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Cloning, sequence analysis, and characterization of the genes involved in isoprimeverose metabolism in Lactobacillus pentosus

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Author: Chaillou, S. · Lokman, B.C. · Leer, R.J. · Posthuma, C. · Postma, P.W. · Pouwels, P.H.
Source:Journal of Bacteriology, 9, 180, 2312-2320
Identifier: 234452
Keywords: disaccharide · article · bacterial gene · bacterial metabolism · carbohydrate metabolism · fermentation · gene control · gene expression regulation · lactobacillus · molecular cloning · nonhuman · nucleotide sequence · priority journal · regulon · repressor gene · Amino Acid Sequence · Bacterial Proteins · Base Sequence · Carrier Proteins · Cell Compartmentation · Cloning, Molecular · Disaccharides · Genes, Bacterial · Glycosides · Lactobacillus · Molecular Sequence Data · Operon · Sequence Analysis, DNA · Sequence Homology, Amino Acid · Substrate Specificity · Symporters · Xylose · Xylosidases · Bacteria (microorganisms) · Lactobacillus pentosus · Posibacteria · Prokaryota


Two genes, xylP and xylQ, from the xylose regulon of Lactobacillus pentosus were cloned and sequenced. Together with the repressor gene of the regulon, xylR, the xylPQ genes form an operon which is inducible by xylose and which is transcribed from a promoter located 145 bp upstream of xylP. A putative xylR binding site (xylO) and a cre-like element, mediating CcpA- dependent catabolite repression, were found in the promoter region. L. pentosus mutants in which both xylP and xylQ (LPE1) or only xylQ (LPE2) was inactivated retained the ability to ferment xylose but were impaired in their ability to ferment isoprimeverose (α-D-xylopyranosyl, (1,6)-D- glucopyranose). Disruption of xylQ resulted specifically in the loss of a membrane-associated α-xylosidase activity when LPE1 or LPE2 cells were grown on xylose. In the membrane fraction of wild-type bacteria α-xylosidase could catalyze the hydrolysis of isoprimeverose and p-nitrophenyl-α-D- xylopyranoside with apparent K(m) and V(max) values of 0.2 mM and 446 nmol/min/mg of protein, and 1.3 mM and 54 nmol/min/mg of protein, respectively. The enzyme could also hydrolyze the α-xylosidic linkage in xyloglucan oligosaccharides, but neither methyl-α-D-xylopyranoside nor α- glucosides were substrates. Glucose repressed the synthesis of α-xylosidase fivefold, and 80% of this repression was released in an L. pentosus ΔccpA mutant. The α-xylosidase gene was also expressed in the absence of xylose when xylR was disrupted. Chemicals/CAS: alpha-D-xylosidase, EC 3.2.1.-; Bacterial Proteins; Carrier Proteins; Disaccharides; Glycosides; isoprimeverose cation symporter, Lactobacillus; isoprimeverose, 534-98-5; Symporters; Xylose; Xylosidases, EC 3.2.1.-