Unraveling the Mechanism for the Viability Deficiency of Shewanella oneidensis oxyR Null Mutant

Abstract

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.

Defining the binding determinants of Shewanella oneidensis OxyR: Implications for the link between the contracted OxyR regulon and adaptation

“…2B). These data are in excellent agreement with previous findings (18,19). In ΔSooxyR/SoOxyR L197P , the amount of KatB produced was drastically increased, migrating as the supercomplex on the gel (Fig.…”

“…In SoOxyR, the counterparts of these two cysteines are Cys-203 and Cys-212 respectively; mutation of either residue to serine locks SoOxyR into the reduced form (19,24). In addition, SoOxyR L197P mutant is found to be locked into the oxidized state (25) (18,19). Because the promoter is slightly leaky (26,27) Fig.…”

“…Clearly, the S. oneidensis OxyR resembles those of Neisseria, working as both a repressor and an activator. Nevertheless, it carries novel features that have never been observed from OxyRs studied to date, including compromised resistance to exogenous H 2 O 2 , intertwined regulation with OhrR, and non-exchangeable functionality with E. coli OxyR (18)(19)(20)23).…”

“…Nevertheless, three unexpected phenotypes resulting from the SooxyR disruption were observed (11). First, although the SooxyR mutant under non-stress conditions degrades H 2 O 2 more rapidly, it still carries defects in both viability (plating defect) due to increased sensitivity to H 2 O 2 and growth (18,19,20), phenotypes reported in bacteria with OxyR being a positive regulator, such as E. coli (21; 22). Second, SoOxyR is also able to respond to organic peroxides (OP), functionally intertwined with OhrR, a regulator specific for OP (23).…”

“…Because of the potential application in bioremediation, biogeochemical circulation of minerals and bioelectricity, these bacteria have been intensively studied, especially the model species S. oneidensis (18). The S. oneidensis OxyR (SoOxyR) resembles those of Neisseria in that it represses expression of catalase gene katB under non-stress conditions but functions as an activator for all of its target genes when cells are challenged by H 2 O 2 (19).…”