1 Department of Chemical Engineering, University of Washington, Seattle, WA 98195-2125, USA
2 Department of Microbiology, University of Washington, Seattle, WA 98195-2125, USA
Correspondence
Mary E. Lidstrom
lidstrom{at}u.washington.edu
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ABSTRACT |
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INTRODUCTION |
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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. 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., 1998
, 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
). The gene mxaG encodes cytochrome cL (Anderson & Lidstrom, 1988
; Nunn et al., 1989
). The gene mxaD encodes the 17 kDa periplasmic protein that directly or indirectly stimulates the interaction between methanol dehydrogenase and cytochrome cL (Toyama et al., 2003
), and the other genes in this region are involved in either assembly or regulatory functions (Anthony, 2000
; Lidstrom & Tsygankov, 1991
). Two other regulatory genes (mxbDM) (Springer et al., 1997
) and genes required for synthesis of the PQQ prosthetic group, pqqABC/DE (Morris et al., 1994
; Toyama et al., 1997
), are found at the mxb locus. The other two known pqq genes (pqqFG) reside within another cluster in the genome (Springer et al., 1996
). 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/response regulator pairs (MxbDM and MxcQE) and the fifth is predicted to encode an additional response regulator (MxaB) (Nunn & Lidstrom, 1986a
, b
; Springer et al., 1997
, 1998
, 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
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). Little is known concerning the role of this consensus sequence in M. extorquens AM1. In other organisms, multiple A- or T-tracts function as upstream recognition elements, increasing promoter activity (Cheema et al., 1999
) or facilitating DNA curvature for the binding of regulatory proteins (Aiyar et al., 1998
). In this study we have investigated the function of this sequence upstream of Mox promoters by site-directed mutagenesis.
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METHODS |
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Media and growth conditions.
E. coli stains were grown on LuriaBertani (LB) (Sambrook et al., 1989) broth or solid medium made by addition of 1·5 % agar (Difco). M. extorquens AM1 was grown aerobically at 30 °C either in liquid or on agar plates using a minimal salts medium (Fulton et al., 1984
) containing either 0·5 % (v/v) methanol or 0·4 % (w/v) succinate. Where necessary, media were supplemented with appropriate antibiotics, all of which were obtained from Sigma. Ampicillin (Ap) and kanamycin (Km) were used at 50 µg ml1 for both E. coli and M. extorquens AM1. Rifamycin (Rif) (50 µg ml1) was routinely added to plates for growth of M. extorquens AM1. Tetracycline (Tc) was added at final concentration of 12·5 µg ml1 for E. coli and 10 µg ml1 for M. extorquens AM1. Transformations of E. coli were performed using commercially available cells (JM109 and TOP10, from Promega and Invitrogen, respectively).
Generation of directed mutations.
Data from the M. extorquens AM1 genome project (http://www.integratedgenomics.com/genomereleases.html#list6) were used to design PCR primers to amplify the putative promoter of mxcQ. Other promoters containing the multiple A-tract region were amplified by PCR using chromosomal DNA of wild-type M. extorquens AM1 as a template and were cloned into pCR2.1 TOPO. Site-directed mutagenesis was performed using the Quick Change site-directed mutagenesis kit (Stratagene) according to the supplier's instructions. The mutations were verified by sequencing (Department of Biochemistry, University of Washington) and were subcloned into pCM130 as either EcoRIEcoRI or BamHIHindIII fragments in the correct orientation such that xylE was expressed from the cloned promoter. The pCM130 constructions were transferred into wild-type M. extorquens AM1 by triparental matings (Chistoserdov et al., 1994a) with the helper plasmid pRK2073. For chromosomal insertion constructions, the respective fragments downstream of genes of interest were cloned into pCM184 using SacISacII or ApaISacI sites and the fragments upstream of the genes of interest were cloned at the NdeIBglII or NdeIKpnI sites. The resulting plasmid DNA was electroporated into E. coli S17-1. These strains were used as donors in biparental matings with wild-type M. extorquens AM1, and KmRTcS progeny were identified on minimal medium agar plates containing succinate as described previously (Chistoserdov et al., 1994b
). In all cases, the identity of the double-crossover mutants was confirmed by diagnostic PCR using chromosomal DNA as template.
