bCytophaga hutchinsonii specializes in cellulose digestion by employing a collection of novel cell-associated proteins. Here, we identified a novel gene locus, CHU_1276, that is essential for C. hutchinsonii cellulose utilization. Disruption of CHU_1276 in C. hutchinsonii resulted in complete deficiency in cellulose degradation, as well as compromised assimilation of cellobiose or glucose at a low concentration. Further analysis showed that CHU_1276 was an outer membrane protein that could be induced by cellulose and low concentrations of glucose. Transcriptional profiling revealed that CHU_1276 exerted a profound effect on the genome-wide response to both glucose and Avicel and that the mutant lacking CHU_1276 displayed expression profiles very different from those of the wild-type strain under different culture conditions. Specifically, comparison of their transcriptional responses to cellulose led to the identification of a gene set potentially regulated by CHU_1276. These results suggest that CHU_1276 plays an essential role in cellulose utilization, probably by coordinating the extracellular hydrolysis of cellulose substrate with the intracellular uptake of the hydrolysis product in C. hutchinsonii. Cytophaga hutchinsonii is a common cellulolytic soil bacterium that belongs to the phylum Bacteroidetes (1-4). A unique strategy is thought to be used by C. hutchinsonii to digest crystalline cellulose and to utilize filter paper as the sole carbon source. The mechanism, however, is largely unknown. 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 (6-8). 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). Recently, endoglucanases that are processive and may act as functional equivalents of exocellulases, and cellulose binding proteins (CBP) on the outer membrane (OM), have been identified in C. hutchinsonii (9-11), though their exact roles in cellulose digestion remain to be determined.Active import of oligosaccharides released by cell surface enzymes across the outer membrane has been proven to be crucial in the efficient utilization of polysaccharide substrates by many members of the phylum Bacteroidetes (12). This strategy has been well illustrated for starch utilization by the intestinal anaerobe Bacteroides thetaiotaomicron and involves a series of starch utilization system (Sus) proteins, specifically the cell surface lipoprotein SusD and the porin SusC, responsible for the binding and transport of oligosaccharides (13-16). For C. hutchinsonii, previo...

Cytophaga hutchinsonii, a member of the phylum Bacteroidetes, employs a novel collection of cell-associated proteins to digest crystalline cellulose. Other Bacteroidetes rely on cell surface proteins related to the starch utilization system (Sus) proteins SusC and SusD to bind oligosaccharides and import them across the outer membrane for further digestion. These bacteria typically produce dozens of SusC-like porins and SusD-like oligosaccharide-binding proteins to facilitate utilization of diverse polysaccharides. C. hutchinsonii specializes in cellulose digestion and its genome has only two susC-like genes and two susD-like genes. Single and multiple gene deletions were constructed to determine if the susC-like and susD-like genes have roles in cellulose utilization. A mutant lacking all susC-like and all susD-like genes digested cellulose and grew on cellulose as well as wild-type cells. Further, recombinantly expressed SusD-like proteins CHU_0547 and CHU_0554 failed to bind cellulose or β-glucan hemicellulosic polysaccharides. The results suggest that the Bacteroidetes Sus paradigm for polysaccharide utilization may not apply to the cellulolytic bacterium C. hutchinsonii.

Cytophaga hutchinsonii is a gliding cellulolytic bacterium that is ubiquitously distributed in soil. The mechanism by which C. hutchinsonii achieves cellulose digestion, however, is still largely unknown. In this study, we obtained a C. hutchinsonii mutant that was defective in utilizing filter paper or Avicel as the sole carbon source by transposon mutagenesis. The interrupted gene locus, CHU_2981, encodes a hypothetical protein with only 130 amino acids. Cell fractionation and western blot detection of CHU_2981 fused with a C-terminal green fluorescence protein (GFP) indicated that CHU_2981 is located in the periplasm. The CHU_2981-disrupted mutant cells exhibited a significant growth defect on Avicel but not on glucose and cellobiose. The absence of CHU_2981 also resulted in a significant defect in colony spreading and individual cell motility compared to wild-type cells. Further analysis demonstrated that the CHU_2981-disrupted mutant cells exhibited a different profile of cellulose-absorbed outer membrane proteins from that of wild-type cells, in which protein varieties and amounts were markedly decreased. Our results showed that CHU_2981, the periplasmic non-cellulolytic protein, plays an important role in both cellulose utilization and cell motility probably by being involved in the appropriate production of outer membrane proteins.

Cytophaga hutchinsonii, an aerobic cellulolytic soil bacterium, is capable of degrading crystalline cellulose and gliding over surface rapidly. The involved mechanisms, however, are largely unknown. Here, we used the mariner-based transposon HimarEm1 to screen for C. hutchinsonii mutants deficient in utilizing filter paper as the sole carbon source. A novel gene locus, chu_1719, encoding a hypothetical protein was identified, whose inactivation resulted in a compromised growth of C. hutchinsonii on filter paper. Further analysis revealed that disruption of chu_1719 suppressed colony spreading but had no significant effect on Avicel degradation in liquid medium. Carboxymethylcellulase (CMCase) activity of the mutant membrane proteins was reduced by about 40% as compared with the wild-type strain. Moreover, profiles of cellulose-adsorbed outer membrane proteins were significantly different between the mutant and wild-type (WT) strains. These results suggest that chu_1719 plays an important role in controlling the spreading motility and cellulose utilization probably by affecting the appropriate production of membrane proteins in C. hutchinsonii.