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. 1997 Mar;179(5):1598–1608. doi: 10.1128/jb.179.5.1598-1608.1997

Negative regulation of L-arabinose metabolism in Bacillus subtilis: characterization of the araR (araC) gene.

I Sá-Nogueira 1, L J Mota 1
PMCID: PMC178872  PMID: 9045819

Abstract

The Bacillus subtilis araC locus, mapped at about 294 degrees on the genetic map, was defined by mutations conferring an Ara- phenotype to strains bearing the metabolic araA, araB, and araD wild-type alleles (located at about 256 degrees on the genetic map) and by mutants showing constitutive expression of the three genes. In previous work, it has been postulated that the gene in which these mutations lie exerts its effect on the ara metabolic operon in trans, and this locus was named araC by analogy to the Escherichia coli regulatory gene. Here, we report the cloning and sequencing of the araC locus. This region comprises two open reading frames with divergently arranged promoters, the regulatory gene, araC, encoding a 41-kDa polypeptide, and a partially cloned gene, termed araE, which most probably codes for a permease involved in the transport of L-arabinose. The DNA sequence of araC revealed that its putative product is very similar to a number of bacterial negative regulators (the GalR-LacI family). However, a helix-turn-helix motif was identified in the N-terminal region by its identity to the consensus signature sequence of another group of repressors, the GntR family. The lack of similarity between the predicted primary structure of the product encoded by the B. subtilis regulatory gene and the AraC regulator from E. coli and the apparently different modes of action of these two proteins lead us to propose a new name, araR, for this gene. The araR gene is monocistronic, and the promoter region contains -10 and -35 regions (as determined by primer extension analysis) similar to those recognized by RNA polymerase containing the major vegetative cell sigma factor sigmaA. An insertion-deletion mutation in the araR gene leads to constitutive expression of the L-arabinose metabolic operon. We demonstrate that the araR gene codes for a negative regulator of the ara operon and that the expression of araR is repressed by its own product.

