Semirational engineering of Cytophaga hutchinsonii polyphosphate kinase for developing a cost-effective, robust, and efficient adenosine 5'-triphosphate regeneration system.

IMPORTANCE: The adenosine 5'-triphosphate (ATP) regeneration system can significantly reduce the cost of many biocatalytic processes. Numerous studies have endeavored to utilize the ATP regeneration system based on Cytophaga hutchinsonii PPK (ChPPK). However, the wild-type ChPPK enzyme possesses limitations such as low enzymatic activity, poor stability, and limited substrate tolerance, impeding its application in catalytic reactions. To enhance the performance of ChPPK, we employed a semi-rational design approach to obtain the variant ChPPK/A79G/S106C/I108F/L285P. The enzymatic kinetic parameters and the catalytic performance in the synthesis of nicotinamide mononucleotide demonstrated that the variant ChPPK/A79G/S106C/I108F/L285P exhibited superior enzymatic properties than the wild-type enzyme. All data indicated that our engineered ATP regeneration system holds inherent potential for implementation in biocatalytic processes.

Glycosyltransferase-Related Protein GtrA Is Essential for Localization of Type IX Secretion System Cargo Protein Cellulase Cel9A and Affects Cellulose Degradation in Cytophaga hutchinsonii.

The Gram-negative bacterium Cytophaga hutchinsonii digests cellulose through a novel cellulose degradation mechanism. It possesses the lately characterized type IX secretion system (T9SS). We recently discovered that N-glycosylation of the C-terminal domain (CTD) of a hypothetical T9SS substrate protein in the periplasmic space of C. hutchinsonii affects protein secretion and localization. In this study, green fluorescent protein (GFP)-CTDCel9A recombinant protein was found with increased molecular weight in the periplasm of C. hutchinsonii. Site-directed mutagenesis studies on the CTD of cellulase Cel9A demonstrated that asparagine residue 900 in the D-X-N-X-S motif is important for the processing of the recombinant protein. We found that the glycosyltransferase-related protein GtrA (CHU_0012) located in the cytoplasm of C. hutchinsonii is essential for outer membrane localization of the recombinant protein. The deletion of gtrA decreased the abundance of the outer membrane proteins and affected cellulose degradation by C. hutchinsonii. This study provided a link between the glycosylation system and cellulose degradation in C. hutchinsonii. IMPORTANCE N-Glycosylation systems are generally limited to some pathogenic bacteria in prokaryotes. The disruption of the N-glycosylation pathway is related to adherence, invasion, colonization, and other phenotypic characteristics. We recently found that the cellulolytic bacterium Cytophaga hutchinsonii also has an N-glycosylation system. The cellulose degradation mechanism of C. hutchinsonii is novel and mysterious; cellulases and other proteins on the cell surface are involved in utilizing cellulose. In this study, we identified an asparagine residue in the C-terminal domain of cellulase Cel9A that is necessary for the processing of the T9SS cargo protein. Moreover, the glycosyltransferase-related protein GtrA is essential for the localization of the GFP-CTDCel9A recombinant protein. Deletion of gtrA affected cellulose degradation and the abundance of outer membrane proteins. This study enriched the understanding of the N-glycosylation system in C. hutchinsonii and provided a link between N-glycosylation and cellulose degradation, which also expanded the role of the N-glycosylation system in bacteria.

Some novel features of strong promoters discovered in Cytophaga hutchinsonii.

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.

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

Cytophaga hutchinsonii can efficiently degrade crystalline cellulose, in which the cell surface cellulases secreted by the type IX secretion system (T9SS) play important roles, but the degradation mechanism remains unclear, and the anchor mechanism of cellulases on the outer membrane in C. hutchinsonii has not been studied. Here, chu_2177 was identified by transposon mutagenesis and was proved to be indispensable for cellulose utilization in C. hutchinsonii. Disruption of chu_2177 resulted in O-antigen deficiency and chu_177 could confer O-antigen ligase activity upon an Escherichia coli waal mutant, indicating that chu_2177 encoded the O-ntigen ligase. Moreover, deletion of chu_2177 caused defects in cellulose utilization, cell motility, biofilm formation, and stress resistance. Further study showed that the endoglucanase activity was markedly decreased in the outer membrane but was increased in the culture fluid without chu_2177. Western blot proved that endoglucanase CHU_1336 was not located on the outer membrane but was released in the culture fluid of the Δ2177 mutant. Further proteomics analysis showed that many cargo proteins of T9SS were missing in the outer membrane of the Δ2177 mutant. Our study revealed that the deletion of chu_2177 affected the localization of many T9SS cargo proteins including cellulases on the outer membrane of C. hutchinsonii.

