Survival of Anaerobic Fe 2+ Stress Requires the ClpXP Protease

Shewanella oneidensis strain MR-1 is a versatile bacterium capable of respiring extracellular, insoluble ferric oxide minerals under anaerobic conditions. The respiration of iron minerals results in the production of soluble ferrous ions, which at high concentrations are toxic to living organisms. It is not fully understood how Fe2+ is toxic to cells anaerobically, nor is it fully understood howS. oneidensis is able to resist high levels of Fe2+ . Here we describe the results of a transposon mutant screen and subsequent deletion of the genesclpX andclpP inS. oneidensis , which demonstrate that the protease ClpXP is required for anaerobic Fe2+ resistance. Many cellular processes are known to be regulated by ClpXP, including entry into stationary phase, envelope stress response, and turnover of stalled ribosomes. However, none of these processes appears to be responsible for mediating anaerobic Fe2+ resistance inS. oneidensis . Protein trapping studies were performed to identify ClpXP targets inS. oneidensis under Fe2+ stress, implicating a wide variety of protein targets.Escherichia coli strains lackingclpX orclpP also display increased sensitivity to Fe2+ anaerobically, indicating Fe2+ resistance may be a conserved role for the ClpXP protease system. Hypotheses regarding the potential role(s) of ClpXP during periods of high Fe2+ are discussed. We speculate that metal-containing proteins are misfolded under conditions of high Fe2+ and that the ClpXP protease system is necessary for their turnover.IMPORTANCE Prior to the evolution of cyanobacteria and oxygenic photosynthesis, life arose and flourished in iron-rich oceans. Today, aqueous iron-rich environments are less common, constrained to low-pH conditions and anaerobic systems such as stratified lakes and seas, digestive tracts, subsurface environments, and sediments. The latter two ecosystems often favor dissimilatory metal reduction, a process that produces soluble Fe2+ from iron oxide minerals. Dissimilatory metal-reducing bacteria must therefore have mechanisms to tolerate anaerobic Fe2+ stress, and studying resistance in these organisms may help elucidate the basis of toxicity.Shewanella oneidensis is a model dissimilatory metal-reducing bacterium isolated from metal-rich sediments. Here we demonstrate a role for ClpXP, a protease system widely conserved in bacteria, in anaerobic Fe2+ resistance in bothS. oneidensis andEscherichia coli .