A Two-Component System Regulates the Expression of an ABC Transporter for Xylo-Oligosaccharides inGeobacillus stearothermophilus

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 ATP-binding cassette (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 nonphosphorylated fused response regulator, His 6 -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 revealed direct repeat sequences of a GAAA-like motif. In vitro transcriptional assays and quantitative reverse transcription-PCR demonstrated that xynE transcription is activated 140-fold in the presence of 1.5 M XynC. The His 6 -tagged sugar-binding lipoprotein (XynE) of the ABC transporter interacted with different xylosaccharides, as demonstrated by isothermal titration calorimetry. The change in the heat capacity of binding (⌬C p ) for XynE with xylotriose suggests a 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 transport system for xylo-oligosaccharides 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.

Biochemical and Structural Insights into Xylan Utilization by the Thermophilic Bacterium Caldanaerobius polysaccharolyticus

“…have uncharacterized homologs of XBP1 ranging in amino acid identity from 74 to 87%, the most closely related protein with demonstrated activity is XynE from G. stearothermophilus (52% identity). Similar to XBP1, GsXynE binds preferentially to xylotriose, although it prefers xylotetraose over xylobiose, which is in contrast to XBP1 (8). The gene encoding GsXynE is located within a 39.7-kb gene cluster containing hemicellulose utilization genes (6), and its expression is regulated by a nearby two-component system (8).…”

“…In addition to possessing the N-terminal (␣/␤) 8 and C-terminal ␤-sandwich domains characteristic of GH 3 enzymes (57), Xyl3A also has a C-terminal fibronectin repeat 3-like (FnIII-like) domain (supplemental Fig. S1).…”

“…To facilitate a concerted action of these enzymes for hemicellulose degradation, several microorganisms have evolved gene clusters encoding the different hemicellulolytic enzymes (1,(3)(4)(5)(6). The transport mechanism for xylan degradation products has been fairly well described in bacteria such as Streptomyces lividans and Geobacillus stearothermophilus (7,8). However, our knowledge in this area of sugar metabolism by bacteria is still limited.…”

Two distinct arabinofuranosidases contribute to arabino-oligosaccharide degradation in Bacillus subtilis

“…However, Bacillus species and other aerobic bacteria secrete a moderate number of enzymes that attack the polysaccharide backbone, releasing quite large oligosaccharides (Numan & Bhosle, 2006;. The complete hydrolysis of these products is accomplished by intracellular or membrane-associated enzymes (Beylot et al, 2001a;Shulami et al, 2007). Thus, the cytoplasmic nature of AbfA and Abf2 in B. subtilis might represent an adaptive advantage for the bacterium since competing non-hemicellulolytic micro-organisms are unable to utilize the relatively large arabino-oligomers and arabino-oligosaccharides products released by the extracellular enzymes of B. subtilis .…”

Single-cell genomics reveals metabolic strategies for microbial growth and survival in an oligotrophic aquifer

“…4), including endo-1,4-xylanases for the breakdown of xylan into xylose subunits, and xylose isomerase for the conversion of xylose into xylulose. An encoded extracellular solute binding protein has~30 % amino acid similarity to the xylo-oligosaccharide binding XynE protein in Geobacillus stearothermophilus (Shulami et al, 2007), suggesting a role for this protein in xylan/xylose uptake into the cell. Xylulose generated via these reactions is subsequently metabolized via the pentose phosphate and EmbdenMeyerhof-Parnas pathways.…”