Extracellular organic disulfide reduction by Shewanella oneidensis MR-1.

Microbial reduction of organic disulfides affects the macromolecular structure and chemical reactivity of natural organic matter. Currently, the enzymatic pathways that mediate disulfide bond reduction in soil and sedimentary organic matter are poorly understood. In this study, we examined the extracellular reduction of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) by Shewanella oneidensis strain MR-1. A transposon mutagenesis screen performed with S. oneidensis resulted in the isolation of a mutant that lost ~90% of its DTNB reduction activity. Genome sequencing of the mutant strain revealed that the transposon was inserted into the dsbD gene, which encodes for an oxidoreductase involved in cytochrome c maturation. Complementation of the mutant strain with the wild-type dsbD partially restored DTNB reduction activity. Because DsbD catalyzes a critical step in the assembly of multi-heme c-type cytochromes, we further investigated the role of extracellular electron transfer cytochromes in organic disulfide reduction. The results indicated that mutants lacking proteins in the Mtr system were severely impaired in their ability to reduce DTNB. These findings provide new insights into extracellular organic disulfide reduction and the enzymatic pathways of organic sulfur redox cycling.IMPORTANCEOrganic sulfur compounds in soils and sediments are held together by disulfide bonds. This study investigates how Shewanella oneidensis breaks apart extracellular organic sulfur compounds. The results show that an enzyme involved in the assembly of c-type cytochromes as well as proteins in the Mtr respiratory pathway is needed for S. oneidensis to transfer electrons from the cell surface to extracellular organic disulfides. These findings have important implications for understanding how organic sulfur decomposes in terrestrial ecosystems.

Comparison of phage-derived recombinases for genetic manipulation of Pseudomonas species.

IMPORTANCE: The Pseudomonas genus contains many members currently being investigated for applications in biodegradation, biopesticides, biocontrol, and synthetic biology. Though several strains have been identified with beneficial properties, chromosomal manipulations to further improve these strains for commercial applications have been limited due to the lack of efficient genetic tools that have been tested across this genus. Here, we test the recombineering efficiencies of five phage-derived recombinases across three biotechnologically relevant Pseudomonas strains: P. putida KT2440, P. protegens Pf-5, and P. protegens CHA0. These results demonstrate a method to generate targeted mutations quickly and efficiently across these strains, ideally introducing a method that can be implemented across the Pseudomonas genus and a strategy that may be applied to develop analogous systems in other nonmodel bacteria.

The Outer Membrane Cytochrome OmcA Is Essential for Infection of Shewanella oneidensis by a Zebrafish-Associated Bacteriophage.

The microbiota-the mixture of microorganisms in the intestinal tract of animals-plays an important role in host biology. Bacteriophages are a prominent, though often overlooked, component of the microbiota. The mechanisms that phage use to infect susceptible cells associated with animal hosts, and the broader role they could play in determining the substituents of the microbiota, are poorly understood. In this study, we isolated a zebrafish-associated bacteriophage, which we named Shewanella phage FishSpeaker. This phage infects Shewanella oneidensis strain MR-1, which cannot colonize zebrafish, but it is unable to infect Shewanella xiamenensis strain FH-1, a strain isolated from the zebrafish gut. Our data suggest that FishSpeaker uses the outer membrane decaheme cytochrome OmcA, which is an accessory component of the extracellular electron transfer (EET) pathway in S. oneidensis, as well as the flagellum to recognize and infect susceptible cells. In a zebrafish colony that lacks detectable FishSpeaker, we found that most Shewanella spp. are sensitive to infection and that some strains are resistant to infection. Our results suggest that phage could act as a selectivity filter for zebrafish-associated Shewanella and show that the EET machinery can be targeted by phage in the environment. IMPORTANCE Phage exert selective pressure on bacteria that influences and shapes the composition of microbial populations. However, there is a lack of native, experimentally tractable systems for studying how phage influence microbial population dynamics in complex communities. Here, we show that a zebrafish-associated phage requires both the outer membrane-associated extracellular electron transfer protein OmcA and the flagellum to infect Shewanella oneidensis strain MR-1. Our results suggest that the newly discovered phage-FishSpeaker-could exert selective pressure that restricts which Shewanella spp. colonize zebrafish. Moreover, the requirement of OmcA for infection by FishSpeaker suggests that the phage preferentially infects cells that are oxygen limited, a condition required for OmcA expression and an ecological feature of the zebrafish gut.

