Probing cytoplasmic peroxide metabolism in Shewanella oneidensis.

The facultative anaerobe Shewanella oneidensis respires an extensive set of electron acceptors and, as a consequence, can leak electrons to produce reactive oxygen species such as hydrogen peroxide (H2O2). However, the effects of respiration on cytoplasmic redox homeostasis are poorly characterized in comparison. In the present study, the H2O2 sensor HyPer-3 was deployed to interrogate cytoplasmic peroxide levels of both wild-type and gene deletion mutants lacking peroxide scavenging enzymes following exposure to H2O2. HyPer-3 signals were validated in the S. oneidensis wild-type strain and exhibited a dynamic range of 0-250 µM H2O2. As reported by the HyPer-3 sensor, the cytoplasm of H2O2-perturbed mutant strains lacking periplasmic glutathione peroxidase (PgpD) and double deletion mutants lacking catalase (KatB) and bifunctional catalase-peroxidases (KatG1 or KatG2) contained high H2O2 concentrations. The high cytoplasmic H2O2 concentrations correlated with impaired H2O2 removal rates displayed by the mutant strains. Results of the present study provide the first in vivo interrogation of the redox environment of the S. oneidensis cytoplasm with HyPer-3 sensors and indicate that proper redox conditions in minimal growth medium are maintained by the concerted action of both well-known (periplasmic PgpD, cytoplasmic KatB and KatG1) and previously overlooked (cytoplasmic KatG2) peroxidases and catalases.

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

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.

Resistance of perfluorooctanoic acid to degradation by the microbially driven Fenton reaction.

Per- and polyfluoroalkyl substances (PFAS) such as perfluorooctanoic acid (PFOA) have received recent heightened attention as emerging contaminants. Due to widespread application in household products and aqueous film-forming foams, PFAS are globally distributed in the environment, and bioaccumulate in the blood and tissues of mammals including humans. The microbially driven Fenton reaction, a hybrid biotic/abiotic hydroxyl radical (HO•)-generating system, previously degraded a wide variety of persistant organic pollutants. In the present study, the microbially driven Fenton reaction was employed to attempt degradation of PFOA. Batch cultures of the facultatively anaerobic bacteria Shewanella oneidensis were amended with PFOA and Fe(III)-citrate. Under aerobic conditions, S. oneidensis reduced oxygen to hydrogen peroxide (H2O2), while under anaerobic conditions, S. oneidensis reduced Fe(III) to Fe(II). During aerobic-to-anaerobic transition periods, Fe(II) and H2O2 interacted chemically via the Fenton reaction to produce HO• radicals, which in turn interacted with PFOA. Batch reactors were cycled between aerobic and anaerobic phases for four cycles, residual PFOA was extracted via liquid-liquid extraction and analyzed by liquid chromatography combined with tandem mass spectrometry. Unlike degradation of other organic pollutants, PFOA concentrations remained unchanged, which indicated that PFOA was resistant to degradation by the microbially-driven Fenton reaction. Similar to abiotic (purely chemical) Fenton reaction systems, these results most likely reflect the inability of HO• radicals to oxidatively degrade PFOA.

Microbial helpers allow cyanobacteria to thrive in ferruginous waters.

The Great Oxidation Event (GOE) was a rapid accumulation of oxygen in the atmosphere as a result of the photosynthetic activity of cyanobacteria. This accumulation reflected the pervasiveness of O2 on the planet's surface, indicating that cyanobacteria had become ecologically successful in Archean oceans. Micromolar concentrations of Fe2+ in Archean oceans would have reacted with hydrogen peroxide, a byproduct of oxygenic photosynthesis, to produce hydroxyl radicals, which cause cellular damage. Yet, cyanobacteria colonized Archean oceans extensively enough to oxygenate the atmosphere, which likely required protection mechanisms against the negative impacts of hydroxyl radical production in Fe2+ -rich seas. We identify several factors that could have acted to protect early cyanobacteria from the impacts of hydroxyl radical production and hypothesize that microbial cooperation may have played an important role in protecting cyanobacteria from Fe2+ toxicity before the GOE. We found that several strains of facultative anaerobic heterotrophic bacteria (Shewanella) with ROS defence mechanisms increase the fitness of cyanobacteria (Synechococcus) in ferruginous waters. Shewanella species with manganese transporters provided the most protection. Our results suggest that a tightly regulated response to prevent Fe2+ toxicity could have been important for the colonization of ancient ferruginous oceans, particularly in the presence of high manganese concentrations and may expand the upper bound for tolerable Fe2+ concentrations for cyanobacteria.

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

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 ß-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.