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

Seo, Jae Gu; Park, Sae W.; Park, Hyuk; Kim, Seo Y.; Ro, Young Tae; Kim, Eungbin; Cho, Jin W.; Kim, Young Beom

doi:10.1099/mic.0.2007/011965-0

Cited by 8 publications

(8 citation statements)

References 31 publications

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“…Total RNA was isolated from cells harvested at the mid-exponential-growth phase using TRIzol reagent (Invitrogen), as previously described (32). To identify mdoR transcripts in cells grown in SMB-MeOH and SMB-glucose, reverse transcription-PCR (RT-PCR) using two primers, MR-F and MR-R, was performed according to the manufacturer's instructions (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

“…Vectors were introduced into Mycobacterium sp. strain JC1 wild type or an mdoR deletion mutant by electrotransformation according to the method of Seo et al (32).…”

Section: Methodsmentioning

confidence: 99%

“…DNA fragments containing the putative promoter of mdoR were prepared by PCR using primers RF-F and RF-R with 6-mer EcoRI and XhoI sites (underlined, Table 2). The resulting 304-bp fragments were end labeled with [␥- 32 P]ATP and digested with EcoRI or XhoI to prepare strand-specific end-labeled DNA fragments. The end-labeled fragments were mixed with MdoR in the binding buffer used for EMSA.…”

Section: Methodsmentioning

confidence: 99%

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MdoR Is a Novel Positive Transcriptional Regulator for the Oxidation of Methanol in Mycobacterium sp. Strain JC1

Park

Kim

2011

J Bacteriol

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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 ␤-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.Methylotrophic bacteria use reduced carbon compounds containing no carbon-carbon bonds as sole carbon and energy sources (4). The enzyme for the oxidation of methanol to formaldehyde in methanol-oxidizing bacteria is highly expressed in cells growing on methanol, indicating the expression of the gene for this enzyme is regulated according to the presence of methanol (19).The regulation of methanol oxidation in Gram-negative bacteria is known to be variable. At least 26 genes are required for the oxidation of methanol in Methylobacterium extorquens AM1 (23, 37). MxaB, a putative two-component response regulator, and MxbD and MxbM, a putative sensor-regulator pair, are involved in the regulation of methanol oxidation in this bacterium (35,36). Also, MxcQ and MxcE, another putative two component regulatory system, are required for the expression of mxaF, the gene for the large subunit of methanol dehydrogenase (MDH) (37). Further, a methanol-inducible promoter with a multi-A tract sequence upstream of mxaF is essential for the expression of mxaF (22, 39). In Paracoccus denitrificans, genes involved in methanol oxidation are located in the mxa gene cluster (38). A two-component system consisting of MxaY, a putative histidine kinase, and MxaX, a putative response regulator, is involved in the control of MDH expression (11). Another two-component system consisting of FlhR and FlhS also regulates methanol oxidation in Paracoccus denitrificans (12). However, little is known about the regulation of the genes responsible for the oxidation of methanol in Grampositive bacteria. It is only known that the gene for NADdependent MDH in Bacillus methanolicus strain MGA3 is upregulated in cells growing...

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Section: Methodsmentioning

confidence: 99%

“…Vectors were introduced into Mycobacterium sp. strain JC1 wild type or an mdoR deletion mutant by electrotransformation according to the method of Seo et al (32).…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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MdoR Is a Novel Positive Transcriptional Regulator for the Oxidation of Methanol in Mycobacterium sp. Strain JC1

Park

Kim

2011

J Bacteriol

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“…Methylotrophic metabolism in mycobacteria is only poorly understood and, by far, has been best studied in Mycobacterium sp. strain JC1 (21)(22)(23)(24). Interestingly, the most common C 1 assimilation pathways (RuMP and serine) have been suggested to be absent in mycobacteria (21), and mycobacterial species (except M. tuberculosis, which does not grow on methanol) have been shown to be capable of utilizing both CO and methanol as the sole carbon and energy sources by utilizing eukaryotic C 1 assimilation pathways (3,21,(23)(24)(25).…”

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confidence: 99%

Methylotrophy in Mycobacteria: Dissection of the Methanol Metabolism Pathway in Mycobacterium smegmatis

