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

Abstract

The Bacillus polymyxa CF43 leLk 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 le4 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 (Pi-D-fructosyltransferases). Multiple-sequence alignment of 14 of these proteins revealed several previously unreported features. LeIA 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 lek 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 leL4::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.Exopolysaccharides appear to play a role in the early steps of plant colonization by symbiotic or phytopathogenic bacteria such as Rhizobium spp. or Agrobacterium spp., respectively (23). Free-living rhizobacteria belonging to the genus Bacillus have been isolated from the rhizosphere of Graminae spp., including wheat and maize (7,24,26,32

?-Fructosidase superfamily: Homology with some ?-L-arabinases and ?-D-xylosidases

“…Several such frame shifts were described earlier in the sequences of family GH32: Bacillus subtilis sucrase (SwissProt accession number P07819), B. stearothermophilus levanase (P94469), Bacillus sp. L7 levanase (O31411), Debaryomyces occidentalis invertase (P24133), Aspergillus niger invertase (O13388), and Avena sativa invertase (Q43076) 25–30. To our knowledge, in two sequences frame shifts are found for the first time.…”

Structure-Function Relationships of Glucansucrase and Fructansucrase Enzymes from Lactic Acid Bacteria

“…Most of the research has been performed on levansucrases, in particular on enzymes from Bacillus spp. (12,107,189,195) and Zymomonas spp. (71,187,219,220), and, to a lesser extent, Lactobacillus spp.…”

Computational analysis of the fructosyltransferase enzymes in plants, fungi and bacteria

“…Reported and predicted subcellular localization showed that most plant FTases are vacuolar enzymes while sequences without a predicted signal peptide had a nuclear localizacion prediction (Supplementary Table 1). Fungi and bacteria FTases were extracellular enzymes, excepting some sequences for which an intracellular localization prediction was done (Supplementary Table 1), as reported previously (Bezzate et al, 1994;Ryan et al, 2005;Yuan et al, 2006).…”