Search tips
Search criteria 


Logo of jbacterPermissionsJournals.ASM.orgJournalJB ArticleJournal InfoAuthorsReviewers
J Bacteriol. 1994 April; 176(8): 2177–2183.
PMCID: PMC205337

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


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

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.7M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Anagnostopoulos C, Spizizen J. REQUIREMENTS FOR TRANSFORMATION IN BACILLUS SUBTILIS. J Bacteriol. 1961 May;81(5):741–746. [PMC free article] [PubMed]
  • 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. [PMC free article] [PubMed]
  • 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. [PMC free article] [PubMed]
  • 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. [PubMed]
  • 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. [PubMed]
  • Blatch GL, Woods DR. Molecular characterization of a fructanase produced by Bacteroides fragilis BF-1. J Bacteriol. 1993 May;175(10):3058–3066. [PMC free article] [PubMed]
  • Brückner R, Wagner E, Götz F. Characterization of a sucrase gene from Staphylococcus xylosus. J Bacteriol. 1993 Feb;175(3):851–857. [PMC free article] [PubMed]
  • Burne RA, Penders JE. Characterization of the Streptococcus mutans GS-5 fruA gene encoding exo-beta-D-fructosidase. Infect Immun. 1992 Nov;60(11):4621–4632. [PMC free article] [PubMed]
  • Chambert R, Treboul G, Dedonder R. Kinetic studies of levansucrase of Bacillus subtilis. Eur J Biochem. 1974 Jan 16;41(2):285–300. [PubMed]
  • Fouet A, Klier A, Rapoport G. Nucleotide sequence of the sucrase gene of Bacillus subtilis. Gene. 1986;45(2):221–225. [PubMed]
  • 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. [PubMed]
  • Jannière L, Bruand C, Ehrlich SD. Structurally stable Bacillus subtilis cloning vectors. Gene. 1990 Mar 1;87(1):53–61. [PubMed]
  • Klein RD, Poorman RA, Favreau MA, Shea MH, Hatzenbuhler NT, Nulf SC. Cloning and sequence analysis of the gene encoding invertase from the yeast Schwanniomyces occidentalis. Curr Genet. 1989 Sep;16(3):145–152. [PubMed]
  • Kurnit DM. 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. [PubMed]
  • Leigh JA, Coplin DL. Exopolysaccharides in plant-bacterial interactions. Annu Rev Microbiol. 1992;46:307–346. [PubMed]
  • Lemesle-Varloot L, Henrissat B, Gaboriaud C, Bissery V, Morgat A, Mornon JP. Hydrophobic cluster analysis: procedures to derive structural and functional information from 2-D-representation of protein sequences. Biochimie. 1990 Aug;72(8):555–574. [PubMed]
  • 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. [PMC free article] [PubMed]
  • 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. [PubMed]
  • 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. [PMC free article] [PubMed]
  • Moran CP, Jr, Lang N, LeGrice SF, Lee G, Stephens M, Sonenshein AL, 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. [PubMed]
  • Nelson AD, Barber LE, Tjepkema J, Russell SA, Powelson R, Evans HJ. Nitrogen fixation associated with grasses in Oregon. Can J Microbiol. 1976 Apr;22(4):523–530. [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PubMed]
  • Sato Y, Kuramitsu HK. Sequence analysis of the Streptococcus mutans scrB gene. Infect Immun. 1988 Aug;56(8):1956–1960. [PMC free article] [PubMed]
  • Scholle RR, Robb SM, Robb FT, Woods DR. Nucleotide sequence and analysis of the Vibrio alginolyticus sucrase gene (scrB). Gene. 1989 Aug 1;80(1):49–56. [PubMed]
  • 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. [PMC free article] [PubMed]
  • Taussig R, Carlson M. Nucleotide sequence of the yeast SUC2 gene for invertase. Nucleic Acids Res. 1983 Mar 25;11(6):1943–1954. [PMC free article] [PubMed]
  • 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. [PubMed]
  • Vellanoweth RL, Rabinowitz JC. 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. [PubMed]
  • 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. [PubMed]
  • Zabarovsky ER, Allikmets RL. An improved technique for the efficient construction of gene libraries by partial filling-in of cohesive ends. Gene. 1986;42(1):119–123. [PubMed]

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