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. 1992 May;174(9):3049–3055. doi: 10.1128/jb.174.9.3049-3055.1992

Catabolite repression of the xyl operon in Bacillus megaterium.

T Rygus 1, W Hillen 1
PMCID: PMC205960  PMID: 1569031

Abstract

We characterized catabolite repression of the genes encoding xylose utilization in Bacillus megaterium. A transcriptional fusion of xylA encoding xylose isomerase to the spoVG-lacZ indicator gene on a plasmid with a temperature-sensitive origin of replication was constructed and efficiently used for single-copy replacement cloning in the B. megaterium chromosome starting from a single transformant. In the resulting strain, beta-galactosidase expression is 150-fold inducible by xylose and 14-fold repressed by glucose, showing that both regulatory effects occur at the level of transcription. Insertion of a kanamycin resistance gene into xylR encoding the xylose-dependent repressor leads to the loss of xylose-dependent regulation and to a small drop in the efficiency of glucose repression to eightfold. Deletion of 184 bp from the 5' part of the xylA reading frame reduces glucose repression to only twofold. A potential glucose-responsive element in this region is discussed on the basis of sequence similarities to other glucose-repressed genes in Bacillus subtilis. The sequence including the glucose-responsive element is also necessary for repression exerted by the carbon sources fructose and mannitol. Their efficiencies of repression correlate to the growth rate of B. megaterium, as is typical for catabolite repression. Glycerol, ribose, and arabinose exert only a basal twofold repression of the xyl operon, which is independent of the presence of the cis-active glucose-responsive element within the xylA reading frame.

