Molecular Characterization of a High-Affinity Xylobiose Transporter of Streptomyces thermoviolaceus OPC-520 and Its Transcriptional Regulation

Abstract

Streptomyces thermoviolaceus OPC-520 secretes two types of xylanases (StxI and StxII), an acetyl xylan esterase (StxIII), and an ␣-L-arabinofuranosidase (StxIV) in the presence of xylan. Xylan degradation products (mainly xylobiose) produced by the action of these enzymes entered the cell and were then degraded to xylose by an intracellular ␤-xylosidase (BxlA). A gene cluster involved in xylanolytic system of the strain was cloned and sequenced upstream of and including a BxlA-encoding gene (bxlA). The gene cluster consisted of four different open reading frames organized in the order bxlE, bxlF, bxlG, and bxlA. Reverse transcriptase PCR analysis revealed that the gene cluster is transcribed as polycistronic mRNA. The deduced gene products, comprising BxlE (a sugar-binding lipoprotein), BxlF (an integral membrane protein), and BxlG (an integral membrane protein), showed similarity to components of the bacterial ATP-binding cassette (ABC) transport system; however, the gene for the ATP binding protein was not linked to the bxl operon. The soluble recombinant BxlE protein was analyzed for its binding activity for xylooligosaccharides. The protein showed highlevel affinity for xylobiose (K d ‫؍‬ 8.75 ؋ 10 ؊9 M) and for xylotriose (K d ‫؍‬ 8.42 ؋ 10 ؊8 M). Antibodies raised against the recombinant BxlE recognized the detergent-soluble BxlE isolated from S. thermoviolaceus membranes. The deduced BxlF and BxlG proteins are predicted to be integral membrane proteins. These proteins contained the conserved EAA loop (between the fourth and the fifth membrane-spanning segments) which is characteristic of membrane proteins from binding-protein-dependent ABC transporters. In addition, the bxlR gene located upstream of the bxl operon was cloned and expressed in Escherichia coli. The bxlR gene encoded a 343-residue polypeptide that is highly homologous to members of the GalR/LacI family of bacterial transcriptional regulators. The purified BxlR protein specifically bound to a 4-bp inverted sequence overlapping the ؊10 region of the bxl operon. The binding of BxlR to the site was inhibited specifically by low concentrations of xylobiose. This site was also present in the region located between stxI and stxIV and in the upstream region of stxII. BxlR specifically bound to the regions containing the inverted sequence. These results suggest that BxlR might act as a repressor of the genes involved not only in the uptake system of xylan degradation products but also in xylan degradation of S. thermoviolaceus OPC-520.Streptomyces bacteria are gram positive, soil inhabiting, and filamentous, with a high GϩC content in their DNA. They produce a number of secondary metabolites and extracellular proteins, including enzymes hydrolyzing different types of polysaccharides such as xylan, cellulose, and chitin (12). Unlike cellulose and chitin, xylans have a relatively complex structure consisting of a ␤-1,4-linked D-xylose polymer replaced with L-arabinofuranosyl, glucuronyl, 4-O-methlglucuronyl, and acetyl groups (37). For complete hydroly...

Genome Sequence and Analysis of the Soil Cellulolytic ActinomyceteThermobifida fuscaYX

“…4A). Tfu1617, Tfu1618, and Tfu1619 are components of the xylobiose transporter based on their high similarity to the recently characterized xylobiose transporter from Streptomyces thermoviolaceus (49). Tfu1612 is the secreted xyloglucanase possessing a potential TAT targeting signal peptide characterized by Irwin et al (17).…”

The dasABC Gene Cluster, Adjacent to dasR , Encodes a Novel ABC Transporter for the Uptake of N , N ′-Diacetylchitobiose in Streptomyces coelicolor A3(2)

“…The binding specificities and affinities of the His-tagged DasA protein were determined by using a Biacore X system and carboxymethylated dextran (CM5) sensor chips (GE Healthcare), as reported elsewhere (36,40). GlcNAc, (GlcNAc) 2 , (GlcNAc) 3 3 ] were subjected to the analysis at concentrations ranging from 10 nM to 1 mM.…”

The l -Arabinan Utilization System of Geobacillus stearothermophilus

“…In Gram-positive bacteria, relatively few ATPbinding cassette (ABC) transporters that are involved in the uptake of hemicellulolytic products have been characterized (19). The ABC transport systems, XynEF and BxlEFG, from G. stearothermophilus (68) and Streptomyces thermoviolaceus (72), respectively, were demonstrated to bind xylo-oligosaccharides. The ABC transporter, AraNPQ, from Bacillus subtilis is involved in the uptake of arabino-oligosaccharides (22).…”