Metal Reduction and Protein Secretion Genes Required for Iodate Reduction by Shewanella oneidensis

Abstract

The metal-reducing gammaproteobacterium Shewanella oneidensis reduces iodate (IO₃^-) as an anaerobic terminal electron acceptor. Microbial IO₃^- electron transport pathways are postulated to terminate with nitrate (NO₃^-) reductase, which reduces IO₃^- as an alternative electron acceptor. Recent studies with S. oneidensis, however, have demonstrated that NO₃^- reductase is not involved in IO₃^- reduction. The main objective of the present study was to determine the metal reduction and protein secretion genes required for IO₃^- reduction by Shewanella oneidensis with lactate, formate, or H₂ as the electron donor. With all electron donors, the type I and type V protein secretion mutants retained wild-type IO₃^- reduction activity, while the type II protein secretion mutant lacking the outer membrane secretin GspD was impaired in IO₃^- reduction. Deletion mutants lacking the cyclic AMP receptor protein (CRP), cytochrome maturation permease CcmB, and inner membrane-tethered c-type cytochrome CymA were impaired in IO₃^- reduction with all electron donors, while deletion mutants lacking c-type cytochrome MtrA and outer membrane β-barrel protein MtrB of the outer membrane MtrAB module were impaired in IO₃^- reduction with only lactate as an electron donor. With all electron donors, mutants lacking the c-type cytochromes OmcA and MtrC of the metal-reducing extracellular electron conduit MtrCAB retained wild-type IO₃^- reduction activity. These findings indicate that IO₃^- reduction by S. oneidensis involves electron donor-dependent metal reduction and protein secretion pathway components, including the outer membrane MtrAB module and type II protein secretion of an unidentified IO₃^- reductase to the S. oneidensis outer membrane.IMPORTANCE Microbial iodate (IO₃^-) reduction is a major component in the biogeochemical cycling of iodine and the bioremediation of iodine-contaminated environments; however, the molecular mechanism of microbial IO₃^- reduction is poorly understood. Results of the present study indicate that outer membrane (type II) protein secretion and metal reduction genes encoding the outer membrane MtrAB module of the extracellular electron conduit MtrCAB are required for IO₃^- reduction by S. oneidensis On the other hand, the metal-reducing c-type cytochrome MtrC of the extracellular electron conduit is not required for IO₃^- reduction by S. oneidensis These findings indicate that the IO₃^- electron transport pathway terminates with an as yet unidentified IO₃^- reductase that associates with the outer membrane MtrAB module to deliver electrons extracellularly to IO₃^.

Keywords: Shewanella oneidensis; iodate; iodine; metals; reduction.

FIG 1

Effect of IO₃⁻ concentration on IO₃⁻ reduction activity of S. oneidensis MR-1. IO₃⁻ reduction was performed in M1 medium amended with 20 mM lactate and 250 μM IO₃⁻, ranging from 0.1 to 2 mM iodate at room temperature and 300 rpm. Values are means of triplicate samples from anaerobic incubations. Error bars represent SDs. Some error bars cannot be seen due to small SDs. Symbols: ●, 0.10 mM; ■, 0.25 mM; ⧫, 0.50 mM; ▲, 1.00 mM; ▼, 1.50 mM; ○, 2.00 mM.

FIG 2

IO₃⁻ reduction activity of S. oneidensis wild-type (MR-1) and EEC mutant strains with IO₃⁻ as the electron acceptor and lactate, formate, or H₂ as the electron donor and mtrB-CxxC motif mutants and complemented strains of ΔmtrA and ΔmtrB with pBBRmtrA and pBBRmtrB, respectively (A), and with IO₃⁻ as the electron acceptor and lactate as the electron donor (mutant strains normalized to wild-type levels) (B). Values are means from triplicate samples from anaerobic incubations. Error bars represent SDs. Some error bars cannot be seen due to small SDs.

FIG 3

IO₃⁻ reduction activity of S. oneidensis wild-type (MR-1) and c-type cytochrome and crp mutants with IO₃⁻ as the electron acceptor and lactate, formate, or H₂ as the electron donor and their complemented strains with pBBRcymA, pBBRccmB, and pBBRcrp, respectively (A), and with IO₃⁻ as the electron acceptor and lactate as the electron donor (mutant strains normalized to wild-type levels) (B). Values are means of triplicate samples from anaerobic incubations. Error bars represent SDs. Some error bars cannot be seen due to small SDs.

FIG 4

IO₃⁻ reduction activity of S. oneidensis wild-type (MR-1) and ΔtolC, ΔgspD, and ΔSO3800 protein secretion mutants with IO₃⁻ as the electron acceptor and lactate, formate, or H₂ as the electron donor (mutant strains normalized to wild-type levels). Values are means of triplicate samples from anaerobic incubations. Error bars represent SDs.

FIG 5

Working model of the lactate (MtrAB)-dependent IO₃⁻ reduction electron transport pathway in S. oneidensis, including a comparison with the MtrAB-dependent metal reduction pathway. In both the metal and IO₃⁻ reduction pathways, electrons originating from lactate 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 cytochrome MtrA. At this location in the electron transport chain, the metal and IO₃⁻ reduction pathways diverge and terminate with either metal-reducing c-type cytochrome MtrC or an unknown terminal IO₃⁻ reductase, both of which associate with MtrA and β-barrel protein MtrB. MtrC and the unknown IO₃⁻ reductase are both secreted extracellularly by the type II protein secretion system to form a ternary complex with the MtrAB module on the outside face of the outer membrane. The formate- and H₂-dependent IO₃⁻ reduction pathways are MtrAB-independent and thus are not depicted in this working model.