Study Of Lipid A Structure And Related Genes In Vibrio Parahaemolyticus

Lipid A,also known as endotoxin,is the main component of lipopolysaccharide（LPS）,the outer membrane of Gram-negative bacteria.Lipid A is very important for bacteria’s pathogenic ability and bacteria’s resistance to the external environment.Vibrio parahaemolyticus is a conditional pathogen that exists in seafood and other aquatic products.In recent years,large-scale poisoning incidents caused by V.parahaemolyticus are not uncommon.As a pathogen,the structure and biosynthesis of Lipid A in Vibrio parahaemolyticus have not been studied yet.This paper studies the genes elated to Lipid A biosynthesisin V.parahaemolyticus ATCC33846.The functions of related genes were identified,and the effects of structural changes of Lipid A on the growth status,drug resistance,and permeability of V.parahaemolyticus were studied.The main conclusions are as follows:（1）Through BLAST,it is found that there are four genes that may be related to secondary acylation during Lipid A biosynthesisin V.parahaemolyticus ATCC33846:VP＿RS01045,VP＿RS00880,VP＿RS08405 and VP＿RS12170.（2）E.coli MLK1067 produces the penta-acylated Lipid A lacking the secondary acylation at the C3’position.When the four genes were overexpressed in E.coli MLK1067,a C_12:0 secondary acyl chain was added at the C3’position of Lipid A only in E.coli overexpressing VP＿RS01045,but not VP＿RS00880,VP＿RS08405 or VP＿RS12170.This result indicates that the enzyme encoded by VP＿RS01045 can catalyze the addition of C_12:0 at the C3’position of Lipid A.（3）E.coli MKV15b produces Lipid IV_A,which lacks secondary fatty acid chains at the C2’and C3’positions.When the four genes were overexpressed in E.coli MKV15b,a C_12:0secondary acyl chain was again added at the C3’position in E.coli overexpressing VP＿RS01045,but a C_14:0 secondary acyl chain was added at the C2’positionof Lipid A in E.coli overexpressing VP＿RS00880,VP＿RS08405 or VP＿RS12170.This result indicates that the enzymes encoded by VP＿RS00880,VP＿RS08405 and VP＿RS12170 are also Lipid A secondary acyltransferases,which can catalyze the addition of C_14:0 at the C2’position of Lipid A.（4）Knocked out VP＿RS01045,VP＿RS00880,VP＿RS08405 and VP＿RS12170 in V.parahaemolyticus ATCC33846 respectively,to construct mutant strains VPW001,VPW002,VPW003 and VPW004.By analyzing the Lipid A structure and gene transcription level of the mutant strains,we found that these four genes may have complementary functions in V.parahaemolyticus.The deletion of a single gene has little effect on the structure of Lipid A.（5）The five strains of V.parahaemolyticus ATCC33846,VPW001,VPW002,VPW003and VPW004 were compared in different environments.It was found that the deletion of the VP＿RS08405 gene caused the bacteria to grow slowly at 37℃.After the deletion of VP＿RS01045 or VP＿RS08405,bacteria can grow under p H 5.0 codition.After the deletion of VP＿RS01045,the MIC value of polymyxin B decreased from 50μg·m L^-1 of wild type to 25μg·m L^-1;the MIC value of polymyxin E decreased from 100μg·m L^-1 of wild type to 50μg·m L^-1.The research results of this paper provide a basis for us to further study the structure and biosynthesis of Lipid A in V.parahaemolyticus.