Studies On Molecular Mechanisms Underlying The Impacts Of ArcA On Growth And Cell Envelope Integrity In Shewanella Oneidensis

In?-proteobacterial species,such as Escherichia coli,the Arc two-component system plays a major role in mediating the metabolic transition from aerobiosis to anaerobiosis,and thus is crucial for anaerobic growth but dispensable for aerobic growth.In Shewanella oneidensis,which is well known for its respiratory diversity,Arc represents an exception:it's absence not only affects aerobic growth but also cell envelope integrity.This dissertation explored the mechanism underlying growth and cell envelope defects resulting from the loss of Arc.Important achivements are two folds:i)The growth defect is in part linked to the impaired transportation of oligopeptides and di-tripeptides;ii)Alternative sigma factor?^E(encoded by rpo E)is tightly associated with the cell envelope defect.The previous study revealed that the growth defect resulting from the loss of Arc A(DNA-binding response regulator of the Arc system)is tryptone-dependent.In this study,we demonstrated that the growth defect partly linked to the utilization of oligopeptides and di-tripeptides,and that peptide uptake rather than peptide degradation is significantly affected by the Arc A loss.A systematic characterization of major small peptide uptake systems manifests that ABC peptide transporter Sap and four proton-dependent oligopeptide transporters(POTs)are responsible for transport of oligopeptides and di-tripeptides respectively.Among them,Sap and Dtp A(one of POTs)are responsive to the arc A mutation but only dtp A is under the direct control of Arc A by EMSA and bacterial one-hybrid(B1H)analysis.We further showed that both Sap and Dtp A,when overproduced,improve the growth of the arc A mutant.While the data firmly establish a link between transport of oligopeptides and di-tripeptides and the arc A mutation,other yet-unidentified factors are implicated in the growth defect resulting from the Arc A loss.To survive and thrive in harsh and ever-changing environments,intricate mechanisms have evolved for bacterial cells to monitor perturbations impacting the integrity of their envelope and to mount an appropriate response to contain or repair the damage.In this study,we report in S.oneidensis a previously undescribed mechanism for the envelope defect resulting from the loss of Arc,a two-component transcriptional regulatory system crucial for respiration.We uncovered?^E,a master regulator establishing and maintaining the integrity of the cell envelope in?-proteobacteria,as the determining factor for the cell envelope defect of the arc A mutant.The results of Arc A mutant susceptibility to different antibiotics and agents discovered that the inner membrane and peptidoglycan layer were not significantly compromised,but Arc A mutation results in drastically sensitive to SDS which acts on outer membrane first.Combination with the transcriptome and proteome analysis based on the previous studies,we found that when Arc A is depleted,?^E activity is compromised by enhanced production of anti-?^E protein RseA.As a result,over produced RseA had no effect on the growth of the wild type,but significantly inhibited the growth of?arc A,and this effect was determined?^E-dependent.Surprisingly,S.oneidensis?^E is not essential for viability,but becomes so in the absence of Arc A,which is different from E.coli.However,?^E becomes essential while Arc A mutation,and we also demonstrated that Arc A and?^E are synthetic lethal in S.oneidensis.Further prediction and analysis revealed that transcription of rse operon relies on?⁷⁰ within the rpo E coding sequence in S.oneidensis,which is different with?^Ein E.coli.In addition,we demonstrated that there is an interplay between these two regulators as arc A expression is affected by availability of?^E.In conclusion,this study revealed some factors,oligopeptides and di-tripeptides transporters Sap and Dtp A,that in part explain growth defect caused by the Arc A loss.More importantly,?^E plays a major role on the outer membrane defect of?arc A,which became more important when in Arc A mutant.Overall,our results underscore functional interplay of regulatory systems for envelope stress response:although each of the systems may respond to perturbation of particular components of the envelope,they are functionally intertwined,working together to form an interconnected safety net.