Transcriptional analysis of pqqD and study of the regulation of pyrroloquinoline quinone biosynthesis in Methylobacterium extorquens AM1

Abstract

Methanol dehydrogenase, the enzyme that oxidizes methanol to formaldehyde in gram-negative methylotrophs, contains the prosthetic group pyrroloquinoline quinone (PQQ). To begin to analyze how the synthesis of PQQ is coordinated with the production of other methanol dehydrogenase components, the transcription of one of the key PQQ synthesis genes has been studied. This gene (pqqD) encodes a 29-amino-acid peptide that is thought to be the precursor for PQQ biosynthesis. A unique transcription start site was mapped to a guanidine nucleotide 95 bp upstream of the pqqD initiator codon. RNA blot analysis identified two transcripts, a major one of 240 bases encoding pqqD and a minor one of 1,300 bases encoding pqqD and the gene immediately downstream, pqqG. Both transcripts are present at similar levels in cells grown on methanol and on succinate, but the levels of PQQ are about fivefold higher in cells grown on methanol than in cells grown on succinate. These results suggest that PQQ production is regulated at a level different from the transcription of pqqD. The genes mxbM, mxbD, mxcQ, mxcE, and mxaB are required for transcription of the genes encoding the methanol dehydrogenase subunits and were assessed for their role in PQQ production. PQQ levels were measured in mutants defective in each of these regulatory genes and compared with levels of pqqD transcription, measured with a transcriptional fusion between the pqqD promoter and xylE. The results showed that only a subset of these regulatory genes (mxbM, mxbD, and mxaB) is required for transcription of pqqD, and only mxbM and mxbD mutants affected the final levels of PQQ significantly.Methylobacterium extorquens AM1 is a facultative methylotrophic bacterium capable of growing on single-carbon compounds such as methanol and methylamine as well as multicarbon compounds such as succinate (13). Methanol is oxidized to formaldehyde by the periplasmic enzyme methanol dehydrogenase, and then the formaldehyde is either assimilated into the cell or is oxidized to CO 2 , with the generation of energy. The methanol dehydrogenase contains pyrroloquinoline quinone (PQQ) as the prosthetic group (2).A complex array of genes is involved in methanol oxidation (14, 15) in M. extorquens AM1, and functions have been determined for a number of them. mxaF encodes the large subunit of methanol dehydrogenase, mxaI encodes the methanol dehydrogenase small subunit, and mxaG encodes the cytochrome c L structural polypeptide (1,22,23). mxaA, mxaK, and mxaL are involved in the insertion of calcium into the active site of methanol dehydrogenase (22-24). mxbM, mxbD, mxcQ, mxcE, and mxaB are genes required for transcription of methanol oxidation genes (14,20,22,23,29).The transcriptional regulation of the mxaF promoter in M. extorquens AM1 regulatory mutants has been studied, and in the wild-type strain, a sixfold increase in mxaF transcription was found in cells grown on methanol compared with cells grown on succinate. In strains defective in mxcQ, mxcE, mxbM, mxbD, or mxaB, the transcription f...

Molecular analysis of mxbD and mxbM, a putative sensor-regulator pair required for oxidation of methanol in Methylobacterium extorquens AM1

“…2), are apparently transcribed separately from mxbDM. A gap of 325 bp exists between mx6M and pqqD, and a transcriptional start site for pqqD has been identified within this region (Ramamoorthi & Lidstrom, 1995). The mxbD-xylE fusions showed that the 229 bp region upstream of the translation start of mxbD contains sufficient information to allow transcription under inducing conditions.…”

“…In these Methylobacterium strains, this second sensor kinase-response regulator pair (MxbDM) is required for normal expression of mxaF mxbD and mxbM in M . extorquens AM1 (Xu et a[., 1993), pqqD (Ramamoorthi & Lidstrom, 1995) and mxaW (Xu et al, 1993), and is also apparently involved in the negative regulation of expression of cytochrome c553 (Springer et al, 1995). However, MxcQE are not required for mxaW or pqqD expression.…”

“…In addition, in M . extorquens AM1, MxbD, MxbM and MxaB have been shown to be required for normal expression of a pqqD-xylE fusion (Ramamoorthi & Lidstrom, 1995), and in M . organophilum XX, MxbD and MxbM are required for expression of an mxaW-xylE fusion (Xu et al, 1993).…”

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

“…A region upstream of the 235 sequence of mxaF, pqqA and mxaW (Anderson et al, 1990;Ramamoorthi & Lidstrom, 1995;Zhang & Lidstrom, 2003) was analysed and found to contain a multiple A sequence. A similar sequence was found upstream of the putative promoter of mxbD (Springer et al, 1997) (Fig.…”

“…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).…”