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

Abstract

ABSTRACTBiological hydrolysis of cellulose above 70°C involves microorganisms that secrete free enzymes, and deploy separate protein systems to adhere to their substrate. Strongly cellulolytic Caldicellulosiruptor bescii is one such extreme thermophile, which deploys modular, multi-functional carbohydrate acting enzymes to deconstruct plant biomass. Additionally, C. bescii also encodes for non-catalytic carbohydrate binding proteins, which likely evolved as a mechanism to compete against other heterotrophs in carbon limited biotopes that these bacteria inhabit. Analysis of the Caldicellulosiruptor pangenome identified a type IV pilus (T4P) locus encoded upstream of the tāpirins, that is encoded for by all Caldicellulosiruptor species. In this study, we sought to determine if the C. bescii T4P plays a role in attachment to plant polysaccharides. The major C. bescii pilin (CbPilA) was identified by the presence of pilin-like protein domains, paired with transcriptomics and proteomics data. Using immuno-dot blots, we determined that the plant polysaccharide, xylan, induced production of CbPilA 10 to 14-fold higher than glucomannan or xylose. Furthermore, we are able to demonstrate that recombinant CbPilA directly interacts with xylan, and cellulose at elevated temperatures. Localization of CbPilA at the cell surface was confirmed by immunofluorescence microscopy. Lastly, a direct role for CbPilA in cell adhesion was demonstrated using recombinant CbPilA or anti-CbPilA antibodies to reduce C. bescii cell adhesion to xylan and crystalline cellulose up to 4.5 and 2-fold, respectively. Based on these observations, we propose that CbPilA and by extension, the T4P, plays a role in Caldicellulosiruptor cell attachment to plant biomass.IMPORTANCEMost microorganisms are capable of attaching to surfaces in part to persist in their environment. Here, we describe that the thermophilic, plant degrading bacterium, Caldicellulosiruptor bescii, uses type IV pili to attach to carbohydrates found in plant biomass. This ability is likely key to survival in environments where carbon sources are limiting, allowing C. bescii to compete against other plant degrading microorganisms. Interestingly, the carbohydrate that induced the highest expression of pilin protein was xylan, a hemicellulose that is not the majority polysaccharide in plant biomass. Not only do we demonstrate a direct interaction of the pilin with the polysaccharides, but also that cell attachment to polysaccharides can be disrupted by the addition of recombinant pilin and notably by antibody neutralization of the native pilin. This mechanism mirrors those recently described in pathogenic Gram-positive bacteria, and further supports the ancient origins of type IV pilus systems.