Reversible Lysine Acetylation Of DnaA Coordinates DNA Replication Initiation In E.coli

Initiation of chromosomal replication and its regulation are critical and fundamental in most cellular organisms.When and whether to initiate DNA replication determines the fate of cells.Cells must promote DNA replication initiation under favorable environments and prevent DNA replication initiation in stressful conditions.In bacteria,the initiation of chromosomal DNA replication is mediated by DnaA.Multiple systems regulate the activity of the replication initiator ATP-DnaA in Escherichia coli including RIDA(Regulatory inactivation of DnaA)and DARS(DnaA-reactivating sequence).As a member of the AAA+ ATPase family,DnaA has conserved Walker A and Walker B motifs that are crucial for ATP/ADP binding.Protein lysine acetylation has emerged as a key posttranslational modification in cellular regulation.Here,we showed that DnaA can be acetylated by Pat(acetyltransferase)and acetyl-phosphate(AcP)and deacetylated by CobB(deacetylase)in vitro.To better understand how DnaA is regulated and what is role of lysine acetylation in DNA replication,we generated a chromosomal knock-in strain,whose dnaA gene was fused with a His-tag sequence at N terminus in E.coli.By using this strategy and affinity purification,we isolated native expressed DnaA from various genetic background and different growth stages.Strikingly,DnaA purified from stationary phase shows higher acetylation level compared with proteins from lag and exponential phase.The absence of Pat or CobB changes the acetylation level of DnaA.Acetate kinase(AckA)and phosphotransacetylase(Pta)reversibly convert acetate and acetyl-CoA via a high-energy acetyl-phosphate(AcP)intermediate.We constructed ackA-pta double deletion mutant and found that disruption of acetyl-phosphate(AcP)synthesis decreased the acetylation level of DnaA.Cell cycle analysis showed that the DNA replication initiation frequency of the cobB deletion muntant,pat deletion mutant and ackA-pta double deletion mutant changed dramaticlly compared with that of the parental strain.We concluded that the CobB/Pat system and AcP participate in the epsilon-N lysine acetylation modification of DnaA and affect the DNA replication initiation frequency.Mass spectrometry revealed that a total of 17 lysine residues of DnaA were acetylated in different growth stages,including a conserved lysine 178 in Walker A motif,which is responsible for ATP binding.The acetylation of lysine 178 inhibits ATP/ADP binding and impairs its ability of binding to oriC by using a site-specifically acetylation strategy.This indicates that the acetylation of lysine 178 weakens the activities of DnaA.We found that the acetylation of lysine 178 decreased its ability of binding to dnaA promoter,and this suggests that the acetylation of DnaA may participate the process of dnaA autoregulation.Moreover,the acetylation of lysine 178 can be removed by CobB.Collectively,our findings indicate that protein lysine acetylation is a novel mechanism to mediate DNA replication initiation and expand our understanding of the regulation mechanisms of DNA replication initiation.