The Role Of CHU₁798in Gliding And Cellulose Degradation Of Cytophaga Hutchinsonii

Cytophaga hutchinsonii is an abundant bacterium that can degrade crystalline cellulose efficiently by a novel unknown mechanism. C. hutchinsonii cells glide rapidly over glass surfaces without flagellum and type IV pili, and till now its mechanism of gliding is not clear. C. hutcinsonii can rapidly digest crystalline cellulose by a novel mechanism, significantly different from most aerobic fungi and anaerobic bacteria. It doec not secrete soluble extracellular cellulolytic enzymes and has no cellulolsme-like structures. C. hutchinsonii cells arrange themselves regularly on cellulose fiber surfaces when they perform the digestion, which indicates cells motility might facilitate cellulose degradation by C. hutchinsonii. The study of the novel mechanism of crystalline cellulose degradation by C. hutchinsonii would improve the understanding of the microbial cellulose utilization strategies. Recently, some genetic manipulation tools of C. hutchinsonii have been gradually established, and these tools provide us an opputunity to study the machanism of gliding and crystalline cellulose degradation by C. hutchinsonii. In this study, three genes related to cells gliding motility were found by Tn4351transposon mutagenesis, and CHU₁798was further studied. The relationship between cell motility and cellulose degradation was also studied.1. Establishment of Tn4351transposon libiary and screening of gliding-related genesTn4351transposon libiary was established by conjugation, and700of the tranoposon mutants were screened based on the characteristics of cells gliding motility and cellolose degradation by C. hutchinsonii. Finally, three Tn4351transposon mutants that lost gliding motility on soft agar were obtained. The inseretion site of Tn4351transoposon was identified by inverse PCR, and three focuses（CHU₁798, CHU₃693, and CHU₃620） were validated. The deleted mutants with inactivated CHU₁798, and CHU₃693were obtained by gene deletion, respectively.2. Funtional study of CHU₁798Tn4351transposon mutagenesis and gene targeting were used to validate a new gene CHU₁798required for individual cell movement over glass surfaces and colony spreading on agar surfaces through gliding motility. CHU1798encodes a hypothetical protein with a CHU_C domain of unknown function. An alignment of C. hutchinsonii CHU1798showed that three bacteria in the phylum Bacteroidetes have its homologs with the sequence identity more than30%and all the proteins had the CHU C domain. These bacteria including C. aurantiaca, S. koreensis and N. koreensis also have homologs of most of the Gld and Spr genes involved in gliding motility of F. johnsoniae, although S. koreensis was previously described as nonmotile. We speculated that these homologous genes of CHU₁798might also play a role in gliding motility of some other members of Bacteroidetes. F. johnsoniae, as a model strain to study gliding motility mechanism in phylum Bacteroidetes, exhibits colony spreading on agar surfaces and cells movement over glass surfaces. Nineteen Gld and Spr proteins that are related to gliding motility have been identified. All the gliding genes have their homologous genes in the genome of C. hutchinsonii. However, till now only one of the homologous gliding proteins （sprP） which was speculated to play a role in the type Ⅸ protein secretion system of the bacterium was proved to play a crucial role in gliding and cellulose degradation of C. hutchinsonii. SprB is the essential adhesion along the cell surface of F. johnsoniae that can be propelled by motor to motivate gliding. But according to the unpublished data in our lab, the homologous SprB didn’t have any effect on gliding motility of colony spreading on agar and individual cells over glass surfaces of C. hutchinsonii. Other reported gliding related genes of C. hutchinsonii such as CHU₀134, CHU₁797and CHU1798in this study have no homologous gene in F. johnsoniae. Besides this, no motility organelle similar to short fibrils of F. johnsoniae has been observed on the cell surface of C. hutchinsonii. These results indicated that C. hutchinsonii might have different gliding machines from F. johnsoniae.Our study showed that the mutant with a disruption of CHU₁798lost the ability to spread on agar surfaces and glide over glass surfaces, but it could still degrade cellulose in liquid culture and on filter paper plate. However, it could not degrade cellulose on Avicel PH101plate. Under the previous condition, bacterial cells were in direct contact with the cellulose. While under the later condition with the cellulose powder buried in the agar, bacterial cells had to moved into the agar to get to contact with the cellulose. These results suggested that gliding motility would assist the cells to approach to the nutrients like cellulose, but was not required for cellulose depolymerization once the cells has got to contact with the substrate.The observation by scanning electron microscopy showed that at the early stage of incubation the mutant cells settled ramdomly on the surface of cellulose fiber which might be caused by the detect of single cells’motility, while the wild type cells arranged themselves regularly along the long axis of the fiber. We speculated that gliding motility could help the cells to move along the fiber to look for suitble place to settel down. But after36hours’ incubation, there were plenty of cells grown on the fiber surfaces. The mutant cells arranged themselves side by side regularly as the same as the wild type cells, indicating that the intereaction between the cells to form parallel arrangement was not affected by the single cells’motility.