Search PubMed⌕ Search

Biomedical subjects

Y D Tsygankov

Publications and source records attributed to Y D Tsygankov.

17 recordsLinked to original sources
PubMed1998

Refined genetic map of the obligate methylotroph Methylobacillus flagellatum.

We present a refined genetic map of the obligate methylotroph Methylobacillus flagellatum. New, Hfr (high-frequency-of-transfer) donors, and pulsed-field gel electrophoresis, were used to determine that M. flagellatum contains one approximately 3.1-Mb circular chromosome, and no plasmids. A correlation between time-of-entry units and DNA length was established. Using in vivo and in vitro cloning, and sequencing, a number of new genetic markers were identified and mapped; in addition, the nature of some of the previously mapped markers was elucidated.

Chromosome Mapping↗
PubMed1997

Regulation of ammonia assimilation in an obligate methylotroph Methylobacillus flagellatum under steady-state and transient growth conditions.

The obligate methylotroph Methylobacillus flagellatum was grown in the presence of different ammonium concentrations and the regulation of the enzymes associated with ammonium assimilation was investigated in steady-state and transient growth regimes. As the medium changed from C-limitation to dual C/N- and finally to N-limitation, the culture passed through three definite growth phases. The NADP(+)-dependent glutamate dehydrogenase (GDH) was present under ammonium limitation of the culture growth (at 2 mmol 1(-1) of ammonium in the growth medium) and increased in response to an increase in nitrogen availability. Glutamine synthetase (GS) and glutamate synthase (GOGAT) activities were negligible during C- and C/N-limitation. In N-limited cells the GOGAT activity increased as the dilution rate increased up to 0.35 h-1, and then sharply dropped. In the N-sufficient cultures both NAD(+)-and NADP(+)-dependent isocitrate dehydrogenase (NAD-ICDH and NADP-ICDH) activities were up-regulated as dilution rate increased, but in the N-limited culture the NAD-ICDH activity was up-regulated whereas NADP-ICDH one was down-regulated. Pulse additions of ammonium and methanol demonstrated the coordinate regulation of the GDH and ICDHs activities. When pulses were added to the C/N-limited cultures, there was an immediate utilization of the nutrients, resulting in an increase in biomass; at the same time the GDH and ICDH activities increased and the GS and GOGAT activities decreased. When the same ammonium/methanol pulse was added into the N-limited culture, there was a 3 h delay in the culture response, after which the substrates were utilized at rates close to the ones shown by the C/N-limited culture after the analogous pulse.

Alanine Dehydrogenase↗
PubMed1996

Two new members of the bio B superfamily: cloning, sequencing and expression of bio B genes of Methylobacillus flagellatum and Corynebacterium glutamicum.

Cloning, characterization and expression of the bio B gene of the obligate methylotrophic bacterium, Methylobacillus flagellatum, are reported. A chromosomal fragment containing bio B has been isolated by complementation of a bio B- mutant of M. flagellatum. Nucleotide (nt) sequence analysis of this fragment revealed the presence of an open reading frame of 966 nt identified as bio B, which is the first gene of the M. flagellatum bio cluster. Gene bio B was expressed in Escherichia coli and M. flagellatum, resulting in efficient conversion of dethiobiotin to biotin. The Corynebacterium glutamicum bio B has also been cloned and sequenced. Comparison of the amino acid sequences derived from known bio B genes allowed us to identify four cysteines participating as putative ligands forming the [2Fe-2S] cluster. Genomic organization of the bio biosynthetic genes shows wide diversity in various bacteria. The results of the database screening suggested that bio B proteins belong to a superfamily of proteins, including biotin and lipoate synthases and some proteins with unidentified functions.

Bacterial Proteins↗
PubMed1996

Identification and characterization of the pqqDGC gene cluster involved in pyrroloquinoline quinone production in an obligate methylotroph Methylobacillus flagellatum.

