The Targets Of Protein Kinase YihE And Its Cellular Pathways In Escherichia Coli

Our previous studies had shown that Echerchia coli protein kinase YihE could protect bacterial cells from antibiotic treatment.The studies on the function of YihE and its cellular pathways would help us to further understand the bacterial stress responses and the development of antibiotic resistance.Proteins PpsA,Pgm,and DnaK,which are involved in important metabolic pathways in cells,were found to be potential targets of YihE through phosphor-proteomic sequencing.Therefore,it is important to verify these targets and analyze their cellular pathways for understanding the function of YihE.In the present work,various methods were used to detect the kinase activity of YihE in vitro.The results showed that YihE could phosphorylate PpsA?Pgm and DnaK in vitro,albeit with very low activity.Meanwhile,all of target proteins were found to be able to phosphorylate themselves.Mutating the auto-phosphorylating site of DnaK and removing the GFP tag on YihE did not affect the YihE activity.Therefore,PpsA?Pgm and DnaK might be the cellular targets of YihE.Compared to the wild type strain,?pgm and ?dnaK were more sensitive to nalidixic acid treatment,while ?ppsA was more sensitive under low concentrations of nalidixic acid,indicating that these genes could protect cells from antibiotic treatment.Since pgm and ppsA genes are involved in glucose-phosphate stress,we also examined the effect of ?MG on the growth of these mutants.The results showed that ?MG treatment affected the growth of the mutants,which suggested that the function of these genes might be related to glucose-phosphate stress.In addition,measurements of pyruvate in the wild-type strain and the ?yihE mutant revealed that the concentration of pyruvate sharply increased in the ?yihE mutant,which implied the consumption of PEP(a postulated signal for glucose-phosphate stress).Thus,we propose that,under the presence of antibiotics,YihE kinase regulates the activities of PpsA and Pgm through phosphorylation to avoid the accumulation of toxic glucose-6-phosphate and the depletion of PEP,which could cause growth inhibition or cell death.The presented work would help us to further understand the metabolic pathways regulated by YihE kinase,which provided a new way for us to study the mechanism of antibiotic resistance through bacterial stress responses.