Cytophaga hutchinsonii chu_2177, encoding the O-antigen ligase, is essential for cellulose degradation

2022 ◽  
Author(s):  
Yahong Tan ◽  
Wenxia Song ◽  
Lijuan Gao ◽  
Weican Zhang ◽  
Xuemei Lu
Keyword(s):  
Cellulose Degradation ◽  
Cytophaga Hutchinsonii ◽  
O Antigen

scholarly journals A Disulfide Oxidoreductase (CHU_1165) Is Essential for Cellulose Degradation by Affecting Outer Membrane Proteins in Cytophaga hutchinsonii

2020 ◽  
Vol 86 (8) ◽  
Author(s):  
Dong Zhao ◽  
Ying Wang ◽  
Sen Wang ◽  
Weican Zhang ◽  
Qingsheng Qi ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Disulfide Bond ◽  
Cellulose Degradation ◽  
Outer Membrane Proteins ◽  
Hypothetical Protein ◽  
Pleiotropic Effect ◽  
Content Type ◽  
Cytophaga Hutchinsonii ◽  
Disulfide Oxidoreductase

ABSTRACT 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 Δ1165 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.

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

2016 ◽  
Vol 82 (6) ◽  
pp. 1933-1944 ◽  
Author(s):  
Hong Zhou ◽  
Xia Wang ◽  
Tengteng Yang ◽  
Weixin Zhang ◽  
Guanjun Chen ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Cellulose Degradation ◽  
Expression Profiles ◽  
Transcriptional Profiling ◽  
Hydrolysis Product ◽  
Transcriptional Responses ◽  
Content Type ◽  
Cytophaga Hutchinsonii

ABSTRACTCytophaga hutchinsoniispecializes in cellulose digestion by employing a collection of novel cell-associated proteins. Here, we identified a novel gene locus, CHU_1276, that is essential forC. hutchinsoniicellulose utilization. Disruption of CHU_1276 inC. hutchinsoniiresulted 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 inC. hutchinsonii.

A T9SS Substrate Involved in Crystalline Cellulose Degradation by Affecting Crucial Cellulose Binding Proteins in Cytophaga hutchinsonii

2021 ◽  
Author(s):  
Lijuan Gao ◽  
Yaru Su ◽  
Wenxia Song ◽  
Weican Zhang ◽  
Qingsheng Qi ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Gene Locus ◽  
Cellulose Degradation ◽  
Degradation Mechanism ◽  
Crystalline Cellulose ◽  
Cellulolytic Bacterium ◽  
Cytophaga Hutchinsonii ◽  
Surface Localization ◽  
Cell Surface Localization ◽  
Cellulose Binding

Cytophaga hutchinsonii is an abundant soil cellulolytic bacterium that uses a unique cellulose degradation mechanism different from those that involve free cellulases or cellulosomes. Though several proteins were identified to be important for cellulose degradation, the mechanism used by C. hutchinsonii to digest crystalline cellulose remains a mystery. In this study, chu_0922 was identified by insertional mutation and gene deletion as an important gene locus indispensable for crystalline cellulose utilization. Deletion of chu_0922 resulted in defect in crystalline cellulose utilization. The Δ 0922 mutant completely lost the ability to grow on crystalline cellulose even with extended incubation, and selectively utilized the amorphous region of cellulose leading to the increased crystallinity. As a protein secreted by the type Ⅸ secretion system (T9SS), CHU_0922 was found to be located on the outer membrane, and the outer membrane localization of CHU_0922 relied on the T9SS. Comparative analysis of the outer membrane proteins revealed that the abundance of several cellulose binding proteins, including CHU_1276, CHU_1277, and CHU_1279, was reduced in the Δ 0922 mutant. Further study showed that CHU_0922 is crucial for the full expression of the gene cluster containing chu_1276 , chu_1277 , chu_1278 , chu_1279 , and chu_1280 ( cel9C ), which is essential for cellulose utilization. Moreover, CHU_0922 is required for the cell surface localization of CHU_3220, a cellulose binding protein that is essential for crystalline cellulose utilization. Our study provides insights into the complex system that C. hutchinsonii uses to degrade crystalline cellulose. IMPORTANCE The widespread aerobic cellulolytic bacterium Cytophaga hutchinsonii , belonging to the phylum Bacteroidetes , utilizes a novel mechanism to degrade crystalline cellulose. No genes encoding proteins specialized in loosening or disruption the crystalline structure of cellulose were identified in the genome of C. hutchinsonii , except for chu_3220 and chu_1557 . The crystalline cellulose degradation mechanism remains enigmatic. This study identified a new gene locus, chu_0922 , encoding a typical T9SS substrate that is essential for crystalline cellulose degradation. Notably, CHU_0922 is crucial for the normal transcription of chu_1276 , chu_1277 , chu_1278 , chu_1279 , and chu_1280 ( cel9C ), which play important roles in the degradation of cellulose. Moreover, CHU_0922 participates in the cell surface localization of CHU_3220. These results demonstrated that CHU_0922 plays a key role in the crystalline cellulose degradation network. Our study will promote the uncovering of the novel cellulose utilization mechanism of C. hutchinsonii.