Pyrroloquinoline quinone is a prosthetic group of bacterial methanol dehydrogenases as well as some alcohol and glucose dehydrogenases. Genes involved in pyrroloquinoline quinone production have previously been cloned from the representatives of the alpha and gamma subdivisions of the Proteobacteria. We report identification and the sequence of the pqqDGC gene cluster in the obligate methylotroph, Methylobacillus flagellatum, which belongs to the beta subdivision. The deduced products of the pqq genes from M. flagellatum appear to be more similar to their counterparts from non-methylotrophic species of the gamma subdivision than to a facultative methylotroph of the alpha subdivision. A non-polar mutation in pqqG was constructed and resulted in a strain impaired in growth on methanol. This mutant accumulated a detectable amount of intracellular pyrroloquinoline quinone, but in contrast to the wild type, did not excrete pyrroloquinoline quinone into the culture medium. The possible role of PqqG is discussed.

Amino Acid Sequence↗
PubMed1993

Formate dehydrogenase from methylotrophic bacterium Pseudomonas sp. 101: gene cloning and expression in Escherichia coli.

Using two degenerate 20- and 23-mer oligonucleotide probes, the gene encoding NAD(+)-dependent formate dehydrogenase (FDH) (EC 1.2.1.2) was shown to lie within a 3.5 kb PstI fragment of the chromosomal DNA of methylotrophic bacterium Pseudomonas sp. 101. A phasmid library was prepared in the lambda pSL5 vector with partial EcoRI-digested DNA from Pseudomonas sp. 101. The 12 clones selected contained three types of phasmid: lambda pFDH1, lambda pFDH2 and lambda pFDH3 (with inserts of 15, 17.6 and 18.5 kb respectively). The inserts contained the same 15 kb EcoRI and 3.5 kb PstI fragments and included the complete FDH gene. Further subcloning of the insert resulted in plasmid pFDH2 and a 2.32 kb HindIII-BgIII fragment. Active enzyme was expressed in Escherichia coli TG1 (pFDH2) strain under control of a lac promoter.

Amino Acid Sequence↗
PubMed1992

Growth and enzymological characteristics of a pink-pigmented facultative methylotroph Methylobacterium sp. MB1.

Growth characteristics of batch and continuous cultures of the pink facultative methylotroph Methylobacterium sp. MB1 were determined. The response of a chemostat culture to a pulse increase of methanol concentration was studied. Malate, succinate and oxaloacetate additions to the methanol-supplemented medium decreased batch culture growth inhibition by methanol. The carotenoid content in cells grown in a chemostat decreased with increasing growth rate. The key enzyme activities of C1-metabolism were measured in a chemostat culture at different dilution rates.

Carotenoids↗
PubMed1991

Oxidative and assimilative enzyme activities in continuous cultures of the obligate methylotroph Methylobacillus flagellatum.

In methanol-limited continuous cultures of the obligate methylotrophic bacterium Methylobacillus flagellatum grown at rates from 0.05 to 0.63 h-1, and also in an oxyturbidostat culture of M. flagellatum growing at the rate of 0.73 h-1, levels of methanol dehydrogenase, enzymes of formaldehyde oxidation (both linear and cyclic) and assimilation (RuMP cycle), a number of intermediary metabolism and TCA cycle enzymes and also 'dye-linked' formaldehyde dehydrogenase were determined. It was shown that the activities of dissimilatory enzymes, with the exception of 'dye-linked' formaldehyde dehydrogenase, decreased with increasing growth rate. Activities of assimilative enzymes and activities of the TCA cycle enzymes detected as well as the 'dye-linked' formaldehyde dehydrogenase activity, increased with increasing growth rate. A periplasmic location was shown for the latter enzyme and a role in formaldehyde detoxification was proposed.

Aldehyde Oxidoreductases↗
PubMed1991

Genetic organization of methylamine utilization genes from Methylobacterium extorquens AM1.

An isolated 5.2-kb fragment of Methylobacterium extorquens AM1 DNA was found to contain a gene cluster involved in methylamine utilization. Analysis of polypeptides synthesized in an Escherichia coli T7 expression system showed that five genes were present. Two of the genes encoded the large and small subunits of methylamine dehydrogenase, and a third encoded amicyanin, the presumed electron acceptor for methylamine dehydrogenase, but the function of the other two genes is not known. The order on the 5.2-kb fragment was found to be large-subunit gene, the two genes of unknown function, small-subunit gene, amicyanin gene. The gene for azurin, another possible electron acceptor in methylamine oxidation, does not appear to be present within this cluster of methylamine utilization genes.

