| Vibrio alginolyticus,a conditional pathogen,which mainly distributed in marine environment and marine organisms.In China,vibriosis is mainly controlled by chemical drugs and antibiotics,as a result,the abuse of drugs in aquaculture conduced the obvious enhancement of bacterial drug resistance.N-acetylglucosamine-1-phosphateuridyltransferase/glucosamine-1-phosphate-acetyltransferase(GlmU),a crucial enzyme in peptidoglycan biosynthesis,might be a potential target in novel antimicrobial drugs invention.Firstly,the acetylated proteome of V.alginolyticus was sequenced through mass spectrometry and a number of virulence related proteins were identified,including the bifunctional enzyme GlmU,which plays important roles in peptidoglycan biosynthesis.Subsequently,GlmU and protein-lysine deacetylase/desuccinylase/lipoamidase(CobB)of V.alginolyticus were expressed and purified in vitro.The acetylation assay showed that CobB could deacetylate GlmU in vitro.In addition,the enzymatic activity of acetylated GlmU was significant decreased,which could be reversed after deacetylation by CobB.To further clear the function of CobB in regulation of enzyme activity of GlmU in vitro,Overlap PCR and homologous recombination technique were used to construct cobB unmarked gene in-frame deletion strain in combination with chloramphenicol(Cm)forward screening method and susceptibility of sac B gene to sucrose.There were no significant difference of growth trend and protein expression level between wild-type strain and cobB gene knockout strain,while the acetylation level of GlmU and peptidoglycan content in cobB gene knockout strain was significantly increased or decreased,respectively.These results indicated that the enzyme activity of GlmU of V.alginolyticus could be regulated by acetylation.In conclusion,the relationship between reversible lysine acetylation and GlmU function of V.alginolyticus was explored in this study,which provided a new idea for further antimicrobial drugs invention. |
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