Reconstructing electron transfer components from an Fe(II) oxidizing bacterium.

Neutrophilic Fe(II) oxidizing bacteria play an important role in biogeochemical processes and have also received attention for multiple technological applications. These micro-organisms are thought to couple their metabolism with extracellular electron transfer (EET) while oxidizing Fe(II) as electron donor outside the cell. Sideroxydans lithotrophicus ES-1 is a freshwater chemolithoautotrophic Fe(II) oxidizing bacterium that is challenging to culture and not yet genetically tractable. Analysis of the S. lithotrophicus ES-1 genome predicts multiple EET pathways, which are proposed to be involved in Fe(II) oxidation, but not yet validated. Here we expressed components of two of the proposed EET pathways, including the Mto and Slit_0867-0870 PCC3 pathways, from S. lithotrophicus ES-1 into Aeromonas hydrophila, an established model EET organism. We demonstrate that combinations of putative inner membrane and periplasmic components from the Mto and Slit_0867-0870 PCC3 pathways partially complemented EET activity in Aeromonas mutants lacking native components. Our results provide evidence for electron transfer functionality and interactions of inner membrane and periplasmic components from the Mto and Slit_0867-0870 PCC3 pathways. Based on these findings, we suggest that EET in S. lithotrophicus ES-1 could be more complicated than previously considered and raises questions regarding directionality of these electron transfer pathways.

Evidence for Quinol Oxidation Activity of ImoA, a Novel NapC/NirT Family Protein from the Neutrophilic Fe(II)-Oxidizing Bacterium Sideroxydans lithotrophicus ES-1.

Sideroxydans species are important chemolithoautotrophic Fe(II)-oxidizing bacteria in freshwater environments and play a role in biogeochemical cycling of multiple elements. Due to difficulties in laboratory cultivation and genetic intractability, the electron transport proteins required for the growth and survival of this organism remain understudied. In Sideroxydans lithotrophicus ES-1, it is proposed that the Mto pathway transfers electrons from extracellular Fe(II) oxidation across the periplasm to an inner membrane NapC/NirT family protein encoded by Slit_2495 to reduce the quinone pool. Based on sequence similarity, Slit_2495 has been putatively called CymA, a NapC/NirT family protein which in Shewanella oneidensis MR-1 oxidizes the quinol pool during anaerobic respiration of a wide range of substrates. However, our phylogenetic analysis using the alignment of different NapC/NirT family proteins shows that Slit_2495 clusters closer to NirT sequences than to CymA. We propose the name ImoA (inner membrane oxidoreductase) for Slit_2495. Our data demonstrate that ImoA can oxidize quinol pools in the inner membrane and is able to functionally replace CymA in S. oneidensis. The ability of ImoA to oxidize quinol in vivo as opposed to its proposed function of reducing quinone raises questions about the directionality and/or reversibility of electron flow through the Mto pathway in S. lithotrophicus. IMPORTANCE Fe(II)-oxidizing bacteria play an important role in biogeochemical cycles. At circumneutral pH, these organisms perform extracellular electron transfer, taking up electrons from Fe(II) outside the cell, potentially through a porin-cytochrome complex in the outer membrane encoded by the Mto pathway. Electrons from Fe(II) oxidation would then be transported to the quinone pool in the inner membrane via periplasmic and inner membrane electron transfer proteins. Directly demonstrating the functionality of genes in neutrophilic iron oxidizers is challenging due to the absence of robust genetic methods. Here, we heterologously expressed a NapC/NirT family tetraheme cytochrome ImoA, encoded by Slit_2495, an inner membrane protein from the Gram-negative Fe(II)-oxidizing bacterium Sideroxydans lithotrophicus ES-1, proposed to be involved in extracellular electron transfer to reduce the quinone pool. ImoA functionally replaced the inner membrane c-type cytochrome CymA in the Fe(III)-reducing bacterium Shewanella oneidensis. We suggest that ImoA may function primarily to oxidize quinol in S. lithotrophicus.

