Sequence and characterization of mxaB, a response regulator involved in regulation of methanol oxidation, and of mxaW, a methanol-regulated gene in Methylobacterium extorquens AM1

Abstract

In the facultative serine cycle methylotroph Methylobacterium extorquens AM1, mxaB is required for regulation of methanol oxidation and is located at the end of a large cluster of methylotrophy genes that begins with mxaF. The sequence of mxaB has been obtained and indicates that the gene product is a member of the response regulator family. None of the open reading frames near mxaB showed sequence identity to sensor kinases. Complementation studies suggest a promoter may be located adjacent to mxaB. Another gene (mxaW) is present immediately upstream of mxaF, divergently transcribed from a methanolinducible promoter. The sequence in the region of mxaW was also obtained. MxaW showed no identity to known proteins. Mutations in mxaW and in an adjacent open reading frame, OrfR, had no effect on growth of M. extorquens AM1 on methanol or other substrates. The MxaW mutant had normal methanol dehydrogenase activity and normal transcription of the mxaF promoter. Therefore, the function of mxaW is unknown. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V.

Development of improved versatile broad-host-range vectors for use in methylotrophs and other Gram-negative bacteria The GenBank accession numbers for the sequences reported in this paper are AF327711, AF327712, AF327713, AF327714, AF327715, AF327716, AF327717, AF327718, AF327719 and AF327720.

“…High reporter activities were obtained with both constructs (Table 2). pCM131 showed an approximately twofold Improved broad-host-range vectors increase in XylE activity in methanol-grown cells as compared to succinate-grown cells, similar to previously reported results (Springer et al, 1998). An analogous increase in βGal activity was not observed with pCM133.…”

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

“…In the fifth case, the mxaW promoter, the sequence exerted a negative effect, as expression increased in the deletion construct. The function of mxaW is not known, since mutants defective in mxaW show no detectable phenotype, including no change in mxaF promoter activity (Springer et al, 1998). However, the promoter is weakly methanol-inducible, so the role of the A-tract sequence in mxaW expression does not appear to involve methanol induction.…”

“…These Mox genes are distributed between three different loci: mxa, mxb and mxc. Fourteen genes (mxaFJGIRSACKLDEHB) transcribed in the same direction, together with an additional gene (mxaW) transcribed in the opposite direction, are located on the mxa gene cluster (Anderson et al, 1990;Morris et al, 1995;Springer et al, 1998Springer et al, , 1995. The large and small subunits of methanol dehydrogenase are encoded by mxaF and mxaI, respectively (Anderson & Lidstrom, 1988; Nunn & Lidstrom, 1986a, b;Nunn et al, 1989).…”

“…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.…”

“…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).…”