Complete genome sequence of iron-oxidizing Stutzerimonas stutzeri strain FeN3W isolated from Catalina Harbor sediment.

Stutzerimonas stutzeri strain FeN3W is an iron-oxidizing bacterium isolated from marine sediment. FeN3W's 5.9 Mb genome encodes complete pathways for glycolysis, gluconeogenesis, TCA cycle, pentose phosphate pathway, and aerobic and anaerobic (nitrate) respiration. The genome contains 32 putative heme-binding proteins predicted to localize to the cell envelope.

Genome sequence of Asaia bogorensis strain SC1 isolated from an Aedes aegypti mosquito crop.

Understanding microbe-host interactions is key to combating disease transmission by mosquitoes. Here, we report the genome sequence of Asaia bogorensis strain SC1 isolated from a human-blood-fed Aedes aegypti mosquito crop. Metabolic pathway characteristics of aerobic respiration were present in the genome, along with multiple putative antibiotic resistance mechanisms.

Complete genome sequence of Halomonas sp. strain M1, a thiosulfate-oxidizing bacterium isolated from a hyperalkaline serpentinizing system, Ney Springs.

We report the full genome sequence of Halomonas sp. strain M1, isolated from a continental high pH serpentinizing spring in northern California, USA. The 3.7 Mb genome has a G + C content of 54.13%, encodes 3,354 protein-coding genes, and provides insights into the metabolic potential for sulfur oxidation.

Targeted rRNA depletion enables efficient mRNA sequencing in diverse bacterial species and complex co-cultures.

IMPORTANCE: Microbes present one of the most diverse sources of biochemistry in nature, and mRNA sequencing provides a comprehensive view of this biological activity by quantitatively measuring microbial transcriptomes. However, efficient mRNA capture for sequencing presents significant challenges in prokaryotes as mRNAs are not poly-adenylated and typically make up less than 5% of total RNA compared with rRNAs that exceed 80%. Recently developed methods for sequencing bacterial mRNA typically rely on depleting rRNA by tiling large probe sets against rRNAs; however, such approaches are expensive, time-consuming, and challenging to scale to varied bacterial species and complex microbial communities. Therefore, we developed EMBR-seq+, a method that requires fewer than 10 short oligonucleotides per rRNA to achieve up to 99% rRNA depletion in diverse bacterial species. Finally, EMBR-seq+ resulted in a deeper view of the transcriptome, enabling systematic quantification of how microbial interactions result in altering the transcriptional state of bacteria within co-cultures.

Complete genome sequence of Roseinatronobacter sp. strain S2, a chemolithoheterotroph isolated from a pH 12 serpentinizing system.

Here, we report the complete genome sequence for Roseinatronobacter sp. S2, a sulfur-oxidizing heterotroph isolated from a serpentinizing system in Northern California. The S2 genome is 4.4 Mb and contains 4,570 protein-encoding genes. This organism contains the genes necessary for sulfur species oxidation and complete ethylmalonyl and pentose phosphate pathways.

Determining resident microbial community members and their correlations with geochemistry in a serpentinizing spring.

Terrestrial serpentinizing systems allow us insight into the realm of alkaliphilic microbial communities driven by geology in a way that is frequently more accessible than their deep subsurface or marine counterparts. However, these systems are also marked by geochemical and microbial community variation due to the interactions of serpentinized fluids with host geology and the surface environment. To separate the transient from the endemic microbes in a hyperalkaline environment, we assessed the Ney Springs terrestrial serpentinizing system microbial community and geochemistry at six time points over the span of a year. Using 16S rRNA gene surveys we observed 93 amplicon sequence variants (ASVs) that were found at every sampling event. This is compared to ~17,000 transient ASVs that were detected only once across the six sampling events. Of the resident community members, 16 of these ASVs were regularly greater than 1% of the community during every sampling period. Additionally, many of these core taxa experienced statistically significant changes in relative abundance with time. Variation in the abundance of some core populations correlated with geochemical variation. For example, members of the Tindallia group, showed a positive correlation with variation in levels of ammonia at the spring. Investigating the metagenome assembled genomes of these microbes revealed evidence of the potential for ammonia generation via Stickland reactions within Tindallia. This observation offers new insight into the origin of high ammonia concentrations (>70 mg/L) seen at this site. Similarly, the abundance of putative sulfur-oxidizing microbes like Thiomicrospira, Halomonas, and a Rhodobacteraceae species could be linked to changes observed in sulfur-oxidation intermediates like tetrathionate and thiosulfate. While these data supports the influence of core microbial community members on a hyperalkaline spring's geochemistry, there is also evidence that subsurface processes affect geochemistry and may impact community dynamics as well. Though the physiology and ecology of these astrobiologically relevant ecosystems are still being uncovered, this work helps identify a stable microbial community that impacts spring geochemistry in ways not previously observed in serpentinizing ecosystems.