PioABC-Dependent Fe(II) Oxidation during Photoheterotrophic Growth on an Oxidized Carbon Substrate Increases Growth Yield.

Microorganisms that carry out Fe(II) oxidation play a major role in biogeochemical cycling of iron in environments with low oxygen. Fe(II) oxidation has been largely studied in the context of autotrophy. Here, we show that the anoxygenic phototroph, Rhodopseudomonas palustris CGA010, carries out Fe(II) oxidation during photoheterotrophic growth with an oxidized carbon source, malate, leading to an increase in cell yield and allowing more carbon to be directed to cell biomass. We probed the regulatory basis for this by transcriptome sequencing (RNA-seq) and found that the expression levels of the known pioABC Fe(II) oxidation genes in R. palustris depended on the redox-sensing two-component system, RegSR, and the oxidation state of the carbon source provided to cells. This provides the first mechanistic demonstration of mixotrophic growth involving reducing power generated from both Fe(II) oxidation and carbon assimilation. IMPORTANCE The simultaneous use of carbon and reduced metals such as Fe(II) by bacteria is thought to be widespread in aquatic environments, and a mechanistic description of this process could improve our understanding of biogeochemical cycles. Anoxygenic phototrophic bacteria like Rhodopseudomonas palustris typically use light for energy and organic compounds as both a carbon and an electron source. They can also use CO2 for carbon by carbon dioxide fixation when electron-rich compounds like H2, thiosulfate, and Fe(II) are provided as electron donors. Here, we show that Fe(II) oxidation can be used in another context to promote higher growth yields of R. palustris when the oxidized carbon compound malate is provided. We further established the regulatory mechanism underpinning this observation.

Light-Induced Patterning of Electroactive Bacterial Biofilms.

Electroactive bacterial biofilms can function as living biomaterials that merge the functionality of living cells with electronic components. However, the development of such advanced living electronics has been challenged by the inability to control the geometry of electroactive biofilms relative to solid-state electrodes. Here, we developed a lithographic strategy to pattern conductive biofilms of Shewanella oneidensis by controlling aggregation protein CdrAB expression with a blue light-induced genetic circuit. This controlled deposition enabled S. oneidensis biofilm patterning on transparent electrode surfaces, and electrochemical measurements allowed us to both demonstrate tunable conduction dependent on pattern size and quantify the intrinsic conductivity of the living biofilms. The intrinsic biofilm conductivity measurements enabled us to experimentally confirm predictions based on simulations of a recently proposed collision-exchange electron transport mechanism. Overall, we developed a facile technique for controlling electroactive biofilm formation on electrodes, with implications for both studying and harnessing bioelectronics.

The potential for bacteria from carbon-limited deep terrestrial environments to participate in chlorine cycling.

Bacteria capable of dehalogenation via reductive or hydrolytic pathways are ubiquitous. Little is known, however, about the prevalence of bacterial dechlorination in deep terrestrial environments with a limited carbon supply. In this study we analyzed published genomes from three deep terrestrial subsurface sites: a deep aquifer in Western Siberia, the Sanford Underground Research Facility in South Dakota, USA, and the Soudan Underground Iron Mine (SUIM) in Minnesota, USA to determine if there was evidence to suggest that microbial dehalogenation was possible in these environments. Diverse dehalogenase genes were present in all analyzed metagenomes, with reductive dehalogenase and haloalkane dehalogenase genes the most common. Taxonomic analysis of both hydrolytic and reductive dehalogenase genes was performed to explore their affiliation; this analysis indicated that at the SUIM site, hydrolytic dehalogenase genes were taxonomically affiliated with Marinobacter species. Because of this affiliation, experiments were also performed with Marinobacter subterrani strain JG233 ('JG233'), an organism containing three predicted hydrolytic dehalogenase genes and isolated from the SUIM site, to determine whether hydrolytic dehalogenation was an active process and involved in growth on a chlorocarboxylic acid. Presence of these genes in genome appears to be functional, as JG233 was capable of chloroacetate dechlorination with simultaneous chloride release. Stable isotope experiments combined with confocal Raman microspectroscopy demonstrated that JG233 incorporated carbon from 13C-chloroacetate into its biomass. These experiments suggest that organisms present in these extreme and often low-carbon environments are capable of reductive and hydrolytic dechlorination and, based on laboratory experiments, may use this capability as a competitive advantage by utilizing chlorinated organic compounds for growth, either directly or after dechlorination.

