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

Meng Zhang1 and Mary E. Lidstrom1,2

1 Departments of Chemical Engineering, University of Washington, Seattle, WA 98195-1750, USA
2 Departments of Microbiology, University of Washington, Seattle, WA 98195-1750, USA

Correspondence
Mary E. Lidstrom
Lidstrom{at}u.washington.edu


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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.


Abbreviations: MDH, methanol dehydrogenase; PQQ, pyrroloquinolone quinone


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Methylobacterium extorquens AM1 is a pink-pigmented facultative methylotroph that can grow on single-carbon compounds such as methanol as sole carbon and energy source. Methanol is oxidized to formaldehyde by the periplasmic enzyme methanol dehydrogenase (MDH). The formaldehyde is consumed inside the cell, either assimilated into biomass through the serine cycle or oxidized to CO2 with the generation of energy (Lidstrom, 1991; Anthony, 2000). MDH is an {alpha}2{beta}2 tetramer with two active sites, each containing a pyrroloquinoline quinone (PQQ) prosthetic group and a calcium atom (Anthony, 2000).

Genetic analysis of M. extorquens AM1 has shown that at least 25 genes are involved in the oxidation of methanol to formaldehyde (Lidstrom, 1991). These genes have been mapped to five gene clusters on the M. extorquens AM1 chromosome: mxa, mxb, pqqABC/DE, pqqFG and mxc. The first of these loci contains a cluster of 14 genes all transcribed in the same direction: mxaFJGIRSACKLDEHB with an additional upstream gene, mxaW, which is divergently transcribed (Anderson et al., 1990; Morris et al., 1995; Springer et al., 1995, 1998). mxaF and mxaI encode the {alpha} and {beta} subunits of MDH, respectively. mxaG encodes the cytochrome cL structural polypeptide (Anderson & Lidstrom, 1988; Nunn & Lidstrom, 1986a, b; Nunn et al., 1989). mxaJ, mxaR, mxaS, mxaD, mxaE and mxaH encode genes of unknown function, thought to be involved in MDH stability and/or assembly (Lidstrom, 1991). mxaACK and L are involved in inserting the calcium into the enzyme (Morris et al., 1995; Richardson & Anthony, 1992). mxaB is a transcriptional regulator of methanol oxidation genes (Springer et al., 1998). The function of mxaW is unknown. Although mutants in mxaW show no phenotype, a methanol-inducible promoter is present upstream of the gene (Springer et al., 1998). In addition, six genes, pqqABC/DE and pqqFG, are required for PQQ biosynthesis in M. extorquens AM1 (Morris et al., 1994; Springer et al., 1996; Toyama et al., 1997). In most PQQ-synthesizing bacteria, pqqC and pqqD are separate genes, but in M. extorquens AM1 we have shown that these genes are fused into a single polypeptide, which we have designated pqqC/D (Toyama et al., 1997). Four more genes are involved in transcriptional regulation of the methanol oxidation system, mxbDM and mxcQE (Springer et al., 1995, 1997).

Although the genes involved in methanol oxidation are identified and sequenced, little is known about their transcriptional organization. Using reporter gene fusions, methanol-inducible promoters have been detected upstream of mxaF, mxaW, mxbD and pqqA (Ramamoorthi & Lidstrom, 1995; Springer et al., 1997) and transcriptional start sites have been mapped for mxaF and pqqA (Anderson et al., 1990; Ramamoorthi & Lidstrom, 1995). In addition, two transcripts were detected by Northern blots in the ppqAB region, a major one encoding pqqA and a minor one encoding pqqAB (Ramamoorthi & Lidstrom, 1995). In this study, we have focused on the promoters and transcriptional organization for the major gene clusters encoding structural genes involved in synthesis of active MDH and in PQQ synthesis.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Chemicals and enzymes.
All chemicals used were analytical grade and obtained from Baker Chemicals or Fisher Scientific. X-Gal was from ISC Bioexpress. Enzymes for molecular biology were purchased from Roche Molecular Biochemicals and New England Biolabs, and used according to the suppliers' instructions. Taq DNA polymerase was obtained from Gibco-BRL.

Media and growth conditions.
Methylobacterium strains were grown at 30 °C on minimum medium described previously (Fulton et al., 1984), containing 0·5 % (v/v) methanol or 0·4 % (w/v) succinate. Escherichia coli strains were grown on Luria–Bertani (LB) broth or solid media (Sambrook et al., 1989) by adding 1·5 % agar (Difco). Appropriate antibiotics, all of which were obtained from Sigma, were added to the following final concentration (mg l-1): tetracycline, 12·5 (10 for M. extorquens AM1); kanamycin, 50; ampicillin, 50; streptomycin, 25.

