The degradation of cellulose and hemicellulose, the main components of the plant cell wall, is a pivotal step in the carbon cycle on Earth and of major importance for the realization of a renewable energy source (biomass) that does not contribute net CO2to the atmosphere.Geobacillus stearothermophiluspossesses an extensive hemicellulolytic system allowing it to grow efficiently on xylan. The key enzyme is a single extracellular xylanase, capable of producing short decorated xylo-oligosaccharides from the natural branched polysaccharide. Gel retardation analyses demonstrated that XylR is a master regulator of the xylanolytic system and xylo-oligosaccharides act as the molecular inducers. The expression of the extracellular xylanase gene (xynA) is also repressed by glucose and other sugarsviaCcpA. In addition thexynAgene appears to be controlled by the transition state regulator AbrB. The xylo-oligosaccharides are transportedviaa specific ABC transporter, which is activated by the response regulator XynC, which is part of a two-component sensing system.
Interestingly, thexynAgene is regulated by quorum sensing at very low cell densities.xynA-mRNA extracted from different cell densities showed that the activation ofxynAis at the transcriptional level. In addition, in culture grown in conditioned medium the xylanase specific activity remains high and constant even at low-cell density, indicating that a diffusible extracellular factor may be involved. To allow the identification of the xylanase activation factor (XAF), we developed a 96-well plate-based assay that allows following cell growth and xylanase activity simultaneously at low-density cultures (~ 0.003 OD600). Initial purification steps of XAF indicate that the compound can bind to a silica-based octadecyl bonded phase (Sep-Pak C18) and can be eluted with 30 % to 60 % acetonitrile.
