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

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 sigma(70) orthologue in M. extorquens AM1.