Adgamov RR, Timchenko NF, Allenov AV
… +1 more, Ermolaeva SA
Mol Gen Mikrobiol Virusol
· 2010 · PMID 20364476
A total of 84 Y. pseudotuberculosis isolates were studied. The isolates were obtained in Russian Federation in 1967-2008. The majority of Y. pseudotuberculosis isolates (n = 55) were of clinical origin and were isolated...A total of 84 Y. pseudotuberculosis isolates were studied. The isolates were obtained in Russian Federation in 1967-2008. The majority of Y. pseudotuberculosis isolates (n = 55) were of clinical origin and were isolated from feces of patients with the clinically and serologically proved diagnosis of pseudotuberculosis/Far East scarlet-like fever. These isolates included 18 isolates obtained from 3 outbreaks. Nine isolates were isolated from the internal organs of wild rodents. Other isolates were obtained from environmental sources. Ten Y. pseudotuberculosis isolates belonged to the serovar III and the other isolates belonged to the serovar I. The sequences of 600 b.p. fragment of the inv gene that encodes 667 through 866 invasin amino acids were determined for all isolates. Totally, 3 allelic variants were found. The most abundant allele 1 was found in 76 isolates. The allele 1 is represented in the database Genbank by the strain IP31758 isolated in the Far East of Russia (Eppinger et al., 2007). The allele 2 differed from allele 1 in 3 positions: G,2299N, O2300N, and O2302N. Substitutions in positions 2299 and 2302 were non-synonymous and resulted in amino acid substitutions Ser768 Thr and Val769 Ala. Six isolates carried allele 2. Allele 3 was found in two isolates different from allele 2 by a synonymous substitution G2324O. This allele is similar to the sequence found in Y. pestis strains, represented in the GenBank. The allelic distribution was not serovar specific: Y. pseudotuberculosis of serovar III and majority of serovar I isolates carried allele 1. The analysis of the allelic distribution among subpopulations formed on the base of a source of isolation revealed a statistically significant difference in spreading of alleles among clinical and wild rodent isolates (p < 0.05). Allele 1 prevailed over clinical isolates (95%), while allele 1 and allele 2 were disseminated equally among rodent isolates (55 % and 45 %, respectively).
Specific features of evolution of chromosomes in mammals and other amniotes are reviewed. Comparative analysis of chromosome architecture revealed nonrandom distribution of chromosome rearrangement sites in genome, proba...Specific features of evolution of chromosomes in mammals and other amniotes are reviewed. Comparative analysis of chromosome architecture revealed nonrandom distribution of chromosome rearrangement sites in genome, probable role of chromosome rearrangement in adaptation, and evolution-mediated selection of conservative chromosome sites. The chromosome sites stable during evolution are saturated with genes contributing to early organism development. Ruptures of these sites are incompatible with organism survival. Further analysis of chromosome evolution requires more information about completely sequenced genomes.
Complete nucleotide sequence of genomic RNA of hepatitis A virus (HAV) rapidly replicating strain MB-7 was determined. Comparison of nucleotide and deduced amino-acid sequences demonstrated the highest level of identity...Complete nucleotide sequence of genomic RNA of hepatitis A virus (HAV) rapidly replicating strain MB-7 was determined. Comparison of nucleotide and deduced amino-acid sequences demonstrated the highest level of identity of MB-7 with strain HAS-15 (above 99%) and high homology with other HAV strains (HM 175/7, CR326, and GBM/HFS) used in production of anti-hepatitis A vaccines. MB-7 was classified as subgenotype IA. Phylogenetic analysis showed that MB-7 is most closely related to the strain HAS-15 and the HAV variants circulating in Russia. Comparative analysis of genomic differences between MB-7 and HAS-15 with other HAV strains revealed among changes characteristic of MB-7 those typical of the described earlier rapidly replicating HAV strains (nt. 149-162 in 5'-untranslated region and changes in the VP3 and 2C genes). These results suggest the functional importance of changes in above-mentioned regions of HAV genome for the increased replication level of MB-7 in vitro.
