Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1994 Apr;176(8):2177–2183. doi: 10.1128/jb.176.8.2177-2183.1994

Cloning, sequencing, and disruption of a levanase gene of Bacillus polymyxa CF43.

S Bezzate 1, M Steinmetz 1, S Aymerich 1
PMCID: PMC205337  PMID: 8157587

Abstract

The Bacillus polymyxa CF43 lelA gene, expressing both sucrose and fructan hydrolase activities, was isolated from a genomic library of B. polymyxa screened in Bacillus subtilis. The gene was detected as expressing sucrose hydrolase activity; B. subtilis transformants did not secrete the lelA gene product (LelA) into the extracellular medium. A 1.7-kb DNA fragment sufficient for lelA expression in Escherichia coli was sequenced. It contains a 548-codon open reading frame. The deduced amino acid sequence shows 54% identity with mature B. subtilis levanase and is similar to other fructanases and sucrases (beta-D-fructosyltransferases). Multiple-sequence alignment of 14 of these proteins revealed several previously unreported features. LelA appears to be a 512-amino-acid polypeptide containing no canonical signal peptide. The hydrolytic activities of LelA on sucrose, levan, and inulin were compared with those of B. subtilis levanase and sucrase, confirming that LelA is indeed a fructanase. The lelA gene in the chromosome of B. polymyxa was disrupted with a chloramphenicol resistance gene (cat) by "inter-gramic" conjugation: the lelA::cat insertion on a mobilizable plasmid was transferred from an E. coli transformant to B. polymyxa CF43, and B. polymyxa transconjugants containing the lelA::cat construct replacing the wild-type lelA gene in their chromosomes were selected directly. The growth of the mutant strain on levan, inulin, and sucrose was not affected.