Bacterial matings.
Triparental matings were performed as described previously (Chistoserdov et al., 1994).

Construction of plasmids for promoter studies.
The PCR products that included the upstream region of the gene to be studied were first cloned into the pCR2.1 TOPO vector. These fragments were then cut and inserted into the appropriate promoter probe vector using the multiple cloning sites in front of the reporter gene.

RT-PCR.
The RT-PCR kit was obtained from Gibco-BRL or Roche Molecular Biochemicals and the experiment was performed according to the suppliers' instructions. The primers used for RT-PCR were designed across the intergenic region of the two genes to be studied, to generate a PCR product of approximately 100–500 bp.

RNA isolation.
Total bacterial RNA was isolated from M. extorquens AM1 cells grown to mid-exponential phase on succinate or methanol, using the Epicentre Technologies RNA purification kit. The concentration and quality of total RNA was analysed using an Agilent Bioanalyser 2100 and an Agilent separations chip by the Center for Expression Arrays (University of Washington, Seattle, WA).

{beta}-Galactosidase assays.
Quantitative analyses of lacZ expression were performed in cell extracts according to Miller (1972). Cell extracts of M. extorquens AM1 were obtained by passing concentrated cell suspension through a French pressure cell at 37 kPa (Aminco) as described by Chistoserdova & Lidstrom (1991).

Catechol-2,3-dioxygenase activity (XylE assays).
Catechol-2,3-dioxygenase was assayed in cell extracts as described by Zukowski et al. (1983).

Transcriptional start site mapping.
The mxaW transcriptional start site was mapped by means of primer extension using the ThermoScript (Gibco-BRL) cDNA synthesis protocol or 1st Strand AMV synthesis kit (Roche) using 8–10 µg total RNA. Primers were labelled with [{gamma}-32P]ATP [6000 Ci mmol-1 (222 TBq mmol-1); NEN], using T4 polynucleotide kinase (Roche). In each case, the primers for the reverse transcription reaction were 18–25 mers and were located at different sites with relation to the start codon.


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Table 1. Bacterial strains and plasmids

 

   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
RT-PCR of mxa and pqq gene clusters
The clustering of genes involved in methanol oxidation in the mxa gene cluster (Fig. 1) and of genes involved in PQQ synthesis in the two pqq gene clusters (Fig. 2) suggested the possibility that the genes in each case might be co-transcribed. However, attempts to obtain clear and reproducible bands with Northern blots have not been successful (data not shown). Therefore, we used RT-PCR across each pair of genes in each cluster to assess the possibility of a contiguous transcript. The first set of genes tested was in the mxa cluster (Fig. 1). In this case, RT-PCR products of the correct size and sequence were obtained across each pair of genes in the entire 14-gene set (mxaFJGIRSACKLDEHB), which covers 12·6 kb. No products were obtained between mxaF and the upstream region, or between mxaB and the downstream region. In each case, controls for DNA contamination of the RNA preparations using direct PCR without the RT step were negative (data not shown). These data suggest that this 14-gene cluster may be transcribed as a single operon.



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Fig. 1. RT-PCR for the intergenic regions of mxa genes. Asterisks indicate positive controls using chromosomal DNA as template; other lanes used cDNA as template; multiple lanes show replicates with different amounts (labelled on top of each lane) of cDNA as template. Size standards are shown on each gel. The arrow under the mxa gene cluster shows the putative transcript. All RT-PCR reactions were repeated with the same results.

 


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Fig. 2. RT-PCR for the intergenic regions of pqq genes. Asterisks indicate positive controls using chromosomal DNA as template; other lanes used cDNA as template; multiple lanes show replicates with the amounts of cDNA on top of each lane. The arrows under the pqq gene clusters show the putative transcripts. All RT-PCR reactions were repeated with the same results.