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Selected References

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  1. Adler K., Beyreuther K., Fanning E., Geisler N., Gronenborn B., Klemm A., Müller-Hill B., Pfahl M., Schmitz A. How lac repressor binds to DNA. Nature. 1972 Jun 9;237(5354):322–327. doi: 10.1038/237322a0. [DOI] [PubMed] [Google Scholar]
  2. Allison S. L., Phillips A. T. Nucleotide sequence of the gene encoding the repressor for the histidine utilization genes of Pseudomonas putida. J Bacteriol. 1990 Sep;172(9):5470–5476. doi: 10.1128/jb.172.9.5470-5476.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Anagnostopoulos C., Spizizen J. REQUIREMENTS FOR TRANSFORMATION IN BACILLUS SUBTILIS. J Bacteriol. 1961 May;81(5):741–746. doi: 10.1128/jb.81.5.741-746.1961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Arantes O., Lereclus D. Construction of cloning vectors for Bacillus thuringiensis. Gene. 1991 Dec 1;108(1):115–119. doi: 10.1016/0378-1119(91)90495-w. [DOI] [PubMed] [Google Scholar]
  5. Archer R. P., Klinefelter D. Relationships between MMPI codetypes and MAC scale elevations in adolescent psychiatric samples. J Pers Assess. 1992 Feb;58(1):149–159. doi: 10.1207/s15327752jpa5801_14. [DOI] [PubMed] [Google Scholar]
  6. Barnell W. O., Yi K. C., Conway T. Sequence and genetic organization of a Zymomonas mobilis gene cluster that encodes several enzymes of glucose metabolism. J Bacteriol. 1990 Dec;172(12):7227–7240. doi: 10.1128/jb.172.12.7227-7240.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Barns S. M., Delwiche C. F., Palmer J. D., Pace N. R. Perspectives on archaeal diversity, thermophily and monophyly from environmental rRNA sequences. Proc Natl Acad Sci U S A. 1996 Aug 20;93(17):9188–9193. doi: 10.1073/pnas.93.17.9188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Beck C. F., Warren R. A. Divergent promoters, a common form of gene organization. Microbiol Rev. 1988 Sep;52(3):318–326. doi: 10.1128/mr.52.3.318-326.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Burland V., Plunkett G., 3rd, Sofia H. J., Daniels D. L., Blattner F. R. Analysis of the Escherichia coli genome VI: DNA sequence of the region from 92.8 through 100 minutes. Nucleic Acids Res. 1995 Jun 25;23(12):2105–2119. doi: 10.1093/nar/23.12.2105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bussey L. B., Switzer R. L. The degA gene product accelerates degradation of Bacillus subtilis phosphoribosylpyrophosphate amidotransferase in Escherichia coli. J Bacteriol. 1993 Oct;175(19):6348–6353. doi: 10.1128/jb.175.19.6348-6353.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dale R. M., McClure B. A., Houchins J. P. A rapid single-stranded cloning strategy for producing a sequential series of overlapping clones for use in DNA sequencing: application to sequencing the corn mitochondrial 18 S rDNA. Plasmid. 1985 Jan;13(1):31–40. doi: 10.1016/0147-619x(85)90053-8. [DOI] [PubMed] [Google Scholar]
  12. Davis E. O., Henderson P. J. The cloning and DNA sequence of the gene xylE for xylose-proton symport in Escherichia coli K12. J Biol Chem. 1987 Oct 15;262(29):13928–13932. [PubMed] [Google Scholar]
  13. DiRusso C. C. Nucleotide sequence of the fadR gene, a multifunctional regulator of fatty acid metabolism in Escherichia coli. Nucleic Acids Res. 1988 Aug 25;16(16):7995–8009. doi: 10.1093/nar/16.16.7995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. ENGLESBERG E., ANDERSON R. L., WEINBERG R., LEE N., HOFFEE P., HUTTENHAUER G., BOYER H. L-Arabinose-sensitive, L-ribulose 5-phosphate 4-epimerase-deficient mutants of Escherichia coli. J Bacteriol. 1962 Jul;84:137–146. doi: 10.1128/jb.84.1.137-146.1962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Englesberg E., Irr J., Power J., Lee N. Positive control of enzyme synthesis by gene C in the L-arabinose system. J Bacteriol. 1965 Oct;90(4):946–957. doi: 10.1128/jb.90.4.946-957.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Farabaugh P. J. Sequence of the lacI gene. Nature. 1978 Aug 24;274(5673):765–769. doi: 10.1038/274765a0. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Fleischmann R. D., Adams M. D., White O., Clayton R. A., Kirkness E. F., Kerlavage A. R., Bult C. J., Tomb J. F., Dougherty B. A., Merrick J. M. Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science. 1995 Jul 28;269(5223):496–512. doi: 10.1126/science.7542800. [DOI] [PubMed] [Google Scholar]