Comparative analysis of amino acid and nucleotides sequences of ORFs located in extended segments of the terminal variable regions in variola virus genome detected a promising locus for viral genotyping according to the...Comparative analysis of amino acid and nucleotides sequences of ORFs located in extended segments of the terminal variable regions in variola virus genome detected a promising locus for viral genotyping according to the geographic origin. This is ORF O1L of VARV. The primers were calculated for synthesis of this ORF fragment by PCR, which makes it possible to distinguish South America-Western Africa genotype from other VARV strains. Subsequent RFLP analysis reliably differentiated Asian strains from African strains (except Western Africa isolates). This method has been tested using 16 VARV strains from various geographic regions. The developed approach is simple, fast and reliable.
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...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.
Umiarov AM, Siniashina LN, Amelina IP
… +4 more, Datsenko KA, Bol'shakova TN, Dobrynina OIu, Karataev GI
Mol Gen Mikrobiol Virusol
· 2010 · PMID 20361664
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 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.
Genomic fingerprinting analysis of plague agent strains of the main subspecies isolated in natural foci of various types in the Russian Federation and neighboring countries suggests their genetic polymorphism, while they...Genomic fingerprinting analysis of plague agent strains of the main subspecies isolated in natural foci of various types in the Russian Federation and neighboring countries suggests their genetic polymorphism, while they are similar in phenotypic properties. The strains of the main subspecies, Y. pesis subsp. Pestis, fall into four genetic variants, each of them being associated with specific carrier species. The conserved genomic fingerprinting profile of each genovariant of Y. pesis subsp. Pestis strains ensures the suggested methodic approach to be promising for the intraspecies differentiation of plague agent strains (including atypical strains). Correlation of genovariants with carrier species permits their application for research into enzootic territories, where carrier change-over takes place.
Fomenko NV, Stronin OV, Khasnatinov MA
… +3 more, Danchinova GA, Bataa J, Gol'tsova NA
Mol Gen Mikrobiol Virusol
· 2009 · PMID 20050161
48 full-length Borrelia garinii and Borrelia afzelii from West Siberia and Mongolia ospA gene nucleotide sequences was determined. Four groups of Borrelia garinii were revealed using the analysis of nucleotide sequences....48 full-length Borrelia garinii and Borrelia afzelii from West Siberia and Mongolia ospA gene nucleotide sequences was determined. Four groups of Borrelia garinii were revealed using the analysis of nucleotide sequences. The most variable ospA gene region was demonstrated to be included in region where the antigenic determinants of protein were encoded. High homology level was shown for nucleotide sequences corresponding to isolates of Borrelia afzelii.
Microbe Russian Anti-Plague Research Institute, Saratov, Russia The literature data and experimental results of the authors on the molecular basis of plague agent interaction with invertebrates are discussed. The details...Microbe Russian Anti-Plague Research Institute, Saratov, Russia The literature data and experimental results of the authors on the molecular basis of plague agent interaction with invertebrates are discussed. The details of the plague agent life cycle, its genome organization, and molecular genetic mechanisms of its survival in flea vector and on the nematode cuticule are discussed. The experimental data about the ability to form biofilms at abiotic and biotic surfaces in the Yersinia pestis strains of the main and non-main subspecies are presented. Mechanisms of horizontal and vertical transmission of plague agent are considered. The suggestion about participation of the new member in the complex parasitic biocenosis (nematode, vector parasite) is put forward.
