A Preliminary Study On The Factors Affecting Adhesion Of Cytophaga Hutchinsonii To Microcrystalline Cellulose And On Cellulose Binding Proteins

Cytophaga hutchinsonii is a widespread aerobic cellulose-degrading bacterium which can degrade crystalline cellulose thoroughly and rapidly.Cytophaga hutchinsonii exhibits a specific cellulose degrading mechanism different from the classic mechanisms reported previously in fungi or anaerobic bacteria. To digest cellulose, Cytophaga hutchinsonii requires the direct contact with the substrate. It does not produce cellulosomes on cell surface or secrete extracellular cellulolytic enzymes. Most of the cellulases encoded by Cytophaga hutchinsonii contain neither carbohydrate-binding modules (CBMs) nor docking domain-like structures. All these characteristics show that cellulose degradation in Cytophaga hutchinsonii is unique and distinctive. Up to now,research on the mechanism of cellulose degradation in Cytophaga hutchinsonii remains to be blank at home and abroad.To uncover the mechanism of cellulose adhesion in Cytophaga hutchinsonii, a preliminary study on factors influencing cellulose adhesion was applied, such as metabolism activity of bacterium, membrane proteins or polysaccharides, and cellulose analogs.We also investigate the specificity of bacterial adhesion as well as the main constituent involved in the adhesion process.The research results showed that the adhesion between Cytophaga hutchinsonii and crystalline cellulose is highly specific. The adhesion is not affected by bacterial metabolism activity and competitive inhibition ofcellobiose and sodium carboxymethyl cellulose as well. The progress is closely related to cell surface proteins, but little affected by cell surface polysaccharides. Specific proteins for crystalline cellulose binding were supposed to exist on cell surface. Based on these results, further study on cell surface proteins related to adhesion is applied.Because of the important role of proteins play in the adhesion progress, we searched for the cellulose binding proteins (CBPs) of Cytophaga hutchinsonii by direct isolation and identification. To study the mechanism of Cytophaga hutchinsonii adhered to cellulose, the identification of these proteins has a very important significance. Nature membrane proteins which binding to cellulose were isolated by SDS-PAGE, and identified by MALDI-TOF mass spectrometry.13 CBPs were obtained together, contains three gliding motility-related proteins, five hypothetical proteins, an outer membrane lipoprotein, aβ-glucosidase, an ATP synthase, a heat shock protein GroEL, and a peptidyl-prolyl cis/trans isomerase. Bioinformatics analyses were carried out, such as prediction of their potential signal peptides and trans-membrane domains.To study the function of cellulose adhesion proteins, we firstly tried to express these proteins inE.coli but hardto acquire active expression. So we considered building the expression system of Cytophaga hutchinsonii for isolation and purification of CBPs and for depth research.The genetic manipulation system is still incomplete in Cytophaga hutchinsonii, so we build the expression system based on self-replicated plasmids that laboratory possessed.Erythrocin resistance gene was used as selective marker gene, and the green fluorescent protein orβ-galactosidase was used as reporter gene in this plasmid. Results showed that it can replicate and express in Cytophaga hutchinsonii. At the same time, the activities of some promoters have been compared, and find out that the promoter of CHU₁284 exhibits the maximal activity.In this paper, we started the research by comparing the cellulose adhesion influenced by different treats;thirteen three cellulose binding proteins were isolated which play an important role in binding process. The expression system ofCytophaga hutchinsonii was built successfully as an important supplement of genetic manipulation system and provides experiment basis for the further study of cellulose adhesion and degradation in Cytophaga hutchinsonii.