Physiologic, Genomic, and Electrochemical Characterization of Two Heterotrophic Marine Sediment Microbes from the Idiomarina Genus.

Extracellular electron transfer (EET), the process that allows microbes to exchange electrons in a redox capacity with solid interfaces such as minerals or electrodes, has been predominantly described in microbes that use iron during respiration. In this work, we characterize the physiology, genome, and electrochemical properties of two obligately heterotrophic marine microbes that were previously isolated from marine sediment cathode enrichments. Phylogenetic analysis of isolate 16S rRNA genes showed two strains, SN11 and FeN1, belonging to the genus Idiomarina. Strain SN11 was found to be nearly identical to I. loihiensis L2-TRT, and strain FeN1 was most closely related to I. maritima 908087T. Each strain had a relatively small genome (~2.8-2.9 MB). Phenotypic similarities among FeN1, SN11, and the studied strains include being Gram-negative, motile, catalase- and oxidase-positive, and rod-shaped. Physiologically, all strains appeared to exclusively use amino acids as a primary carbon source for growth. This was consistent with genomic observations. Each strain contained 17 to 22 proteins with heme-binding motifs. None of these were predicted to be extracellular, although seven were of unknown localization and lacked functional annotation beyond cytochrome. Despite the lack of homology to known EET pathways, both FeN1 and SN11 were capable of sustained electron uptake over time in an electrochemical system linked to respiration. Given the association of these Idiomarina strains with electro-active biofilms in the environment and their lack of autotrophic capabilities, we predict that EET is used exclusively for respiration in these microbes.

Genome-Scale Mutational Analysis of Cathode-Oxidizing Thioclava electrotropha ElOx9T.

Extracellular electron transfer (EET) - the process by which microorganisms transfer electrons across their membrane(s) to/from solid-phase materials - has implications for a wide range of biogeochemically important processes in marine environments. Though EET is thought to play an important role in the oxidation of inorganic minerals by lithotrophic organisms, the mechanisms involved in the oxidation of solid particles are poorly understood. To explore the genetic basis of oxidative EET, we utilized genomic analyses and transposon insertion mutagenesis screens (Tn-seq) in the metabolically flexible, lithotrophic Alphaproteobacterium Thioclava electrotropha ElOx9T. The finished genome of this strain is 4.3 MB, and consists of 4,139 predicted ORFs, 54 contain heme binding motifs, and 33 of those 54 are predicted to localize to the cell envelope or have unknown localizations. To begin to understand the genetic basis of oxidative EET in ElOx9T, we constructed a transposon mutant library in semi-rich media which was comprised of >91,000 individual mutants encompassing >69,000 unique TA dinucleotide insertion sites. The library was subjected to heterotrophic growth on minimal media with acetate and autotrophic oxidative EET conditions on indium tin oxide coated glass electrodes poised at -278 mV vs. SHE or un-poised in an open circuit condition. We identified 528 genes classified as essential under these growth conditions. With respect to electrochemical conditions, 25 genes were essential under oxidative EET conditions, and 29 genes were essential in both the open circuit control and oxidative EET conditions. Though many of the genes identified under electrochemical conditions are predicted to be localized in the cytoplasm and lack heme binding motifs and/or homology to known EET proteins, we identified several hypothetical proteins and poorly characterized oxidoreductases that implicate a novel mechanism(s) for EET that warrants further study. Our results provide a starting point to explore the genetic basis of novel oxidative EET in this marine sediment microbe.

Investigation of microbial metabolisms in an extremely high pH marine-like terrestrial serpentinizing system: Ney Springs.

