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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-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 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.

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 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 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 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+ and Mg 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 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 ., (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, an aerobic soil bacterium which could degrade cellulose, produces yellow flexirubin pigments. In this study, fabZ, annotated as a putative β-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., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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 glides rapidly over surfaces by an unknown mechanism without flagella and type IV pili and it can degrade crystalline cellulose efficiently by a novel mechanism. Tn4351 transposon mutagenesis was used to identify a new gene, CHU_1798, essential for colony spreading on agar surfaces. Further study showed that disruption of CHU_1798 caused non-spreading colonies on both soft and hard agar surfaces and individual cells were partially deficient in gliding on glass surfaces. The CHU_1798 mutant could digest cellulose as long as the cells were in direct contact with the cellulose, but it could not degrade cellulose powder buried in the agar plate. Scanning electron microscopy showed that individual mutant cells arranged irregularly on the cellulose fiber surface at an early stage of incubation, but later showed a regular parallel arrangement when there were plenty of cells and could spread along the cellulose fibers. These results suggest that CHU_1798 plays an important role in the motility of C. hutchinsonii and provide insight into the relation between cell motility and cellulose degradation., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Cytophaga hutchinsonii is a widely distributed cellulolytic bacterium in the phylum Bacteroidetes. 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 in C. hutchinsonii. Unmarked deletion of CHU_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 by C. 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 of C. hutchinsonii., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

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 efficiently degrade crystalline cellulose by an unknown strategy. Genomic analysis suggests the C. hutchinsonii genome lacks homologs to an obvious exoglucanase that previously seemed essential for cellulose degradation. One of the putative endoglucanases, CHU_2103, was successfully expressed in Escherichia coli JM109 and identified as a processive endoglucanase with transglycosylation activity. It could hydrolyze carboxymethyl cellulose (CMC) into cellodextrins and rapidly decrease the viscosity of CMC. When regenerated amorphous cellulose (RAC) was degraded by CHU_2103, the ratio of the soluble to insoluble reducing sugars was 3.72 after 3 h with cellobiose and cellotriose as the main products, indicating that CHU_2103 was a processive endoglucanase. CHU_2103 could degrade cellodextrins of degree of polymerization ≥3. It hydrolyzed p-nitrophenyl β-D-cellodextrins by cutting glucose or cellobiose from the non-reducing end. Meanwhile, some larger-molecular-weight cellodextrins could be detected, indicating it also had transglycosylation activity. Without carbohydrate-binding module (CBM), CHU_2103 could bind to crystalline cellulose and acted processively on it. Site-directed mutation of CHU_2103 demonstrated that the conserved aromatic amino acid W197 in the catalytic domain was essential not only for its processive activity, but also its cellulose binding ability.

Cytophaga hutchinsonii is an aerobic cellulolytic gliding bacterium. The mechanism of its cell motility over surfaces without flagella and type IV pili is not known. In this study, mariner-based transposon mutagenesis was used to identify a new locus CHU_1797 essential for colony spreading on both hard and soft agar surfaces through gliding. CHU_1797 encodes a putative outer membrane protein of 348 amino acids with unknown function, and proteins which have high sequence similarity to CHU_1797 were widespread in the members of the phylum Bacteroidetes. The disruption of CHU_1797 suppressed spreading toward glucose on an agar surface, but had no significant effect on cellulose degradation for cells already in contact with cellulose. SEM observation showed that the mutant cells also regularly arranged on the surface of cellulose fiber similar with that of the wild type strain. These results indicated that the colony spreading ability on agar surfaces was not required for cellulose degradation by C. hutchinsonii. This was the first study focused on the relationship between cell motility and cellulose degradation of 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 a Gram-negative aerobic soil bacterium which can digest crystalline cellulose completely through a strategy different from that of the well-studied cellulolytic aerobic fungi and anaerobic bacteria. However, despite the availability of a published genome sequence, studies on this organism have been very limited because of the lack of a genetic manipulation system. This paper describes the development of replicative oriC plasmids, carrying the replication origin of the C. hutchinsonii chromosome, and an electroporation method for Escherichia coli-C. hutchinsonii shuttle vectors based on oriC plasmids with an efficiency of about 2 × 10(4) transformants per microgram plasmid DNA. Heterologous proteins, including green fluorescent protein and β-galactosidase, were expressed successfully and proved functional in C. hutchinsonii under the control of the CHU_1284 promoter in oriC plasmids. Finally, the gene CHU_0344, encoding the main extracellular protein, was targeted by homologous recombination based on the oriC plasmid. These genetic techniques and tools will provide a means to study the novel cellulose degradation system of C. hutchinsonii.

Objective: To study the morphological changes of Cytophaga hutchinsonii cell during its life circle., Methods: Cytophaga hutchinsonii cell was observed under light microscope, fluorescence microscope and scanning electron microscope. The nucleoids in the cell were stained with fluorescent dye Hoechst33342 and examined by fluorescence microscopy., Results: We discovered that under starvation conditions, the long, flexible rod cell of Cytophaga hutchinsonii would bend and turn into circular cell. The circular cell failed to produce carboxymethyl cellulase. Some of the circular cells might further wind around and turn into tiny spherical cells. The tiny spherical cell similar to the microcyst of sporocytophaga could germ into long flexible rods again under certain circumstances. When growing cultures to logarithmic phase of cell growth, Cytophaga hutchinsonii cell with three nucleoids in it was occasionally observed, which indicated that the two strands of DNA might act differently in the initiation of DNA replication., Conclusion: This is the first detailed description of the formation process of circular cell and tiny spherical cell in the life circle of Cytophaga hutchinsonii. The result will help to further reveal the relation between morphologic change and cellulose degradation ability of the strain.