Functional Analyses Of The Transcription Factor XC0449 And Its Regulation Of Virulence With HpaR In Xanthomonas Campestris Pv.campestris

MarR(multiple antibiotic resistance regulator)family transcription factors(TFs)are widely found in bacteria and archaea,and participate in important physiological and biochemical reactions of various bacteria,including various antibiotic stress,oxidative stress,virulence factors and catabolism of environmental aromatic compounds.Bacteria of Xanthomonas can cause variety of forest disease and pose a great threat to the growth of forest in China.This study used Xanthomonas campestris pv.campestris as research material to analyze the function of MarR family TFs,in order that providing basis and ideas for the relative research of forest disease caused by Xanthomonas.The main results are as follows:1.The Insert inactivated mutant of XC0449,the transcription factor of xanthomonas MarR family,resulted in the inhibition of its growth on the basic culture medium MMX.2.The deletion mutant of transcription factor XC0449(Δ0449)affected the growth of the bacteria on MMX medium and significantly reduced the virulence to the host.3.The ChIP-seq experiment analyses found that XC0449 regulated the downstream gene XC0356,which was an important enzyme(Dihydroxy-acid dehydratase,DHAD)to participate in the synthesis of valine.XC0449 could bind to XC0356 promoter region and positively regulated the expression of XC0356.4.Strain growth experiments showed that addition of valine(100 mM)into the MMX medium,restored the phenotype of A0449 growing on basic medium.Moreover,XC0049 could bind valine,also could be a sensor to respond to valine at different concentrations.5.Asn31 and Asp76 were the crucial sites for the binding of XC0449 and valine.6.From the early research,insertional inactivation of MarR family transcription regulator HpaR(XC2827)resulted in significantly decrease in virulence and increase in the production of the extracellular proteases.We expressed and purified HpaR,XC0449,and other MarR family transcription factor proteins to confirm the physical interaction between HpaR and XC0449 in vitro.Morever,HpaR and XC0449 coordinately act as the transcriptional activators to regulate the expression of the virulence-associated gene XC0705,and eventually controled the bacterial virulence and the production of extracellular proteases.