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.

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.

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.

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.

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.

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.