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.

Abiotic and biotic factors influencing small-scale corn production along a shade spectrum in arid urban agriculture settings.

Urban agriculture may be an avenue to help alleviate strain on the global production of staple crops like corn (Zea mays), but significant knowledge gaps exist regarding the optimization of staple crop production in urban settings, and especially in arid urban settings where different challenges exist for crop success. We sought to assess abiotic and biotic factors that impact sweet corn production in six arid urban agricultural plots with varying levels of shade stress, a known inhibitor of corn production. Corn successfully reached maturity in 50% of the studied plots (n = 18). Microbial richness and diversity were uniformly high in all plot soils and not indicated as a hinderance to corn production nor correlated with corn success. Multiple corn success metrics were positively correlated with average daytime light intensity (r = 0.74 to 0.84) and soil organic matter (r = 0.77 to 0.89), suggesting that these factors are critical aspects of successful corn production. In plots that did not receive optimal light exposure, exceptional soil health and morning vs afternoon sun exposure offset at least some degree of shade stress in these arid urban environments. Corn success metrics were negatively correlated with soil calcium, magnesium, sodium and sulfate (r = -0.71 to -0.90), suggesting that minimizing or mitigating the buildup of salt constituents in soils is critical for successful corn production. Optimizing staple crop production in arid urban agricultural settings supports food chain stability and social and economic security of local communities. This work suggests abiotic and biotic drivers of corn success which can be utilized for crop optimization in these environments.

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.

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.

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.

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.

Subsurface microbial communities as a tool for characterizing regional-scale groundwater flow.

Subsurface microbial community distribution patterns are influenced by biogeochemical and groundwater fluxes and may inform hydraulic connections along groundwater-flow paths. This study examined the regional-scale microbial community of the Death Valley Regional Flow System and evaluated whether subsurface communities can be used to identify groundwater-flow paths between recharge and discharge areas. Samples were collected from 36 sites in three groundwater basins: Pahute Mesa-Oasis Valley (PMOV), Ash Meadows (AM), and Alkali Flat-Furnace Creek Ranch (AFFCR). Microbial diversity within and between communities varied by location, and communities were separated into two overall groups that affiliated with the AM and PMOV/AFFCR basins. Network analysis revealed patterns between clusters of common microbes that represented groundwaters with similar geochemical conditions and largely corroborated hydraulic connections between recharge and discharge areas. Null model analyses identified deterministic and stochastic ecological processes contributing to microbial community assemblages. Most communities were more different than expected and governed by dispersal limitation, geochemical differences, or undominating processes. However, certain communities from sites located within or near the Nevada National Security Site were more similar than expected and dominated by homogeneous dispersal or selection. Overall, the (dis)similarities between the microbial communities of DVRFS recharge and discharge areas supported previously documented hydraulic connections between: (1) Spring Mountains and Ash Meadows; (2) Frenchman and Yucca Flat and Amargosa Desert; and (3) Amargosa Desert and Death Valley. However, only a portion of the flow path between Pahute Mesa and Oasis Valley could be supported by microbial community analyses, likely due to well-associated artifacts in samples from the two Oasis Valley sites. This study demonstrates the utility of combining microbial data with hydrologic, geologic, and water-chemistry information to comprehensively characterize groundwater systems, highlighting both strengths and limitations of this approach.

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.

Evolutionary stasis of a deep subsurface microbial lineage.

Sulfate-reducing bacteria Candidatus Desulforudis audaxviator (CDA) were originally discovered in deep fracture fluids accessed via South African gold mines and have since been found in geographically widespread deep subsurface locations. In order to constrain models for subsurface microbial evolution, we compared CDA genomes from Africa, North America and Eurasia using single cell genomics. Unexpectedly, 126 partial single amplified genomes from the three continents, a complete genome from of an isolate from Eurasia, and metagenome-assembled genomes from Africa and Eurasia shared >99.2% average nucleotide identity, low frequency of SNP's, and near-perfectly conserved prophages and CRISPRs. Our analyses reject sample cross-contamination, recent natural dispersal, and unusually strong purifying selection as likely explanations for these unexpected results. We therefore conclude that the analyzed CDA populations underwent only minimal evolution since their physical separation, potentially as far back as the breakup of Pangea between 165 and 55 Ma ago. High-fidelity DNA replication and repair mechanisms are the most plausible explanation for the highly conserved genome of CDA. CDA presents a stark contrast to the current model organisms in microbial evolutionary studies, which often develop adaptive traits over far shorter periods of time.

Patterns of in situ Mineral Colonization by Microorganisms in a ~60°C Deep Continental Subsurface Aquifer.