Engineering Wired Life: Synthetic Biology for Electroactive Bacteria.

Electroactive bacteria produce or consume electrical current by moving electrons to and from extracellular acceptors and donors. This specialized process, known as extracellular electron transfer, relies on pathways composed of redox active proteins and biomolecules and has enabled technologies ranging from harvesting energy on the sea floor, to chemical sensing, to carbon capture. Harnessing and controlling extracellular electron transfer pathways using bioengineering and synthetic biology promises to heighten the limits of established technologies and open doors to new possibilities. In this review, we provide an overview of recent advancements in genetic tools for manipulating native electroactive bacteria to control extracellular electron transfer. After reviewing electron transfer pathways in natively electroactive organisms, we examine lessons learned from the introduction of extracellular electron transfer pathways into Escherichia coli. We conclude by presenting challenges to future efforts and give examples of opportunities to bioengineer microbes for electrochemical applications.

Oligo-heterotrophic Activity of Marinobacter subterrani Creates an Indirect Fe(II) Oxidation Phenotype in Gradient Tubes.

Autotrophic bacteria utilizing Fe(II) as their energy and electron sources for growth affect multiple biogeochemical cycles. Some chemoheterotrophic bacteria have also been considered to exhibit an Fe(II) oxidation phenotype. For example, several Marinobacter strains have been reported to oxidize Fe(II) based on formation of oxidized iron bands in semi-solid gradient tubes that produce opposing concentration gradients of Fe(II) and oxygen. While gradient tubes are a simple and visually compelling method to test for Fe(II) oxidation, this method alone cannot confirm if, and to what extent, Fe(II) oxidation is linked to metabolism in chemoheterotrophic bacteria. Here we probe the possibility of protein-mediated and metabolic by-product-mediated Fe(II) oxidation in Marinobacter subterrani JG233, a chemoheterotroph previously proposed to oxidize Fe(II). Results from conditional and mutant studies, along with measurements of Fe(II) oxidation rates, suggest M. subterrani is unlikely to facilitate Fe(II) oxidation under microaerobic conditions. We conclude that the Fe(II) oxidation phenotype observed in gradient tubes inoculated with M. subterrani JG233 is a result of oligo-heterotrophic activity, shifting the location where oxygen dependent chemical Fe(II) oxidation occurs, rather than a biologically mediated process. IMPORTANCE Gradient tubes are the most commonly used method to isolate and identify neutrophilic Fe(II)-oxidizing bacteria. The formation of oxidized iron bands in gradient tubes provides a compelling assay to ascribe the ability to oxidize Fe(II) to autotrophic bacteria whose growth is dependent on Fe(II) oxidation. However, the physiological significance of Fe(II) oxidation in chemoheterotrophic bacteria is less well understood. Our work suggests that oligo-heterotrophic activity of certain bacteria may create a false-positive phenotype in gradient tubes by altering the location of the abiotic, oxygen-mediated oxidized iron band. Based on the results and analysis presented here, we caution against utilizing gradient tubes as the sole evidence for the capability of a strain to oxidize Fe(II) and that additional experiments are necessary to ascribe this phenotype to new isolates.

Engineering Biological Electron Transfer and Redox Pathways for Nanoparticle Synthesis.

