The Mechanisms Of Catalysis And Regulation Of Fic Proteins From Pseudomonas Fluorescens Strain 2P24

Bacteria have evolved a rich set of mechanisms to cope with harsh conditions unfavorable for cell proliferation.Through modulate the processes of DNA replication,mRNA transcription or protein translation,bacteria regulate cell growth and form persisters to adapt stresses.The Fic domain is a widely-distributed motif containing conserved sequence HPFx[D/E]GN[G/K]RxxR in prokaryotic and eukaryotic proteins,many of which catalyze the transfer of the AMP moiety of ATP to substrates that often contain an ATPase or GTPase domain.Soil bacterium Pseudomonas fluorescens 2P24 is affected by variants of stress factors,such as heavy metal,drought,higher temperature,and inhibitory factors secreted by bacteria,fungi,animals and plants.Therefore,the colonization and biocontrol activity of P.fluorescens mainly dependent on adaption of adverse conditions in its environment.Three putative non-secreted Fic proteins are present in the genome of P.fluorescens 2P24,however,the cellular targets,catalytic mechanisms and functions of these Fic proteins are still unknown.We showed that the expression of Fic-1 from strain 2P24 in both P.fluorescens and Escherichia coli significantly reduced the yield of plasmid DNA,arrested chromosomal division,and halted the separation of cells.Fic-1 interacted with GyrA,GyrB,LigA,ParE,PolB,PriA and RecX by detecting with bacterial two hybrid system.Fic-1 catalyzed the AMPylation on PfGyrB at Tyr111 and its paralog PfParE at Tyr109,a residue forms a hydrogen bond with the N3 atom of the adenine ring and is critical for ATP hydrolysis by DNA gyrase or topoisomerase IV.Fic-1-dependent AMPylation of GyrB affected the ATP hydrolysis and negative supercoiling activities of DNA gyrase,and triggered the SOS response,which subsequently led to cell filamentation.Fic-1 also promoted the formation of limited and elongated cells when the SOS response was blocked.These data suggested that cell filamentation induced by Fic-1 mainly dependents on the SOS pathway,and on an unidentified pathway as well.Fic-1 exhibited auto-AMPylation activity.Mutation of the Fic-1 auto-AMPylated site greatly reduced its AMPylation activity toward both itself and GyrB.We identified an a-inhibitor protein named anti-Fic-1(AntF),which encoded by a gene immediately upstream of fic-1 and composed canonical type II antitoxin and toxin module with Fic-1.The antF and fic-1 genes transcribed together under a promoter located 25bp upstream of translational initiation site of antF.AntF interacted with Fic-1,inhibited its auto-AMPylation and its AMPylation activity to GyrB in vitro,and blocked Fic-1-mediated inhibition of DNA replication in bacteria.AntF could be digested by Lon protease,but binding with Fic-1 protected AntF from degradation in vitro.Further analysis found that the Fic-2E56G mutant defective in the inhibitory motif was able to AMPylate ParE but not GyrB;In addition,Fic proteins from Yersinia pseudotuberculosis,Staphylococcus aureus modified ParE by AMPylation,however,those from P.aeruginosa,Mycobacterium tuberculosis or Streptococcus pneumoniae modified neither GyrB nor ParE.Cell growth was inhibited and the ratio of anucleate cells was increased by expression of Fic-2 or PfParEY109A in P.fluorescens 2P24.Our results suggest that at least two Fic proteins from P.fluorescens target either GyrB of DNA gyrase or ParE of topoisomerase IV to regulate the essential biological processes such as DNA replication,chromosome division and cell separation in bacteria.The catalytic activities of Fic proteins are strictly regulated under the normal condition and could possibly be released to regulate DNA replication and control cell division,in order to survive under harsh conditions.