The Role And Regulation Of DesABC Efflux Pump In Zeamines Resistance Of Dickeya Zeae

Zeamines are a family of polyamino compounds produced by the casual agent of bacterial foot rot disease,Dickey a zeae.They are not only the important virulent factors of Dickeya zeae but also potent antimicrobial compounds with broad spectrum activity at relatively low concentration to multi-drug resistant bacteria.Unlike several multiple-drug resistant bacteria,our preliminary study showed the D.zeae EC1 could withstand relatively high concentrations of zeamines,suggesting the existance of zeamines resistant mechanisms in D.zeae EC1.This study not only showed the key role of the efflux pump DesABC of D.zeae EC1 in conferring resistance against zeamines but also identified several mechanisms modulating the transcriptional expression of the DesABC system.The main findings are shown as follows:RND family efflux pump confers zeamine resistance to D.zeae EC1.By knocking out the putative transpoter genes in and nearby the zms gene cluster and determing their zeamine sensitivity,we found that deletion of the genes encoding putative ABC transporters with in the zms cluster did not affect the sensitivity of D.zeae EC1 against zeamines.Instead,the desAB genes found in the vicinity of the zms cluster,which encode a putative RND efflux pump,played a key role in zeamine resistance.Using sequence alignment and phylogenic study,DesB,which is required for substrate recognition in DesABC,was wildely distrubuted in Dickeya and associated with zeamine resistance.Substrate specificity assay revealed that the DesB had a narrow substrate range and was specific to zeamines.In addition,DesABC system was found promoting the survival ability of D.zeae EC1 against zeamines.By comparing the growth patterns of wild type strain EC1 and its desB mutant in the LS5 medium optimized for zeamine production,DesABC was found conferring the bacterial resistance against zeamines from the late exponential growth stage when zeamines were accumulated to threshold high level.Zeamines modulated the expression of the desAB genes in Dickeya.By measuring the expression of desAB at different stages during bacterial growth in LS5 medium,the expression level of desAB was shown steadily increasing as the increment of bacterial population and concentration of zeamines.Comparison of the expression levels of desAB in wild type strain EC1 and the desB mutant,together with the changed expression pattern of desAB in the zmsA mutant with or without zeamines,demonstrated that zeamines could modulate desAB expression in D.zeae EC1.Further analysis showed a relatively low concentration of zeamines was sufficient to stimulate the expression of desAB in Dickeya strains.Zeamines and ZmsPQR modulate desAB expression through DzrR.By constructing the reporter strain ΔzmsK(pDesABgfp),and using the transposon mutagenesis,the transposon mutant Dz974 was obtained.The desAB expression level in mutant Dz974 was greatly reduced and could not be rescued by exogenous addition of zeamines.FPNI-PCR and DNA sequencing results indicated that transposon insertion in mutant Dz974 was located within the ORF of dzrR.Bioinformatic analysis showed that dzrR is located downstream of desAB encoding putative two component system response regulator.Further experimental analysis showed that compared with wild type EC1,deletion of dzrR resulted in significantly decreased expression of desAB,increased sensitivity to zeamines,but the yield of zeamines was not significantly changed.These results indicated the DzrR is involved in the regulation of zeamine resistance by modulating desAB expression.The results from comparison of the expression patterns of desAB in wild type strain EC1 and the dzrR mutant under different culture condition indicated that modulation of desAB by DzrR was dependent on zeamines.In addition,the expression level of desAB in mutants of zmsPQR located in the zms gene cluster was significantly increased,which indicated that ZmsPQR regulates the expression of desAB genes.Further studies found that ZmsPQR regulates desAB through DzrR.In summary,this paper first identified the anti-zeamine efflux pump DesABC,and proved that the expression of this efflux pump is co-regulated by zeamines,response regulator DzrR and transporter system ZmsPQR.