MdoR is a novel positive transcriptional regulator for the oxidation of methanol in Mycobacterium sp. strain JC1.

Mycobacterium sp. strain JC1 is able to grow on methanol as a sole source of carbon and energy using methanol:N,N'-dimethyl-4-nitrosoaniline oxidoreductase (MDO) as a key enzyme for methanol oxidation. The second open reading frame (mdoR) upstream of, and running divergently from, the mdo gene was identified as a gene for a TetR family transcriptional regulator. The N-terminal region of MdoR contained a helix-turn-helix DNA-binding motif. An electrophoretic mobility shift assay (EMSA) indicated that MdoR could bind to a mdo promoter region containing an inverted repeat. The mdoR deletion mutant did not grow on methanol, but growth on methanol was restored by a plasmid containing an intact mdoR gene. In DNase I footprinting and EMSA experiments, MdoR bound to two inverted repeats in the putative mdoR promoter region. Reverse transcription-PCR indicated that the mdoR gene was transcribed only in cells growing on methanol, whereas ß-galactosidase assays showed that the mdoR promoter was activated in the presence of methanol. These results indicate that MdoR serves as a transcriptional activator for the expression of mdo and its own gene. Also, MdoR is the first discovered member of the TetR family of transcriptional regulators to be involved in the regulation of the methanol oxidation, as well as to function as a positive autoregulator.

Identification and functional characterization of a gene for the methanol : N,N'-dimethyl-4-nitrosoaniline oxidoreductase from Mycobacterium sp. strain JC1 (DSM 3803).

Mycobacterium sp. strain JC1 is able to grow on methanol as a sole source of carbon and energy using methanol : N,N'-dimethyl-4-nitrosoaniline oxidoreductase (MDO) as a key enzyme for primary methanol oxidation. Purified MDO oxidizes ethanol and formaldehyde as well as methanol. The Mycobacterium sp. strain JC1 gene for MDO (mdo) was cloned, sequenced, and determined to have an open reading frame of 1272 bp. Northern blot and promoter analysis revealed that mdo transcription was induced in cells grown in the presence of methanol. Northern blotting together with RT-PCR also showed that the mdo gene was transcribed as monocistronic mRNA. Primer extension analysis revealed that the transcriptional start site of the mdo gene is located 21 bp upstream of the mdo start codon. An mdo-deficient mutant of Mycobacterium sp. strain JC1 did not grow with methanol as a sole source of carbon and energy.

Cloning, characterization and expression of a gene encoding dihydroxyacetone synthase in Mycobacterium sp. strain JC1 DSM 3803.

Dihydroxyacetone synthase (DHAS) is a key enzyme involved in the assimilation of methanol in Mycobacterium sp. strain JC1 DSM 3803. The structural gene encoding DHAS in Mycobacterium sp. strain JC1 was cloned using random-primed probes synthesized after PCR with synthetic primers based on the amino acid sequences conserved in two yeast DHASs and several transketolases. The cloned gene, dasS, had an ORF of 2193 nt, encoding a protein with a calculated molecular mass of 78,197 Da. The deduced amino acid sequence of dasS contained an internal sequence of Mycobacterium sp. strain JC1 DHAS and exhibited 29.2 and 27.3 % identity with those of Candida boidinii and Hansenula polymorpha enzymes, respectively. Escherichia coli transformed with the cloned gene produced a novel protein with a molecular mass of approximately 78 kDa, which cross-reacted with anti-DHAS antiserum and exhibited DHAS activity. Primer-extension analysis revealed that the transcriptional start site of the gene was the nucleotide A located 31 bp upstream from the dasS start codon. RT-PCR showed that dasS was transcribed as a monocistronic message. Northern hybridization and beta-galactosidase assay with the putative promoter region of dasS revealed that the gene was transcribed only in cells growing on methanol. The expression of dasS in Mycobacterium sp. strain JC1 was free from catabolite repression.

Growth of mycobacteria on carbon monoxide and methanol.

Several mycobacterial strains, such as Mycobacterium flavescens, Mycobacterium gastri, Mycobacterium neoaurum, Mycobacterium parafortuitum, Mycobacterium peregrinum, Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium tuberculosis, and Mycobacterium vaccae, were found to grow on carbon monoxide (CO) as the sole source of carbon and energy. These bacteria, except for M. tuberculosis, also utilized methanol as the sole carbon and energy source. A CO dehydrogenase (CO-DH) assay, staining by activity of CO-DH, and Western blot analysis using an antibody raised against CO-DH of Mycobacterium sp. strain JC1 (formerly Acinetobacter sp. strain JC1 [J. W. Cho, H. S. Yim, and Y. M. Kim, Kor. J. Microbiol. 23:1-8, 1985]) revealed that CO-DH is present in extracts of the bacteria prepared from cells grown on CO. Ribulose bisphosphate carboxylase/oxygenase (RubisCO) activity was also detected in extracts prepared from all cells, except M. tuberculosis, grown on CO. The mycobacteria grown on methanol, except for M. gastri, which showed hexulose phosphate synthase activity, did not exhibit activities of classic methanol dehydrogenase, hydroxypyruvate reductase, or hexulose phosphate synthase but exhibited N,N-dimethyl-4-nitrosoaniline-dependent methanol dehydrogenase and RuBisCO activities. Cells grown on methanol were also found to have dihydroxyacetone synthase. Double immunodiffusion revealed that the antigenic sites of CO-DHs, RuBisCOs, and dihydroxyacetone synthases in all mycobacteria tested are identical with those of the Mycobacterium sp. strain JC1 enzymes.