Mapping the Enzyme Machineries of Cellulolytic Soil-Dwelling Bacteroidetes

Bacteria from the phylum Bacteroidetes are regarded as proficient degraders of complex carbohydrates, but most species are limited to soluble glycans, e.g. hemicelluloses and pectins. Two aerobic Bacteroidetes members, Cytophaga hutchinsonii and Sporocytophaga myxococcoides, have however been known as proficient cellulose metabolizers for decades, but do not conform to the known mechanisms of enzymatic cellulose conversion. Neither species encodes cellobiohydrolases or lytic polysaccharide monooxygenases, and no apparent\ua0\ua0 complexed systems such as cellulosomes have been identified.\ua0\ua0 Many Bacteroidetes species utilize so-called polysaccharide utilization loci (PULs) which encode the necessary enzymes, binding proteins, sugar transporters and regulatory elements for target polysaccharides, but also these are absent in the genomes of C. hutchinsonii and S. myxococcoides. Mutagenesis studies instead point toward the Type IX secretion system being a crucial factor in polysaccharide turnover, and it is also tightly linked to their rapid gliding motility. In order to shed light on the enigmatic cellulolytic systems of these bacteria, we have used quantitative proteomics to map which proteins they produce during growth on cellulose and pectin, respectively, and determined the proteins’ cellular locations. Both bacteria produced similar yet distinct arrays of mostly unstudied putative cellulases during growth, and interestingly, cellulolytic activity was detected not only in the extracellular fraction and outer membrane but also intracellularly. In addition, several glycoside hydrolase family 8 (GH8) enzymes, that have previously been overlooked as potential cellulases in these species, were found to be both abundant and selectively produced during growth on cellulose. These GH8-containing proteins, which comprise large regions of unknown function and range between ~1100-2800 amino acids in total, are currently being functionally characterized to clarify their roles in cellulose turnover