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Selected References

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  1. Boylan S. A., Chun K. T., Edson B. A., Price C. W. Early-blocked sporulation mutations alter expression of enzymes under carbon control in Bacillus subtilis. Mol Gen Genet. 1988 May;212(2):271–280. doi: 10.1007/BF00334696. [DOI] [PubMed] [Google Scholar]
  2. Ferrari F. A., Nguyen A., Lang D., Hoch J. A. Construction and properties of an integrable plasmid for Bacillus subtilis. J Bacteriol. 1983 Jun;154(3):1513–1515. doi: 10.1128/jb.154.3.1513-1515.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Fisher S. H., Sonenshein A. L. Control of carbon and nitrogen metabolism in Bacillus subtilis. Annu Rev Microbiol. 1991;45:107–135. doi: 10.1146/annurev.mi.45.100191.000543. [DOI] [PubMed] [Google Scholar]
  4. Fouet A., Sonenshein A. L. A target for carbon source-dependent negative regulation of the citB promoter of Bacillus subtilis. J Bacteriol. 1990 Feb;172(2):835–844. doi: 10.1128/jb.172.2.835-844.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Geissendörfer M., Hillen W. Regulated expression of heterologous genes in Bacillus subtilis using the Tn10 encoded tet regulatory elements. Appl Microbiol Biotechnol. 1990 Sep;33(6):657–663. doi: 10.1007/BF00604933. [DOI] [PubMed] [Google Scholar]
  6. Gärtner D., Geissendörfer M., Hillen W. Expression of the Bacillus subtilis xyl operon is repressed at the level of transcription and is induced by xylose. J Bacteriol. 1988 Jul;170(7):3102–3109. doi: 10.1128/jb.170.7.3102-3109.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Heilmann H. J., Mägert H. J., Gassen H. G. Identification and isolation of glucose dehydrogenase genes of Bacillus megaterium M1286 and their expression in Escherichia coli. Eur J Biochem. 1988 Jun 15;174(3):485–490. doi: 10.1111/j.1432-1033.1988.tb14124.x. [DOI] [PubMed] [Google Scholar]
  8. Henkin T. M., Grundy F. J., Nicholson W. L., Chambliss G. H. Catabolite repression of alpha-amylase gene expression in Bacillus subtilis involves a trans-acting gene product homologous to the Escherichia coli lacl and galR repressors. Mol Microbiol. 1991 Mar;5(3):575–584. doi: 10.1111/j.1365-2958.1991.tb00728.x. [DOI] [PubMed] [Google Scholar]
  9. Hillen W., Klock G., Kaffenberger I., Wray L. V., Reznikoff W. S. Purification of the TET repressor and TET operator from the transposon Tn10 and characterization of their interaction. J Biol Chem. 1982 Jun 10;257(11):6605–6613. [PubMed] [Google Scholar]
  10. Itaya M., Kondo K., Tanaka T. A neomycin resistance gene cassette selectable in a single copy state in the Bacillus subtilis chromosome. Nucleic Acids Res. 1989 Jun 12;17(11):4410–4410. doi: 10.1093/nar/17.11.4410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jacob S., Allmansberger R., Gärtner D., Hillen W. Catabolite repression of the operon for xylose utilization from Bacillus subtilis W23 is mediated at the level of transcription and depends on a cis site in the xylA reading frame. Mol Gen Genet. 1991 Oct;229(2):189–196. doi: 10.1007/BF00272155. [DOI] [PubMed] [Google Scholar]
  12. Kreuzer P., Gärtner D., Allmansberger R., Hillen W. Identification and sequence analysis of the Bacillus subtilis W23 xylR gene and xyl operator. J Bacteriol. 1989 Jul;171(7):3840–3845. doi: 10.1128/jb.171.7.3840-3845.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Miwa Y., Fujita Y. Determination of the cis sequence involved in catabolite repression of the Bacillus subtilis gnt operon; implication of a consensus sequence in catabolite repression in the genus Bacillus. Nucleic Acids Res. 1990 Dec 11;18(23):7049–7053. doi: 10.1093/nar/18.23.7049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Palva I., Pettersson R. F., Kalkkinen N., Lehtovaara P., Sarvas M., Söderlund H., Takkinen K., Käriäinen L. Nucleotide sequence of the promoter and NH2-terminal signal peptide region of the alpha-amylase gene from Bacillus amyloliquefaciens. Gene. 1981 Oct;15(1):43–51. doi: 10.1016/0378-1119(81)90103-7. [DOI] [PubMed] [Google Scholar]
  15. Rothstein D. M., Devlin P. E., Cate R. L. Expression of alpha-amylase in Bacillus licheniformis. J Bacteriol. 1986 Nov;168(2):839–842. doi: 10.1128/jb.168.2.839-842.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Rygus T., Hillen W. Inducible high-level expression of heterologous genes in Bacillus megaterium using the regulatory elements of the xylose-utilization operon. Appl Microbiol Biotechnol. 1991 Aug;35(5):594–599. doi: 10.1007/BF00169622. [DOI] [PubMed] [Google Scholar]
  17. Rygus T., Scheler A., Allmansberger R., Hillen W. Molecular cloning, structure, promoters and regulatory elements for transcription of the Bacillus megaterium encoded regulon for xylose utilization. Arch Microbiol. 1991;155(6):535–542. doi: 10.1007/BF00245346. [DOI] [PubMed] [Google Scholar]
  18. Rüther U. pUR 250 allows rapid chemical sequencing of both DNA strands of its inserts. Nucleic Acids Res. 1982 Oct 11;10(19):5765–5772. doi: 10.1093/nar/10.19.5765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Shimotsu H., Henner D. J. Construction of a single-copy integration vector and its use in analysis of regulation of the trp operon of Bacillus subtilis. Gene. 1986;43(1-2):85–94. doi: 10.1016/0378-1119(86)90011-9. [DOI] [PubMed] [Google Scholar]
  20. Soberon X., Covarrubias L., Bolivar F. Construction and characterization of new cloning vehicles. IV. Deletion derivatives of pBR322 and pBR325. Gene. 1980 May;9(3-4):287–305. doi: 10.1016/0378-1119(90)90328-o. [DOI] [PubMed] [Google Scholar]
  21. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  22. Sussman M. D., Vary P. S., Hartman C., Setlow P. Integration and mapping of Bacillus megaterium genes which code for small, acid-soluble spore proteins and their protease. J Bacteriol. 1988 Oct;170(10):4942–4945. doi: 10.1128/jb.170.10.4942-4945.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Vary P. S., Halsey W. F. Host-range and partial characterization of several new bacteriophages for Bacillus megaterium QM b1551. J Gen Virol. 1980 Nov;51(Pt 1):137–146. doi: 10.1099/0022-1317-51-1-137. [DOI] [PubMed] [Google Scholar]
  24. Von Tersch M. A., Robbins H. L. Efficient cloning in Bacillus megaterium: comparison to Bacillus subtilis and Escherichia coli cloning hosts. FEMS Microbiol Lett. 1990 Aug;58(3):305–309. doi: 10.1111/j.1574-6968.1990.tb13994.x. [DOI] [PubMed] [Google Scholar]
  25. Weickert M. J., Chambliss G. H. Site-directed mutagenesis of a catabolite repression operator sequence in Bacillus subtilis. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6238–6242. doi: 10.1073/pnas.87.16.6238. [DOI] [PMC free article] [PubMed] [Google Scholar]

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