A Type IX Secretion System 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 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 Δ0922 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 Δ0922 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 the CTD in the secretion and localization of T9SS substrates in C. hutchinsonii were studied by fusing the green fluorescent protein (GFP) with the CTD from CHU_2708. The CTD is necessary for the secretion of GFP by C. hutchinsonii T9SS. The GFP-CTDCHU_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 CTDCHU_2708 suggested 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 evidence that the CTD as the signal of T9SS was N-glycosylated in the periplasm of C. hutchinsonii. IMPORTANCE The bacterial N-glycosylation system has previously been found only 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. In this study, 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 the CTD appears to play an important role in affecting T9SS substrate transportation and localization. This study enriched our understanding of the widespread existence and multiple biological roles of N-glycosylation in bacteria.

Responses of bacterial communities and organic matter degradation in surface sediment to Macrobrachium nipponense bioturbation.

The excessive accumulation of organic matter (OM) in sediments in aquaculture ponds is a potential environmental threat due to the risk of endogenous water pollution and eutrophication. From the perspective of inhibiting OM accumulation to prevent endogenous water pollution, the present study investigated the OM degradation states, variations of bacterial communities and basic environmental factors in sediments with/without Macrobrachium nipponense treatment/control groups in triplicate for effects of bioturbation on OM degradation in 90-day incubation. The total organic carbon (TOC) and total nitrogen (TN) in the M. nipponense treatment were higher than in the control at the 30th and 60th days, while no significant differences between treatment and control were found at the end of the experiment. Significantly higher oxidation-reduction potential (ORP) and more extensively degraded OM were observed in the M. nipponense treatment. Eleven significantly differential bacterial taxa were enriched in the sediments of M. nipponense treatment, of which eight (Actinobacteria, Chitinophagales, Chitinophagaceae, Flavihumibacter, Marinilabiliaceae, Cytophaga xylanolytica group, Christensenellaceae, and Christensenellaceae R-7 group) were significantly correlated with at least two OM degradation indicators. The functional groups chemoheterotrophy, aerobic chemoheterotrophy, xylanolysis, ureolysis, and intracellular parasites were enhanced by M. nipponense and were negatively correlated with OM degradation indictors. Overall, the M. nipponense bioturbation effectively increased the ORP to provide better conditions for OM degradation, altered the taxonomic composition and functional groups to enhance the bacterial ability for OM degradation, and finally promoted the OM degradation of the surface sediment in an artificial aquaculture system.

International Committee on Systematics of Prokaryotes: Subcommittee on the Taxonomy of Aerobic Bacteroidetes (formerly Flavobacterium and Cytophaga-like bacteria).

Minutes of the meeting: 15 November 2017 (Skype meeting).

Identification of a unique 1,4-ß-D-glucan glucohydrolase of glycoside hydrolase family 9 from Cytophaga hutchinsonii.

Cytophaga hutchinsonii is an aerobic cellulolytic soil bacterium that rapidly digests crystalline cellulose. The predicted mechanism by which C. hutchinsonii digests cellulose differs from that of other known cellulolytic bacteria and fungi. The genome of C. hutchinsonii contains 22 glycoside hydrolase (GH) genes, which may be involved in cellulose degradation. One predicted GH with uncertain specificity, CHU_0961, is a modular enzyme with several modules. In this study, phylogenetic tree of the catalytic modules of the GH9 enzymes showed that CHU_0961 and its homologues formed a new group (group C) of GH9 enzymes. The catalytic module of CHU_0961 (CHU_0961B) was identified as a 1,4-ß-D-glucan glucohydrolase (EC 3.2.1.74) that has unique properties compared with known GH9 cellulases. CHU_0961B showed highest activity against barley glucan, but low activity against other polysaccharides. Interestingly, CHU_0961B showed similar activity against ρ-nitrophenyl ß-D-cellobioside (ρ-NPC) and ρ-nitrophenyl ß-D-glucopyranoside. CHU_0961B released glucose from the nonreducing end of cello-oligosaccharides, ρ-NPC, and barley glucan in a nonprocessive exo-type mode. CHU_0961B also showed same hydrolysis mode against deacetyl-chitooligosaccharides as against cello-oligosaccharides. The kcat/Km values for CHU_0961B against cello-oligosaccharides increased as the degree of polymerization increased, and its kcat/Km for cellohexose was 750 times higher than that for cellobiose. Site-directed mutagenesis showed that threonine 321 in CHU_0961 played a role in hydrolyzing cellobiose to glucose. CHU_0961 may act synergistically with other cellulases to convert cellulose to glucose on the bacterial cell surface. The end product, glucose, may initiate cellulose degradation to provide nutrients for bacterial proliferation in the early stage of C. hutchinsonii growth. KEY POINTS: • CHU_0961 and its homologues formed a novel group (group C) of GH9 enzymes. • CHU_0961 was identified as a 1,4-ß-d-glucan glucohydrolase with unique properties. • CHU_0961 may play an important role in the early stage of C. hutchinsonii growth.