The ovine foot rot is a severe infectious disease of sheep. Dichelobacter nodosus is an essential pathogen of this disease. An obligatory anaerobic gram-negative rod-shaped microorganism has slow rate of accumulating bac...The ovine foot rot is a severe infectious disease of sheep. Dichelobacter nodosus is an essential pathogen of this disease. An obligatory anaerobic gram-negative rod-shaped microorganism has slow rate of accumulating bacterial density and fastidious growth requirements. This causes obstacles to vaccine production and makes it difficult to diagnose the disease. The diagnosis in this case is more expensive. Fimbriae (or pili) are one of the major factors of virulence of D. nodosus. Their antigenic and immunogenic properties make them good vaccine components for elevated immunogenicity. Since the nucleotide sequence of the fimA gene encoding fimbrial subunit was determined, attempts to produce recombinant pili were undertaken. The production of the genetic-engineering fimbriae would allow the price of the vaccines to be reduced and their manufacture to be simplified. The vaccine immunogenicity is increased in this case. At first, E. coli was selected as an expression system, but morphogenetic expression of the pili was not achieved on its surface because of some differences in the biogenesis and structure of fimbriae from D. nodosus. Successful morphogenesis of the pili was achieved in Pseudomonas aeruoginosa, which had closest similarity in the structure of pili. The level of the immunity obtained after immunization of the sheep with recombinant pili was similar to the level of the immunity after native pili or whole cells of D. nodosus had been used. This review contains information regarding the recombinant strains of Pseudomonas aeruoginosa obtained using fimbriae of D. nodosus and expression of pilin genes in different bacterial systems.
Fusion gene consisting of dextran-binding domain from Leuconostoc mesenteroides subsp. Mesenteroides (DBD) and human recombinant interferon-beta (IFN-beta) incorporated between the nucleotide sequence encoding for the re...Fusion gene consisting of dextran-binding domain from Leuconostoc mesenteroides subsp. Mesenteroides (DBD) and human recombinant interferon-beta (IFN-beta) incorporated between the nucleotide sequence encoding for the recognition site of human enteropeptidase (DDDDK) was installed and constructed in Escherichia coli. The overproducing strain of the chimeric protein DBD-IFN-beta consisting of the IFN-beta, spacer including 10 GS-repeats, human enteropeptidase recognition site, and dextran-binding domain from Leuconostoc mesenteroides was constructed. Free human recombinant interferon-beta was obtained as a result of treatment of the chimeric protein DBD-IFN-beta immobilized on sephadex G-25 with human enteropeptidase. The ability of free and immobilized protein to protect human cells from viral infection was demonstrated. The developed approach can be used for purification of the recombinant proteins with different biological activity and possible construction of new immunostimulating and antiviral drugs, growth factors, anti-cancer drugs, etc.
Vedernikov VE, Ivanov MK, Prasolova MA
… +2 more, Kandrushin EV, Dymshits GM
Mol Gen Mikrobiol Virusol
· 2009 · PMID 20017361
Hepatitis C virus (HCV) infection is a major cause of severe liver disease including liver cirrhosis and hepatocellular carcinoma. Genotyping became fundamental in the clinical assessment of patients with hepatitis C bec...Hepatitis C virus (HCV) infection is a major cause of severe liver disease including liver cirrhosis and hepatocellular carcinoma. Genotyping became fundamental in the clinical assessment of patients with hepatitis C because the genotype of the hepatitis C virus determines the chance of therapeutic response and duration of treatment. We developed a new real-time PCR assay for genotyping of HCV with increased specificity due to a novel approach to dual-labeled probe design using oligodeoxyinosine linkers. The assay allows genotypes 1, 2, 3 to be distinguished and genotypes 4-6 with high specificity to be blocked. The analytical sensitivity (150 IU/ml) can be implemented. Of the 285 clinical samples genotyped using the developed assay, 45% were genotype 1; 6%, genotype 2; 49%, genotype 3. No discordant results with 5-UTR sequencing and commercial genotyping assays were obtained.