Effects of the histone-like protein HU on cellulose degradation and biofilm formation of Cytophaga hutchinsonii

2018 ◽  
Vol 102 (15) ◽  
pp. 6593-6611 ◽  
Author(s):  
Zhiwei Guan ◽  
Ying Wang ◽  
Lijuan Gao ◽  
Weican Zhang ◽  
Xuemei Lu
Keyword(s):  
Biofilm Formation ◽  
Cellulose Degradation ◽  
Cytophaga Hutchinsonii

scholarly journals FLP-FRT-Based Method To Obtain Unmarked Deletions ofCHU_3237(porU) and Large Genomic Fragments of Cytophaga hutchinsonii

2014 ◽  
Vol 80 (19) ◽  
pp. 6037-6045 ◽  
Author(s):  
Ying Wang ◽  
Zhiquan Wang ◽  
Jing Cao ◽  
Zhiwei Guan ◽  
Xuemei Lu
Keyword(s):  
Cellulose Degradation ◽  
Degradation Mechanism ◽  
Signal Peptidase ◽  
Crystalline Cellulose ◽  
Cellulolytic Bacterium ◽  
Efficient Tool ◽  
Content Type ◽  
Cytophaga Hutchinsonii ◽  
Recombination System ◽  
Colony Spreading

ABSTRACTCytophaga hutchinsoniiis a widely distributed cellulolytic bacterium in the phylumBacteroidetes. 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 inC. hutchinsonii. Unmarked deletion ofCHU_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 byC. 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 ofC. hutchinsonii.

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

2017 ◽  
Vol 8 ◽  
Author(s):  
Xinfeng Bai ◽  
Xifeng Wang ◽  
Sen Wang ◽  
Xiaofei Ji ◽  
Zhiwei Guan ◽  
...  
Keyword(s):  
Cellulose Degradation ◽  
Cytophaga Hutchinsonii ◽  
Functional Studies

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

2014 ◽  
Vol 80 (15) ◽  
pp. 4511-4518 ◽  
Author(s):  
Xiaofei Ji ◽  
Ying Wang ◽  
Cong Zhang ◽  
Xinfeng Bai ◽  
Weican Zhang ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Cell Surface ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Cellulose Degradation ◽  
Outer Membrane Proteins ◽  
Cellulolytic Enzymes ◽  
Content Type ◽  
Cytophaga Hutchinsonii ◽  
Glucosidase Activity

ABSTRACTCytophaga hutchinsoniiis 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 β-glucosidase activity was distributed on the cell surface ofC. 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 byC. hutchinsonii. An outer membrane protein, CHU_1277, was disrupted by insertional mutation. Although the mutant maintained the same endoglucanase activity and most of the β-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 β-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 β-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 byC. hutchinsonii.

scholarly journals Cytophaga hutchinsonii SprA and SprT Are Essential Components of the Type IX Secretion System Required for Ca2+ Acquisition, Cellulose Degradation, and Cell Motility

2021 ◽  
Vol 12 ◽  
Author(s):  
Lijuan Gao ◽  
Yahong Tan ◽  
Weican Zhang ◽  
Qingsheng Qi ◽  
Xuemei Lu
Keyword(s):  
Cell Surface ◽  
Protein Secretion ◽  
Cellulose Degradation ◽  
Secretion System ◽  
Gliding Motility ◽  
Uptake System ◽  
Cytophaga Hutchinsonii ◽  
Essential Components ◽  
Single Deletion ◽  
Type Ix Secretion System