  19. Fujita Y., Fujita T., Miwa Y., Nihashi J., Aratani Y. Organization and transcription of the gluconate operon, gnt, of Bacillus subtilis. J Biol Chem. 1986 Oct 15;261(29):13744–13753. [PubMed] [Google Scholar]
  20. Gärtner D., Degenkolb J., Ripperger J. A., Allmansberger R., Hillen W. Regulation of the Bacillus subtilis W23 xylose utilization operon: interaction of the Xyl repressor with the xyl operator and the inducer xylose. Mol Gen Genet. 1992 Apr;232(3):415–422. doi: 10.1007/BF00266245. [DOI] [PubMed] [Google Scholar]
  21. Haydon D. J., Guest J. R. A new family of bacterial regulatory proteins. FEMS Microbiol Lett. 1991 Apr 15;63(2-3):291–295. doi: 10.1016/0378-1097(91)90101-f. [DOI] [PubMed] [Google Scholar]
  22. Haydon D. J., Quail M. A., Guest J. R. A mutation causing constitutive synthesis of the pyruvate dehydrogenase complex in Escherichia coli is located within the pdhR gene. FEBS Lett. 1993 Dec 20;336(1):43–47. doi: 10.1016/0014-5793(93)81605-y. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Hueck C., Kraus A., Hillen W. Sequences of ccpA and two downstream Bacillus megaterium genes with homology to the motAB operon from Bacillus subtilis. Gene. 1994 May 27;143(1):147–148. doi: 10.1016/0378-1119(94)90621-1. [DOI] [PubMed] [Google Scholar]
  25. Igo M. M., Losick R. Regulation of a promoter that is utilized by minor forms of RNA polymerase holoenzyme in Bacillus subtilis. J Mol Biol. 1986 Oct 20;191(4):615–624. doi: 10.1016/0022-2836(86)90449-3. [DOI] [PubMed] [Google Scholar]
  26. Kadner R. J., Murphy G. P., Stephens C. M. Two mechanisms for growth inhibition by elevated transport of sugar phosphates in Escherichia coli. J Gen Microbiol. 1992 Oct;138(10):2007–2014. doi: 10.1099/00221287-138-10-2007. [DOI] [PubMed] [Google Scholar]
  27. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  28. Lepesant J. A., Dedonder R. Métabolisme du L-arabinose chez Bacillus subtilis Marburg Ind-168. C R Acad Sci Hebd Seances Acad Sci D. 1967 Jun 5;264(23):2683–2686. [PubMed] [Google Scholar]
  29. Martin-Verstraete I., Débarbouillé M., Klier A., Rapoport G. Mutagenesis of the Bacillus subtilis "-12, -24" promoter of the levanase operon and evidence for the existence of an upstream activating sequence. J Mol Biol. 1992 Jul 5;226(1):85–99. doi: 10.1016/0022-2836(92)90126-5. [DOI] [PubMed] [Google Scholar]
  30. Martin I., Débarbouillé M., Ferrari E., Klier A., Rapoport G. Characterization of the levanase gene of Bacillus subtilis which shows homology to yeast invertase. Mol Gen Genet. 1987 Jun;208(1-2):177–184. doi: 10.1007/BF00330439. [DOI] [PubMed] [Google Scholar]
  31. Mauzy C. A., Hermodson M. A. Structural homology between rbs repressor and ribose binding protein implies functional similarity. Protein Sci. 1992 Jul;1(7):843–849. doi: 10.1002/pro.5560010702. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Meng L. M., Kilstrup M., Nygaard P. Autoregulation of PurR repressor synthesis and involvement of purR in the regulation of purB, purC, purL, purMN and guaBA expression in Escherichia coli. Eur J Biochem. 1990 Jan 26;187(2):373–379. doi: 10.1111/j.1432-1033.1990.tb15314.x. [DOI] [PubMed] [Google Scholar]
  33. Moran C. P., Jr, Lang N., LeGrice S. F., Lee G., Stephens M., Sonenshein A. L., Pero J., Losick R. Nucleotide sequences that signal the initiation of transcription and translation in Bacillus subtilis. Mol Gen Genet. 1982;186(3):339–346. doi: 10.1007/BF00729452. [DOI] [PubMed] [Google Scholar]