 
Similar experiments were not carried out for mxaW, because it is flanked by genes transcribed in the opposite orientation and must be a single-gene transcript. However, the two pqq gene clusters involved in PQQ synthesis have been analysed. For pqqABC/DE, it had previously been shown that pqqA and pqqB were co-transcribed (Ramamoorthi & Lidstrom, 1995). Positive bands of the correct size were obtained for the other two intergenic regions of this cluster, but not for the upstream and downstream regions, suggesting that these genes constitute an operon transcribed by the single promoter upstream of pqqA (Fig. 2). Little is known about the other PQQ cluster containing pqqFG. The genome sequence showed a group of six genes in this region, all transcribed in the same direction, including pqqFG (Fig. 2). The first of these is predicted to encode an isoleucyl tRNA synthetase and the sixth a dioxygenase, while the other two (orf181 and orf219) are ORFs of unknown function. Positive products for the orf181–orf219–pqqF–pqqG–dioxygenase intergenic regions were also obtained but not for the region between the gene predicted to encode isoleucyl tRNA synthetase and orf181, suggesting that the last five genes might constitute an operon (Fig. 2).

Promoter analysis for mxa and pqq intergenic regions
The RT-PCR results suggested that the three gene clusters analysed might each be transcribed as single transcripts. However, a positive RT-PCR product could be obtained between two transcripts if the transcripts overlap. Therefore, we screened the larger intergenic regions for promoter activity, using xylE as a reporter, first with the promoter probe vector pCM76 and later with a low background vector (pCM130). The regions screened were those upstream of mxaJ, mxaG, mxaI, mxaR, mxaS, mxaE, mxaH, mxaB, pqqF, pqqG and orf219. However, no significant activity above background was found for any of these constructs, suggesting that no promoter was present in these intergenic regions (data not shown).

Analysis of mxaF and mxaW promoter regions
It had previously been shown that a 0·4 kb region between mxaF and mxaW has full activity compared to a 1·5 kb region that had been analysed previously (Marx & Lidstrom, 2001) and the transcriptional start site had been previously mapped to a position 168 bp upstream of the translational start site (Anderson et al., 1990). To more precisely define the promoter region, a number of smaller fragments were tested for promoter activity, using the xylE reporter in both pCM76 and pCM130, and the results are shown in Fig. 3. A fragment covering 100 bp upstream of the transcriptional start site showed full activity, a fragment covering 89 bp upstream showed intermediate activity, and a fragment covering 61 bp upstream showed activity at the vector background level. Therefore, the full promoter activity appeared to require a region approximately 90–100 bp upstream of the transcriptional start site and no activity could be detected when the -10, -35 region alone was present.



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Fig. 3. The M. extorquens AM1 mxa promoter region showing the DNA fragments tested for promoter activity, the catechol dioxygenase and {beta}-galactosidase activities from those fragments, and the transcriptional start sites. The transcriptional start site for mxaF was published previously (Anderson et al., 1990). The data on each line show the bp upstream of the transcription start site of mxaF or translation start site of mxaW.

 
The 0·4 kb region between mxaF and mxaW has methanol-inducible xylE activity when cloned in the orientation opposite to mxaF (Marx & Lidstrom, 2001; Springer et al., 1998), suggesting the promoter of mxaW is also within this 0·4 kb region but divergently transcribed compared to the 14-gene mxa cluster (Fig. 3). Since the transcriptional start site of mxaW was not known, we mapped it and found it to be 52 bp upstream of the mxaW translational start site (Fig. 4). The region encompassing the mxaW promoter was further defined by subcloning into a vector that targets low activity promoters (pCM132 using lacZ as a reporter), and found to be between 92 and 146 bp upstream of the transcriptional start site (Fig. 3). Since the distance between the two transcriptional start sites is 148 bp, the -10, -35 regions of the two promoters do not overlap. However, the regions shown to be essential for both promoters do overlap (Fig. 3).



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Fig. 4. Transcriptional start site mapping for mxaW and orf181. G, A, T and C are sequencing lanes. PE, primer-extension reaction. The nucleotides labelled with * are the transcriptional start sites.

 
Analysis of pqq cluster promoter regions
The RT-PCR and promoter cloning data suggested that the two pqq gene clusters were each transcribed from a single promoter upstream of pqqA (for the pqqABC/DE cluster) and orf181 (for the orf181–orf219–pqqFG–dioxygenase cluster). The transcriptional start site upstream of pqqA had previously been determined to be 94 bp upstream of the translational start site (Ramamoorthi & Lidstrom, 1995) (Fig. 5). We also mapped the transcriptional start site for orf181 and found it to be 101 bp upstream of the translational start site of orf181 (Figs 4 and 5). Subclones were used to narrow down the promoter region for the pqqA and orf181 clusters, which were found to lie within 46 bp and 225 bp upstream of the transcriptional start site, respectively (Fig. 5).