Ney Springs, a continental serpentinizing spring in northern California, has an exceptionally high reported pH (12.4) for a naturally occurring water source. With high conductivity fluids, it is geochemically more akin to marine serpentinizing systems than other terrestrial locations. Our geochemical analyses also revealed high sulfide concentrations (544 mg/L) and methane emissions (83% volume gas content) relative to other serpentinizing systems. Thermodynamic calculations were used to investigate the potential for substrates resulting from serpentinization to fuel microbial life, and were found to support the energetic feasibility of sulfate reduction, anaerobic methane oxidation, denitrification, and anaerobic sulfide oxidation within this system. Assessment of the microbial community via 16S rRNA taxonomic gene surveys and metagenome sequencing revealed a community composition dominated by poorly characterized members of the Izemoplasmatales and Clostridiales. The genomes of these dominant taxa point to a fermentative lifestyle, though other highly complete (>90%) metagenome assembled genomes support the potential for organisms to perform sulfate reduction, sulfur disproportionation and/or sulfur oxidation (aerobic and anaerobic). Two chemolithoheterotrophs identified in the metagenome, a Halomonas sp. and a Rhodobacteraceae sp., were isolated and shown to oxidize thiosulfate and were capable of growth in conditions up to pH 12.4. Despite being characteristic products of serpentinization reactions, little evidence was seen for hydrogen and methane utilization in the Ney Springs microbial community. Hydrogen is not highly abundant and could be consumed prior to reaching the spring community. Other metabolic strategies may be outcompeted by more energetically favorable heterotrophic or fermentation reactions, or even inhibited by other compounds in the spring such as ammonia. The unique geochemistry of Ney Springs provides an opportunity to study how local geology interacts with serpentinized fluids, while its microbial community can better inform us of the metabolic strategies employed in hyperalkaline environments.

Sediment Disturbance Negatively Impacts Methanogen Abundance but Has Variable Effects on Total Methane Emissions.

Methane emissions from aquatic ecosystems are increasingly recognized as substantial, yet variable, contributions to global greenhouse gas emissions. This is in part due to the challenge of modeling biologic parameters that affect methane emissions from a wide range of sediments. For example, the impacts of fish bioturbation on methane emissions in the literature have been shown to result in a gradient of reduced to enhanced emissions from sediments. However, it is likely that variation in experimental fish density, and consequently the frequency of bioturbation by fish, impacts this outcome. To explore how the frequency of disturbance impacts the levels of methane emissions in our previous work we quantified greenhouse gas emissions in sediment microcosms treated with various frequencies of mechanical disturbance, analogous to different levels of activity in benthic feeding fish. Greenhouse gas emissions were largely driven by methane ebullition and were highest for the intermediate disturbance frequency (disturbance every 7 days). The lowest emissions were for the highest frequency treatment (3 days). This work investigated the corresponding impacts of disturbance treatments on the microbial communities associated with producing methane. In terms of total microbial community structure, no statistical difference was observed in the total community structure of any disturbance treatment (0, 3, 7, and 14 days) or sediment depth (1 and 3 cm) measured. Looking specifically at methanogenic Archaea however, a shift toward greater relative abundance of a putatively oxygen-tolerant methanogenic phylotype (ca. Methanothrix paradoxum) was observed for the highest frequency treatments and at depths impacted by disturbance (1 cm). Notably, quantitative analysis of ca. Methanothrix paradoxum demonstrated no change in abundance, suggesting disturbance negatively and preferentially impacted other methanogen populations, likely through oxygen exposure. This was further supported by a linear decrease in quantitative abundance of methanogens (assessed by qPCR of the mcrA gene), with increased disturbance frequency in bioturbated sediments (1 cm) as opposed to those below the zone of bioturbation (3 cm). However, total methane emissions were not simply a function of methanogen populations and were likely impacted by the residence time of methane in the lower frequency disturbance treatments. Low frequency mechanical disruption results in lower methane ebullition compared to higher frequency treatments, which in turn resulted in reduced overall methane release, likely through enhanced methanotrophic activities, though this could not be identified in this work. Overall, this work contributes to understanding how animal behavior may impact variation in greenhouse gas emissions and provides insight into how frequency of disturbance may impact emissions.

Complete Genome Sequence of Halomonas sp. Strain FeN2, a Novel Cathode-Oxidizing Bacterium Isolated from Catalina Harbor Sediments.

