Studies On Functional Mechanisms Of Trx And AhpC In Oxidative Stress Response In Shewanella Oneidensis

In natural environments,reactive oxygen species（ROS）are ubiquitous because they can be generated by both intracellular and extracellular reactions.ROS affect cell physiology at multiple levels by reacting with a variety of biological macromolecules,including DNA,proteins,and lipids.Oxidative stress,imposed by excessive ROS,is a major survival challenge that all aerobic organisms have to face.In response to oxidative stress,bacteria have evolved complex and varied strategies,including induceable expression of ROS scavenging enzymes,such as catalases（CAT）,superoxide dismutases（SOD）,and NADH-dependent peroxidase AhpCF,regulation of the abundance and activity of thioredoxin（Trx）and glutaredoxin（Grx）antioxidant systems,and activation of dedicated responsive regulators for coordinated regulation.Our previous studies found that the mechanisms that Shewanella oneidensis,a Gram-negative facultative anaerobic bacterium,exploits to respond to oxidative stress carry many novel features,including:the interaction between Trx and Grx systems and the Oxy R and OhrR regulators affects survival;the abilities of AhpC to scavenge hydrogen peroxide（H₂O₂）and organic peroxides（OPs）are substantially different.This dissertation took these features as a starting point to systematically analyze the physiological effects and underpinning mechanisms of Trx and Grx systems in oxidative stress response of S.oneidensis and to elucidate the molecular mechanism that explains the difference of AhpC in scavenging activity for H₂O₂ and OPs.The Trx and Grx systems are profoundly involved in bacterial response to H₂O₂,but to date,we know surprisingly little about the roles of these systems in response to ROS other than H₂O₂.This study proved that Trx1（trx A）is the major thiol-disulfide redox enzyme and that in its absence a Grx system becomes essential under normal physiological conditions.Although overshadowed by Trx1 in the wild type,Trx2（trx C）can fully compensate for the Trx1 loss when overproduced.In addition,Trx1 is physiologically linked to dual-activity（inhibitory and activating regulation of kat B,which encodes a major catalase）Oxy R and repressor-only OhrR.The absence of Trx1causes the intracellular redox potential to reach an oxidizing level that prevents Oxy R from functioning as a repressor,thereby partially relieving the repression of Oxy R on the kat B gene.More importantly,Trx1 plays a critical role in the cellular response to OPs mainly by mediating the redox status of OhrR.We confirmed that while none of the trx and grx genes are Oxy R-dependent,the expression of trx A and trx C can be affected by OhrR indirectly.Further investigations demonstrated that the depletion of glutathione（GSH）is likely the cue to trigger induced expression of trx A and trx C,although the direct regulatory proteins for these genes are unknown.AhpC is a bacterial representative of 2-Cys peroxiredoxins（Prxs）with broad substrate specificity and robust functional plasticity.The broad substrate specificity of AhpC makes it a junction for the cross-talk between Oxy R and OhrR and its functional plasticity has the potential to link Trx/Grx and Oxy R-OhrR.However,the molecular basis underpinning these two important attributes of AhpC remains unclear.By assessing AhpC’s suppressing effect on the plating defect of the?kat B strain,we showed that successful suppression can be achieved only when AhpCF is produced in a dose-and time-dependent manner through a complex mechanism involving activation of Oxy R,transcription attenuation,and translation reduction.By analyzing AhpC truncation variants,we demonstrated that reactivity with OPs rather than H₂O₂ is resilient to mutagenesis,implying that OPs reduction is the core catalytic function of AhpC.Intact AhpC can be recycled only by its cognate reductase AhpF,and AhpC variants lacking the Prx domain or the extreme C-terminal five residues become promiscuous electron acceptors from the thioredoxin reductase（Trx R）and the GSH reductase（Gor）in addition to AhpF,implicating an additional dimension to functional plasticity of AhpC.Finally,we revealed that the catalytic activity of S.oneidensis AhpC is less affected by mutations than that of its Escherichia coli counterpart.In conclusion,this dissertation provides an in-depth study of the thioredoxin Trx and the peroxidase AhpC in the oxidative stress response of S.oneidensis.Our results underscore the particular importance of Trx1 in the bacterial OPs stress response,and suggest that the physiological roles of bacterial AhpCs are adapted to different oxidative challenges,depending on the organism,and that its functional plasticity is even more extensive than previously reported.These results not only contribute to improving the survivability of S.oneidensis in adverse environments through genetic modification,but also provide a new entry point for promoting the practical application of S.oneidensis in bio-energy and material transformation due to the coupling of these proteins with extracellular electron transport.