Iodate Reduction by Shewanella oneidensis Requires Genes Encoding an Extracellular Dimethylsulfoxide Reductase

Abstract

Microbial iodate (IO₃ ^-) reduction is a major component of the iodine biogeochemical reaction network in anaerobic marine basins and radioactive iodine-contaminated subsurface environments. Alternative iodine remediation technologies include microbial reduction of IO₃ ^- to iodide (I^-) and microbial methylation of I^- to volatile gases. The metal reduction pathway is required for anaerobic IO₃ ^- respiration by the gammaproteobacterium Shewanella oneidensis. However, the terminal IO₃ ^- reductase and additional enzymes involved in the S. oneidensis IO₃ ^- electron transport chain have not yet been identified. In this study, gene deletion mutants deficient in four extracellular electron conduits (EECs; ΔmtrA, ΔmtrA-ΔmtrDEF, ΔmtrA-ΔdmsEF, ΔmtrA-ΔSO4360) and DMSO reductase (ΔdmsB) of S. oneidensis were constructed and examined for anaerobic IO₃ ^- reduction activity with either 20 mM lactate or formate as an electron donor. IO₃ ^- reduction rate experiments were conducted under anaerobic conditions in defined minimal medium amended with 250 μM IO₃ ^- as anaerobic electron acceptor. Only the ΔmtrA mutant displayed a severe deficiency in IO₃ ^- reduction activity with lactate as the electron donor, which suggested that the EEC-associated decaheme cytochrome was required for lactate-dependent IO₃ ^- reduction. The ΔmtrA-ΔdmsEF triple mutant displayed a severe deficiency in IO₃ ^- reduction activity with formate as the electron donor, whereas ΔmtrA-ΔmtrDEF and ΔmtrA-ΔSO4360 retained moderate IO₃ ^- reduction activity, which suggested that the EEC-associated dimethylsulfoxide (DMSO) reductase membrane-spanning protein DmsE, but not MtrA, was required for formate-dependent IO₃ ^- reduction. Furthermore, gene deletion mutant ΔdmsB (deficient in the extracellular terminal DMSO reductase protein DmsB) and wild-type cells grown with tungsten replacing molybdenum (a required co-factor for DmsA catalytic activity) in defined growth medium were unable to reduce IO₃ ^- with either lactate or formate as the electron donor, which indicated that the DmsAB complex functions as an extracellular IO₃ ^- terminal reductase for both electron donors. Results of this study provide complementary genetic and phenotypic evidence that the extracellular DMSO reductase complex DmsAB of S. oneidensis displays broad substrate specificity and reduces IO₃ ^- as an alternate terminal electron acceptor.

Keywords: DMSO reductase; Shewanella oneidensis; anaerobic respiration; bioremediation; formate metabolism; iodate reduction; molybdopterin; radioactive iodine.

FIGURE 1

Mtr paralogs of extracellular electron conduits (EEC) system responsible for anaerobic respiration in S. oneidensis. PEC, periplasmic electron carrier; IOMP, integral outer membrane β-barrel protein; ETR, extracellular terminal reductase.

FIGURE 2

IO₃^– reduction activity of Shewanella oneidensis wild-type (MR-1) and ΔmtrA, ΔmtrAΔmtrDEF,ΔmtrAΔdmsEF, and ΔmtrAΔSO4360 mutants with IO₃^– as the electron acceptor and lactate (A) or formate (B) as the electron donor and their IO₃^– reduction rate. Values are means of triplicate samples from anaerobic incubations. Error bars represent one standard deviation.

FIGURE 3

Effect of molybdenum (Mo) substitution with tungsten (W) on IO₃^– reduction activity of Shewanella oneidensis with IO₃^– as the electron acceptor and (A) lactate or (B) formate as the electron donor. Values are means of triplicate samples from anaerobic incubations. Error bars represent one standard deviation.

FIGURE 4

IO₃^– reduction activity of Shewanella oneidensis wild-type (MR-1) and ΔdmsB, ΔdmsB + pBBRdmsB,ΔdmsB + pBBR1MCS, MR-1 + pBBR1MCS strains with IO₃^– as the electron acceptor and (A) lactate or (B) formate as the electron donor and their IO₃^– reduction rate. Values are means of triplicate samples from anaerobic incubations. Error bars represent one standard deviation.

FIGURE 5

Hypothetical working model of the lactate (MtrAB)- and formate (DmsEF)-dependent IO₃^– reduction electron transport pathways in Shewanella oneidensis. In the IO₃^– reduction pathways, electrons originating from lactate dehydrogenase or formate dehydrogenase located at the head end of the electron transport chain are transferred to the inner membrane-localized menaquinone pool and subsequently to CymA, which facilitates electron transfer across the periplasmic space to decaheme c-type cytochromes MtrA or DmsE, respectively. At this location in the electron transport chain, the IO₃^– reduction pathways diverge to MtrAB (lactate as electron donor) or DmsEF (formate as electron donor) and terminate with DmsA and DmsB, both of which associate with MtrA (or DmsE) and β-barrel protein MtrB (or DmsF). DmsA and DmsB are both secreted extracellularly by the type II protein secretion system to form a ternary complex with the MtrAB (or DmsEF) EEC modules on the outside face of the outer membrane (Gralnick et al., 2006).