2018

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The mycobacteria comprise both pathogenic and nonpathogenic bacteria. Although several features related to pathogenicity in various mycobacterial species, such as , have been studied in great detail, methylotrophy, i.e., the ability of an organism to utilize single-carbon (C) compounds as the sole source of carbon and energy, has remained largely unexplored in mycobacteria. Reports are available that suggest that mycobacteria, including and, are capable of utilizing alternative C compounds to meet their carbon and energy requirements. However, physiological pathways that are functional in mycobacteria to utilize such carbon compounds are only poorly understood. Here we report the identification and characterization of the gene products required for establishing methylotrophy in We present,-dimethyl--nitrosoaniline (NDMA)-dependent methanol oxidase (Mno) as the key enzyme that is essential for the growth of on methanol. We show that Mno has both methanol and formaldehyde dehydrogenase activities Further, is able to utilize methanol even in the absence of the major formaldehyde dehydrogenase MscR, which suggests that Mno is sufficient to dissimilate methanol and the resulting formaldehyde Finally, we show that devoid of phosphoenolpyruvate carboxykinase, which has been shown to fix CO in , does not grow on methanol, suggesting that the final step of methanol utilization requires CO fixation for biomass generation. Our work here thus forms the first comprehensive report that explores methylotrophy in a mycobacterial species. Methylotrophy, the ability to utilize single-carbon (C) compounds as the sole carbon and energy sources, is only poorly understood in mycobacteria. Both pathogenic and nonpathogenic mycobacteria, including , are capable of utilizing C compounds to meet their carbon and energy requirements, although the precise pathways are not well studied. Here we present a comprehensive study of methylotrophy in With several genetic knockouts, we have dissected the entire methanol metabolism pathway in We show that while methanol dissimilation in differs from that in other mycobacterial species, the concluding step of CO fixation is similar to that in It is therefore both interesting and important to examine mycobacterial physiology in the presence of alternative carbon sources.

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“…Total RNA was isolated from cells harvested at the mid-exponential growth phase using TRIzol reagent (Invitrogen), according to the method previously described by Seo et al (2007). Northern blots were performed by the method described in Sambrook et al (1989).…”

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confidence: 99%

Expression and regulation of ribulose 1,5-bisphosphate carboxylase/oxygenase genes in Mycobacterium sp. strain JC1 DSM 3803

Lee

Park

et al. 2009

J Microbiol.

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Ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) is the key enzyme of the Calvin reductive pentose phosphate cycle. Two sets of structural genes (cbbLS-1 and -2) for form I RubisCO have been previously identified in the Mycobacterium sp. strain JC1, which is able to grow on carbon monoxide (CO) or methanol as sole sources of carbon and energy. Northern blot and reverse transcriptase PCR showed that the cbbLS-1 and -2 genes are expressed in cells grown on either carbon monoxide (CO) or methanol, but not in cells grown in nutrient broth. A promoter assay revealed that the cbbLS-2 promoter has a higher activity than the cbbLS-1 promoter in both CO- and methanol-grown cells, and that the activities of both promoters were higher in CO-grown cells than in methanol-grown cells. A gel mobility shift assay and footprinting assays showed that CbbR expressed in Escherichia coli from a cbbR gene, which is located downstream of cbbLS-1 and transcribed in the same orientation as that of the cbbLS genes, specifically bound to the promoter regions of the cbbLS-1 and -2 genes containing inverted repeat sequence. A DNase I footprinting assay revealed that CbbR protected positions -59 to -3 and -119 to -78 of the cbbLS-1 and -2 promoters, respectively. Overexpression of CbbR induced the transcription of RubisCO genes in Mycobacterium sp. strain JC1 grown in nutrient broth. Our results suggest that the CbbR product from a single cbbR gene may positively regulate two cbbLS operons in the Mycobacterium sp. strain JC1 as is the case for Rhodobacter sphaeroides and Cupriavidus necator.

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

Cited by 8 publications

References 31 publications

MdoR Is a Novel Positive Transcriptional Regulator for the Oxidation of Methanol in Mycobacterium sp. Strain JC1

MdoR Is a Novel Positive Transcriptional Regulator for the Oxidation of Methanol in Mycobacterium sp. Strain JC1

Methylotrophy in Mycobacteria: Dissection of the Methanol Metabolism Pathway in Mycobacterium smegmatis

Expression and regulation of ribulose 1,5-bisphosphate carboxylase/oxygenase genes in Mycobacterium sp. strain JC1 DSM 3803

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