Minimal inhibiting AgNO3 concentration (MICs) in the gram-negative bacteria Escherichia coli K12, Serratia proteamaculans 94, and Serratia liquefaciens MG1 were found to be on the average within the range of 0.075-0.3 mi...Minimal inhibiting AgNO3 concentration (MICs) in the gram-negative bacteria Escherichia coli K12, Serratia proteamaculans 94, and Serratia liquefaciens MG1 were found to be on the average within the range of 0.075-0.3 microg/ml, and for Pseudomonas aeruginosa PAO1 and P. chlororaphis 449, 0.15-0.3 microg/ml. Biofilm formation in Escherichia coli AB1157 and S. Proteamaculans 94 was completely inhibited at an AgNO3 concentration of 0.3 microg/ml, and in Pseudomonas aeruginosa PAO1, at 0.6 microg/mlAgNO3. Mutations in E. coli genes encoding for global regulators of gene expression, such as sigma S and sigma N subunits of RNA polymerase, catabolite repression protein CRP, and Lon protease, had no marked effect on the sensitivity of cells to silver. The wild-type E. coli strains and strains deficient in excision repair (uvrA, uvrB), SOS-repair or recombination (recA, lexA, recBC, recF mutants) did not differ in their silver sensitivity. This suggests that the sensitivity of bacteria to silver does not correlate with DNA lesions that could be repaired by these repair and recombination systems. E. coli mutant strains deficient in porins OmpF or OmpC, were 3-4-fold more resistant to silver ions as compared with the wild-type strain. Experiments with pME6863 plasmid harboring the gene of N-acyl-homoserine lactonase AiiA demonstrated that Quorum Sensing regulation (QS) did not participate in the control of S. proteamaculans 94 and P. chlororaphis 449 silver sensitivity. The same conclusion was drawn from the comparison of AgNO3 MICs for the S. liquefaciens wild-type strain and a mutant strain deficient in QS.
Kuznetsovskiĭ AV, Migova LV, Fedorov IuN
… +6 more, Kibirev IaI, Vorob'ev AA, Darmov IV, Borisevich IV, Savinykh AV, Gorin OV
Mol Gen Mikrobiol Virusol
· 2009 · PMID 20017359
The genotyping variety of 5 known anthracis vaccine strains using 18 variable loci of the chromosomal localization taken from a microbe culture collection of 48 Research Institute of Ministry of Defense was revealed in t...The genotyping variety of 5 known anthracis vaccine strains using 18 variable loci of the chromosomal localization taken from a microbe culture collection of 48 Research Institute of Ministry of Defense was revealed in the research. The stability of the VNTR-loci was shown to be inherited from the B. anthracis strains with common origin and an opportunity of their gene-identification application. The gene profile of each analyzed vaccine strain using every 18 polymorphic loci was determined and the amplification products were sequenced. The variation of electrophoretic mobility of the amplifiers was found to be caused by the presence of the replication elements with various numbers of copies in their structure.
The specificity of DNA-methyltransferase M.Bsc4I was defined in cellular lysate of Bacillus schlegelii 4. For this purpose, we used methylation sensitivity of restriction endonucleases, and also modeling of methylation....The specificity of DNA-methyltransferase M.Bsc4I was defined in cellular lysate of Bacillus schlegelii 4. For this purpose, we used methylation sensitivity of restriction endonucleases, and also modeling of methylation. The modeling consisted in editing sequences of DNA using replacements of methylated bases and their complementary bases. The substratum DNA processed by M.Bsc4I also were used for studying sensitivity of some restriction endonucleases to methylation. Thus, it was shown that M.Bsc4I methylated 5'-Cm4CNNNNNNNGG-3' and the overlapped dcm-methylation blocked its activity. The offered approach can appear universal enough and simple for definition of specificity of DNA-methyltransferases.
DNA fragments encoding for two collagen binding decapeptides from the human von Willebrand factor (vWF-H1 and vWF-H2) were cloned in the Escherichia coil culture. Overproducing strains of the chimeric proteins vWF(H1)-CB...DNA fragments encoding for two collagen binding decapeptides from the human von Willebrand factor (vWF-H1 and vWF-H2) were cloned in the Escherichia coil culture. Overproducing strains of the chimeric proteins vWF(H1)-CBD and vWF(H2)-CBD consisting of the corresponding decapeptide, Gly-Ser spacer and a cellulose binding domain (CBD) from Anaerocellum thermophilum were constructed. Using one-stage purification on cellulose, the highly purified samples of vWF(H1)-CBD and vWF(H2)-CBD proteins were obtained and the ability of these proteins to bind collagen was studied. These constructions are planned to be used for development of the recombinant collagen binding proteins with different biological activities, which, in its turn, will be used for development of the new generation products and materials for medicine, such as different kinds of implants, the coats, etc.
