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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 have been identified as 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 defects 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 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.

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 , can rapidly digest crystalline cellulose using a novel strategy. In this study, the deletion mutant of Bacteroidetes , can rapidly digest crystalline cellulose using a novel strategy. In this study, the deletion mutant of chu_0174 ( gldN T9SS, and is indispensable for cellulose degradation, motility, and secretion of C-terminal domain (CTD) proteins. Notably, the Δ C. hutchinsonii T9SS, and is indispensable for cellulose degradation, motility, and secretion of C-terminal domain (CTD) proteins. Notably, the Δ gldN mutant showed significant growth defects in Ca 2+ - and Mg 2+ -deficient media. These growth defects could be relieved by the addition of Ca 2+ or Mg 2+ The intracellular concentrations of Ca 2+ and Mg 2+ were markedly reduced in Δ gldN These results demonstrated that GldN is essential for the acquisition of trace amounts of Ca 2+ and Mg 2+ , especially for Ca 2+ Moreover, an outer membrane efflux protein, CHU_2807, which was decreased in abundance on the outer membrane of Δ gldN , is essential for normal growth in PY6 medium. The reduced intracellular accumulation of Ca 2+ and Mg 2+ in the Δ 2807 mutant indicated that CHU_2807 is involved in the uptake of trace amounts of Ca 2+ and Mg 2+ This study provides insights into the role of T9SS in metal ion assimilation in C. hutchinsonii uses a novel but poorly understood strategy to utilize crystalline cellulose. Recent studies showed that a T9SS exists in IMPORTANCE and is involved in cellulose degradation and motility. However, the main components of the Cytophaga 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 Ca 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 Ca 2+ and Mg 2+ ions under Ca 2+ - and Mg 2+ -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 Ca 2+ 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 2+ 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 ., (Copyright © 2020 American Society for Microbiology.)

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_1165 His 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 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 Cytophaga 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 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 ., (Copyright © 2020 American Society for Microbiology.)

The crystalline region of cellulose is the main barrier to the utilization of crystalline cellulose. Cytophaga hutchinsonii actively digests the crystalline region of cellulose by an unknown mechanism. Transposon mutagenesis was done to identify a novel gene locus chu_1557, which is required for efficient disruption of the crystalline region of cellulose, and the absence of CHU_1557 resulted in decreased glucose assimilation efficiency. The defect of the mutant in the disruption of the crystalline region of cellulose was partially retained by additional glucose or pre-culturing the mutant in a low glucose concentration medium which could improve its glucose absorption efficiency. These results suggested that extracellular glucose has important roles in the disruption of crystalline cellulose by C. hutchinsonii. Further study showed that the expression of an outer membrane protein CHU_3732 was downregulated by the absence of CHU_1557 in a low glucose concentration medium. CHU_3732 was involved in uptake of glucose and its expression was induced by a low concentration of glucose. CHU_3732 was predicted to be a porin, so we inferred that it may work as a glucose transport channel in the outer membrane. Based on these results, we deduced that CHU_1557 played a role in the process of glucose assimilation and its disruption affected the expression of other proteins related to glucose transportation such as CHU_3732, and then affected the cell growth in a low glucose concentration medium and disruption of the crystalline region of cellulose.

Cytophaga hutchinsonii, belonging to Bacteroidetes, is speculated to use a novel cell-contact mode to digest cellulose. In this study, we identified a histone-like protein HU, CHU_2750, in C. hutchinsonii, whose transcription could be induced by crystalline but not amorphous cellulose. We constructed a CHU_2750-deleted mutant and expressed CHU_2750 in Escherichia coli to study the gene's functions. Our results showed that although the deletion of CHU_2750 was not lethal to C. hutchinsonii, the mutant displayed an abnormal filamentous morphology, loose nucleoid, and obvious defects in the degradation of crystalline cellulose and cell motility. Further study indicated that the mutant displayed significantly decreased cell surface and intracellular endoglucanase activities but with β-glucosidase activities similar to the wild-type strain. Analyses by real-time quantitative PCR revealed that the transcription levels of many genes involved in cellulose degradation and/or cell motility were significantly downregulated in the mutant. In addition, we found that CHU_2750 was important for biofilm formation of C. hutchinsonii. The main extracellular components of the biofilm were analyzed, and the results showed that the mutant yielded significantly less exopolysaccharide but more extracellular DNA and protein than the wild-type strain. Collectively, our findings demonstrated that CHU_2750 is important for cellulose degradation, cell motility, and biofilm formation of C. hutchinsonii by modulating transcription of certain related genes, and it is the first identified transcriptional regulator in these processes of C. hutchinsonii. Our study shed more light on the mechanisms of cellulose degradation, cell motility, and biofilm formation by C. hutchinsonii.

Cytophaga hutchinsonii is a gram-negative bacterium that can efficiently degrade crystalline cellulose by a novel strategy without cell-free cellulases or cellulosomes. Genomic analysis implied that C. hutchinsonii had endoglucanases and β-glucosidases but no exoglucanases which could processively digest cellulose and produce cellobiose. In this study, BglA was functionally expressed in Escherichia coli and found to be a β-glucosidase with wide substrate specificity. It can hydrolyze pNPG, pNPC, cellobiose, and cellodextrins. Moreover, unlike most β-glucosidases whose activity greatly decreases with increasing length of the substrate chains, BglA has similar activity on cellobiose and larger cellodextrins. The K m values of BglA on cellobiose, cellotriose, and cellotetraose were calculated to be 4.8 × 10 -2 , 5.6 × 10 -2 , and 5.3 × 10 -2  mol/l, respectively. These properties give BglA a great advantage to cooperate with endoglucanases in C. hutchinsonii in cellulose degradation. We proposed that C. hutchinsonii could utilize a simple cellulase system which consists of endoglucanases and β-glucosidases to completely digest amorphous cellulose into glucose. Moreover, BglA was also found to be highly tolerant to glucose as it retained 40 % activity when the concentration of glucose was 100 times higher than that of the substrate, showing potential application in the bioenergy industry.

