Molecular characterization of a high-affinity xylobiose transporter of Streptomyces thermoviolaceus OPC-520 and its transcriptional regulation.
Streptomyces thermoviolaceus OPC-520 secretes two types of xylanases (StxI and StxII), an acetyl xylan esterase (StxIII), and an alpha-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 beta-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 high-level affinity for xylobiose (K(d) = 8.75 x 10(-9) M) and for xylotriose (K(d) = 8.42 x 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.
Tsujibo
Hiroshi
H
Department of Microbiology, Osaka University of Pharmaceutical Sciences, Takatsuki, Osaka 569-1094, Japan. tsujibo@gly.oups.ac.jp
Kosaka
Mitsuo
M
Ikenishi
Sadao
S
Sato
Takaji
T
Miyamoto
Katsushiro
K
Inamori
Yoshihiko
Y
D018528:ATP-Binding Cassette Transporters / Q000235:genetics*; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004187:Disaccharides / Q000378:metabolism*; D008969:Molecular Sequence Data; D005810:Multigene Family; D016366:Open Reading Frames; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D014995:Xylosidases / Q000235:genetics*
D018528:ATP-Binding Cassette Transporters; D004187:Disaccharides; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase; C004173:xylobiose