Regulatory Roles And Mechanism Of Cytidine Repressor On Survival Adaptability Of Pantoea Agglomerans YS19

Bacteria evolve a variety of signal regulatory mechanisms in order to survive diverse environments.As an important signal molecule,indole regulates many physiological and biochemical behaviors in different strains,,but the regulatory mechanism is still unknown.Therefore,the capture of the key proteins involved in indole signal regulatory pathway is of great importance to enrich the understandings of complex regulation network and the regulation mechanisms mediated by indole.A predominant rice endophyte model Pantoea agglomerans YS19 and its ?cytR mutant were selected to investigate the regulating effect of cytR mutation on the survival adaptability of YS19.The stress-resistant ability of the ?cytR mutant was found to be affected.The mutation weakened the ability to form biofilm,as well as the resistance to heavy metal ions(zinc),acid and tetracyclin.Furthermore,cytR mutation decreased strain's survival ability in sulfur-starved environment.In sulfur-free M9 medium,the survival rate of ?cytR was reduced by 18.6% compared with that of the wild type YS19,which was only approximate to the survival rate of YS19(decreased by 16.6%)after the addition of indole;Whereas,indole had little effect on the survival rate of ?cytR which was 39.13%(0.5 m M indole)and 42.63%(0 m M indole),respectively.These results implicated that cytR played an important role in indole-mediated signal transduction,namely,indole-mediated sulfur starvation resistance is partly through cytR regulation.As a result,cytR is a vital factor participated in the regulation of survival adaptability in YS19.In order to find stress resistant genes that was regulated by of cytR,we analyzed the whole-cell protein expression in YS19 and the ?cytR mutant with SDS-PAGE and found a differential band located between 17-26 k D.The protein is only expressed in wild type yet not expressed in the mutant,suggesting a positive regulation by by Cyt R.After identification by MS analysis,six proteins with the function of adversity-resisting and similar MW were found.Interestingly,all the genes were transcriptionally regulated by Cyt R as identified by q PCR.Since cytR was involved in the survival adaptability regulation of numerous bacteria that was down-regulated by indole,how indole was sensed by strains baecames as a question.Then,the regulatory role of indole on the cytR promoter was investigated.Through cloning of cytR promoter and constructing a recombinant plasmid using gfp as the report gene,it was identified that cytR promoter was was down-regulated by indole and up-regulated by cytidine.However,when indole and cytidine were both added,the two chemicals worked in a synergistic manner.Based on this,a model in which the cytR promoter sensed indole to play an stress-resistant role was proposed and validated under tetracycline and sulfur starvation stess.The above phenomena proved that the sense of indole in bacteria was likely to occur at the transcriptional level.Indole-sense gene cytR was transcriptionally regulated by indole was finally discussed in our investigation.This study enriched our knowledge on cytR regulation in YS19.Besides,some new genes in indole-mediated signal pathways and their response patterns to indole were initially obtained,which will refresh our understandings towards cytR and indole-mediated signal pathways.