The Biochemical Mechanism Of Histidine Kinase VgrS In Response To Metal Ions In Xanthomonas

Transition metals are critical nutrients for all organisms,including bacterial pathogens.The perception and transport of metal ions play an important role in regulating host antigen immunity and bacterial pathogenicity.The two-component system（TCS）in bacteria can sense the stimulation of external metal ions and control the adaptive response of cells.In TCS,transmembrane histidine kinase（HK）activates or inhibits the expression of target genes by phosphorylating intracellular response regulatory protein（RR）in response to extracellular stimuli of metal ions.However,the biochemical mechanism of metal recognition and signal transmission is yet to be resolved in detail.Previously research showed that TCS VgrSR in Xanthomonas campestris can sense iron limitation and regulate the Fe³⁺balance in vivo.In this subject,33 conserved amino acid residues in VgrS signal sensing region were substituted into Alanine by using site-directed mutagenesis to detect the mechanism of VgrS sensing metal ions and phosphorylation signal transmission.We identified nine residues（N2,K36,L40,E46,W47,R74,K75,Y85,and D86）of the 33 single point mutants that play an important role in growth under iron stress,and the swimming mobility of six residues（N2,L40,W47,R74,K75,and Y85）decreased compared with that of wild type under iron stress.Biochemical analyses andβ-glucuronidase assays showed that except for W47A and R74A,seven single-point substitution mutations of nine residues decreased the iron-binding affinity and iron-deficiency-induced protein phosphorylation.Our results demonstrated that VgrS senses zinc as well.Phenotypic screening of 33 conserved site mutants showed that the substitutions N2A,L40A,and K75A affected growth under zinc stress,swimming mobility,direct binding of zinc,and zinc-induced attenuation of autokinase activity.Structure prediction and molecular dynamics analyses indicated that L40,E46,and K75 are located on the surface of a ligand-binding pocket,and responsible for direct binding with metal ions.Upon metal binding,the rigidity of signal sensing protein enhances,especially at the C-terminalα-helix.Combined with the results of genetic and biochemical experiments,the rigid structure of the protein inhibited the VgrS auto-phosphoryaltion,and subsequently attenuated the regulating capacity of VgrR.Our results clarified the vital mechanism of metal ions in VgrS conformational change and phosphorylation signal transmission.It provides insight into how a phytopathogenic bacterium recognizes distinct metal ions to modulate the metal homeostasis and pathogenesis.These findings may have applications in the development of new metal-containing anti-bacterial agents.