We report the complete, closed, circular genome of Halomonas sp. strain FeN2, a metabolically versatile electrotroph that was isolated from Catalina Harbor sediments. The 4.8-Mb genome contains 4,286 protein-coding genes and has complete glycolytic, tricarboxylic acid, glyoxylate, pentose phosphate, and reductive pentose phosphate pathways. FeN2 also contains genes for aerobic and anaerobic (denitrification) respiration.

Identification of a pathway for electron uptake in Shewanella oneidensis.

Extracellular electron transfer (EET) could enable electron uptake into microbial metabolism for the synthesis of complex, energy dense organic molecules from CO2 and renewable electricity1-6. Theoretically EET could do this with an efficiency comparable to H2-oxidation7,8 but without the need for a volatile intermediate and the problems it causes for scale up9. However, significant gaps remain in understanding the mechanism and genetics of electron uptake. For example, studies of electron uptake in electroactive microbes have shown a role for the Mtr EET complex in the electroactive microbe Shewanella oneidensis MR-110-14, though there is substantial variation in the magnitude of effect deletion of these genes has depending on the terminal electron acceptor used. This speaks to the potential for previously uncharacterized and/or differentially utilized genes involved in electron uptake. To address this, we screened gene disruption mutants for 3667 genes, representing ≈99% of all nonessential genes, from the S. oneidensis whole genome knockout collection using a redox dye oxidation assay. Confirmation of electron uptake using electrochemical testing allowed us to identify five genes from S. oneidensis that are indispensable for electron uptake from a cathode. Knockout of each gene eliminates extracellular electron uptake, yet in four of the five cases produces no significant defect in electron donation to an anode. This result highlights both distinct electron uptake components and an electronic connection between aerobic and anaerobic electron transport chains that allow electrons from the reversible EET machinery to be coupled to different respiratory processes in S. oneidensis. Homologs to these genes across many different genera suggesting that electron uptake by EET coupled to respiration could be widespread. These gene discoveries provide a foundation for: studying this phenotype in exotic metal-oxidizing microbes, genetic optimization of electron uptake in S. oneidensis; and genetically engineering electron uptake into a highly tractable host like E. coli to complement recent advances in synthetic CO2 fixation15.

Electrochemical evidence for in situ microbial activity at the Deep Mine Microbial Observatory (DeMMO), South Dakota, USA.

The subsurface is Earth's largest reservoir of biomass. Micro-organisms are the dominant lifeforms in this habitat, but the nature of their in situ activities remains largely unresolved. At the Deep Mine Microbial Observatory (DeMMO) located in the Sanford Underground Research Facility (SURF) in Lead, South Dakota (USA), we performed in situ electrochemical incubations designed to assess the potential for deep groundwater microbial communities to utilize extracellular electron transfer to support microbial respiration. DeMMO 4 was chosen for its stable geochemistry and microbial community. Graphite and indium tin oxide electrodes poised at -200 mV versus SHE were incubated along with open circuit controls and various minerals in a parallel flow reactor that split access to fluids across different treatments. From the patterns of net current over time (fluctuating between anodic and cathodic currents over the course of a few days to weeks) and the catalytic features measured using periodic cyclic voltammetry, evidence of both oxidative and reductive microbe-electrode interactions was observed. The predominant catalytic activity ranged from -210 to -120 mV. The observed temporal variability in electrochemical activity was unexpected given the documented stability in major geochemical parameters. This suggests that the accessed fluids are more heterogeneous in electrochemically active microbial populations than previously predicted from the stable community composition. As previously reported, the fracture fluid and surface-attached microbial communities at SURF differed significantly. However, only minimal differences in community composition were observed between poised potential electrodes, open circuit electrodes, and mineral incubations. These data support that in this environment the ability to attach to surfaces is a stronger driver of microbial community structure than the type or reactivity of the surface. We demonstrate that insight into specific activities can be gained from electrochemical methods, specifically chronoamperometry coupled with routine cyclic voltammetry, which provide a sensitive approach to evaluate microbial activities in situ.

Differences in Applied Redox Potential on Cathodes Enrich for Diverse Electrochemically Active Microbial Isolates From a Marine Sediment.