Many species of bacteria are naturally capable of types of electron transport not observed in eukaryotic cells. Some species live in environments containing heavy metals not typically encountered by cells of multicellular organisms, such as arsenic, cadmium, and mercury, leading to the evolution of enzymes to deal with these environmental toxins. Bacteria also inhabit a variety of extreme environments, and are capable of respiration even in the absence of oxygen as a terminal electron acceptor. Over the years, several of these exotic redox and electron transport pathways have been discovered and characterized in molecular-level detail, and more recently synthetic biology has begun to utilize these pathways to engineer cells capable of detecting and processing a variety of metals and semimetals. One such application is the biologically controlled synthesis of nanoparticles. This review will introduce the basic concepts of bacterial metal reduction, summarize recent work in engineering bacteria for nanoparticle production, and highlight the most cutting-edge work in the characterization and application of bacterial electron transport pathways.

Evidence of a Streamlined Extracellular Electron Transfer Pathway from Biofilm Structure, Metabolic Stratification, and Long-Range Electron Transfer Parameters.

A strain of Geobacter sulfurreducens, an organism capable of respiring solid extracellular substrates, lacking four of five outer membrane cytochrome complexes (extABCD+ strain) grows faster and produces greater current density than the wild type grown under identical conditions. To understand cellular and biofilm modifications in the extABCD+ strain responsible for this increased performance, biofilms grown using electrodes as terminal electron acceptors were sectioned and imaged using electron microscopy to determine changes in thickness and cell density, while parallel biofilms incubated in the presence of nitrogen and carbon isotopes were analyzed using NanoSIMS (nanoscale secondary ion mass spectrometry) to quantify and localize anabolic activity. Long-distance electron transfer parameters were measured for wild-type and extABCD+ biofilms spanning 5-µm gaps. Our results reveal that extABCD+ biofilms achieved higher current densities through the additive effects of denser cell packing close to the electrode (based on electron microscopy), combined with higher metabolic rates per cell compared to the wild type (based on increased rates of 15N incorporation). We also observed an increased rate of electron transfer through extABCD+ versus wild-type biofilms, suggesting that denser biofilms resulting from the deletion of unnecessary multiheme cytochromes streamline electron transfer to electrodes. The combination of imaging, physiological, and electrochemical data confirms that engineered electrogenic bacteria are capable of producing more current per cell and, in combination with higher biofilm density and electron diffusion rates, can produce a higher final current density than the wild type. IMPORTANCE Current-producing biofilms in microbial electrochemical systems could potentially sustain technologies ranging from wastewater treatment to bioproduction of electricity if the maximum current produced could be increased and current production start-up times after inoculation could be reduced. Enhancing the current output of microbial electrochemical systems has been mostly approached by engineering physical components of reactors and electrodes. Here, we show that biofilms formed by a Geobacter sulfurreducens strain producing ∼1.4× higher current than the wild type results from a combination of denser cell packing and higher anabolic activity, enabled by an increased rate of electron diffusion through the biofilms. Our results confirm that it is possible to engineer electrode-specific G. sulfurreducens strains with both faster growth on electrodes and streamlined electron transfer pathways for enhanced current production.

Survival of the first rather than the fittest in a Shewanella electrode biofilm.

For natural selection to operate there must exist heritable variation among individuals that affects their survival and reproduction. Among free-living microbes, where differences in growth rates largely define selection intensities, competitive exclusion is common. However, among surface attached communities, these dynamics become less predictable. If extreme circumstances were to dictate that a surface population is immortal and all offspring must emigrate, the offspring would be unable to contribute to the composition of the population. Meanwhile, the immortals, regardless of reproductive capacity, would remain unchanged in relative abundance. The normal cycle of birth, death, and competitive exclusion would be broken. We tested whether conditions required to set up this idealized scenario can be approximated in a microbial biofilm. Using two differentially-reproducing strains of Shewanella oneidensis grown on an anode as the sole terminal electron acceptor - a system in which metabolism is obligately tied to surface attachment - we found that selection against a slow-growing competitor is drastically reduced. This work furthers understanding of natural selection dynamics in sessile microbial communities, and provides a framework for designing stable microbial communities for industrial and experimental applications.

