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

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.

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

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.

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

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.

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

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.

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

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.

An Extracytoplasmic Function Sigma Factor Controls Cellulose Utilization by Regulating the Expression of an Outer Membrane Protein inCytophaga hutchinsonii

ABSTRACTThe common soil cellulolytic bacterium known asCytophaga hutchinsoniimakes use of a unique but poorly understood strategy in order to utilize cellulose. While several genes have been identified as being an active part of the utilization of cellulose, the mechanism(s) by whichC. hutchinsoniiboth (i) senses its environment and (ii) regulates the expression of those genes are not as yet known. In this study, we identified and characterized the geneCHU_3097encoding an extracytoplasmic function (ECF) σ factor (σcel1), the disruption of which compromisedC. hutchinsoniicellulose assimilation to a large degree. The σcel1and its putative partner anti-σcel1, encoded by theCHU_3096gene found immediately downstream fromCHU_3097, copurifiedin vitro. The σcel1was discovered to be associated with inner membrane when cells were cultured on glucose and yet was partially released from the membrane in response to cellulose. This release was found to occur on glucose when the anti-σcel1was absent. Transcriptome analyses found a σcel1-regulated, cellulose-responsive gene regulon, within which an outer membrane protein encoding the geneCHU_1276, essential for cellulose utilization, was discovered to be significantly downregulated byCHU_3097disruption. The expression of CHU_1276 almost fully restored cellulose utilization to theCHU_3097mutant, demonstrating that CHU_1276 represents a critical regulatory target of σcel1. In this way, our study provided insights into the role of an ECF σ factor in coordinating the cellulolytic response ofC. hutchinsonii.IMPORTANCEThe common cellulolytic bacteriumCytophaga hutchinsoniiuses a unique but poorly understood strategy in order to make use of cellulose. Throughout the process of cellulosic biomass breakdown, outer membrane proteins are thought to play key roles; this is evidenced by CHU_1276, which is required for the utilization of cellulose. However, the regulatory mechanism of its expression is not yet known. We found and characterized an extracytoplasmic function σ factor that is involved in coordinating the cellulolytic response ofC. hutchinsoniiby directly regulating the expression ofCHU_1276. This study makes a contribution to our understanding of the regulatory mechanism used byC. hutchinsoniiin order to adjust its genetic programs and so deal with novel environmental cues.