The diversity of microbially mediated redox processes that occur in marine sediments is likely underestimated, especially with respect to the metabolisms that involve solid substrate electron donors or acceptors. Though electrochemical studies that utilize poised potential electrodes as a surrogate for solid substrate or mineral interactions have shed some much needed light on these areas, these studies have traditionally been limited to one redox potential or metabolic condition. This work seeks to uncover the diversity of microbes capable of accepting cathodic electrons from a marine sediment utilizing a range of redox potentials, by coupling electrochemical enrichment approaches to microbial cultivation and isolation techniques. Five lab-scale three-electrode electrochemical systems were constructed, using electrodes that were initially incubated in marine sediment at cathodic or electron-donating voltages (five redox potentials between -400 and -750 mV versus Ag/AgCl) as energy sources for enrichment. Electron uptake was monitored in the laboratory bioreactors and linked to the reduction of supplied terminal electron acceptors (nitrate or sulfate). Enriched communities exhibited differences in community structure dependent on poised redox potential and terminal electron acceptor used. Further cultivation of microbes was conducted using media with reduced iron (Fe0, FeCl2) and sulfur (S0) compounds as electron donors, resulting in the isolation of six electrochemically active strains. The isolates belong to the genera Vallitalea of the Clostridia, Arcobacter of the Epsilonproteobacteria, Desulfovibrio of the Deltaproteobacteria, and Vibrio and Marinobacter of the Gammaproteobacteria. Electrochemical characterization of the isolates with cyclic voltammetry yielded a wide range of midpoint potentials (99.20 to -389.1 mV versus Ag/AgCl), indicating diverse metabolic pathways likely support the observed electron uptake. Our work demonstrates culturing under various electrochemical and geochemical regimes allows for enhanced cultivation of diverse cathode-oxidizing microbes from one environmental system. Understanding the mechanisms of solid substrate oxidation from environmental microbes will further elucidation of the ecological relevance of these electron transfer interactions with implications for microbe-electrode technologies.

Methane-Linked Mechanisms of Electron Uptake from Cathodes by Methanosarcina barkeri.

The Methanosarcinales, a lineage of cytochrome-containing methanogens, have recently been proposed to participate in direct extracellular electron transfer interactions within syntrophic communities. To shed light on this phenomenon, we applied electrochemical techniques to measure electron uptake from cathodes by Methanosarcina barkeri, which is an important model organism that is genetically tractable and utilizes a wide range of substrates for methanogenesis. Here, we confirm the ability of M. barkeri to perform electron uptake from cathodes and show that this cathodic current is linked to quantitative increases in methane production. The underlying mechanisms we identified include, but are not limited to, a recently proposed association between cathodes and methanogen-derived extracellular enzymes (e.g., hydrogenases) that can facilitate current generation through the formation of reduced and diffusible methanogenic substrates (e.g., hydrogen). However, after minimizing the contributions of such extracellular enzymes and using a mutant lacking hydrogenases, we observe a lower-potential hydrogen-independent pathway that facilitates cathodic activity coupled to methane production in M. barkeri Our electrochemical measurements of wild-type and mutant strains point to a novel and hydrogenase-free mode of electron uptake with a potential near -484 mV versus standard hydrogen electrode (SHE) (over 100 mV more reduced than the observed hydrogenase midpoint potential under these conditions). These results suggest that M. barkeri can perform multiple modes (hydrogenase-mediated and free extracellular enzyme-independent modes) of electrode interactions on cathodes, including a mechanism pointing to a direct interaction, which has significant applied and ecological implications.IMPORTANCE Methanogenic archaea are of fundamental applied and environmental relevance. This is largely due to their activities in a wide range of anaerobic environments, generating gaseous reduced carbon that can be utilized as a fuel source. While the bioenergetics of a wide variety of methanogens have been well studied with respect to soluble substrates, a mechanistic understanding of their interaction with solid-phase redox-active compounds is limited. This work provides insight into solid-phase redox interactions in Methanosarcina spp. using electrochemical methods. We highlight a previously undescribed mode of electron uptake from cathodes that is potentially informative of direct interspecies electron transfer interactions in the Methanosarcinales.

Self-standing Electrochemical Set-up to Enrich Anode-respiring Bacteria On-site.

