Research On CHU₀344Protein Secretion Mechanism And CHU₁796Function In Cytophaga Hutchinsonii

The most serious challenges hunman facing for survival in the21st century are the energy crisis and environmental pollution problems. To explore sustainable green energy to replace oil and other fossil resources has become urgent need. Biomass is the most abundant renewable resource, and its main ingredient is lignocellulose. People have paid more and more attention to the utilization of lignocellulose.To reveal the efficient cellulose degradation mechanisms in the nature can help us to better utilize cellulose resources.Cytophaga hutchinsonii is a Gram-negative aerobic bacterium. It could degrade crystalline cellulose rapidly and completely by using a novel strategy different from （?） most aerobic fungi and anaerobic bacteria. It dose not secrete soluble extracellular cellulolytic enzymes and has no cellulosome-like structure. A novel protein secretion system por secretion system （PorSS） which transport main extracellular proteases across the outer membrane has been researched in Porphyromonas gingivalis. Bioinformatics analysis showed that these por genes also exist in C.hutchinsonii, but the functions are unknown. Besides, the mechanism of its cell motility over surfaces （?） without flagella and pili is not known. In this theis, we explored the secretion mechanism of CHU₀344protein, and secretion of CHU₀344influenced by two por homologue genes CHU₀170（porP） and CHU₃237（porU） was also studied. Besides, CHU₁796involved in cellulose degradation and gliding was further studied.CHU₀344, the main extracellular secretion protein, has specific ability of adhesion to cellulose. Bioinformatics analysis showed it had a CTD structure in C-terminal. It is possible that CHU₀344protein can be transport through por secretion system （PorSS）. In our reseach, we found CHU₀344protein was located on outer membrane, as well as secreted into extracellular. The result of western blot for extracellular proteins showed that CHU₀344protein had two bands of different molecular weight which were between60-80kDa and approximately40kDa, respectively. There were two expression up-regulated proteins:CHU₀878and CHU₁608in mutant△0344strain. These two proteins had unknown function but both had CTD structure. We speculated that the two proteins may be compensation for CHU₀344protein. In order to verify the function of the CTD, we knocked out por-secre-tail of C-terminal in CHU₀344, and named the mutant strain△0344-pst. The western blot result of extracellular proteins showed that CHU₀344protein bands were disappeared in△0344-pst strain. It is seemed that the structure of the CTD had a certain influence on CHU₀344protein secretion.The complete set of por genes existed in C.hutchinsonii, but the functions were still unknown. The influence on CHU₀344protein secretion of the two homologous genes CHU₀170（porP） and CHU₃237（porU） was studied to better explore the secretion mechanism of CHU₀344protein.The western blot experiment of extracellular proteins in CHU₀170mutant strain （△0170） showed CHU₀170affected the secretion of CHU₀344protein in some extent. The research results of△3237mutant strain displayed that CHU₀344protein with the two different molecular weight bands disappeared in△3237strains. However, we found a band whose molecular weight was over90kDa in the extracellular protein of△3237.It was probably the original CHU₀344protein with CTD uncut, which suggested CHU₃237can cut down the CTD of CHU₀344protein. Mass spectrum identification of extracellular proteins showed lots of proteins with putative CTDs were absent from the△3237mutant. Therefore, CHU₃237affected the secretion of CHU₀344protein, as well as other proteins transported by PorSS.Sequence alignment displayed that CHU₁796had high sequence similarity with proteins containing OmpA/MotB domains, while MotB domains existed in flagella. We hypothesized that CHU1796was associated with gliding in C.hutchinsonii. For research the function of CHU₁796, we got the mutant of CHU₁796（△1796） and conducted a series of phenotypic experients. The results revealed that there was no difference between wild type and mutant strain in growth and filter paper degradation. However, diffusion capacity on the surface of the hard and soft agar, as well as gliding ability on the surface of the glass, was certain affected in mutant strain. But a single gene CHU₁796influenced the gliding of C.hutchinsonii. Further research may be better to reveal the movement mechanism of C.hutchinsonii, so as to better understand the relationship between the gliding and cellulose degradation.