1887

Abstract

A multiple A-tract sequence has been identified in the promoter regions for the , , , and genes involved in methanol oxidation in AM1, a facultative methylotroph. Site-directed mutagenesis was exploited to delete or change this conserved sequence. Promoter- transcriptional fusions were used to assess promoter activity in these mutants. A fiftyfold drop in the XylE activity was observed for the and promoters without this sequence, and a five- to sixfold drop in the XylE activity was observed for the and promoters without this sequence. Mutants were generated in the chromosomal copies in which this sequence was either deleted or altered, and these mutants were unable to grow on methanol. When one of these sequences was added to P of , which is a weak constitutive promoter in AM1, the activity increased two- to threefold. These results suggest that this sequence is essential for normal expression of these genes in AM1, and may serve as a general enhancer element for genetic constructs in this bacterium.

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2005-11-01
2019-11-12
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References

  1. Aiyar, S. E., Gourse, R. L. & Ross, W. ( 1998; ). Upstream A-tracts increase bacterial promoter activity through interactions with the RNA polymerase alpha subunit. Proc Natl Acad Sci U S A 95, 14652–14657.[CrossRef]
    [Google Scholar]
  2. 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.
    [Google Scholar]
  3. 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, 173–176.[CrossRef]
    [Google Scholar]
  4. Anthony, C. ( 1982; ). The Biochemistry of Methylotrophs. London: Academic Press.
  5. Anthony, C. ( 2000; ). Methanol dehydrogenase, a PQQ-containing quinoprotein dehydrogenase. Subcell Biochem 35, 73–117.
    [Google Scholar]
  6. Cheema, A. K., Choudhury, N. R. & Das, H. K. ( 1999; ). A- and T-tract-mediated intrinsic curvature in native DNA between the binding site of the upstream activator NtrC and the nifLA promoter of Klebsiella pneumoniae facilitates transcription. J Bacteriol 181, 5296–5302.
    [Google Scholar]
  7. Chistoserdov, A. Y., McIntire, W. S., Mathews, F. S. & Lidstrom, M. E. ( 1994a; ). Organization of the methylamine utilization (mau) genes in Methylophilus methylotrophus W3A1-NS. J Bacteriol 176, 4073–4080.
    [Google Scholar]
  8. Chistoserdov, A. Y., Chistoserdova, L. V., McIntire, W. S. & Lidstrom, M. E. ( 1994b; ). Genetic organization of the mau gene cluster in Methylobacterium extorquens AM1: complete nucleotide sequence and generation and characteristics of mau mutants. J Bacteriol 176, 4052–4065.
    [Google Scholar]
  9. Chistoserdova, L., Chen, S. W., Lapidus, A. & Lidstrom, M. E. ( 2003; ). Methylotrophy in Methylobacterium extorquens AM1 from a genomic point of view. J Bacteriol 185, 2980–2987.[CrossRef]
    [Google Scholar]
  10. 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, 1648–1652.[CrossRef]
    [Google Scholar]
  11. 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, 718–723.
    [Google Scholar]
  12. Lidstrom, M. E. ( 1991; ). The methylotrophic bacteria. In The Prokaryotes II. Edited by A. Balows and others. New York: Springer.
  13. Lidstrom, M. E. & Tsygankov, Y. D. ( 1991; ). Molecular genetics of methylotrophic bacteria. Biotechnology 18, 273–304.
    [Google Scholar]
  14. 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, 2065–2075.
    [Google Scholar]
  15. Marx, C. J. & Lidstrom, M. E. ( 2002; ). Broad-host-range cre-lox system for antibiotic marker recycling in gram-negative bacteria. Biotechniques 33, 1062–1067.
    [Google Scholar]
  16. Marx, C. J., O'Brien, B. N., Breezee, J. & Lidstrom, M. E. ( 2003; ). Novel methylotrophy genes of Methylobacterium extorquens AM1 identified by using transposon mutagenesis including a putative dihydromethanopterin reductase. J Bacteriol 185, 669–673.[CrossRef]
    [Google Scholar]
  17. 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, 1746–1755.
    [Google Scholar]
  18. 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, 6825–6831.
    [Google Scholar]
  19. Nunn, D. N. & Lidstrom, M. E. ( 1986a; ). Phenotypic characterization of 10 methanol oxidation mutant classes in Methylobacterium sp. strain AM1. J Bacteriol 166, 591–597.
    [Google Scholar]
  20. 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, 581–590.
    [Google Scholar]
  21. Nunn, D. N., Day, D. & Anthony, C. ( 1989; ). The second subunit of methanol dehydrogenase of Methylobacterium extorquens AM1. Biochem J 260, 857–862.
    [Google Scholar]
  22. Peel, D. & Quayle, J. R. ( 1961; ). Microbial growth on C1 compounds. 1. Isolation and characterization of Pseudomonas AM1. Biochem J 81, 465–469.
    [Google Scholar]
  23. 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, 206–211.
    [Google Scholar]
  24. Saito, H. & Miura, K. I. ( 1963; ). Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim Biophys Acta 72, 619–629.[CrossRef]
    [Google Scholar]
  25. Sambrook, J., Fritsch, E. F. & Maniatis, T. ( 1989; ). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  26. 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, 2985–2993.[CrossRef]
    [Google Scholar]
  27. Springer, A. L., Ramamoorthi, R. & Lidstrom, M. E. ( 1996; ). Characterization and nucleotide sequence of pqqE and pqqF in Methylobacterium extorquens AM1. J Bacteriol 178, 2154–2157.
    [Google Scholar]
  28. 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, 1737–1744.[CrossRef]
    [Google Scholar]
  29. 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, 119–124.[CrossRef]
    [Google Scholar]
  30. Tagami, H. & Aiba, H. ( 1999; ). An inactive open complex mediated by an UP element at Escherichia coli promoters. Proc Natl Acad Sci U S A 96, 7202–7207.[CrossRef]
    [Google Scholar]
  31. 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, 595–602.[CrossRef]
    [Google Scholar]
  32. Toyama, H., Inagaki, H., Matsushita, K., Anthony, C. & Adachi, O. ( 2003; ). The role of the MxaD protein in the respiratory chain of Methylobacterium extorquens during growth on methanol. Biochim Biophys Acta 1647, 372–375.[CrossRef]
    [Google Scholar]
  33. Zhang, M. & Lidstrom, M. E. ( 2003; ). Promoters and transcripts for genes involved in methanol oxidation in Methylobacterium extorquens AM1. Microbiology 149, 1033–1040.[CrossRef]
    [Google Scholar]
  34. 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, 1101–1105.[CrossRef]
    [Google Scholar]
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