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Fig. 5. The M. extorquens AM1 pqq regions, showing the DNA fragments tested for promoter activity, the catechol dioxygenase activities from those fragments, and the transcriptional start sites for both clusters. The transcriptional start site for pqqA was published previously (Ramamoorthi & Lidstrom, 1995). The data on each line show the number of bp upstream of the transcription start site for pqqA or the translation start site for orf181.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Twenty-five genes are known to be involved in methanol oxidation to formaldehyde in M. extorquens AM1, found in five gene clusters. In this study, we have analysed transcripts and promoter regions for the three gene clusters encoding structural genes involved in the synthesis of active MDH and in PQQ synthesis, involving 21 of the known methanol oxidation genes. The mxa cluster contains 15 genes, most of which are required for growth on methanol and for active methanol oxidation. In this study, we have shown that the 15 genes are transcribed by two divergent methanol-inducible promoters, one for mxaW and the other for mxaFJGIRSACKLDEHB. The genes immediately downstream of mxaW and mxaB are transcribed in the opposite orientation and are not involved in methanol oxidation (Springer et al., 1998). Our results from this study suggest that the mxa region is expressed as two transcripts, one for mxaW and one for the other 14 genes. The two regions important for transcription apparently overlap.

Likewise, we have shown that the two gene clusters involved in PQQ synthesis are also each transcribed as a single transcript and each contains a single upstream promoter. pqqFG appears to be co-transcribed with three other genes of unknown function; orf181, orf219 and a gene predicted to encode a dioxygenase. Genes with identity to orf181, orf219 and the putative dioxygenase are found in the genomes of four bacteria known to synthesize PQQ and containing the other known PQQ genes, Sinorhizobium meliloti, Mesorhizobium loti, Pseudomonas aeruginosa and Rhodopseudomonas palustris with identities, respectively, of 23 %, 23 %, 24 %, 28 % to orf181; 23 %, 21 %, 40 %, 30 % to orf219; and 60 %, 63 %, 29 %, 37 % to the gene encoding the dioxygenase. Although the role of these genes in PQQ synthesis is unknown, their co-transcription with pqqFG in M. extorquens AM1 suggests they may be involved.

An alignment of these four methanol-inducible promoters (Fig. 6) does not show an obvious consensus sequence in the -10, -35 regions or upstream within the defined promoter regions. The -35 regions show similarity to the E. coli {sigma}70 -35 consensus (Fig. 6), but the -10 regions are more divergent. The promoter region of mxaF in a closely related strain, Methylobacterium organophilum XX, was investigated previously (Xu et al., 1993). A region of dyad symmetry was found between 30 and 50 bp upstream of the transcriptional start site in this strain. However, this structure is not present in M. extorquens AM1, even though the overall sequences in this region between mxaF and mxaW are very similar in both strains. The only obvious conserved region upstream of the -35 region within the defined M. extorquens AM1 promoter regions is a hexanucleotide, AAGAAA. A similar hexanucleotide has been previously suggested as a potential regulatory site in M. organophilum XX, based on its presence upstream of mxaF in that organism (Xu et al., 1993). Site-directed mutagenesis will be required to address the sequences important both in the -10, -35 regions and upstream regions.



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Fig. 6. Alignment of the -10 and -35 sequences of mxaF, mxaW, pqqA and orf181.

 
Five regulatory genes are known that are all required for detectable expression of the mxaF promoter, mxbDM, mxcQE and mxaB (Springer et al., 1995, 1997). mxbD and mxcQ are predicted to encode sensor kinases, while mxaB, mxbM and mxcE are all predicted to encode response regulators. Of these, only mxbDM are required for normal expression of the mxaW and pqqA promoters, while mxcQE are required for elevated expression of mxbDM (Ramamoorthi & Lidstrom, 1995; Springer et al., 1997). The precise regions required for this regulatory cascade are not yet known, but must reside within the promoter regions identified in this study.


   ACKNOWLEDGEMENTS
 
This work was supported by a grant from the DOE (DEFG03-96ER20226).


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Anderson, D. J. & Lidstrom, M. E. (1988). The moxFG region encodes four polypeptides in the methanol-oxidizing bacterium Methylobacterium sp. strain AM1. J Bacteriol 170, 2254–2262.[Medline]

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Chistoserdova, L. & Lidstrom, M. E. (1991). Hydroxypyruvate reductase from Methylobacterium extorquens AM1. J Bacteriol 173, 7228–7232.[Medline]

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Received 6 November 2002; revised 3 January 2003; accepted 3 January 2003.



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