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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
and
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 LuriaBertani (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 100500 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).
-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 810 µg total RNA. Primers were labelled with [-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 1825 mers and were located at different sites with relation to the start codon.
|
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
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 90100 bp upstream of the transcriptional start site and no activity could be detected when the -10, -35 region alone was present.
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
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.
|
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Anderson, D. J., Morris, C. J., Nunn, D. N., Anthony, C. & Lidstrom, M. E. (1990). Nucleotide sequence of the Methylobacterium extorquens AM1 moxF and moxJ genes involved in methanol oxidation. Gene 90, 173176.[CrossRef][Medline]
Anthony, C. (2000). Methanol dehydrogenase, a PQQ-containing quinoprotein dehydrogenase. Subcell Biochem 35, 73117.[Medline]
Chistoserdov, A. Y., Chistoserdova, L. V., McIntire, W. S. & Lidstrom, M. E. (1994). Genetic organization of the mau gene cluster in Methylobacterium extorquens AM1: complete nucleotide sequence and generation and characteristics of mau mutants. J Bacteriol 176, 40524065.[Abstract]
Chistoserdova, L. & Lidstrom, M. E. (1991). Hydroxypyruvate reductase from Methylobacterium extorquens AM1. J Bacteriol 173, 72287232.[Medline]
Figurski, D. H. & Helinski, D. R. (1979). Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A 76, 16481652.[Abstract]
Fulton, G. L., Nunn, D. N. & Lidstrom, M. E. (1984). Molecular cloning of a malyl coenzyme A lyase gene from Pseudomonas sp. strain AM1, a facultative methylotroph. J Bacteriol 160, 718723.[Medline]
Lidstrom, M. E. (1991). The methylotrophic bacteria. In: The Prokaryotes, 2nd edn, pp. 441445. Edited by A. Balows & others. New York: Springer.
Marx, C. J. & Lidstrom, M. E. (2001). Development of improved versatile broad-host-range vectors for use in methylotrophs and other Gram-negative bacteria. Microbiology 147, 20652075.
Miller, J. H. (1972). Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Morris, C. J., Biville, F., Turlin, E., Lee, E., Ellermann, K., Fan, W. H., Ramamoorthi, R., Springer, A. L. & Lidstrom, M. E. (1994). Isolation, phenotypic characterization, and complementation analysis of mutants of Methylobacterium extorquens AM1 unable to synthesize pyrroloquinoline quinone and sequences of pqqD, pqqG, and pqqC. J Bacteriol 176, 17461755.[Abstract]
Morris, C. J., Kim, Y. M., Perkins, K. E. & Lidstrom, M. E. (1995). Identification and nucleotide sequences of mxaA, mxaC, mxaK, mxaL, and mxaD genes from Methylobacterium extorquens AM1. J Bacteriol 177, 68256831.[Abstract]
Nunn, D. N. & Lidstrom, M. E. (1986a). Phenotypic characterization of 10 methanol oxidation mutant classes in Methylobacterium sp. strain AM1. J Bacteriol 166, 591597.[Medline]
Nunn, D. N. & Lidstrom, M. E. (1986b). Isolation and complementation analysis of 10 methanol oxidation mutant classes and identification of the methanol dehydrogenase structural gene of Methylobacterium sp. strain AM1. J Bacteriol 166, 581590.[Medline]
Nunn, D. N., Day, D. & Anthony, C. (1989). The second subunit of methanol dehydrogenase of Methylobacterium extorquens AM1. Biochem J 260, 857862.[Medline]
Peel, D. & Quayle, J. R. (1961). Microbial growth on C1 compounds. 1. Isolation and characterization of Pseudomonas AM1. Biochem J 81, 465469.
Ramamoorthi, R. & Lidstrom, M. E. (1995). Transcriptional analysis of pqqD and study of the regulation of pyrroloquinoline quinone biosynthesis in Methylobacterium extorquens AM1. J Bacteriol 177, 206211.[Abstract]
Richardson, I. W. & Anthony, C. (1992). Characterization of mutant forms of the quinoprotein methanol dehydrogenase lacking an essential calcium ion. Biochem J 287, 709715.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Springer, A. L., Chou, H. H., Fan, W. H., Lee, E. & Lidstrom, M. E. (1995). Methanol oxidation mutants in Methylobacterium extorquens AM1: identification of new genetic complementation groups. Microbiology 141, 29852993.[Abstract]
Springer, A. L., Ramamoorthi, R. & Lidstrom, M. E. (1996). Characterization and nucleotide sequence of pqqE and pqqF in Methylobacterium extorquens AM1. J Bacteriol 178, 21542157.[Abstract]
Springer, A. L., Morris, C. J. & Lidstrom, M. E. (1997). Molecular analysis of mxbD and mxbM, a putative sensor-regulator pair required for oxidation of methanol in Methylobacterium extorquens AM1. Microbiology 143, 17371744.[Abstract]
Springer, A. L., Auman, A. J. & Lidstrom, M. E. (1998). 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. FEMS Microbiol Lett 160, 119124.[CrossRef][Medline]
Toyama, H., Chistoserdova, L. & Lidstrom, M. E. (1997). Sequence analysis of pqq genes required for biosynthesis of pyrroloquinoline quinone in Methylobacterium extorquens AM1 and the purification of a biosynthetic intermediate. Microbiology 143, 595602.[Abstract]
Xu, H. H., Viebahn, M. & Hanson, R. S. (1993). Identification of methanol-regulated promoter sequences from the facultative methylotrophic bacterium Methylobacterium organophilum XX. J Gen Microbiol 139, 743752.[Medline]
Zukowski, M. M., Gaffney, D. F., Speck, D., Kauffmann, M., Findeli, A., Wisecup, A. & Lecocq, J. P. (1983). Chromogenic identification of genetic regulatory signals in Bacillus subtilis based on expression of a cloned Pseudomonas gene. Proc Natl Acad Sci U S A 80, 11011105.[Abstract]
Received 6 November 2002;
revised 3 January 2003;
accepted 3 January 2003.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |