Functional Research On CHU₀344Protein And CHU₀099Protein In Cytophaga Hutchinsonii

Energy, resources, and environmental problems are the most difficult challenges facing the development of human survival in the21st century, are also the main factors restricting China’s social and economic development. In the long run, the oil resources will move rapidly to the brink of depletion in the first half of this century and the task of the development of sustainable use of alternative resources are very tight. Biomass （biomass energy）, in the form of solar energy to chemical energy in the form stored in the biomass energy, directly or indirectly derived from the photosynthesis of green plants can be converted to conventionalsolid, liquid and gaseous inexhaustible fuels.It is a renewable energy source and the only renewable carbon source on earth. Cellulose is the main part of biomass resources （accounting for more than80%of the total） with low price and an adequate supply. But till now it has not yet been fully exploited.Cytophaga hutchinsonii is a Gram-negative bacterium belonging to the Cytophaga-Flavobacterium-Bacteroides group which is widely present in nature. C. hutchinsonii could degrade crystalline cellulose rapidly and completely. However, its genome sequence does not code for any homologues of either processive cellulases, including exo-cellulases and processive endo-cellulases, or dockerin and cohesion which are important components of cellulosome. So, it utilizes an unknown strategy to degrade crystalline celluloses, which is different from both the free cellulases mechanism used by most aerobic microorganisms and the cellulosome mechanism used by most anaerobic bacteria. This unknown mechanism may be helpful to improve the efficiency from crystalline cellulose into biofuel.In this thesis, we focus on the extracellular secretion protein （CHU₀344） and the resuscitation promoting factor （CHU0099） in C. hutchinsonii. We preliminary research on the biological function of CHU₀344protein and CHU₀099protein, in order to further reveals the unique extracellular secretion protein of C. hutchinsonii and the physiological characteristics of the mechanism.The CHU₀344protein is the main secretion protein of C. hutchinsonii which has the ability of adhesion to the microcrystalline cellulose. Bioinformatic analysis shows that there is no signal peptide but a CPD-like C-terminal domain in chu₀344. It is possible that CHU0344protein can be transport into the extracellular through a novel protein transport system named SSpor. Using protein expression and purification technology we collect quantity more than high-purity CHU₀344protein. Comparing the nature of the fusion protein and native protein and the results of the characteristics of natural protein and fusion protein with the combination of microcrystalline cellulose, the structure of the two proteins are quite different. CHU₀344natural protein is a nucleic acid binding protein and the protein thiol exists as the free sulfhydryl state with no significant disulfide bond strucrure.The Western blot results showed that CHU0344is mainly localized in the cell surface or secreted into the extracellular. Heterologous expression of the fusion protein does not combine with nucleic acid but disulfide structure is formed. These results show that the maturation process of CHU₀344in C. hutchinsonii and E. coli are significantly different.The basis for further study of C. hutchinsonii new extracellular protein secretion mechanism is also provided. CHU₀344mutant is successfully constructed by the gene knockout technology and the physiological growth experiments results show that mutant decrease the exercise capacity in solid media.In decline phase the C. hutchinsonii cells morphology changes significantly, the activity also decreases, suggesting that there may be resting body in special phase. Biomformatics analysis shows that there are some certain similar sequences between CHU₀099and resuscitation promoting factor of Salmonella. Homology modeling shows the CHU₀099protein tertiary structure has a high degree of similarity with the Rpf protein of Salmonella.It is speculated that CHU₀099is a kind of resuscitation promoting factor. Using protein expression and purification technology we collect quantity more than high-purity CHU₀099protein and results show that CHU₀099recombinant protein can promote the growth of the wild strains of C. hutchinsonii recovery from senescent cells. This class Rpf protein is found in the in Bacteroides for the first time.More and more details of degradation of cellulose by C. hutchinsonii are understood through the primary researches in microbiology and biochemistry. These researches would be of great help for the future study.