A Two-Component System Regulates the Expression of an ABC Transporter for Xylo-oligosaccharides In Geobacillus stearothermophilus

Department of Biotechnology and Food Engineering and Institute of Catalysis Science and Technology, Technion-Israel Institute of Technology, Haifa 32000, Israel, Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA 02111, USA

^* To whom correspondence should be addressed. Email: yshoham{at}tx.technion.ac.il.

	Abstract

Geobacillus stearothermophilus T-6 utilizes an extensive and highly regulated hemicellulolytic system. The genes comprising the xylanolytic system are clustered in a 39.7-kb chromosomal segment. This segment contains a 6-kb transcriptional unit (xynDCEFG) coding for a potential two-component system (xynDC) and an ABC transport system (xynEFG). The xynD promoter region contains a 16-bp inverted repeat resembling the operator site for the xylose repressor, XylR. XylR was found to bind specifically to this sequence and binding was efficiently prevented in vitro in the presence of xylose. The ABC transport system was shown to comprise an operon of three genes (xynEFG) that is transcribed from its own promoter. The non-phosphorylated fused response regulator, His₆-XynC, bound to a 220 bp fragment, corresponding to the xynE operator. DNase I footprinting analysis showed four protected zones that cover the -53 and the +34 regions and direct repeat sequences of GAAA-like motif. In vitro transcriptional assays and quantitative RT-PCR demonstrated that xynE transcription is activated 140-fold in the presence of 1.5 µM XynC. The His₆-tagged sugar-binding lipoprotein (XynE) of the ABC transporter interacted with different xylosaccharides as demonstrated by isothermal titration calorimetry (ITC). The change in the heat capacity of binding (Cp) for XynE with xylotriose suggests stacking interaction in the binding site that can be provided by a single Trp residue and a sugar moiety. Taken together, our data show that XynEFG constitutes an ABC transporter system for xylooligosachrides and that its transcription is negatively regulated by XylR and activated by the response regulator XynC, which is part of a two-component sensing system.