for MccR51 production and immunity are located in a continuous 11.1 kb DNA fragment. These genes were cloned in pUC19 and pACYC184 plasmid vectors. Deletion derivatives and Tn5 insertion mutant plasmids which determined no microcin synthesis and immunity were obtained. Analysis of clones' phenotypes and physical mapping of mutant plasmids demonstrated that the 5 kb DNA fragment was indispensable for microcin production. The region of about 4.6 kb confers complete immunity of the producing strains, while partial immunity is provided by 1.8-1.9 kb DNA fragment.

Anti-Bacterial Agents↗

PubMed1990

[Microcin R51 and a plasmid determining its synthesis].

A new microcin produced by an Citrobacter R51 strain has been detected. This antibiotic has been shown to inhibit the growth of a number of Gram-negative as well as Gram-positive strains of bacteria on the minimal medium plates. The properties of partially purified microcin were characterized. Constitutive synthesis of microcin is determined by a conjugative plasmid. The genes of microcin synthesis and immunity were cloned on a plasmid and plasmid vehicles. A physical map of the 12 kb fragment coding for the production of microcin R51 and immunity to this antibiotic is presented.

Anti-Bacterial Agents↗

PubMed1988

[Molecular cloning of plasmid ColIb-P9 genes responsible for its mutator function].

The localization of plasmid ColIb-P9 muc genes mediating the plasmids protective and mutagenesis-increasing activity has been determined. The increase of muc genes dose by cloning them within the multicopy vector has been shown to repress the mutator function of the plasmid. No essential homology has been revealed between ColIb-P9 muc gene nucleotide sequences, pKM101 muc genes with a similar function, and umuDC chromosome genes. It has been shown that the synthesis of 38 KD protein is essential for the manifestation of the mutator function of the plasmid.

Bacteriocin Plasmids↗

PubMed1987

[The ColIb-CA53 plasmid suppresses umuC- and umuD-mutations in Escherichia coli K-12].

The presence of the ColIa-CA53 plasmid in umuC and umuD mutant Escherichia coli K-12 cells restores their mutability under UV irradiation to a level that even exceeds that of the isogenic umuC+umuD+ strains, as well as increases their resistance to the lethal effects of UV irradiation. The ColIb-P9 plasmid which suppresses the umuC mutant phenotype, as we have shown earlier, acts in the same manner with respect to the umuD mutant cells. The results of the study demonstrate that both plasmids encode products that are functionally similar to those of the chromosomal genes umuC and umuD. The plasmids ColIa-CA53, ColIb-P9 and pKM101 are shown to have practically the same effect upon the mutagenesis and survival of the umuC, umuD mutant cells.

Escherichia coli↗