Abstract

Isolation and expression of the xynB gene and its product, XynB, a consistent component of the Clostridium cellulovorans cellulosome. J. Bacteriol. 186: 8347-8355. 2004. S.O. Han, H. Yukawa, M. Inui and R.H. Doi.

The nucleotide sequence of the Clostridium cellulovorans xynBgene, which encodes the XynB xylanase, consists of 1,821 bpand encodes a protein of 607 amino acids with a molecular weightof 65,976. XynB contains a typical N-terminal signal peptideof 29 amino acid residues, followed by a 147-amino-acid sequencethat is homologous to the family 4-9 (subfamily 9 in family4) carbohydrate-binding domain. Downstream of this domain isa family 10 catalytic domain of glycosyl hydrolase. The C terminusseparated from the catalytic domain by a short linker sequencecontains a dockerin domain responsible for cellulosome assembly.The XynB sequence from mass spectrometry and N-terminal aminoacid sequence analyses agreed with that deduced from the nucleotidesequence. XynB was highly active toward xylan, but not activetoward carboxymethyl cellulose. The enzyme was optimally activeat 40°C and pH 5.0. Northern hybridizations revealed thatxynB is transcribed as a monocistronic 1.9-kb mRNA. RNA ligase-mediatedrapid amplification of 5' cDNA ends by PCR (RLM-5'RACE PCR)analysis of C. cellulovorans RNA identified a single transcriptionalstart site of xynB located 47 bp upstream from the first nucleotideof the translation initiation codon. Alignment of the xynB promoterregion provided evidence for highly conserved sequences thatexhibited strong similarity to the sigma(A) consensus promoter sequencesof gram-positive bacteria. Expression of xynB mRNA increasedfrom early to middle exponential phase and decreased duringthe early stationary phase when the cells were grown on cellobiose.No alternative promoter was observed by RLM-5'RACE PCR and reversetranscriptase PCR analyses during expression. The analysis ofthe products from xylan hydrolysis by thin-layer chromatography indicated its endoxylanase activity. The results suggest that XynB is a consistent and major cellulosomal enzyme during growthon cellulose or xylan.

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