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 Fe ²⁺ is toxic to cells anaerobically, nor is it fully understood how S. oneidensis is able to resist high levels of Fe ²⁺ . Here we describe the results of a transposon mutant screen and subsequent deletion of the genes clpX and clpP in S. oneidensis, which demonstrate that the protease ClpXP is required for anaerobic Fe ²⁺ 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 Fe ²⁺ resistance in S. oneidensis. Protein trapping studies were performed to identify ClpXP targets in S. oneidensis under Fe ²⁺ stress, implicating a wide variety of protein targets. Escherichia coli strains lacking clpX or clpP also display increased sensitivity to Fe ²⁺ anaerobically, indicating Fe ²⁺ resistance may be a conserved role for the ClpXP protease system. Hypotheses regarding the potential role(s) of ClpXP during periods of high Fe ²⁺ are discussed. We speculate that metal-containing proteins are misfolded under conditions of high Fe ²⁺ and that the ClpXP protease system is necessary for their turnover.