Novel Microbial Groups Drive Productivity in an Archean Iron Formation.

Deep subsurface environments are decoupled from Earth's surface processes yet diverse, active, and abundant microbial communities thrive in these isolated environments. Microbes inhabiting the deep biosphere face unique challenges such as electron donor/acceptor limitations, pore space/fracture network limitations, and isolation from other microbes within the formation. Of the few systems that have been characterized, it is apparent that nutrient limitations likely facilitate diverse microbe-microbe interactions (i.e., syntrophic, symbiotic, or parasitic) and that these interactions drive biogeochemical cycling of major elements. Here we describe microbial communities living in low temperature, chemically reduced brines at the Soudan Underground Mine State Park, United States. The Soudan Iron mine intersects a massive hematite formation at the southern extent of the Canadian Shield. Fractured rock aquifer brines continuously flow from exploratory boreholes drilled circa 1960 and are enriched in deuterium compared to the global meteoric values, indicating brines have had little contact with surface derived waters, and continually degas low molecular weight hydrocarbons C1-C4. Microbial enrichments suggest that once brines exit the boreholes, oxidation of the hydrocarbons occur. Amplicon sequencing show these borehole communities are low in diversity and dominated by Firmicute and Proteobacteria phyla. From the metagenome assemblies, we recovered approximately thirty genomes with estimated completion over 50%. Analysis of genome taxonomy generally followed the amplicon data, and highlights that several of the genomes represent novel families and genera. Metabolic reconstruction shows two carbon-fixation pathways were dominant, the Wood-Ljungdahl (acetogenesis) and Calvin-Benson-Bassham (via RuBisCo), indicating that inorganic carbon likely enters into the microbial foodweb with differing carbon fractionation potentials. Interestingly, methanogenesis is likely driven by Methanolobus and suggests cycling of methylated compounds and not H2/CO2 or acetate. Furthermore, the abundance of sulfate in brines suggests cryptic sulfur cycling may occur, as we detect possible sulfate reducing and thiosulfate oxidizing microorganisms. Finally, a majority of the microorganisms identified contain genes that would allow them to participate in several element cycles, highlighting that in these deep isolated systems metabolic flexibility may be an important life history trait.

Engineering Cooperation in an Anaerobic Coculture.

Over the past century, microbiologists have studied organisms in pure culture, yet it is becoming increasingly apparent that the majority of biological processes rely on multispecies cooperation and interaction. While little is known about how such interactions permit cooperation, even less is known about how cooperation arises. To study the emergence of cooperation in the laboratory, we constructed both a commensal community and an obligate mutualism using the previously noninteracting bacteria Shewanella oneidensis and Geobacter sulfurreducens Incorporation of a glycerol utilization plasmid (pGUT2) enabled S. oneidensis to metabolize glycerol and produce acetate as a carbon source for G. sulfurreducens, establishing a cross-feeding, commensal coculture. In the commensal coculture, both species coupled oxidative metabolism to the respiration of fumarate as the terminal electron acceptor. Deletion of the gene encoding fumarate reductase in the S. oneidensis/pGUT2 strain shifted the coculture with G. sulfurreducens to an obligate mutualism where neither species could grow in the absence of the other. A shift in metabolic strategy from glycerol catabolism to malate metabolism was associated with obligate coculture growth. Further targeted deletions in malate uptake and acetate generation pathways in S. oneidensis significantly inhibited coculture growth with G. sulfurreducens The engineered coculture between S. oneidensis and G. sulfurreducens provides a model laboratory system to study the emergence of cooperation in bacterial communities, and the shift in metabolic strategy observed in the obligate coculture highlights the importance of genetic change in shaping microbial interactions in the environment.IMPORTANCE Microbes seldom live alone in the environment, yet this scenario is approximated in the vast majority of pure-culture laboratory experiments. Here, we develop an anaerobic coculture system to begin understanding microbial physiology in a more complex setting but also to determine how anaerobic microbial communities can form. Using synthetic biology, we generated a coculture system where the facultative anaerobe Shewanella oneidensis consumes glycerol and provides acetate to the strict anaerobe Geobacter sulfurreducens In the commensal system, growth of G. sulfurreducens is dependent on the presence of S. oneidensis To generate an obligate coculture, where each organism requires the other, we eliminated the ability of S. oneidensis to respire fumarate. An unexpected shift in metabolic strategy from glycerol catabolism to malate metabolism was observed in the obligate coculture. Our work highlights how metabolic landscapes can be expanded in multispecies communities and provides a system to evaluate the evolution of cooperation under anaerobic conditions.