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

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 BacteroidetesCytophaga 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. hutchinsoniiIMPORTANCE 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.

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.IMPORTANCECytophaga 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.

A Bifunctional Polyphosphate Kinase Driving the Regeneration of Nucleoside Triphosphate and Reconstituted Cell-Free Protein Synthesis.

Reconstituted cell-free protein synthesis systems (e.g., the PURE system) allow the expression of toxic proteins, hetero-oligomeric protein subunits, and proteins with noncanonical amino acids with high levels of homogeneity. In these systems, an artificial ATP/GTP regeneration system is required to drive protein synthesis, which is accomplished using three kinases and phosphocreatine. Here, we demonstrate the replacement of these three kinases with one bifunctional Cytophaga hutchinsonii polyphosphate kinase that phosphorylates nucleosides in an exchange reaction from polyphosphate. The optimized single-kinase system produced a final sfGFP concentration (∼530 µg/mL) beyond that of the three-kinase system (∼400 µg/mL), with a 5-fold faster mRNA translation rate in the first 90 min. The single-kinase system is also compatible with the expression of heat-sensitive firefly luciferase at 37 °C. Potentially, the single-kinase nucleoside triphosphate regeneration approach developed herein could expand future applications of cell-free protein synthesis systems and could be used to drive other biochemical processes in synthetic biology which require both ATP and GTP.

Identification of a cell-surface protein involved in glucose assimilation and disruption of the crystalline region of cellulose by Cytophaga hutchinsonii.

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.

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

The common soil cellulolytic bacterium known as Cytophaga hutchinsonii makes 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 which C. hutchinsonii both (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 gene CHU_3097 encoding an extracytoplasmic function (ECF) σ factor (σcel1), the disruption of which compromised C. hutchinsonii cellulose assimilation to a large degree. The σcel1 and its putative partner anti-σcel1, encoded by the CHU_3096 gene found immediately downstream from CHU_3097, copurified in vitro The σcel1 was 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-σcel1 was absent. Transcriptome analyses found a σcel1-regulated, cellulose-responsive gene regulon, within which an outer membrane protein encoding the gene CHU_1276, essential for cellulose utilization, was discovered to be significantly downregulated by CHU_3097 disruption. The expression of CHU_1276 almost fully restored cellulose utilization to the CHU_3097 mutant, 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 of C. hutchinsoniiIMPORTANCE The common cellulolytic bacterium Cytophaga hutchinsonii uses 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 of C. hutchinsonii by directly regulating the expression of CHU_1276 This study makes a contribution to our understanding of the regulatory mechanism used by C. hutchinsonii in order to adjust its genetic programs and so deal with novel environmental cues.

Solution NMR structure of CHU_1110 from Cytophaga hutchinsonii, an AHSA1 protein potentially involved in metal ion stress response.

We report the solution nuclear magnetic resonance (NMR) structure of CHU_1110 from Cytophaga hutchinsonii. CHU_1110 contains three α-helices and one antiparallel ß-sheet, forming a large cavity in the center of the protein, which are consistent with the structural characteristics of AHSA1 protein family. This protein shows high structural similarities to the prokaryotic proteins RHE_CH02687 from Rhizobium etli and YndB from Bacillus subtilis, which can bind with flavinoids. Unlike these two homologs, CHU_1110 shows no obvious interaction with flavonoids in NMR titration experiments. In addition, no direct interaction has been observed between CHU_1110 and ATP, although many homologous sequences of CHU_1110 have been annotated as ATPase. Combining the analysis of structural similarity of CHU_1110 and genomic context of its encoding gene, we speculate that CHU_1110 may be involved in the stress response of bacteria to heavy metal ions, even though its specific biological functions that need to be further investigated.