Azurin↗
PubMed1991

Nucleotide sequence of the amicyanin gene from Methylobacterium extorquens AM1.

The gene for amicyanin from the methylotrophic bacterium, Methylobacterium extorquens AM1 was identified. It encodes a protein consisting of 119 amino acids with a molecular weight of 12,609 kDa. The amino acid sequence shows the presence of a typical leader sequence and signal peptidase recognition site. Two putative hairpin structures were found, one located directly behind the amicyanin gene and another located 50 bp upstream. The same sequence AAAATCCC was found near the start codons for the small subunit of methylamine dehydrogenase and amicyanin, but its significance is not known.

Amino Acid Sequence↗
PubMed1990

Cloning and sequencing of the structural gene for the small subunit of methylamine dehydrogenase from Methylobacterium extorquens AM1: evidence for two tryptophan residues involved in the active center.

In two independent clone libraries, clones were identified that hybridized with oligonucleotide probes based on N- or C-terminal polypeptide sequence of the small subunit of methylamine dehydrogenase from Methylobacterium extorquens AM1. Plasmids from all clones had in common a 5.2 kb Bam HI-HindIII DNA fragment. A 0.57 kb SacII-BclI subfragment that hybridized to the oligonucleotide probes was sequenced. Nucleotide sequence analysis coincided with polypeptide sequence data in the structural part of the small subunit with a single contradiction: amino acid 17 is Asp rather than Asn. The two amino acids that are involved in the active center which had not been determined from previous polypeptide sequencing proved to be tryptophans.

Amino Acid Sequence↗
PubMed1990

Cloning, sequence and expression in Escherichia coli of the Methylobacillus flagellatum recA gene.

By means of interspecific complementation of an Escherichia coli recA- mutation with phasmids containing a gene bank from an obligate methylotroph, Methylobacillus flagellatum (Mf), the recA+ gene from this bacterium was identified. When expressed in an E. coli recA- host, it can function in recombination, DNA repair, and prophage induction. The nucleotide sequence of the gene has been determined. The coding region consists of 1032 bp specifying 344 amino acids. The deduced RecA protein structure shows a striking homology with RecA from other bacteria, except for the C-terminal region and some residues which were proposed to be responsible for the coprotease ability of RecA proteins. The region preceding the recA-Mf gene start codon has no SOS box--the LexA repressor binding site. Expression of the recA-Mf gene in E. coli proved to be DNA-damage independent.

Amino Acid Sequence↗
PubMed1990

Mapping of pgi and gpd genes involved in C-1 assimilation in the obligate methylotroph Methylobacillus flagellatum.

Homologous matings with plasmids R68.45 and pULB113, and also with Hfr type donor were employed for mapping pgi and gpd genes involved in C-1 metabolism in the obligate methylotroph Methylobacillus flagellatum. A preliminary map of the late chromosomal region was constructed on the basis of these experimental results. The C-1 markers were linked to methionine and leucine auxotrophy and nalidixic acid resistance markers. The phenomenon of retrotransfer, or shuttle transfer of chromosomal markers by Inc P1 plasmids, revealed earlier, was demonstrated for M. flagellatum.

Chromosome Mapping↗
PubMed1990

Genetic mapping of the obligate methylotroph Methylobacillus flagellatum: characteristics of prime plasmids and mapping of the chromosome in time-of-entry units.

A pULB113 (RP4::mini-Mu cts) plasmid was used to generate a library of prime plasmids carrying fragments of the Methylobacillus flagellatum genome. The genes carried by these prime plasmids were identified by complementation after transfer to suitably marked Escherichia coli and Pseudomonas aeruginosa strains. The hybrid plasmids were used for complementation mapping with a range of E. coli, M. flagellatum, and P. aeruginosa mutants. A preliminary map of the M. flagellatum genome section with seven groups of linked markers was obtained. Three of seven groups contain an overlapping sequence of cloned genes and can be considered as one large group of linked genes. A high-frequency-of-recombination donor of M. flagellatum (strain MFK64) mobilized the chromosome in a polarized manner from a single transfer origin. The donor was used to construct a time-of-entry map of the M. flagellatum chromosome. This was achieved by determining the time of entry of six randomly dispersed markers, four of which are included in known groups of linked markers. The linear map of M. flagellatum reported here consists of 44 markers.