Anaerobic respiration coupled with electron transport to insoluble minerals (referred to as extracellular electron transport [EET]) is thought to be critical for microbial energy production and persistence in many subsurface environments, especially those lacking soluble terminal electron acceptors. While EET-capable microbes have been successfully isolated from various environments, the diversity of bacteria capable of EET is still poorly understood, especially in difficult-to-sample, low energy or extreme environments, such as many subsurface ecosystems. Here, we describe an on-site electrochemical system to enrich EET-capable bacteria using an anode as a respiratory terminal electron acceptor. This anode is connected to a cathode capable of catalyzing abiotic oxygen reduction. Comparing this approach with electrocultivation methods that use a potentiostat for poising the electrode potential, the two-electrode system does not require an external power source. We present an example of our on-site enrichment utilized in an alkaline pond at the Cedars, a terrestrial serpentinization site in Northern California. Prior attempts to cultivate mineral reducing bacteria were unsuccessful, which is likely due to the low-biomass nature of this site and/or the low relative abundance of metal reducing microbes. Prior to implementing our two-electrode enrichment, we measured the vertical profile of dissolved oxygen concentration. This allowed us to place the carbon felt anode and platinum-electroplated carbon felt cathode at depths that would support anaerobic and aerobic processes, respectively. Following on-site incubation, we further enriched the anodic electrode in the laboratory and confirmed a distinct microbial community compared to the surface-attached or biofilm communities normally observed at the Cedars. This enrichment subsequently led to the isolation of the first electrogenic microbe from the Cedars. This method of on-site microbial enrichment has the potential to greatly enhance the isolation of EET-capable bacteria from low biomass or difficult to sample habitats.

Variation in electrode redox potential selects for different microorganisms under cathodic current flow from electrodes in marine sediments.

Extracellular electron transport (EET) is a microbial process that allows microorganisms to transport electrons to and from insoluble substrates outside of the cell. Although progress has been made in understanding how microbes transfer electrons to insoluble substrates, the process of receiving electrons has largely remained unexplored. We investigated redox potentials favourable for donating electrons to dissolved and insoluble components in Catalina Harbor marine sediment by combining electrochemical techniques with geochemistry and molecular methods. Working electrodes buried in sediment microcosms were poised at seven redox potentials between -300 and -750 mV versus Ag/AgCl using a three-electrode system. In electrode biofilms recovered after 2-month incubations, overall community diversity increased with more negative redox potentials. Abundances of known EET-capable groups (e.g., Alteromonadales and Desulfuromonadales) varied with redox potential. Motility and chemotaxis genes were found in greater abundance in electrode communities, suggesting a possible selective advantage of these pathways for colonization and utilization of the electrode. Our enrichments demonstrated the validity of this approach in capturing groups known, as well as novel groups (e.g., Campylobacterales) that perform EET. The diverse nature of the enriched cathode communities suggest that insoluble substrate oxidation may be a critical, although poorly described microbial metabolic process in marine sediment.

Tracking Electron Uptake from a Cathode into Shewanella Cells: Implications for Energy Acquisition from Solid-Substrate Electron Donors.

While typically investigated as a microorganism capable of extracellular electron transfer to minerals or anodes, Shewanella oneidensis MR-1 can also facilitate electron flow from a cathode to terminal electron acceptors, such as fumarate or oxygen, thereby providing a model system for a process that has significant environmental and technological implications. This work demonstrates that cathodic electrons enter the electron transport chain of S. oneidensis when oxygen is used as the terminal electron acceptor. The effect of electron transport chain inhibitors suggested that a proton gradient is generated during cathode oxidation, consistent with the higher cellular ATP levels measured in cathode-respiring cells than in controls. Cathode oxidation also correlated with an increase in the cellular redox (NADH/FMNH2) pool determined with a bioluminescence assay, a proton uncoupler, and a mutant of proton-pumping NADH oxidase complex I. This work suggested that the generation of NADH/FMNH2 under cathodic conditions was linked to reverse electron flow mediated by complex I. A decrease in cathodic electron uptake was observed in various mutant strains, including those lacking the extracellular electron transfer components necessary for anodic-current generation. While no cell growth was observed under these conditions, here we show that cathode oxidation is linked to cellular energy acquisition, resulting in a quantifiable reduction in the cellular decay rate. This work highlights a potential mechanism for cell survival and/or persistence on cathodes, which might extend to environments where growth and division are severely limited.IMPORTANCE The majority of our knowledge of the physiology of extracellular electron transfer derives from studies of electrons moving to the exterior of the cell. The physiological mechanisms and/or consequences of the reverse processes are largely uncharacterized. This report demonstrates that when coupled to oxygen reduction, electrode oxidation can result in cellular energy acquisition. This respiratory process has potentially important implications for how microorganisms persist in energy-limited environments, such as reduced sediments under changing redox conditions. From an applied perspective, this work has important implications for microbially catalyzed processes on electrodes, particularly with regard to understanding models of cellular conversion of electrons from cathodes to microbially synthesized products.