Full text

PDF
2177

Images in this article

2180Image
on p.2180

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. 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]
  2. Aslanidis C., Schmid K., Schmitt R. Nucleotide sequences and operon structure of plasmid-borne genes mediating uptake and utilization of raffinose in Escherichia coli. J Bacteriol. 1989 Dec;171(12):6753–6763. doi: 10.1128/jb.171.12.6753-6763.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Aymerich S., Gonzy-Tréboul G., Steinmetz M. 5'-noncoding region sacR is the target of all identified regulation affecting the levansucrase gene in Bacillus subtilis. J Bacteriol. 1986 Jun;166(3):993–998. doi: 10.1128/jb.166.3.993-998.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Aymerich S., Steinmetz M. Cloning and preliminary characterization of the sacS locus from Bacillus subtilis which controls the regulation of the exoenzyme levansucrase. Mol Gen Genet. 1987 Jun;208(1-2):114–120. doi: 10.1007/BF00330431. [DOI] [PubMed] [Google Scholar]
  5. Aymerich S., Steinmetz M. Specificity determinants and structural features in the RNA target of the bacterial antiterminator proteins of the BglG/SacY family. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10410–10414. doi: 10.1073/pnas.89.21.10410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Blatch G. L., Woods D. R. Molecular characterization of a fructanase produced by Bacteroides fragilis BF-1. J Bacteriol. 1993 May;175(10):3058–3066. doi: 10.1128/jb.175.10.3058-3066.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brückner R., Wagner E., Götz F. Characterization of a sucrase gene from Staphylococcus xylosus. J Bacteriol. 1993 Feb;175(3):851–857. doi: 10.1128/jb.175.3.851-857.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Burne R. A., Penders J. E. Characterization of the Streptococcus mutans GS-5 fruA gene encoding exo-beta-D-fructosidase. Infect Immun. 1992 Nov;60(11):4621–4632. doi: 10.1128/iai.60.11.4621-4632.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chambert R., Treboul G., Dedonder R. Kinetic studies of levansucrase of Bacillus subtilis. Eur J Biochem. 1974 Jan 16;41(2):285–300. doi: 10.1111/j.1432-1033.1974.tb03269.x. [DOI] [PubMed] [Google Scholar]
  10. Fouet A., Klier A., Rapoport G. Nucleotide sequence of the sucrase gene of Bacillus subtilis. Gene. 1986;45(2):221–225. doi: 10.1016/0378-1119(86)90258-1. [DOI] [PubMed] [Google Scholar]
  11. Itaya M., Yamaguchi I., Kobayashi K., Endo T., Tanaka T. The blasticidin S resistance gene (bsr) selectable in a single copy state in the Bacillus subtilis chromosome. J Biochem. 1990 Jun;107(6):799–801. doi: 10.1093/oxfordjournals.jbchem.a123128. [DOI] [PubMed] [Google Scholar]
  12. Jannière L., Bruand C., Ehrlich S. D. Structurally stable Bacillus subtilis cloning vectors. Gene. 1990 Mar 1;87(1):53–61. doi: 10.1016/0378-1119(90)90495-d. [DOI] [PubMed] [Google Scholar]
  13. Klein R. D., Poorman R. A., Favreau M. A., Shea M. H., Hatzenbuhler N. T., Nulf S. C. Cloning and sequence analysis of the gene encoding invertase from the yeast Schwanniomyces occidentalis. Curr Genet. 1989 Sep;16(3):145–152. doi: 10.1007/BF00391470. [DOI] [PubMed] [Google Scholar]
  14. Kurnit D. M. Escherichia coli recA deletion strains that are highly competent for transformation and for in vivo phage packaging. Gene. 1989 Oct 30;82(2):313–315. doi: 10.1016/0378-1119(89)90056-5. [DOI] [PubMed] [Google Scholar]
  15. Leigh J. A., Coplin D. L. Exopolysaccharides in plant-bacterial interactions. Annu Rev Microbiol. 1992;46:307–346. doi: 10.1146/annurev.mi.46.100192.001515. [DOI] [PubMed] [Google Scholar]
  16. Lemesle-Varloot L., Henrissat B., Gaboriaud C., Bissery V., Morgat A., Mornon J. P. Hydrophobic cluster analysis: procedures to derive structural and functional information from 2-D-representation of protein sequences. Biochimie. 1990 Aug;72(8):555–574. doi: 10.1016/0300-9084(90)90120-6. [DOI] [PubMed] [Google Scholar]
  17. Lindberg T., Granhall U. Isolation and characterization of dinitrogen-fixing bacteria from the rhizosphere of temperate cereals and forage grasses. Appl Environ Microbiol. 1984 Oct;48(4):683–689. doi: 10.1128/aem.48.4.683-689.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Mavingui P., Laguerre G., Berge O., Heulin T. Genetic and Phenotypic Diversity of Bacillus polymyxa in Soil and in the Wheat Rhizosphere. Appl Environ Microbiol. 1992 Jun;58(6):1894–1903. doi: 10.1128/aem.58.6.1894-1903.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Nelson A. D., Barber L. E., Tjepkema J., Russell S. A., Powelson R., Evans H. J. Nitrogen fixation associated with grasses in Oregon. Can J Microbiol. 1976 Apr;22(4):523–530. doi: 10.1139/m76-078. [DOI] [PubMed] [Google Scholar]
  22. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sato Y., Kuramitsu H. K. Sequence analysis of the Streptococcus mutans scrB gene. Infect Immun. 1988 Aug;56(8):1956–1960. doi: 10.1128/iai.56.8.1956-1960.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Scholle R. R., Robb S. M., Robb F. T., Woods D. R. Nucleotide sequence and analysis of the Vibrio alginolyticus sucrase gene (scrB). Gene. 1989 Aug 1;80(1):49–56. doi: 10.1016/0378-1119(89)90249-7. [DOI] [PubMed] [Google Scholar]
  25. Steinmetz M., Le Coq D., Aymerich S. Induction of saccharolytic enzymes by sucrose in Bacillus subtilis: evidence for two partially interchangeable regulatory pathways. J Bacteriol. 1989 Mar;171(3):1519–1523. doi: 10.1128/jb.171.3.1519-1523.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Taussig R., Carlson M. Nucleotide sequence of the yeast SUC2 gene for invertase. Nucleic Acids Res. 1983 Mar 25;11(6):1943–1954. doi: 10.1093/nar/11.6.1943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Trieu-Cuot P., Carlier C., Poyart-Salmeron C., Courvalin P. Shuttle vectors containing a multiple cloning site and a lacZ alpha gene for conjugal transfer of DNA from Escherichia coli to gram-positive bacteria. Gene. 1991 Jun 15;102(1):99–104. doi: 10.1016/0378-1119(91)90546-n. [DOI] [PubMed] [Google Scholar]
  28. Vellanoweth R. L., Rabinowitz J. C. The influence of ribosome-binding-site elements on translational efficiency in Bacillus subtilis and Escherichia coli in vivo. Mol Microbiol. 1992 May;6(9):1105–1114. doi: 10.1111/j.1365-2958.1992.tb01548.x. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Zabarovsky E. R., Allikmets R. L. An improved technique for the efficient construction of gene libraries by partial filling-in of cohesive ends. Gene. 1986;42(1):119–123. doi: 10.1016/0378-1119(86)90158-7. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES