SGBP-B-like bimodular cellulose-binding protein CHU_1279 is essential for cellulose utilization by Cytophaga...

The widespread cellulolytic specialist Cytophaga hutchinsonii belonging to the phylum Bacteroidetes adopted a unique cellulose utilization strategy that did not conform to the known cellulose-degrading paradigms involving free cellulases or cellulosomes. The strategy used by C. hutchinsonii still remains largely unclear. In this study, we showed that chu_1279 within the chu_1276-chu_1280 gene cluster, which has been previously shown to be important for cellulose utilization by C. hutchinsonii, encodes an outer membrane protein, and its elimination prohibited bacterial growth on cellulose. Structural prediction revealed that CHU_1279 is a surface glycan-binding protein B (SGBP-B)-like protein comprising two putative carbohydrate-binding module (CBM)-like domains. Further analyses verified that recombinant CHU_1279 displayed significant cellulose-binding protein, and its C-terminal domain is predominantly responsible for cellulose binding. Expression of the C-terminal domain but not the N-terminal domain restored cellulose utilization of ∆chu_1279. Moreover, site-directed mutagenesis analyses identified three aromatic residues important for cellulose binding of the recombinant CHU_1279 protein. The defective cellulose utilization of ∆chu_1279 cells otherwise could be recovered by CHU_1279 variants with significantly damaged cellulose-binding capability. Sequence analyses revealed that orthologs of CHU_1279 as well as the atypical polysaccharide utilization loci (PUL) constituted by the gene cluster chu_1276-chu_1280 are also present in two other cellulolytic Bacteroidetes bacteria, Cytophaga aurantiaca and Sporocytophaga myxococcoides, which are closely related to C. hutchinsonii. Our results contribute to unveiling the unique mechanism underlying the efficient cellulose utilization by C. hutchinsonii and similar cellulolytic bacteria.IMPORTANCEMost members of the phylum Bacteroidetes are highly competitive and efficient degraders of complex polysaccharides largely ascribed to their employment of a SusC-like system encoded by a polysaccharide utilization locus (PUL). However, characterization of PULs is limited to those responsible for utilization of (semi)soluble glycans. PULs involved in the utilization of cellulose, the most abundant renewable polymer, have not been identified and functionally characterized yet. We demonstrated that chu_1279 in the cellulolytic specialist C. hutchinsonii encodes an SGBP-B-like protein that is required for cellulose utilization, supporting that the gene cluster chu_1276-chu_1280 in C. hutchinsonii encodes an atypical PUL system dedicated to cellulose assimilation. Further analyses showed that this atypical PUL system is also present in two other cellulolytic Bacteroidetes bacteria. This study not only contributes to unveiling the unusual cellulose utilization strategy adopted by C. hutchinsonii and similar cellulolytic bacteria but also helps expand our understanding of atypical PULs for nutrient acquisition by cellulolytic bacteria.