The type IX secretion system (T9SS) is a novel protein secretion system, which is found in and confined to the phylum Bacteroidetes. T9SS is involved in the secretion of virulence factors, cell surface adhesins, and complex biopolymer degrading enzymes to the cell surface or extracellular medium. Cytophaga hutchinsonii is a widely distributed bacterium, which is able to efficiently digest cellulose and rapidly glide along the solid surfaces. C. hutchinsonii has a full set of orthologs of T9SS components. However, the functions of most homologous proteins have not been verified. In C. hutchinsonii, CHU_0029 and CHU_2709 are similar in sequence to Flavobacterium johnsoniae T9SS components SprA and SprT, respectively. In this study, the single deletion mutants of chu_0029 (sprA) and chu_2709 (sprT) were obtained using a complex medium with the addition of Ca2+ and Mg2+. Single deletion of sprA or sprT resulted in defects in cellulose utilization and gliding motility. Moreover, the ΔsprA and ΔsprT mutants showed growth defects in Ca2+- and Mg2+-deficient media. The results of ICP-MS test showed that both the whole cell and intracellular concentrations of Ca2+ were dramatically reduced in the ΔsprA and ΔsprT mutants, indicating that SprA and SprT are both important for the assimilation of trace amount of Ca2+. While the assimilation of Mg2+ was not obviously influenced in the ΔsprA and ΔsprT mutants. Through proteomics analysis of the cell surface proteins of the wild type and mutants, we found that the ΔsprA and ΔsprT mutants were defective in secretion of the majority of T9SS substrates. Together, these results indicate that SprA and SprT are both essential components of C. hutchinsonii T9SS, which is required for protein secretion, Ca2+ acquisition, cellulose degradation, and gliding motility in C. hutchinsonii. Our study shed more light on the functions of SprA and SprT in T9SS, and further proved the link between the T9SS and Ca2+ uptake system.

N -glycosylation of a cargo protein C-terminal domain recognized by the type IX secretion system in Cytophaga hutchinsonii affects protein secretion and localization

2021 ◽  
Author(s):  
Shuaishuai Xie ◽  
Yahong Tan ◽  
Wenxia Song ◽  
Weican Zhang ◽  
Qingsheng Qi ◽  
...  
Keyword(s):  
Cell Motility ◽  
Cellulose Degradation ◽  
Secretion System ◽  
Crystalline Cellulose ◽  
Homologous Gene ◽  
Cell Resistance ◽  
Cytophaga Hutchinsonii ◽  
Terminal Domain ◽  
Cargo Protein ◽  
Type Ix Secretion System

Cytophaga hutchinsonii is a Gram-negative bacterium belonging to the phylum Bacteroidetes . It digests crystalline cellulose with an unknown mechanism, and possesses a type IX secretion system (T9SS) that can recognize the C-terminal domain (CTD) of the cargo protein as a signal. In this study, the functions of CTD in the secretion and localization of T9SS substrates in C. hutchinsonii were studied by fusing the green fluorescent protein (GFP) with CTD from CHU_2708. CTD is necessary for the secretion of GFP by C. hutchinsonii T9SS. The GFP-CTD CHU_2708 fusion protein was found to be glycosylated in the periplasm with a molecular mass about 5 kDa higher than that predicted from its sequence. The glycosylated protein was sensitive to peptide- N -glycosidase F which can hydrolyze N -linked oligosaccharides. Analyses of mutants obtained by site-directed mutagenesis of asparagine residues in the N-X-S/T motif of CTD CHU_2708 suggest that N -glycosylation occurred on the CTD. CTD N- glycosylation is important for the secretion and localization of GFP-CTD recombinant proteins in C. hutchinsonii . Glycosyltransferase encoding gene chu_3842 , a homologous gene of Campylobacter jejuni pglA , was found to participate in the N -glycosylation of C. hutchinsonii . Deletion of chu_3842 affected cell motility, cellulose degradation, and cell resistance to some chemicals. Our study provided the evidence that CTD as the signal of T9SS was N -glycosylated in the periplasm of C. hutchinsonii . IMPORTANCE The bacterial N -glycosylation system has previously only been found in several species of Proteobacteria and Campylobacterota , and the role of N -linked glycans in bacteria is still not fully understood. C. hutchinsonii has a unique cell-contact cellulose degradation mechanism, and many cell surface proteins including cellulases are secreted by the T9SS. Here, we found that C. hutchinsonii , a member of the phylum Bacteroidetes , has an N -glycosylation system. Glycosyltransferase CHU_3842 was found to participate in the N -glycosylation of C. hutchinsonii proteins, and had effects on cell resistance to some chemicals, cell motility, and cellulose degradation. Moreover, N -glycosylation occurs on the CTD translocation signal of T9SS. The glycosylation of CTD apears to play an important role in affecting T9SS substrates transportation and localization. This study enriched our understanding of the widespread existence and multiple biological roles of N -glycosylation in bacteria.

scholarly journals Cytophaga hutchinsonii gldN, Encoding a Core Component of the Type IX Secretion System, Is Essential for Ion Assimilation, Cellulose Degradation, and Cell Motility

2020 ◽  
Vol 86 (11) ◽  
Author(s):  
Lijuan Gao ◽  
Zhiwei Guan ◽  
Peng Gao ◽  
Weican Zhang ◽  
Qingsheng Qi ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Metal Ion ◽  
Cellulose Degradation ◽  
Secretion System ◽  
Crystalline Cellulose ◽  
Core Component ◽  
Content Type ◽  
Cytophaga Hutchinsonii ◽  
Growth Defects ◽  
Type Ix Secretion System

ABSTRACT 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.

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