Yuri D. Tsygankov

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.

Two new members of the BioB superfamily: cloning, sequencing and expression of bioB 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.

Oxidative and assimilative enzyme activities in continuous cultures of the obligate methylotrophMethylobacillus flagellatum

In methanol-limited continuous cultures of the obligate methylotrophic bacteriumMethylobacillus flagellatum grown at rates from 0.05 to 0.63 h-1, and also in an oxyturbidostat culture ofM. 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.

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 l-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-hours 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.

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.