Analysis Of Five Genes Which Might Be Regulated By The Sensor XC3670 In Xanthomonas Campestris Pv.campestris

Two-component systems(hrpG,rpfC-rpfG,colS-colR)play essential role in signal transduction and function respectively in pathogen-host interaction,quorum sensing and stress tolerance.The Xcc 8004(Xanthomonas campestris pv.campestris 8004)genome encodes 106 TCS genes,most of them with unknown function.Previously,our lab found that a TCS XC3670-XC3669 was greatly involved in pathogenicity.To characterize the detail function and regulatory mechanism of the putative TCS,we tried to identify the downstream genes which are regulated by it.According to the results of two-dimensional electrophoresis mass spectrometry,this work,we chose five candidate genes(XC1163,XC1308,XC2810,XC2847,XC2921),which showed different express pattens between XC3670 mutant and wildtype strains,for further investigation by deletion mutation/complete-ment and translational fusion GUS reporter.XC1163 is annotated as a DNA-binding protein.We constructed the deletion mutant of XC1163 and analyzed its phenotype;the results showed that this gene may be involved in metal and SDS tolerance.In silico analyses by NNPP and SAK promoter prediction tool,we presume its ?70 promoter:ATGACA as-35 sequence,TCTATT as-10 sequence and separately by 17bp.Transcriptional/translational fusion GUS reporter revealed that XC1163 may be negatively regulated by XC3670 at transcriptional level in both NYGB and MMX medium.XC1308 is annotated as a bi-function protein Hpr K/P.We constructed its deletion mutation and complimentary strain for further phenotype analyzation.The result revealed that XC1308 might be involved in the production of extracellular amylase,cellulase,protease,EPS and it also might be involved in in vitro vegetative growth.Using RT-PCR,we confirmed that XC1308 and XC1309 are in the same transcriptional unit.Further experiment is required to fully understand the structure and regulatory relation of this operon.XC2810 encodes a putative protein with PolyA_pol domain and Gly-zipper domain.We presume it may be a membrane protein,which has a signal peptide and a TMH domain predicted by SignalP 4.0 and TMHMM 2.0 tool.Mutant phenotype analysis revealed that XC2847 is not involved in in vitrol growth,EPS and extracellular enzyme production,tolerance,pathogenicity and HR.Although no conserved ?70 promoter was predicted,transcriptional/translational fusion GUS reporter revealed that XC2810 may be positively regulated by XC3670 at transcriptional level.However,further information is required to confirm it.XC2921 encodes a putative protein with unknown function domain.Mutant phenotype analysis revealed that XC2921 is not involved in in vitrol growth,EPS and extracellular enzyme production,tolerance,pathogenicity and HR.Transcriptional/translational fusion GUS reporter revealed that XC2921 may be positively regulated by XC3670 at both transcriptional and translational level.XC2847 encodes a putative protein with Pectate lyase motif and Periplasmic copper-binding motif.By gene mutation and trans-complement we foud that,XC2847 is not only involved in metal and SDS tolerance,but also affects pathogenicity and HR.We presume that XC2847 may encode a novel protein with both metal tolerance and pathogenicity activity.