Caldicellulosiruptor bescii regulates pilus expression in response to the polysaccharide, xylan

Biological hydrolysis of cellulose at high temperatures relies on microorganisms that either secrete free enzymes or assemble cellulosomes. While these enzymatic systems appear to be opposites of one another, they may share an underlying mechanism of attachment. Extreme thermophile Caldicellulosiruptor bescii is highly cellulolytic, due in part to its freely secreted modular, multi-functional carbohydrate acting enzymes. Additionally, C. bescii also employs non-catalytic carbohydrate binding proteins, which likely evolved as a mechanism to compete against other heterotrophs in the carbon limited biotopes that these bacteria inhabit. Prior analysis of the Caldicellulosiruptor pangenome identified that a type IV pilus (T4P) locus is conserved among all Caldicellulosiruptor species. Interestingly, T4P loci are evolutionarily divergent between the highly and weakly cellulolytic members of the genus Caldicellulosiruptor . In this study, we sought to determine if C. bescii T4P plays a role in attachment to plant polysaccharides. Based on pilin-like protein domains, transcriptomics and protein expression data, we identified the major pilin (pilA) encoded for by the C. bescii genome. Using immunodot blots, we identified xylan as the main inducer of PilA production, in comparison to other representative plant polysaccharides. The extracellular location of PilA was further confirmed by immunofluorescence microscopy. Furthermore, recombinant PilA specifically disrupted C. bescii cell adhesion to xylan and crystalline cellulose in competitive cell binding assays. Based on these observations, we propose that PilA, the major C. bescii pilin, and by extension the T4P, plays a role in Caldicellulosiruptor cell attachment to plant biomass.