The Rearch Of KfiA Gene Which Synthesis EcN Capsular Polysaccharide

E.coli Nissle 1917(EcN)is a kind of intestinal probiotics and its serotype is O6: K5:H1.In the intestinal tract,EcN reduce pathogen colonization by extracellular capsule polysaccharide.In addition,the main component of capsular polysaccharide is heparosan that is anticoagulant drugs.Therefore,it is vital significance to research on the synthesis way of capsular polysaccharide in the EcN.We mainly research the capsular polysaccharide synthesis approach of EcN strains and analyze the biosynthesis of capsular polysaccharide mechanism by molecular biology techniques.We detect capsular polysaccharide which is extracted,purified in EcN by 1H NMR,and determine the chemical shift of characteristic peak is about 2ppm.Next,we successfully construct kfi A mutant strains by λ-red reorganization system,and test the growth curve of mutant strains,finding that lack of kfiA gene does not significantly affect the growth of bacteria.Testing the capsular polysaccharide of EcN and EcNΔkfiA by 1H NMR,we find kfi A mutant strains can’t synthesize capsular polysaccharide.In order to further verify the conclusion,we complement kfi A gene in its mutation.We use NMR to detect the capsular polysaccharide in EcN,EcNΔkfiA,EcNΔkfiA IntΦ80::pAH162-Kps MP-kfiA and EcNΔkfiA IntΦ80::pAH162,finding that EcN strains can synthesize capsular polysaccharide only in the presence of kfiA.