Novel Outer Membrane Protein Involved in Cellulose and Cellooligosaccharide Degradation by Cytophaga hutchinsonii

Abstract

bCytophaga hutchinsonii is an aerobic cellulolytic soil bacterium which was reported to use a novel contact-dependent strategy to degrade cellulose. It was speculated that cellooligosaccharides were transported into the periplasm for further digestion. In this study, we reported that most of the endoglucanase and ␤-glucosidase activity was distributed on the cell surface of C. hutchinsonii. Cellobiose and part of the cellulose could be hydrolyzed to glucose on the cell surface. However, the cell surface cellulolytic enzymes were not sufficient for cellulose degradation by C. hutchinsonii. An outer membrane protein, CHU_1277, was disrupted by insertional mutation. Although the mutant maintained the same endoglucanase activity and most of the ␤-glucosidase activity, it failed to digest cellulose, and its cellooligosaccharide utilization ability was significantly reduced, suggesting that CHU_1277 was essential for cellulose degradation and played an important role in cellooligosaccharide utilization. Further study of cellobiose hydrolytic ability of the mutant on the enzymatic level showed that the ␤-glucosidase activity in the outer membrane of the mutant was not changed. It revealed that CHU_1277 played an important role in assisting cell surface ␤-glucosidase to exhibit its activity sufficiently. Studies on the outer membrane proteins involved in cellulose and cellooligosaccharide utilization could shed light on the mechanism of cellulose degradation by C. hutchinsonii.

An Outer Membrane Protein Involved in the Uptake of Glucose Is Essential for Cytophaga hutchinsonii Cellulose Utilization

“…C. hutchinsonii may thus adopt an alternative mode of utilization in which the cell surface enzymes of C. hutchinsonii digest cellulose extracellularly to cellobiose and glucose, eliminating the need for SusC-like and SusD-like proteins to import longer oligosaccharides. A recent study showed that extracted outer membrane proteins containing endoglucanase and ␤-glucosidase activities had the ability to hydrolyze cellulose to glucose in vitro (6). Interference with the efficient hydrolysis of cellobiose into glucose resulting from the absence of an outer membrane protein, CHU_1277, was further found to compromise the ability of C. hutchinsonii to assimilate cellulose (6).…”

“…A recent study showed that extracted outer membrane proteins containing endoglucanase and ␤-glucosidase activities had the ability to hydrolyze cellulose to glucose in vitro (6). Interference with the efficient hydrolysis of cellobiose into glucose resulting from the absence of an outer membrane protein, CHU_1277, was further found to compromise the ability of C. hutchinsonii to assimilate cellulose (6). In the present study, we identified CHU_1276, a gene locus upstream of CHU_1277, whose disruption not only compromised the efficient hydrolysis of cellobiose, but also affected the uptake of glucose at a low concentration.…”

“…This result suggests that the absence of CHU_1276 exerts a profound effect on the response to and assimilation of glucose by C. hutchinsonii. Third, cellulose digestion by C. hutchinsonii occurs in a cell contact-dependent mode, and outer membrane proteins are thus considered to play important roles in both adhering to substrate cellulose and coordinating cellulose depolymerization and the uptake of hydrolytic products (6,7,18,25,26). Not surprisingly, a significant number of genes encoding outer membrane proteins and transporter proteins were among these differentially expressed genes affected by the absence of CHU_1276 (see Data Set S6 in the supplemental material).…”

“…The wild-type and mutant strains were cultured at 28°C for 36 h in PY10 with 0.4% or 0.1% (wt/vol) glucose as the sole carbon source. The cells were then harvested by centrifugation at 6,000 ϫ g for 10 min, washed with Na 2 HPO 4 -KH 2 PO 4 buffer (50 mM, pH 6.8), and resuspended in the same buffer (6). The suspensions were mixed with equal volumes of buffer with glucose or cellobiose at a final concentration of 0.1% or 0.4% (wt/vol).…”

“…Most other wellstudied cellulolytic microorganisms apply one of two strategies, the extracellular-free-cellulase system or the cell surface-anchored multiprotein cellulosome, to achieve the efficient degradation of cellulose (5). C. hutchinsonii has been assumed to use a third but poorly understood strategy involving cell surface cellulases that produce cellulo-oligosaccharides that are directly transported into the periplasm for further digestion (6)(7)(8). Analysis of the genomic sequences of C. hutchinsonii revealed that all the annotated endoglucanases lack recognizable cellulose binding modules (CBMs), and no obvious homologs of cellobiohydrolases are present (3).…”