Safety evaluation of two α-amylase enzyme preparations derived from Bacillus licheniformis expressing an α-amylase gene from Cytophaga species.

A safety assessment was conducted for a symthetic variant Cytophaga sp. α-amylase enzyme expressed in Bacillus licheniformis and formulated into two distinct product formats: whole broth (a preparation in which the production organism is completely inactivated, but containing residual cell debris) and clarified preparation (from which the production organism is completely removed). The enzyme was improved via modern biotechnology techniques for use in the endohydrolysis of starch, glycogen, related polysaccharides and oligosaccharides. Applications range from carbohydrate processing, including the manufacture of sweeteners, fermentation to produce organic acids, amino acids and their salts, and potable or fuel alcohol, with resulting co-products (distillers' grains and corn gluten feed/meal) destined for use in animal feed. The toxicological studies summarized in this article (90-day rodent oral gavage and in vitro genotoxicity studies) noted no test article-related adverse effects and thus substantiate the safety of the α-amylase in not only the clarified form but also as a whole-broth preparation. Consistent with the decision tree analysis for enzymes produced with modern biotechnology techniques, this paper provides supporting information that this variant amylase with homology to an amylase from a potentially pathogenic organism (Cytophaga sp.) can be safely produced in an expression host that belongs to a Safe Strain Lineage, for safe use as processing aid to manufacture human and animal food.

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

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 ß-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.

Chemical shift assignments of CHU_1110: an AHSA1-like protein from Cytophaga hutchinsonii.

AHSA1 protein family is one of the four largest families in the Bet v1-like protein superfamily. The functions and structures of proteins in AHSA1 family are still largely unknown. CHU_1110 with 167 amino acids and a molecular weight of 19.2 kDa is a member of the AHSA1 family from Cytophaga hutchinsonii, a soil bacterium known for its ability to digest crystalline cellulose. Here we report the complete 1H, 13C and 15N chemical shift assignments of CHU_1110. The secondary structural elements of CGL2373 are consistent with the canonical AHSA1 structure. However the sequence identity of CHU_1110 with other members of AHSA1 family with functional and structural reports, such as RHE_CH02687 from Rhizobium etli, Aha1 from Homo sapiens and Yndb from Bacillus subtilis, are very low, which may suggest a different function of CHU_1110. Our chemical shift assignments of CHU_1110 are essential for the following structural and functional research of CHU_1110.

Identification of a fabZ gene essential for flexirubin synthesis in Cytophaga hutchinsonii.

Cytophaga hutchinsonii, an aerobic soil bacterium which could degrade cellulose, produces yellow flexirubin pigments. In this study, fabZ, annotated as a putative ß-hydroxyacyl-(acyl carrier protein) (ACP) dehydratase gene, was identified by insertional mutation and gene deletion as an essential gene for flexirubin pigment synthesis. The availability of a FabZ mutant that fails to produce flexirubin allowed us to investigate the biological role of the pigment in C. hutchinsonii. Loss of flexirubin made the FabZ mutant more sensitive to UV radiation, oxidative stress and alkaline stress than the wild type.

The unusual cellulose utilization system of the aerobic soil bacterium Cytophaga hutchinsonii.

Cellulolytic microorganisms play important roles in global carbon cycling and have evolved diverse strategies to digest cellulose. Some are 'generous,' releasing soluble sugars from cellulose extracellularly to feed both themselves and their neighbors. The gliding soil bacterium Cytophaga hutchinsonii exhibits a more 'selfish' strategy. It digests crystalline cellulose using cell-associated cellulases and releases little soluble sugar outside of the cell. The mechanism of C. hutchinsonii cellulose utilization is still poorly understood. In this review, we discuss novel aspects of the C. hutchinsonii cellulolytic system. Recently developed genetic manipulation tools allowed the identification of proteins involved in C. hutchinsonii cellulose utilization. These include periplasmic and cell-surface endoglucanases and novel cellulose-binding proteins. The recently discovered type IX secretion system is needed for cellulose utilization and appears to deliver some of the cellulolytic enzymes and other proteins to the cell surface. The requirement for periplasmic endoglucanases for cellulose utilization is unusual and suggests that cello-oligomers must be imported across the outer membrane before being further digested. Cellobiohydrolases or other predicted processive cellulases that play important roles in many other cellulolytic bacteria appear to be absent in C. hutchinsonii. Cells of C. hutchinsonii attach to and glide along cellulose fibers, which may allow them to find sites most amenable to attack. A model of C. hutchinsonii cellulose utilization summarizing recent progress is proposed.