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 NaN 3 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., Importance: 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., (Copyright © 2016 American Society for Microbiology.)

Cytophaga hutchinsonii is a Gram-negative bacterium that can degrade crystalline cellulose efficiently with an unknown strategy. Genomic analysis suggested it lacks exoglucanases which are found in many cellulolytic organisms and most of the cellulases in C. hutchinsonii lack recognizable carbohydrate-binding modules (CBMs). CHU_1280, speculated to be an endoglucanase belonging to glycoside hydrolase family 9 (GH9) in C. hutchinsonii, was functionally expressed in Escherichia coli, and evidence was presented suggesting that it may be a processive endoglucanase. In the absence of Ca(2+), CHU_1280 was inactive. But in the presence of Ca(2+), it had a specific activity of 600 U/μmol with carboxymethyl cellulose (CMC) as the substrate. With Ca(2+), CHU_1280 hydrolyzed regenerated amorphous cellulose (RAC) with nearly 80 % of the reducing ends appearing in the soluble fraction, suggesting it degraded cellulose in a processive way. CHU_1280 could bind to cellulose without recognizable CBMs and its binding ability was also Ca(2+)-dependent. Ca(2+) could stabilize the catalytic domain at high temperature, but the denaturation temperature of the whole protein was decreased. C. hutchinsonii might have an exoglucanase-independent cellulases system which included endoglucanases, processive endoglucanases, and β-glucosidases.

Cytophaga 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.

Cytophaga hutchinsonii is a Gram-negative gliding bacterium, which can rapidly degrade crystalline cellulose via a novel strategy without any recognizable processive cellulases. Its mechanism of cellulose binding and degradation is still a mystery. In this study, the mutagenesis of C. hutchinsonii with the mariner-based transposon HimarEm3 and gene complementation with the oriC-based plasmid carrying the antibiotic resistance gene cfxA or tetQ were reported for the first time to provide valuable tools for mutagenesis and genetic manipulation of the bacterium. Mutant A-4 with a transposon mutation in gene CHU_0134, which encodes a putative thiol-disulfide isomerase exhibits defects in cell motility and cellulose degradation. The cellulose binding ability of A-4 was only half of that of the wild-type strain, while the endo-cellulase activity of the cell-free supernatants and on the intact cell surface of A-4 decreased by 40%. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of proteins binding to cellulose in the outer membrane showed that most of them were significantly decreased or disappeared in A-4 including some Gld proteins and hypothetical proteins, indicating that these proteins might play an important role in cell motility and cellulose binding and degradation by the bacterium.

Objective: The aim of the study was to understand the mechanism of Cytophaga hutchinsonii adhension to cellulose., Methods: The effects of different factors on the bacterial adhesion to cellulose were studied, including bacterial age, pH, temperature, cell surface charge, cell viability, cell surface protein, extracellular polysaccharides, and cellulose derivates., Results: Treatments with heat and protease reduced the adhesion remarkably. But treatments with NaN3, formalin, glutaraldehyde, Congo red and NaIO4 had only slight effect on the adhesion. The adhension of Cytophaga hutchinsonii cells to microcrystalline cellulose was specific and not inhibited by cellobiose or carboxymethyl cellulose., Conclusion: The adhesion of Cytophaga hutchinsonii to cellulose was closely related to cell surface proteins, while cellular metabolic activity and extracellular polysaccharides had only slight effect on it. It is speculated that there might be some specific cellulose binding proteins on the cell surface.

Cytophaga hutchinsonii is an important Gram-negative bacterium belonging to the Bacteroides phylum that can efficiently degrade cellulose. But the promoter that mediates the initiation of gene transcription has been unknown for a long time. In this study, we determined the transcription start site (TSS) of C. hutchinsonii by 5′ rapid amplification of cDNA ends (5′RACE). The promoter structure was first identified as TAAT and TATTG which are located -5 and -31 bp upstream of TSS, respectively. The function of -5 and -31 regions and the spacer length of the promoter Pchu_1284 were explored by site directed ligase-independent mutagenesis (SLIM). The results showed that the promoter activities were sharply decreased when the TTG motif was mutated into guanine (G) or cytosine (C). Interestingly, we found that the strong promoter was accompanied with many TTTG motifs which could enhance the promoter activities within certain copies. These characteristics were different from other promoters of Bacteriodes species. Furthermore, we carried out genome scanning analysis for C. hutchinsonii and another Bacteroides species by Perl6.0. The results indicated that the promoter structure of C. hutchinsonii possessed more unique features than other species. Also, the screened inducible promoter Pchu_2268 was used to overexpress protein CHU_2196 with a molecular weight of 120 kDa in C. hutchinsonii. The present study enriched the promoter structure of Bacteroidetes species and also provided a novel method for the highly expressed large protein (cellulase) in vivo, which was helpful to elucidate the unique cellulose degradation mechanism of C. hutchinsonii. Key points • The conserved structure of strong promoter of C. hutchinsonii was elucidated. • Two novel regulation motifs of TTTG and AATTATG in the promoter were discovered. • A new method for induced expression of cellulase in vivo was established. • Helpful for explained the unique cellulose degradation mechanism of C. hutchinsonii. [ABSTRACT FROM AUTHOR]

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 Δ sprA and Δ sprT 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 Δ sprA and Δ sprT 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 Δ sprA and Δ sprT mutants. Through proteomics analysis of the cell surface proteins of the wild type and mutants, we found that the Δ sprA and Δ sprT 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. [ABSTRACT FROM AUTHOR]