Functional Study Of Genes In Cellulose Degradation And Gliding Of Cytophaga Hutchinsonii

Since the beginning of twenty-first century, the problems of energy crisis and environmental pollution are increasingly serious and seriously threaten human’s health and sustainable development. Exploration sustainable green energy to replace oil, coal and other fossil resources has become the urgent need. Lignocellulose, the main component of plant biomass, is the most abundant renewable resource on the earth. More and more attention was paid to the utilization of lignocellulose. However, cellulose is difficult for enzymatic degradation due to its compact structure and the presence of crystalline region. At present, low efficiency and high cost are general problem in cellulose bioconversion. Exploitation the efficient cellulose degradation mechanisms in the nature to resolve the efficient degradation of crystalline cellulose is the key of the lignocellulose utilization.Cytophaga hutchinsonii is an abundant aerobic cellulolytic bacterium that can rapidly digest crystalline cellulose using a novel strategy different from most aerobic fungi and anaerobic bacteria. It does not secrete soluble extracellular cellulolytic enzymes and has no cellulosome-like structure. Besides, the mechanism of its cell motility over surfaces without flagella and type IV pili is not known. C. hutchinsonii cells align themselves along cellulose fiber regularly when they digest them suggesting cell motility might facilitate cellulose digestion. The study of the novel mechanism of crystalline cellulose degradation by C. hutchinsonii would improve the understanding of the microbial cellulose utilization strategies, and also conducive to take advantage of cellulosic biomass. However, due to the lack of genetic manipulation tools, few study focused on the cellulose degradation and gliding mechanisms of C. hutchinsonii. In this study, gene functional study in C. hutchinsonii through transposon mutagenesis and complementation was first developed, and these genetic techniques provided an opportunity to understand the details of the novel cellulose degradation and gliding mechanisms of C. hutchinsonii. Several functional genes involved in cellulose degradation and gliding were further studied, and the utilization of cellulosic substrates of C. hutchinsonii was also discussed.1. Development of transposon mutagenesis and complementationTransposon mutagenesis is effective to study gene function. In this study, two kinds of transposons, Tn4351and HimarEm3, were translocated into C. hutchinsonii through conjugation. HimarEm3was derived from HimarEm1. The transformation frequency would be increased with addition of20μg/ml kanamycin in conjugation medium, and transformation frequency of3.0×10-7or1.2×10-6was finally achieved for Tn4351or HimarEm3, respectively. Complicated insertion of Tn4351in F. johnsoniae was reported. The insertion of HimarEm3in C. hutchinsonii was studied by southern blot, and the results showed that most transformants had a single insertion in the genome. This demonstrated that transposon HimarEm3was an beneficial and convenient tool to study gene function in C. hutchinsonii.The erythromycin resistance gene was the only reported selective marker gene used in C. hutchinsonii, which was an limitation for further genetic manipulation in C. hutchinsonii. In order to find more selective genes functional in C. hutchinsonii, several antibiotic resistance genes were tried to express in C. hutchinsonii. The results showed that cfxA and tetO were functional in C. hutchinsonii. The complemented plasmids carrying cfxA or tetQ for erythromycin insertion were constructed, and gene functional study through transposon mutagenesis and complementation was available.2. Functional study of CHU₀134Direct contact with the cellulose was necessary for effective cellulose degradation by C. hutchinsonii. However, genomic analysis showed that C. hutchinsonii does not possess cellulosome, and most of the encoded cellulase has no carbohydrate-binding module （CBM）. The mutant A-4with significantly reduced cellulose binding ability was constructed through transposon mutagenesis The relative adhesion rate of the mutant was only a half of that of the wild-type strain. The mutant A-4was deficient in cellulose degradation and colony spreading on agar. Cellulase assay showed that the endocellulase activity of the cell-free supernatants and on the intact cell surface of A-4decreased by40%. Sodium dodecyl sulfate polyacrylamide gel electrophoresis （SDS-PAGE） of proteins binding to cellulose in the outer membrane showed that most of them were significantly decreased or disappeared in A-4including some Gld proteins （CHU₀171,0173,0174） and hypothetical proteins （CHU₃654,1277,3655）. It was speculated that these proteins might play an important role in cellulose binding and degradation by C. hutchinsonii.The site of HimarEm3insertion of A-4was analyzed by Southern blot and inverse PCR, and the results showed that the transposon was inserted in gene CHU₀134. The CHU₀134complement strain could restore cell motility, cellulose adhesion and degradation to nearly the level of the wild-type strain indicating that these phenotypic defects of the A-4was caused by mutation in CHU₀134. CHU₀134is annotated to encode a thiol-disulfide isomerase since there was a putative TlpA-like family/thioredoxin （TRX）-like super-family domain containing a Cys-X-X-Cys motif （C369-G-H-C372） in its C-terminal. Thiol-disulfide isomerase can catalyze the isomerization of disulfide bonds in protein which are important for the structure and stability of many extra-cytoplasmic proteins. Sequence analysis of proteins with a sequence similar to CHU0134showed that the most of these proteins belong to bacteria in the phylum Bacteroidetes with nearly the same conserved motif C-G-H-C, and the function of these proteins needs further study.3. Functional study of CHU₁277CHU₁277encoded a cellulose binding outer membrane protein, and inactived by insertional mutagenesis. The SDS-PAGE of cellulose binding outer membrane protein revealed that CHU₁277was absent in the CHU₁277disrupted mutant, indicating that CHU₁277was inactived. The result of RT-PCR showed that gene transcription of the adjacent genes of CHU₁277was not affected. The disruption of