Chromosome Mapping↗
PubMed1989

Construction of Hfr-like donors of the obligate methanol-oxidizing bacterium Methylobacillus flagellatum KT.

The transposon-loaded plasmid pAS8-121, incapable of autonomous replication in Gram-negative bacteria of non-enteric group, was transferred to Methylobacillus flagellatum KT wild type strain MFK1. The transconjugants arose at a frequency of 10(-7) per donor cell. The majority of the transconjugants tested exhibited the transfer of all selected chromosomal markers at rather high (10(-4)-10(-6) per donor cell) but similar frequencies. Only one of the obtained donors, designated MFK 64, was capable of mobilizing M. flagellatum KT chromosome in a polarized manner. The integrated nature of the plasmid in this and other MFK1 (pAS8-121) derivatives was supported by the results of DNA-DNA hybridization.

Conjugation, Genetic↗
PubMed1986

Broad host range vectors derived from an RSF1010::Tn1 plasmid.

Plasmid vector derivatives of the IncQ/P4 plasmid RSF1010 available for cloning DNA into a broad range of bacterial species were constructed. The plasmid pAYC31 constructed for the positive selection of inserted fragments contains part of transposon Tn1 inserted into the sequence of the gene sul. Gene aph transcription in pAYC31 can be initiated from the promoter for the transposase gene tnpA which is under the negative control of the gene tnpR product (Heffron, 1983). The insertion of a BamHI fragment, or of fragments generated by Sau3A, BclI, BglII, or XhoII digestion into the unique BamHI site within the gene tnpR sequence, leads to initiation of transcription from the promoter of the tnpA gene toward the aph gene. Expression of the aph gene upon insertion of a BamHI restriction fragment provides a positive selection for hybrid plasmids by plating the transformed bacteria on media with streptomycin. Versatile cloning vectors pAYC32 of 9.7 kb in length and pAYC39 of 11.3 kb in length were also constructed. Insertion into the BamHI site of vector pAYC32 of a 1.6-kb BglII fragment that contains the lambda cos site produced cosmid vectors pAYC51 and pAYC52. The two 11.3-kb cosmids differ only by the orientation of the 1.6-kb BglII fragment. By insertion into the BamHI site of pAYC32 of a BglII-BamHI fragment of plasmid pHC79 that contains the gene tet and lambda cos site cosmid vector pAYC53 was constructed. Vector pAYC31 was used to construct a gene bank from the chromosomal DNA of an obligate methylotrophic strain Methylomicrobium flagellatum KT.

Chromosome Mapping↗
PubMed1985

Specific-purpose broad-host-range vectors.

Several plasmid derivatives of broad-host-range Inc P4 plasmid RSF1010 were constructed and characterized. Vector pAYC30 was constructed by insertion in vivo into the genome of RSF1010 the Hgr transposon Tn501, originating from the plasmid pVS1 of Pseudomonas aeruginosa. Plasmids with inserts of PstI or SacI fragments may be selected by inactivation of genes sul and aph, respectively. The cloning at unique site SalGI leads to the appearance of HgCl2--sensitive transformants. Versatile cloning vector pAYC1 consists of two replicons, RSF1010 and plasmid pMZ7, a derivative of R6K. The constructed plasmid is 16.9 kb in length and determines resistance to five drugs. Two promoter-probe broad-host-range vectors, pAYC36 and pAYC37, were obtained by replacing a small segment from the DNA sequence of the aph gene promoter of previously described plasmid pAYC32 with the polylinker from plasmid pUC19. Therefore, vector plasmids retained the intact gene aph (Smr); however, they have Sms phenotype because of the insertional inactivation of the promoter. The genetic structure of promoter-probe vectors allows one to select clones, containing hybrid plasmids with an active promoter for gene aph expression.

Chromosome Mapping↗