  34. Pascal M., Kunst F., Lepesant J. A., Dedonder R. Characterization of two sucrase activities in Bacillus subtilis Marburg. Biochimie. 1971;53(10):1059–1066. doi: 10.1016/s0300-9084(71)80193-1. [DOI] [PubMed] [Google Scholar]
  35. Quail M. A., Dempsey C. E., Guest J. R. Identification of a fatty acyl responsive regulator (FarR) in Escherichia coli. FEBS Lett. 1994 Dec 19;356(2-3):183–187. doi: 10.1016/0014-5793(94)01264-4. [DOI] [PubMed] [Google Scholar]
  36. Schwacha A., Bender R. A. Nucleotide sequence of the gene encoding the repressor for the histidine utilization genes of Klebsiella aerogenes. J Bacteriol. 1990 Sep;172(9):5477–5481. doi: 10.1128/jb.172.9.5477-5481.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Shatwell K. P., Charalambous B. M., McDonald T. P., Henderson P. J. Cloning, sequencing, and expression of the araE gene of Klebsiella oxytoca 8017, which encodes arabinose-H+ symport activity. J Bacteriol. 1995 Sep;177(18):5379–5380. doi: 10.1128/jb.177.18.5379-5380.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Stoner C., Schleif R. The araE low affinity L-arabinose transport promoter. Cloning, sequence, transcription start site and DNA binding sites of regulatory proteins. J Mol Biol. 1983 Dec 25;171(4):369–381. doi: 10.1016/0022-2836(83)90035-9. [DOI] [PubMed] [Google Scholar]
  40. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  41. Sullivan M. A., Yasbin R. E., Young F. E. New shuttle vectors for Bacillus subtilis and Escherichia coli which allow rapid detection of inserted fragments. Gene. 1984 Jul-Aug;29(1-2):21–26. doi: 10.1016/0378-1119(84)90161-6. [DOI] [PubMed] [Google Scholar]
  42. Sá-Nogueira I., Nogueira T. V., Soares S., de Lencastre H. The Bacillus subtilis L-arabinose (ara) operon: nucleotide sequence, genetic organization and expression. Microbiology. 1997 Mar;143(Pt 3):957–969. doi: 10.1099/00221287-143-3-957. [DOI] [PubMed] [Google Scholar]
  43. Sá-Nogueira I., Paveia H., de Lencastre H. Isolation of constitutive mutants for L-arabinose utilization in Bacillus subtilis. J Bacteriol. 1988 Jun;170(6):2855–2857. doi: 10.1128/jb.170.6.2855-2857.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sá-Nogueira I., de Lencastre H. Cloning and characterization of araA, araB, and araD, the structural genes for L-arabinose utilization in Bacillus subtilis. J Bacteriol. 1989 Jul;171(7):4088–4091. doi: 10.1128/jb.171.7.4088-4091.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tinoco I., Jr, Borer P. N., Dengler B., Levin M. D., Uhlenbeck O. C., Crothers D. M., Bralla J. Improved estimation of secondary structure in ribonucleic acids. Nat New Biol. 1973 Nov 14;246(150):40–41. doi: 10.1038/newbio246040a0. [DOI] [PubMed] [Google Scholar]
  46. Valentin-Hansen P., Larsen J. E., Højrup P., Short S. A., Barbier C. S. Nucleotide sequence of the CytR regulatory gene of E. coli K-12. Nucleic Acids Res. 1986 Mar 11;14(5):2215–2228. doi: 10.1093/nar/14.5.2215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Weickert M. J., Adhya S. A family of bacterial regulators homologous to Gal and Lac repressors. J Biol Chem. 1992 Aug 5;267(22):15869–15874. [PubMed] [Google Scholar]
  48. 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]
  49. Woese C. R. Bacterial evolution. Microbiol Rev. 1987 Jun;51(2):221–271. doi: 10.1128/mr.51.2.221-271.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  51. Zhang C. C., Durand M. C., Jeanjean R., Joset F. Molecular and genetical analysis of the fructose-glucose transport system in the cyanobacterium Synechocystis PCC6803. Mol Microbiol. 1989 Sep;3(9):1221–1229. doi: 10.1111/j.1365-2958.1989.tb00272.x. [DOI] [PubMed] [Google Scholar]
  52. von Wilcken-Bergmann B., Müller-Hill B. Sequence of galR gene indicates a common evolutionary origin of lac and gal repressor in Escherichia coli. Proc Natl Acad Sci U S A. 1982 Apr;79(8):2427–2431. doi: 10.1073/pnas.79.8.2427. [DOI] [PMC free article] [PubMed] [Google Scholar]

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