Dataset Information

Functional Studies of ?-Glucosidases of Cytophaga hutchinsonii and Their Effects on Cellulose Degradation.

ABSTRACT: Cytophaga hutchinsonii can rapidly digest crystalline cellulose without free cellulases or cellulosomes. Its cell-contact cellulose degradation mechanism is unknown. In this study, the four ?-glucosidase (bgl) genes in C. hutchinsonii were singly and multiply deleted, and the functions of these ?-glucosidases in cellobiose and cellulose degradation were investigated. We found that the constitutively expressed BglB played a key role in cellobiose utilization, while BglA which was induced by cellobiose could partially make up for the deletion of bglB. The double deletion mutant ?bglA/bglB lost the ability to digest cellobiose and could not thrive in cellulose medium, indicating that ?-glucosidases were important for cellulose degradation. When cultured in cellulose medium, a small amount of glucose accumulated in the medium in the initial stage of growth for the wild type, while almost no glucose accumulated for ?bglA/bglB. When supplemented with a small amount of glucose, ?bglA/bglB started to degrade cellulose and grew in cellulose medium. We inferred that glucose might be essential for initiating cellulose degradation, and with additional glucose, C. hutchinsonii could partially utilize cellulose without ?-glucosidases. We also found that there were both cellulose binding cells and free cells when cultured in cellulose. Since direct contact between C. hutchinsonii cells and cellulose is necessary for cellulose degradation, we deduced that the free cells which were convenient to explore new territory in the environment might be fed by the adherent cells which could produce cello-oligosaccharide and glucose into the environment. This study enriched our knowledge of the cellulolytic pathway of C. hutchinsonii.

SUBMITTER: Bai X

PROVIDER: S-EPMC5288383 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Publications

Functional Studies of β-Glucosidases of Cytophaga hutchinsonii and Their Effects on Cellulose Degradation.

Bai Xinfeng X Wang Xifeng X Wang Sen S Ji Xiaofei X Guan Zhiwei Z Zhang Weican W Lu Xuemei X

Frontiers in microbiology 20170202

Cytophaga hutchinsonii can rapidly digest crystalline cellulose without free cellulases or cellulosomes. Its cell-contact cellulose degradation mechanism is unknown. In this study, the four β-glucosidase (bgl) genes in C. hutchinsonii were singly and multiply deleted, and the functions of these β-glucosidases in cellobiose and cellulose degradation were investigated. We found that the constitutively expressed BglB played a key role in cellobiose utilization, while BglA which was in ...[more]

PMID: 28210251

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Project description:Cytophaga hutchinsonii cells can bind to the surface of insoluble cellulose and degrade it by utilizing a novel cell contact-dependent mechanism, in which the outer membrane proteins may play important roles. In this study, the deletion of a gene locus, chu_1165, which encodes a hypothetical protein with 32% identity with TlpB, a disulfide oxidoreductase in Flavobacterium psychrophilum, caused a complete cellulolytic defect in C. hutchinsonii Further study showed that cells of the ?1165 strain could not bind to cellulose, and the levels of many outer membrane proteins that can bind to cellulose were significantly decreased. The N-terminal region of CHU_1165 is anchored to the cytoplasmic membrane with five predicted transmembrane helices, and the C-terminal region is predicted to stretch to the periplasm and has a similar thioredoxin (Trx) fold containing a Cys-X-X-Cys motif that is conserved in disulfide oxidoreductases. Recombinant CHU_1165His containing the Cys-X-X-Cys motif was able to reduce the disulfide bonds of insulin in vitro Site-directed mutation showed that the cysteines in the Cys-X-X-Cys motif and at residues 106 and 108 were indispensable for the function of CHU_1165. Western blotting showed that CHU_1165 was in an oxidized state in vivo, suggesting that it may act as an oxidase to catalyze disulfide bond formation. However, many of the decreased outer membrane proteins that were essential for cellulose degradation contained no or one cysteine, and mutation of the cysteine in these proteins did not affect cellulose degradation, indicating that CHU_1165 may have an indirect or pleiotropic effect on the function of these outer membrane proteins.IMPORTANCE Cytophaga hutchinsonii can rapidly digest cellulose in a contact-dependent manner, in which the outer membrane proteins may play important roles. In this study, a hypothetical protein, CHU_1165, characterized as a disulfide oxidoreductase, is essential for cellulose degradation by affecting the cellulose binding ability of many outer membrane proteins in C. hutchinsonii Disulfide oxidoreductases are involved in disulfide bond formation. However, our studies show that many of the decreased outer membrane proteins that were essential for cellulose degradation contained no or one cysteine, and mutation of cysteine did not affect their function, indicating that CHU_1165 did not facilitate the formation of a disulfide bond in these proteins. It may have an indirect or pleiotropic effect on the function of these outer membrane proteins. Our study provides an orientation for exploring the proteins that assist in the appropriate conformation of many outer membrane proteins essential for cellulose degradation, which is important for exploring the novel mechanism of cellulose degradation in C. hutchinsonii.