CHU₁277caused drastic effect on the growth parameters on cellulosic substrate. The mutant failed to digest cellulose and its oligosaccharides utilization ability was significantly reduced, indicating that CHU₁277was essential for cellulose degradation and played an important role in cellooligosaccharide utilization by C. hutchinsonii.Cellulase assay showed that endocellulase activity of the mutant was almost the same as that of the wild-type strain, while β-glucosidase activity on the cell surface was decreased by30%. The cellobiose hydrolytic activity of the mutant cells was significant reduced according to the analysis of the cellobiose hydrolysis of the intact cells, which was consistent with the poor growth of the mutant in the cellobiose medium. However, the outer membrane proteins extracted from the mutant cells exhibited similar β-glucosidase activity and cellobiose hydrolytic activity to that of the wild-type strain in vitro. These results implied that the quantity and activity of β-glucosidase on the mutant cell surface was actually unreduced, and low activity was exhibited on the cell surface may be caused by the disruption of CHU1277. We speculated that the direct or indirect interaction with CHU₁277might be necessary for the cell surface β-glucosidases to achieve sufficient activity.The study of cellulase activity and cellulose hydrolytic activity of the outer membrane proteins suggested that the cellulolytic enzymes of the mutant were almost integral. This implied that cellulolytic enzymes were not sufficient for cellulose utilization by C. hutchinsonii. Some other non-enzymatic proteins were necessary for effective cellulose degradation. CHU₁277was the first cell-surface protein proved to be essential for cellulose degradation by C. hutchinsonii, and further study of the function of CHU₁277in cellulose degradation would help to clarify the novel mechanism of cellulose degradation by C. hutchinsonii.4. Cellobiose utilization of C. hutchinsoniiCellobiose is one of the few substrates that can be used by C. hutchinsonii as the sole carbon and energy source, which is also the main intermediate product of cellulose degradation. The cellobiose utilization of C. hutchinsonii was first studied. Most of the β-glucosidase activity was located on the cell surface of C. hutchinsonii from the enzyme assay, although the genomic analysis showed that all the candidate β-glucosidases were predicted to be located in the periplasmic space. Native PAGE proved there was an active β-glucosidase band in the sample of outer membrane proteins of C. hutchinsonii, and significant cellobiohydrolase activity was detected on the cell surface and in the outer membrane proteins of C. hutchinsonii.After inoculation of C. hutchinsonii in cellobiose medium, cellobiose was gradually hydrolyzed and glucose was accumulated. Cellobiose in the culture was almost completely hydrolyzed before the mid-exponential phase, and glucose was accumulated to a high concentration. Then, glucose was the sole carbon source to provide further growth of the bacterium. This indicated that cellobiose was hydrolyzed into glucose by β-glucosidases on the cell surface was the main way of cellobiose utilization by C. hutchinsonii.5. Cellulose degradation on the cell surface of C. hutchinsoniiWe first detected that cellulose could be hydrolyzed to glucose on the cell surface of C. hutchinsonii through the analysis of cellulose hydrolyzate by intact cells. With addition of glucono-δ-lactone or NaN3to repress β-glucosidase activity or inhibit energy production through the respiratory chain reaction, we found the process of cellulose degradation on the cell surface of C. hutchinsonii. Cellulose was first hydrolyzed to cellooligosaccharides by cell-surface endocellulases, and cellooligosaccharides could be further hydrolyzed to glucose by cell-surface glucosidases. The extracted outer membrane proteins containing free cellulases were also proved to have ability to hydrolyze cellulose in vitro with glucose as the only product, indicating that cellulose would be hydrolyzed to glucose with the role of endocellulases and β-glucosidases in the outer membrane.Cellulose degradation on the cell surface was different from the possible model proposed by wilson that individual cellulose molecules were removed from cellulose fibers by an outer membrane protein complex and transported into the periplasmic space, then degraded by endoglucanases there. Transient accumulation of cellooligosaccharides was detected in cellulose degradation by intact cells leading to the doubt whether it is the main way of cellulose utilization of C. hutchinsonii. Nevertheless, cellulose could be hydrolyzed on the cell surface and cellulosic hydrolyzate also could be assimilated by the cells of C. hutchinsonii. Assimilation and translocation of cellulose hydrolyzate of the C. hutchinsonii cells need to be further studied.6. Functional study of CHU₁797Transposon mutagenesis and complementation were used to identify a new locus, CHU₁797, essential for colony spreading on agar surfaces. CHU₁797encodes a putative outer-membrane protein of348amino acids with unknown function. Proteins which have high sequence similarity to CHU₁797were widespread in the members of the phylum Bacteroidetes, almost had a DUF3308domain and located on the membrane from the protein subcellular localization prediction. These proteins might be related to gliding motility, and this speculation needs more experimental evidence.The disruption of CHU₁797suppressed spreading toward glucose on an agar surface, but had no significant effect on cellulose degradation for cells already in contact with cellulose. The gliding of the mutant cells on the glass surface was not affected, and the mutant cells also regularly arranged on the surface of cellulose fiber similar with that of the wild-type strain from the SEM observation. These results indicated that the colony spreading ability on agar surfaces was not required for gliding of the C. hutchinsonii cells on the glass surface and cellulose degradation. This implied that the mechanism of spreading and orderly arrangement of cells on the surface of cellulose fiber was different from the mechanism underlying colony spreading over agar surfaces, but may be related to the motility of individual cells over wet glass surfaces.