Epitope mapping of the major envelope glycoprotein E2 of classical swine fever virus (CSFV) is important for our understanding of E2 and also for development of the CSFV-specific diagnostic assays and epitope- or peptide...Epitope mapping of the major envelope glycoprotein E2 of classical swine fever virus (CSFV) is important for our understanding of E2 and also for development of the CSFV-specific diagnostic assays and epitope- or peptide-based marker vaccines. Previous competitive binding studies showed that monoclonal antibodies raised against E2 protein of CSFV detected 8 individual epitopes. At the present study using a set of synthetic peptides covering the full sequences of E2 glycoprotein five linear non-overlap B-cells epitopes were identified. The identified sequences of 12 strains of the CSFV and 5 other pestiviruses were aligned. The data obtained could be useful for improvement of the CSFV diagnostic systems and studies of amino acid substitutions and their influence on antigenic properties of the CSFV E2 protein.
Structural and functional analysis of the araN gene involved in regulation of expression of diagnostically significant symptom (arabinose fermentation) was performed in the Yersinia pestis microorganism. Lack of arabinos...Structural and functional analysis of the araN gene involved in regulation of expression of diagnostically significant symptom (arabinose fermentation) was performed in the Yersinia pestis microorganism. Lack of arabinose fermentation in the Altai substrain, Hissar substrain, and Talas strains was shown to be due to solitary nucleotide insert into the araN gene. The insert is in the position 763 bp. The strains of the main, Caucasian, and Ulege substrains do not contain this mutation of the araN gene. The absence of the mutation correlates with ability to ferment arabinose.
Savel'eva NV, Zagriadskaia IuE, Klimashevskaia SV
… +4 more, Puzyrev VF, Burkov AN, Obriadina AP, Ulanova TI
Mol Gen Mikrobiol Virusol
· 2009 · PMID 19705778
Amino acid sequence of the Chlamydia trachomatis major outer membrane protein (MOMP) was modeled using a series of the recombinant proteins containing six 100 aa fragments of MOMP overlapped in 30 aa. Testing of recombin...Amino acid sequence of the Chlamydia trachomatis major outer membrane protein (MOMP) was modeled using a series of the recombinant proteins containing six 100 aa fragments of MOMP overlapped in 30 aa. Testing of recombinant antigens in EIA showed that proteins containing MOMP fragments comprising 191-286 and 191-354 aa regions had the greatest activity in the reaction with anti- C. trachomatis positive serum samples. The data obtained allows the conclusion about the possibility of the use of presented recombinant proteins for development of diagnostic test for anti- C. trachomatis antibodies detection to be made.
The method of the VNTR-analysis in a locus (5'-NAAA-3')n was studied using samples of the DNA of 73 strains Y. pestis ssp. altaica, isolated in Gorno-Altaisk and 65 strains Y. pestis ssp. Pestis, allocated in Tuva natura...The method of the VNTR-analysis in a locus (5'-NAAA-3')n was studied using samples of the DNA of 73 strains Y. pestis ssp. altaica, isolated in Gorno-Altaisk and 65 strains Y. pestis ssp. Pestis, allocated in Tuva natural foci of plague. It was demonstrated that strains from the Gorno-Altaisk population of plague microbes possessed genomic polymorphism in the locus given a marker that is reflected in variability of the size of an amplicon from 250 up to 275 bp, correlating with places of isolation of strains in separate epizootic loci. Strains Y. pestis ssp. pestis from the Tuva population, on the contrary, are characterized by conservatism in the locus 5'-NAAA-3' (the size of an amplicon was 261 bp). Providing selective direct sequencing of amplicons, exact values of their sizes and number of repeats of a tetranucleotide 5'-NAAA-3' were determined.