Construction And Analysis Of Lipopolysaccharide Synthesis Gene Deletion Mutant Of Strain B29

The functions of lipopolysaccharide (LPS) in some gram-negativebacteria have been reported in the literatures, But there is no suchresearch on bacteria from the genus of Enterobacter. This study attemptsto construct the waaL and waaG deletion mutants of Enterobactercloacae B29, which was isolated from the fecal sample of a severe obeseman. To understand the differences of LPS structure and activity betweenmutant strain and wild strain. According to principle of homologousrecombination,We obtained the upstream and downstream sequences ofwaaL and waaG gene by PCR using chromosomal DNA of B29astemplate and constructured the knock-out vectors pKNG101△waaL andpKNG101△waaG by inserting the connected fragments into the wide hostsuicide plasmids. Then we transfered the knock-out vectors into E.coliSM10by electroporation and co-cultured it with B29. Antibiotics and sucrose pressure culture were applied for waaL and waaG gene mutantstrain selection. We verified the mutant strains by multiple PCR,ERIC-PCR, PCR-DGGE and DNA sequencing methods and comparedthe differences of biological characteristics such as lipopolysaccharidestructure, endotoxin activity, growth rate between the parent strain B29and two knock-out mutant strains. B29△waaL and B29△waaG weresuccessfully constructured. The result of silver-stained polyacrylamidegels showed that the lipopolysaccharide chemotypes of B29wassmooth-type structure, while the size of LPS in mutant strains wereobviously smaller than wild-type strain, especially in B29△waaG. TheLAL tests indicated that the endotoxin activity of two mutant strains werelower than the wild type strain. The strain B29△waaG has a lower growthrate when compared with the wild type B29from the growth curvedetermination. The constructured waaL and waaG gene deletion mutantswhich have changed LPS structure and decreased endotoxin activity willfaciliate further studies on what functions B29LPS should have ininducing the obesity and insulin resistance of germ-free mice or humanbeings.