Engineering lithoheterotrophy in an obligate chemolithoautotrophic Fe(II) oxidizing bacterium.

Neutrophilic Fe(II) oxidizing bacteria like Mariprofundus ferrooxydans are obligate chemolithoautotrophic bacteria that play an important role in the biogeochemical cycling of iron and other elements in multiple environments. These bacteria generally exhibit a singular metabolic mode of growth which prohibits comparative "omics" studies. Furthermore, these bacteria are considered non-amenable to classical genetic methods due to low cell densities, the inability to form colonies on solid medium, and production of copious amounts of insoluble iron oxyhydroxides as their metabolic byproduct. Consequently, the molecular and biochemical understanding of these bacteria remains speculative despite the availability of substantial genomic information. Here we develop the first genetic system in neutrophilic Fe(II) oxidizing bacterium and use it to engineer lithoheterotrophy in M. ferrooxydans, a metabolism that has been speculated but not experimentally validated. This synthetic biology approach could be extended to gain physiological understanding and domesticate other bacteria that grow using a single metabolic mode.

Evidence for Horizontal and Vertical Transmission of Mtr-Mediated Extracellular Electron Transfer among the Bacteria.

Some bacteria and archaea have evolved the means to use extracellular electron donors and acceptors for energy metabolism, a phenomenon broadly known as extracellular electron transfer (EET). One such EET mechanism is the transmembrane electron conduit MtrCAB, which has been shown to transfer electrons derived from metabolic substrates to electron acceptors, like Fe(III) and Mn(IV) oxides, outside the cell. Although most studies of MtrCAB-mediated EET have been conducted in Shewanella oneidensis MR-1, recent investigations in Vibrio and Aeromonas species have revealed that the electron-donating proteins that support MtrCAB in Shewanella are not as representative as previously thought. This begs the question of how widespread the capacity for MtrCAB-mediated EET is, the changes it has accrued in different lineages, and where these lineages persist today. Here, we employed a phylogenetic and comparative genomics approach to identify the MtrCAB system across all domains of life. We found mtrCAB in the genomes of numerous diverse Bacteria from a wide range of environments, and the patterns therein strongly suggest that mtrCAB was distributed through both horizontal and subsequent vertical transmission, and with some cases indicating downstream modular diversification of both its core and accessory components. Our data point to an emerging evolutionary story about metal-oxidizing and -reducing metabolism, demonstrates that this capacity for EET has broad relevance to a diversity of taxa and the biogeochemical cycles they drive, and lays the foundation for further studies to shed light on how this mechanism may have coevolved with Earth's redox landscape. IMPORTANCE While many metabolisms make use of soluble, cell-permeable substrates like oxygen or hydrogen, there are other substrates, like iron or manganese, that cannot be brought into the cell. Some bacteria and archaea have evolved the means to directly "plug in" to such environmental electron reservoirs in a process known as extracellular electron transfer (EET), making them powerful agents of biogeochemical change and promising vehicles for bioremediation and alternative energy. Yet the diversity, distribution, and evolution of EET mechanisms are poorly constrained. Here, we present findings showing that the genes encoding one such EET system (mtrCAB) are present in a broad diversity of bacteria found in a wide range of environments, emphasizing the ubiquity and potential impact of EET in our biosphere. Our results suggest that these genes have been disseminated largely through horizontal transfer, and the changes they have accrued in these lineages potentially reflect adaptations to changing environments.

Electrolocation? The evidence for redox-mediated taxis in Shewanella oneidensis.

