Shewanella oneidensis is an important model organism for its versatility of anaerobic respiration. CymA, a cytoplasmic membrane-bound tetraheme c-type cytochrome, plays a central role in anaerobic respiration by transferring electrons from the quinone pool to a variety of terminal reductases. Although loss of CymA results in defect in respiration of many electron acceptors (EAs), a significant share of the capacity remains in general. In this study, we adopted a transposon random mutagenesis method in a cymA null mutant to identify substituent(s) of CymA with respect to nitrite and nitrate respiration. A total of 87 insertion mutants, whose ability to reduce nitrite was further impaired, were obtained. Among the interrupted genes, the petABC operon appeared to be the most likely candidate given the involvement of the cytochrome bc1 complex that it encodes in electron transport. Subsequent analyses not only confirmed that the complex and CymA were indeed functionally overlapping in nitrate/nitrite respiration but also revealed that both proteins were able to draw electrons from ubiquinone and menaquinone. Furthermore, we found that expression of the bc1 complex was affected by oxygen but not nitrate or nitrite and by global regulators ArcA and Crp in an indirect manner.

Oxidative stresses triggered by reactive oxygen species (ROS) that damage various cellular components are unavoidable for virtually all living organisms. In defense, microorganisms have evolved sophisticated mechanisms to sense, respond to, and battle against ROS. Shewanella oneidensis, an important research model for applied and environmental microbes, employs OxyR to mediate the response to H 2 O 2 by derepressing the production of the major H 2 O 2 scavenger KatB as a major means toward these goals. Surprisingly, despite enhanced H 2 O 2 degradation, the oxyR mutant carries a viability deficiency phenotype (plating defect), which can be suppressed by the addition of exogenous iron species. Experiments showed that the defect was not due to iron starvation. Rather, multiple lines of evidence suggested that H 2 O 2 generated abiotically in lysogeny broth (LB) is responsible for the defect by quickly killing mutant cells. We then showed that the iron species suppressed the plating defect by two distinct mechanisms, either as an H 2 O 2 scavenger without involving living cells or as an environmental cue to stimulate an OxyR-independent response to help cells cope with oxidative stress. Based on the suppression of the plating defect by overproduction of H 2 O 2 scavengers in vivo, we propose that cellular components that are vulnerable to H 2 O 2 and responsible for the defect may reside outside the cytoplasm. IMPORTANCEIn bacteria, OxyR is the major regulator controlling the cellular response to H 2 O 2 . The loss of OxyR results in reduced viability in many species, but the underlying mechanism is unknown. We showed in S. oneidensis that this defect was due to H 2 O 2 generated abiotically in LB. We then showed that this defect could be corrected by the addition of Fe 2؉ or catalase to the LB or increased intracellular production of catalase. Further analyses revealed that Fe 2؉ was able not only to decompose H 2 O 2 directly but also to stimulate the activity of OxyR-independent H 2 O 2 -scavenging enzymes. Our data indicate that iron species play a previously underappreciated role in protecting cells from H 2 O 2 in environments.

Hydrogen sulfide (H2S) has been recognized as a physiological mediator with a variety of functions across all domains of life. In this study, mechanisms of endogenous H2S generation in Shewanella oneidensis were investigated. As a research model with highly diverse anaerobic respiratory pathways, the microorganism is able to produce H2S by respiring on a variety of sulfur-containing compounds with SirACD and PsrABC enzymatic complexes, as well as through cysteine degradation with three enzymes, MdeA, SO_1095, and SseA. We showed that the SirACD and PsrABC complexes, which are predominantly, if not exclusively, responsible for H2S generation via respiration of sulfur species, do not interplay with each other. Strikingly, a screen for regulators controlling endogenous H2S generation by transposon mutagenesis identified global regulator Crp to be essential to all H2S-generating processes. In contrast, Fnr and Arc, two other global regulators that have a role in respiration, are dispensable in regulating H2S generation via respiration of sulfur species. Interestingly, Arc is involved in the H2S generation through cysteine degradation by repressing expression of the mdeA gene. We further showed that expression of the sirA and psrABC operons is subjected to direct regulation of Crp, but the mechanisms underlying the requirement of Crp for H2S generation through cysteine degradation remain elusive.

c ␤-Lactam antibiotics were the earliest discovered and are the most widely used group of antibiotics that work by inactivating penicillin-binding proteins to inhibit peptidoglycan biosynthesis. As one of the most efficient defense strategies, many bacteria produce ␤-lactam-degrading enzymes, ␤-lactamases, whose biochemical functions and regulation have been extensively studied. A signal transduction pathway for ␤-lactamase induction by ␤-lactam antibiotics, consisting of the major peptidoglycan recycling enzymes and the LysR-type transcriptional regulator, AmpR, has been recently unveiled in some bacteria. Because inactivation of some of these proteins, especially the permease AmpG and the ␤-hexosaminidase NagZ, results in substantially elevated susceptibility to the antibiotics, these have been recognized as potential therapeutic targets. Here, we show a contrasting scenario in Shewanella oneidensis, in which the homologue of AmpR is absent. Loss of AmpG or NagZ enhances ␤-lactam resistance drastically, whereas other identified major peptidoglycan recycling enzymes are dispensable. Moreover, our data indicate that there exists a parallel signal transduction pathway for ␤-lactamase induction, which is independent of either AmpG or NagZ.

Oxidative stress is one of the major challenges that Shewanella encounter routinely because they thrive in redox-stratified environments prone to reactive oxygen species (ROS) formation, letting alone that ROS can be generated endogenously. As respiration is the predominant process for endogenous ROS, regulators mediating respiration have been demonstrated and/or implicated to play a role in oxidative stress response. In our efforts to unveil the involvement of global regulators for respiration in the oxidative stress response, we found that loss of the Arc system increases S. oneidensis sensitivity to H2O2 whereas neither Fnr nor Crp has a significant role. A comparison of transcriptomic profiles of the wild-type and its isogenic arcA mutant revealed that the OxyR regulon is independent of the Arc system. We then provided evidence that the enhanced H2O2 sensitivity of the arcA mutant is due to an increased H2O2 uptake rate, a result of a cell envelope defect. Although one of three proteases of the ArcA regulon when in excess is partially accountable for the envelope defect, the major contributors remain elusive. Overall, our data indicate that the Arc system influences the bacterial cell envelope biosynthesis, a physiological aspect that has not been associated with the regulator before.