Characterization Of Genes Relevant To Biosynthesis Of Inner Core Of Lipopolysaccharide In Cronobacter Sakazakii

Cronobacter sakazakii is a Gram-negative bacillus that is widespread in nature and causes blood flow and central nervous system diseases.It can cause illness in people with low immunity,such as the elderly and infants.Research showed that Cronobacter sakazakii had a high mortality rate.The lipopolysaccharide structure of Cronobacter sakazakii is mainly composed of three parts: lipid A,core polysaccharide and O-antigen,and is closely related to the pathogenicity of the strain.Compared with E.coli,the structure and regulation of lipopolysaccharide in Cronobacter sakazakii is not very clear and needs further investigation.In this study,we investigated some of the genes involved in the structure of the core polysaccharide in the lipopolysaccharide structure of C.sakazaki BAA-894.Through homologous comparison of the genes,SDS-PAGE and LC-MS analysis of the LPS structure of the mutants,the genes responsible for the biosynthesis of the core polysaccharides were identified and the structure of inner core of lipopolysaccharide in C.sakazakii was preliminarily explored.In addition,the effects of structural changes of lipopolysaccharide on the strains under different medium conditions were analyzed.The main research results are as follows:(1)The results of protein homology alignment showed that an operon containing four genes ESA_RS18945,ESA_RS18950,ESA_RS18955,and ESA_RS18960 is responsible for the biosynthesis of the inner core of lipopolysaccharide in C.sakazakii.The proteins encoded by these four genes are homologous to Waa Q,Waa C,Waa F,and Waa D in E.coli,responsible for L-glycero-D-mannoheptose addition on the inner core of lipopolysaccharide.In addition,no genes with high homology were found in the outer core polysaccharide,indicating that the outer core polysaccharides of C.sakazakii may be completely different from E.coli.(2)The structures of lipopolysaccharide and Kdo2-lipid A extracted from mutant strains?RS18945,?RS18950,?RS18955 and ?RS18960 were analyzed by SDS-PAGE and LC-MS,respectively.The results indicated that ?RS18945 synthesized lipopolysaccharide with similar length to the wild type BAA-894,suggesting that the enzyme encoded by ESA_RS18945 functions on the side chain of lipopolysaccharide and have similar function to E.coli Waa Q.?RS18950 and ?RS18960 only synthesized Kdo2-lipid A,confirming that enzymes encoded by ESA_RS18950 and ESA_RS18960 have similar functions to E.coli Waa C and Waa D,respectively.Hep-Kdo2-lipid A with a phosphoethanolamine was produced in ?RS18955,suggesting that the enzyme encoded by ESA_RS18955 has similar function to E.coli Waa F.(3)The mutant strains of C.sakazakii with truncated lipopolysaccharide structure secreted a large amount of extracellular polysaccharides in M9 medium,and E.coli with intact lipopolysaccharide structure could also secrete a large amount of extracellular polysaccharides in M9 medium.The results showed that the nutrient deficient growth environment and the changes of lipopolysaccharide on the main chain structure would activate the strains to secrete extracellular polysaccharides.It indicated that the secretion of extracellular polysaccharides was regulated by complex internal regulation.(4)Compared with wild type C.sakazakii BAA-894,the mutant strains ?RS18950,?RS18955 and ?RS18960 with truncated lipopolysaccharide had great changes in cell growth rate and cell morphology.The truncation of lipopolysaccharide structure also had an important effect on the outer membrane of the strain,including improving the hydrophobicity of the cell,enhancing the autoaggregation of the strain,increasing the permeability of the outer membrane and reducing the desiccation tolerance of the strain.In addition,it could enhance the sensitivity to hydrophobic antibiotics and reduce the sensitivity to hydrophilic macromolecular antibiotics.