Regulatory Mechanism Of Lipopolysaccharide On Colanic Acid Biosynthesis In Escherichia Coli

Lipopolysaccharide(LPS)and Colanic acid(CA)are both cellular polysaccharides.In E.coli,Lipopolysaccharide is the main component of cell membrane and CA is an anionic heteropolysaccharide secreted to the outside of cells.The previous study has confirmed that respective deletion of some single genes in waa gene cluster in E.coli W3110 could induction the secretion of CA.This study further explored the influence of different core polysaccharide structures on the synthesis of CA under different nutritional conditions,and preliminarily proposed the regulation mechanism of polysaccharide in E.coli cells.The main conclusions are as follows:(1)When the wild type MG1655 and other 9 mutant strains with defects in LPS core biosynthesis were grown in the glucose-rich M9 or LBG medium at 30℃,ΔF,ΔP,ΔG,Δ(L-Q)ΔF,Δ(L-Q)and Δ(L-G)could produce CA on different levels and Δ(L-Q)and Δ(L-G)produced the most amount of CA.But MG1655,Δ(D-Q),Δ(F-Q)and Δ(C-Q)cells didn’t produce CA significantly.When the 10 strains were grown in LB medium at 30℃,none of them produced visible CA.The above experiments show that the synthesis of CA in E.coli is closely related to the structure of LPS and glucose availability.(2)MG1655,ΔF,ΔP,Δ(D-Q),Δ(F-Q),Δ(C-Q),Δ(L-Q)ΔF and Δ(L-Q)cells were grown to the mid-log phase and cultured in M9 or LB medium,using MG1655 as a control.The transcriptome result showed that compared with MG1655,the genes of wca gene cluster and the key regulted gene rcsA in LPS mutant strains were transcriptionally up-regulated no matter grown in M9 or LB medium,but only some of these strains grown in M9 produced significant amount of CA,there must be reasons why CA were produced in these strains other than the expression of the genes relavant to CA production.(3)The precursor sugars for CA synthesis are all derived from glucose 6-phosphate.In the glucose-rich M9 or LBG medium,the intracellular glucose 6-phosphate level of Δ(L-Q)was significantly higher than that of wild-type strain MG1655.Replacing glucose in M9 medium with glycerol as the sole carbon source,the relevant mutant strains reduced the ability to synthesize CA.Combined with the different core polysaccharide structures of the mutant strains,CA production may be relavant to the accumulation of the common precursors of LPS and CA biosynthesis.(4)Deletion of genes relavant to LPS synthesis not only activates the CA production,but also effects the membrane structures.Transcriptome analysis confirmed that the changes of LPS structures in E.coli would affect the synthesis and assembly of bacterial flagella.What’smore,the defects of LPS increased the sensitivity of E.coli cells to four hydrophobic antibiotics: erythromycin,neomycin,clindamycin and rifampicin.And the sensitivity to antibiotics usually increases gradually with the truncation of the core polysaccharide chain.Based on above results,CA biosynthesis in E.coli is dependent on lipopolysaccharide structure and glucose availability.Which may be related to the accumulation and utilization of LPS and CA common precursors.This study analyzed the factors affecting the synthesis of CA in E.coli MG1655 from its LPS and external nutritional conditions,and also provided a basis for exploring the mutual regulation between polysaccharides in different environments.