Promoters and transcripts for genes involved in methanol oxidation in Methylobacterium extorquens AM1

Abstract

Twenty-five genes are involved in methanol oxidation to formaldehyde by the methanol dehydrogenase system in the facultative methylotroph Methylobacterium extorquens AM1 organized in five gene clusters. RT-PCR was used to assess the transcripts for the main gene clusters that encode methanol dehydrogenase and proteins required for its activity (mxaFGJIRSACKLDEHB), and the enzymes that are required for the synthesis of the methanol dehydrogenase prosthetic group, pyrroloquinoline quinone (pqqABC/DE and the pqqFG cluster). In both cases, positive bands were obtained corresponding to mRNA spanning each of the genes in the cluster, but not across the first and last genes and the gene immediately upstream or downstream of the cluster, respectively. These results suggest that these three gene clusters are each transcribed as a single operon. Confirmation was obtained by cloning a number of intergenic regions into a promoter probe vector. None of these regions showed significant promoter activity. Promoter regions were analysed for mxaF, pqqA, orf181 upstream of pqqFG, and mxaW, a gene located upstream of mxaF and divergently transcribed. The promoter regions for these genes were defined to within 100, 46, 124 and 146 bp, respectively, and the two unknown transcriptional start sites were determined, for mxaW and orf181. Alignment of these promoter regions suggests that they all may be transcribed by the s 70 orthologue in M. extorquens AM1.

Identification of an upstream regulatory sequence that mediates the transcription of mox genes in Methylobacterium extorquens AM1

“…Although no consensus promoter sequence could be identified from this small set, these promoters all shared similarity with the Escherichia coli s 70 promoter consensus sequence (Zhang & Lidstrom, 2003).…”

“…At least 25 genes have been identified to be involved in the methanol oxidation reaction in M. extorquens AM1 (Lidstrom, 1991;Zhang & Lidstrom, 2003). These Mox genes are distributed between three different loci: mxa, mxb and mxc.…”

“…Methanol oxidation is carried out by the enzyme methanol dehydrogenase, which is a quinoprotein using pyrroloquinoline quinone (PQQ) as a prosthetic group, and is an a 2 b 2 heterotetramer coupled to a specific cytochrome c accepter (Anthony, 1982). Methanol dehydrogenase activity and proteins have been shown to be regulated by carbon source in M. extorquens AM1, with three-to sixfold higher levels in the presence of methanol than during growth on multicarbon substrates in the absence of C 1 compounds (Nunn & Lidstrom, 1986a, b).At least 25 genes have been identified to be involved in the methanol oxidation reaction in M. extorquens AM1 (Lidstrom, 1991;Zhang & Lidstrom, 2003). These Mox genes are distributed between three different loci: mxa, mxb and mxc.…”

“…Two additional regulatory genes, mxcQE, are located in the mxc gene cluster. Of the five Mox regulatory genes identified (mxaB, mxbDM, and mxcQE), four are predicted to encode two sets of sensor-kinase/ (Nunn & Lidstrom, 1986a, b;Springer et al, 1997Springer et al, , 1998Springer et al, , 1995.Each of the Mox gene clusters has been shown to constitute a single transcriptional unit, and promoter regions have been identified for all of the Mox gene clusters except the mxcQE pair (Zhang & Lidstrom, 2003). Each of these Mox promoters has been shown to be expressed at higher levels during growth on methanol than on succinate (Zhang & Lidstrom, 2003).…”

“…Transcriptional start sites have been determined for all of the promoters except the mxbDM and mxcQE pair (Anderson et al, 1990;Ramamoorthi & Lidstrom, 1995;Zhang & Lidstrom, 2003). Although no consensus promoter sequence could be identified from this small set, these promoters all shared similarity with the Escherichia coli s 70 promoter consensus sequence (Zhang & Lidstrom, 2003).The regulation of expression of these Mox promoters by carbon source as well as the presence of putative response regulators required for transcription of the Mox promoters suggested that a regulatory region might be present upstream of the promoters involved either in the increased expression during growth on methanol, or in the basal expression that disappears in the Mox regulatory mutants. A putative regulatory sequence was identified, a multiple A-tract sequence in the promoter region of most genes involved in methanol oxidation (Zhang & Lidstrom, 2003).…”