Shewanella oneidensis is a dissimilatory metal reducing bacterium and model for extracellular electron transfer (EET), a respiratory mechanism in which electrons are transferred out of the cell. In the last 10 years, migration to insoluble electron acceptors for EET has been shown to be nonrandom and tactic, seemingly in the absence of molecular or energy gradients that typically allow for taxis. As the ability to sense, locate, and respire electrodes has applications in bioelectrochemical technology, a better understanding of taxis in S. oneidensis is needed. While the EET conduits of S. oneidensis have been studied extensively, its taxis pathways and their interplay with EET are not yet understood, making investigation into taxis phenomena nontrivial. Since S. oneidensis is a member of an EET-encoding clade, the genetic circuitry of taxis to insoluble acceptors may be conserved. We performed a bioinformatic analysis of Shewanella genomes to identify S. oneidensis chemotaxis orthologs conserved in the genus. In addition to the previously reported core chemotaxis gene cluster, we identify several other conserved proteins in the taxis signaling pathway. We present the current evidence for the two proposed models of EET taxis, "electrokinesis" and flavin-mediated taxis, and highlight key areas in need of further investigation.

Plasmid copy number variation of a modular vector set in Shewanella oneidensis MR-1.

To advance synthetic biology approaches that utilize S. oneidensis as host for biotechnology applications, we have investigated the variation in plasmid copy number of a modular vector set resulting from distinct origins of replication under different conditions. The replicons yielded a ≈9X-fold range for plasmid copy number variation in S. oneidensis (while the same origins yielded a ≈3X-fold range in Escherichia coli). This provides a sizeable range to control gene expression levels in S. oneidensis for synthetic biology applications. In addition, plasmid harboring the pBBR1 origin resulted in stable copy numbers in S. oneidensis under different conditions (mid-logarithmic, stationary, multi-plasmid). This may enable the realization of synthetic circuits in S. oneidensis where predictable, quantitative behavior is desired (in either single- or double-plasmid contexts).

A Hybrid Extracellular Electron Transfer Pathway Enhances the Survival of Vibrio natriegens.

Vibrio natriegens is the fastest-growing microorganism discovered to date, making it a useful model for biotechnology and basic research. While it is recognized for its rapid aerobic metabolism, less is known about anaerobic adaptations in V. natriegens or how the organism survives when oxygen is limited. Here, we describe and characterize extracellular electron transfer (EET) in V. natriegens, a metabolism that requires movement of electrons across protective cellular barriers to reach the extracellular space. V. natriegens performs extracellular electron transfer under fermentative conditions with gluconate, glucosamine, and pyruvate. We characterized a pathway in V. natriegens that requires CymA, PdsA, and MtrCAB for Fe(III) citrate and Fe(III) oxide reduction, which represents a hybrid of strategies previously discovered in Shewanella and Aeromonas Expression of these V. natriegens genes functionally complemented Shewanella oneidensis mutants. Phylogenetic analysis of the inner membrane quinol dehydrogenases CymA and NapC in gammaproteobacteria suggests that CymA from Shewanella diverged from Vibrionaceae CymA and NapC. Analysis of sequenced Vibrionaceae revealed that the genetic potential to perform EET is conserved in some members of the Harveyi and Vulnificus clades but is more variable in other clades. We provide evidence that EET enhances anaerobic survival of V. natriegens, which may be the primary physiological function for EET in VibrionaceaeIMPORTANCE Bacteria from the genus Vibrio occupy a variety of marine and brackish niches with fluctuating nutrient and energy sources. When oxygen is limited, fermentation or alternative respiration pathways must be used to conserve energy. In sedimentary environments, insoluble oxide minerals (primarily iron and manganese) are able to serve as electron acceptors for anaerobic respiration by microorganisms capable of extracellular electron transfer, a metabolism that enables the use of these insoluble substrates. Here, we identify the mechanism for extracellular electron transfer in Vibrio natriegens, which uses a combination of strategies previously identified in Shewanella and Aeromonas We show that extracellular electron transfer enhanced survival of V. natriegens under fermentative conditions, which may be a generalized strategy among Vibrio spp. predicted to have this metabolism.