Project description:Cytophaga hutchinsonii is a Gram-negative bacterium that can efficiently degrade crystalline cellulose by a unique mechanism different from the free cellulase or cellulosome strategy. In this study, chu_3220, encoding the hypothetical protein CHU_3220 (205 kDa), was identified by insertional mutation and gene deletion as the first gene essential for degradation of the crystalline region but not the amorphous region of cellulose by C. hutchinsonii A chu_3220 deletion mutant was defective in the degradation of crystalline cellulose and increased the degree of crystallinity of Avicel PH101 but could still degrade amorphous cellulose completely. CHU_3220 was found to be located on the outer surface of the outer membrane and could bind to cellulose. It contains 15 PbH1 domains and a C-terminal domain (CHU_C) that was proved to be critical for the localization of CHU_3220 on the cell surface and the function of CHU_3220 in crystalline cellulose degradation. Moreover, the degradation of crystalline cellulose was intact-cell dependent and inhibited by NaN3 Further study showed that chu_3220 was induced by cellulose and that the endoglucanase activity on the cell surface was significantly reduced without chu_3220 Real-time PCR revealed that the transcription of most genes encoding endoglucanases located on the cell surface was decreased in the chu_3220 deletion mutant, indicating that chu_3220 might also play a role in the regulation of the expression of some endoglucanases. Cytophaga hutchinsonii could efficiently degrade crystalline cellulose with a unique mechanism without cellulosomes and free cellulases. It lacks proteins that are thought to play important roles in disruption of the crystalline region of cellulose, including exoglucanases, lytic polysaccharide monooxygenases, expansins, expansin-like proteins, or swollenins, and most of its endoglucanases lack carbohydrate binding modules. The mechanism of the degradation of crystalline cellulose is still unknown. In this study, chu_3220 was identified as the first gene essential for the degradation of the crystalline region but not the amorphous region of cellulose. CHU_3220 is a high-molecular-weight protein located on the outer surface of the outer membrane and could bind to cellulose. We proposed that CHU_3220 might be an essential component of a protein complex on the cell surface in charge of the decrystallization of crystalline cellulose. The degradation of crystalline cellulose by C. hutchinsonii was not only dependent on intact cells but also required the energy supplied by the cells. This was obviously different from other known cellulose depolymerization system. Our study has shed more light on the novel strategy of crystalline cellulose degradation by C. hutchinsonii.

Project description:The type IX secretion system (T9SS), which is involved in pathogenicity, motility, and utilization of complex biopolymers, is a novel protein secretion system confined to the phylum Bacteroidetes Cytophaga hutchinsonii, a common cellulolytic soil bacterium belonging to the phylum Bacteroidetes, can rapidly digest crystalline cellulose using a novel strategy. In this study, the deletion mutant of chu_0174 (gldN) was obtained using PY6 medium supplemented with Stanier salts. GldN was verified to be a core component of C. hutchinsonii T9SS, and is indispensable for cellulose degradation, motility, and secretion of C-terminal domain (CTD) proteins. Notably, the ΔgldN mutant showed significant growth defects in Ca2+- and Mg2+-deficient media. These growth defects could be relieved by the addition of Ca2+ or Mg2+ The intracellular concentrations of Ca2+ and Mg2+ were markedly reduced in ΔgldN These results demonstrated that GldN is essential for the acquisition of trace amounts of Ca2+ and Mg2+, especially for Ca2+ Moreover, an outer membrane efflux protein, CHU_2807, which was decreased in abundance on the outer membrane of ΔgldN, is essential for normal growth in PY6 medium. The reduced intracellular accumulation of Ca2+ and Mg2+ in the Δ2807 mutant indicated that CHU_2807 is involved in the uptake of trace amounts of Ca2+ and Mg2+ This study provides insights into the role of T9SS in metal ion assimilation in C. hutchinsonii IMPORTANCE The widespread Gram-negative bacterium Cytophaga hutchinsonii uses a novel but poorly understood strategy to utilize crystalline cellulose. Recent studies showed that a T9SS exists in C. hutchinsonii and is involved in cellulose degradation and motility. However, the main components of the C. hutchinsonii T9SS and their functions are still unclear. Our study characterized the function of GldN, which is a core component of the T9SS. GldN was proved to play vital roles in cellulose degradation and cell motility. Notably, GldN is essential for the acquisition of Ca2+ and Mg2+ ions under Ca2+- and Mg2+-deficient conditions, revealing a link between the T9SS and the metal ion transport system. The outer membrane abundance of CHU_2807, which is essential for Ca2+ and Mg2+ uptake in PY6 medium, was affected by the deletion of GldN. This study demonstrated that the C. hutchinsonii T9SS has extensive functions, including cellulose degradation, motility, and metal ion assimilation, and contributes to further understanding of the function of the T9SS in the phylum Bacteroidetes.

Dataset Information

Functional Studies of ?-Glucosidases of Cytophaga hutchinsonii and Their Effects on Cellulose Degradation.

Publications

Functional Studies of β-Glucosidases of <i>Cytophaga hutchinsonii</i> and Their Effects on Cellulose Degradation.

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