Isolation and Characterization of a Shewanella putrefaciens MR-1 Electron Transport Regulator etrA Mutant: Reassessment of the Role of EtrA

Abstract

Shewanella putrefaciens MR-1 has emerged as a good model to study anaerobic respiration and electron transport-linked metal reduction. Its remarkable respiratory plasticity suggests the potential for a complex regulatory system to coordinate electron acceptor use in the absence of O ₂ . It had previously been suggested that EtrA (electron transport regulator A), an analog of Fnr (fumarate nitrate regulator) from Escherichia coli , may regulate gene expression for anaerobic electron transport. An etrA knockout strain (ETRA-153) was isolated from MR-1 using a gene replacement strategy. Reverse transcription-PCR analysis of total RNA demonstrated the loss of the etrA mRNA in ETRA-153. ETRA-153 cells retained the ability to grow on all electron acceptors tested, including fumarate, trimethylamine N -oxide (TMAO), thiosulfate, dimethyl sulfoxide, ferric citrate, nitrate, and O ₂ , as well as the ability to reduce ferric citrate, manganese(IV), nitrate, and nitrite. EtrA is therefore not necessary for growth on, or the reduction of, these electron acceptors. However, ETRA-153 had reduced initial growth rates on fumarate and nitrate but not on TMAO. The activities for fumarate and nitrate reductase were lower in ETRA-153, as were the levels of fumarate reductase protein and transcript. ETRA-153 was also deficient in one type of ubiquinone. These results are in contrast to those previously reported for the putative etrA mutant METR-1. Molecular analysis of METR-1 indicated that its etrA gene is not interrupted; its reported phenotype was likely due to the use of inappropriate anaerobic growth conditions.