A Proteomic Analysis of Fibre Degradation and Assimilation by Butyrivibrio Proteoclasticus
Butyrivibrio proteoclasticus B316T is a Gram-positive, lignocellulose degrading bacterium that is prevalent in the rumen of animals grazing pasture, and is one of only a few rumen microbes known to degrade and utilise xylan in vitro. Xylan is a hemicellulose that comprises up to 45% of the polysaccharide component of ruminant forages. Often as little as 30% of the total energy content of forages is utilised by the ruminant due to poor hemicellulose degradation by the fibrolytic rumen microbes. An opportunity exists to improve forage degradation in the rumen, which is predicted to improve the productivity of forage fed ruminants. A clearer understanding of the strategies employed by fibrolytic rumen microbes to degrade and utilise lignocellulose is important in realising this goal. Almost 10% of the B. proteoclasticus genome encodes proteins involved in polysaccharide metabolism and transport, which includes 134 fibrolytic enzymes that are active upon plant fibre. Many of these are clustered into one of 36 polysaccharide utilisation loci that also contain transmembrane transporters, transcriptional regulators, environmental sensors and genes involved in further polysaccharide metabolism. Gel-based and gel-free proteomic analyses of the cytosolic, cell-associated, and secreted fractions of cells grown on xylan were used to identify proteins involved in the degradation, assimilation, and metabolism of hemicellulose. A set of 416 non-redundant proteins were identified, which included 12 extracellular and 24 cytosolic polysaccharidases, and 59 proteins involved in the uptake and further metabolism of polysaccharide degradation products, many of which were substrate-binding protein components of ATP-driven transporter systems. In cells grown on xylan, several of these proteins displayed significant protein abundance changes relative to cells grown on the monomeric sugar xylose, in a pattern that reflected the growth substrates used. A model of xylan degradation by B. proteoclasticus based on these results hypothesises that B. proteoclasticus attacks the xylan backbone and main substituent groups of hemicellulose in the extracellular space, assimilates the xylooligosaccharides and performs the final stages of degradation within the cell. These results provide insight into a xylan degrading enzyme system that has evolved to efficiently degrade and utilise hemicellulose, extend our understanding of the enzymes that are likely to play important roles in hemicellulose degradation, and support the notion that Butyrivibrio species are important contributors to rumen fibre degradation.