Construction of altered Ptac-xylE vector.
pKF03 was constructed by annealing the overlapping oligos KfptacAf (5'-CTAGTAAGAAATCTGAAATGAGCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAT-3') and KfptacAr (5'-CTAGATCCACACATTATACGAGCCGATGATTAATTGTCAACAGCTCAGATTCTTA-3') to form the 61 bp SpeIXbaI fragment ptacA, which was then cloned into the MCS of pCM130. pKF01, containing an unaltered tac promoter, was constructed for comparison by cloning a 263 bp BglIIHindIII fragment into the MCS of pCM130.
DNA manipulations.
Plasmid DNA isolation, restriction enzyme digestion, ligation, and E. coli transformation were carried out by standard protocols (Sambrook et al., 1989). The chromosomal DNA of M. extorquens AM1 was isolated as described by Saito & Miura (1963)
.
Catechol 2,3-dioxygenase assays.
The activity of XylE was determined in M. extorquens AM1 in crude extracts as described previously (Zhang & Lidstrom, 2003; Zukowski et al., 1983
).
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RESULTS |
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Use of the A-tract sequence as an enhancer element
The importance of the A-tract sequence in transcription of mox promoters suggested that it might serve as an enhancer element for other promoters in M. extorquens AM1. In order to test this hypothesis, the mxaF A-tract sequence, AAGAAA, was inserted 17 bp upstream of the E. coli lac promoter in the promoter-probe vector pCM130. The lac promoter had been previously shown to be a weak promoter in M. extorquens AM1 (Marx & Lidstrom, 2001). The cloned tac promoter had an activity of 5660 mU, while the construct containing the AAGAAA showed an increase to 128180 mU. For comparison, the mxaF promoter showed an activity of 350 mU under these conditions. These results suggest that this sequence can be used to significantly increase promoter activity of non-C1 promoters in this bacterium, but supports the hypothesis that the high activity of the mxaF promoter is only partly due to the A-tract sequence.
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DISCUSSION |
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In all cases except the pqqA promoter, the effect was the same whether the sequence was deleted or altered to all Gs. However, in the case of the pqqA promoter, altering the sequence to all Gs resulted in activities similar to those of the wild-type grown on succinate, in cells grown on either succinate or methanol. These results suggest that the loss of activity in these pqqA promoter deletion constructs had more to do with the spacing upstream of the promoter than the actual sequence. Previous work has defined the regions responsible for full promoter activity for each of these promoters to within 20200 bp upstream of the 35 sequence, depending on the promoter (Zhang & Lidstrom, 2003), but no other consensus sequence or hairpin structure is conserved in that region of these promoters.
Multiple A-tract sequences act as enhancer elements for promoters in many bacterial systems, including the very highly expressed rrn promoters as well as others (Aiyar et al., 1998; Cheema et al., 1999
). In some cases these have been shown to bind the
subunit of RNA polymerase (Aiyar et al., 1998
), and act as positive transcriptional elements via their DNA-curvature characteristics. Therefore, it is possible that the sequence we have identified is analogous to one of these elements. In support of a more general role, addition of this sequence to an E. coli promoter that is poorly transcribed in M. extorquens AM1 (Plac) did increase promoter activity two- to threefold in M. extorquens AM1. In other bacteria, these elements are present in tracts of multiple sets of A-rich or T-rich sequences and in our case these promoters all contain only one upstream A-rich sequence. In one case in which a single A-tract was studied, for the malT promoter of E. coli, deletion of the sequence had little effect on transcription but moving the spacing enhanced transcription (Tagami & Aiba, 1999
). The fact that the addition of this sequence alone did not generate promoter activity at the same high level as the mxaF promoter suggests that other elements in the mxaF promoter region contribute to the high activity. Since no other conserved elements exist upstream of mxaF promoters in other bacteria (Zhang & Lidstrom, 2003
), it is likely that the high activity is due to spacing of the A-rich sequence and/or sequences within the 10/35 region of the promoter.
Alternatively, it is possible that this sequence represents a binding site for a transcriptional regulator. However, so far all attempts to demonstrate binding of the three predicted DNA-binding proteins in the Mox system (MxaB, MxbM and MxcE) to these promoters have been unsuccessful (M. Franke, A. Stover & M. E. Lidstrom, unpublished). Therefore, it is possible that this sequence is either not a site for protein binding, or is a site for an as-yet-unidentified regulator.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 31 May 2005;
revised 3 August 2005;
accepted 4 August 2005.
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