Thioclava electrotropha sp. nov., a versatile electrode and sulfur-oxidizing bacterium from marine sediments.

A taxonomic and physiologic characterization was carried out on Thioclava strain ElOx9T, which was isolated from a bacterial consortium enriched on electrodes poised at electron donating potentials. The isolate is Gram-negative, catalase-positive and oxidase-positive; the cells are motile short rods. The bacterium is facultatively anaerobic with the ability to utilize nitrate as an electron acceptor. Autotrophic growth with H2 and S0 (oxidized to sulfate) was observed. The isolate also grows heterotrophically with organic acids and sugars. Growth was observed at salinities from 0 to 10% NaCl and at temperatures from 15 to 41 °C. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain belongs in the genus Thioclava; it had the highest sequence similarity of 98.8 % to Thioclava atlantica 13D2W-2T, followed by Thioclava dalianensis DLFJ1-1T with 98.5 % similarity, Thioclava pacifica TL 2T with 97.7 % similarity, and then Thioclava indica DT23-4T with 96.9 %. All other sequence similarities were below 97 % to characterized strains. The digital DNA-DNA hybridization estimated when compared to T. atlantica 13D2W-2T, T. dalianensis DLFJ1-1T, T. pacifica TL 2T and T. indica DT23-4T were 15.8±2.1, 16.7+2.1, 14.3±1.9 and 18.3±2.1 %. The corresponding average nucleotide identity values between these strains were determined to be 65.1, 67.8, 68.4 and 64.4 %, respectively. The G+C content of the chromosomal DNA is 63.4 mol%. Based on these results, a novel species Thioclava electrotropha sp. nov. is proposed, with the type strain ElOx9T (=DSM 103712T=ATCC TSD-100T).

Biomarkers' Responses to Reductive Dechlorination Rates and Oxygen Stress in Bioaugmentation Culture KB-1TM.

Using mRNA transcript levels for key functional enzymes as proxies for the organohalide respiration (OHR) rate, is a promising approach for monitoring bioremediation populations in situ at chlorinated solvent-contaminated field sites. However, to date, no correlations have been empirically derived for chlorinated solvent respiring, Dehalococcoides mccartyi (DMC) containing, bioaugmentation cultures. In the current study, genome-wide transcriptome and proteome data were first used to confirm the most highly expressed OHR-related enzymes in the bioaugmentation culture, KB-1TM, including several reductive dehalogenases (RDases) and a Ni-Fe hydrogenase, Hup. Different KB-1™ DMC strains could be resolved at the RNA and protein level through differences in the sequence of a common RDase (DET1545-like homologs) and differences in expression of their vinyl chloride-respiring RDases. The dominant strain expresses VcrA, whereas the minor strain utilizes BvcA. We then used quantitative reverse-transcriptase PCR (qRT-PCR) as a targeted approach for quantifying transcript copies in the KB-1TM consortium operated under a range of TCE respiration rates in continuously-fed, pseudo-steady-state reactors. These candidate biomarkers from KB-1TM demonstrated a variety of trends in terms of transcript abundance as a function of respiration rate over the range: 7.7 × 10-12 to 5.9 × 10-10 microelectron equivalents per cell per hour (µeeq/cell∙h). Power law trends were observed between the respiration rate and transcript abundance for the main DMC RDase (VcrA) and the hydrogenase HupL (R² = 0.83 and 0.88, respectively), but not transcripts for 16S rRNA or three other RDases examined: TceA, BvcA or the RDase DET1545 homologs in KB1TM. Overall, HupL transcripts appear to be the most robust activity biomarker across multiple DMC strains and in mixed communities including DMC co-cultures such as KB1TM. The addition of oxygen induced cell stress that caused respiration rates to decline immediately (>95% decline within one hour). Although transcript levels did decline, they did so more slowly than the respiration rate observed (transcript decay rates between 0.02 and 0.03 per hour). Data from strain-specific probes on the pangenome array strains suggest that a minor DMC strain in KB-1™ that harbors a bvcA homolog preferentially recovered following oxygen stress relative to the dominant, vcrA-containing strain.