Characterization of a family 5 glycoside hydrolase isolated from the outer membrane of cellulolytic Cytophaga hutchinsonii

Cao

et al. 2014

Cytophaga hutchinsonii is a widely distributed cellulolytic bacterium in the phylum Bacteroidetes. It can digest crystalline cellulose rapidly without free cellulases or cellulosomes. The mechanism of its cellulose utilization remains a mystery. We developed an efficient method based on a linear DNA double-crossover and FLP-FRT recombination system to obtain unmarked deletions of both single genes and large genomic fragments in C. hutchinsonii. Unmarked deletion of CHU_3237 (porU), an ortholog of the C-terminal signal peptidase of a type IX secretion system (T9SS), resulted in defects in colony spreading, cellulose degradation, and protein secretion, indicating that it is a component of the T9SS and that T9SS plays an important role in cellulose degradation by C. hutchinsonii. Furthermore, deletions of four large genomic fragments were obtained using our method, and the sizes of the excised fragments varied from 9 to 19 kb, spanning from 6 to 22 genes. The customized FLP-FRT method provides an efficient tool for more rapid progress in the cellulose degradation mechanism and other physiological aspects of C. hutchinsonii.

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confidence: 68%

Section: Discussionmentioning

confidence: 99%

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FLP-FRT-Based Method To Obtain Unmarked Deletions of CHU _ 3237 ( porU ) and Large Genomic Fragments of Cytophaga hutchinsonii

Cao

et al. 2014

“…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)(10)(11), though their exact roles in cellulose digestion remain to be determined.…”

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confidence: 99%

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

Zhou

Yang

et al. 2016

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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...

“…It was speculated that C. hutchinsonii cells use a contact-dependent digestion mode to utilize cellulose, but details of the mechanism were still unknown. Recently, several genetic manipulation techniques, including transposon mutagenesis and gene disruption with replicative plasmids or suicide vectors, have been developed (12)(13)(14)(15) which offer a great opportunity to explore the mysterious mechanism of cellulose degradation by C. hutchinsonii. In a previous study, we found that a cellulose utilization-deficient mutant lost some cellulose binding proteins in the outer membrane.…”

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confidence: 99%

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

Zhang

et al. 2014

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.