The microbial ecology of the deep biosphere is difficult to characterize, owing in part to sampling challenges and poorly understood response mechanisms to environmental change. Pre-drilled wells, including oil wells or boreholes, offer convenient access, but sampling is frequently limited to the water alone, which may provide only a partial view of the native diversity. Mineral heterogeneity demonstrably affects colonization by deep biosphere microorganisms, but the connections between the mineral-associated and planktonic communities remain unclear. To understand the substrate effects on microbial colonization and the community response to changes in organic carbon, we conducted an 18-month series of in situ experiments in a warm (57°C), anoxic, fractured carbonate aquifer at 752 m depth using replicate open, screened cartridges containing different solid substrates, with a proteinaceous organic matter perturbation halfway through this series. Samples from these cartridges were analyzed microscopically and by Illumina (iTag) 16S rRNA gene libraries to characterize changes in mineralogy and the diversity of the colonizing microbial community. The substrate-attached and planktonic communities were significantly different in our data, with some taxa (e.g., Candidate Division KB-1) rare or undetectable in the first fraction and abundant in the other. The substrate-attached community composition also varied significantly with mineralogy, such as with two Rhodocyclaceae OTUs, one of which was abundant on carbonate minerals and the other on silicic substrates. Secondary sulfide mineral formation, including iron sulfide framboids, was observed on two sets of incubated carbonates. Notably, microorganisms were attached to the framboids, which were correlated with abundant Sulfurovum and Desulfotomaculum sp. sequences in our analysis. Upon organic matter perturbation, mineral-associated microbial diversity differences were temporarily masked by the dominance of putative heterotrophic taxa in all samples, including OTUs identified as Caulobacter, Methyloversatilis, and Pseudomonas. Subsequent experimental deployments included a methanogen-dominated stage (Methanobacteriales and Methanomicrobiales) 6 months after the perturbation and a return to an assemblage similar to the pre-perturbation community after 9 months. Substrate-associated community differences were again significant within these subsequent phases, however, demonstrating the value of in situ time course experiments to capture a fraction of the microbial assemblage that is frequently difficult to observe in pre-drilled wells.

The aeroponic rhizosphere microbiome: community dynamics in early succession suggest strong selectional forces.

In the last decade there has been increased interest in the manipulation of rhizosphere microbial communities in soilless systems (hydroponics) through the addition of plant growth promoting microbes (PGPMs) to increase plant nutrition, lower plant stress response, and control pathogens. This method of crop management requires documenting patterns in communities living in plant roots throughout the growing season to inform decisions on timing of application and composition of the supplemental PGPM consortium. As a contribution to this effort, we measured changes in the bacterial community through early succession (first 26 days) in plant root biofilms growing in an indoor commercial aeroponic system where roots were sprayed with a mist of nutrient-amended water. By 12 days following seed germination, a root-associated community had established that was distinct from the source communities found circulating in the system. Successional patterns in the community over the following 2 weeks (12-26 days) included changes in abundance of bacterial groups that have been documented in published literature as able to utilize plant root exudates, release plant hormones, or augment nutrient availability. Six bacterial families/genera (Hydrogenophilaceae, Rhizobium, Legionellaceae, Methylophilus, Massilia, or Herbaspirillum) were the most abundant in each root sample, comprising 8-37% of the microbiome. Given the absence of soil-associated microbial communities in hydroponic systems, they provide an ideal design for isolating plant-microbial interactions and identifying key components possibly contributing to plant health.

Four Draft Single-Cell Genome Sequences of Novel, Nearly Identical Kiritimatiellaeota Strains Isolated from the Continental Deep Subsurface.

The recently proposed bacterial phylum Kiritimatiellaeota represents a globally distributed monophyletic clade distinct from other members of the Planctomycetes, Verrucomicrobia, and Chlamydiae (PVC) superphylum. Here, we present four phylogenetically distinct single-cell genome sequences from within the Kiritimatiellaeota lineage sampled from deep continental subsurface aquifer fluids of the Death Valley Regional Flow System in the United States.

A comparative study of prokaryotic diversity and physicochemical characteristics of Devils Hole and the Ash Meadows Fish Conservation Facility, a constructed analog.

Devils Hole is the sole natural habitat of the critically endangered Devils Hole pupfish (Cyprinodon diabolis). To establish a backup population, the Ash Meadows Fish Conservation Facility (AMFCF), a full-scale replica of the uppermost 6.7 m of Devils Hole, was constructed by management agencies in the mid-2010s. Despite rigorous efforts to mimic the bathymetric and physical details of the Devils Hole environment, the biogeochemistry and microbiology of the AMFCF refuge tank remain largely unaddressed. We evaluated water physicochemistry and employed Illumina DNA sequencing of 16S rRNA gene libraries to evaluate planktonic and benthic bacterial and archaeal community composition within their respective physicochemical contexts in Devils Hole and AMFCF on the same day. Major ion concentrations were consistent between the two systems, but water temperature and dissolved oxygen dynamics differed. Bioavailable nitrogen (primarily nitrate) was 5x lower in AMFCF. Devils Hole and AMFCF nitrogen:phosphorus molar ratios were 107:1 and 22:1, indicative of different nutrient control mechanisms. Both sites are microbiologically diverse, with over 40 prokaryotic phyla represented at each, with 37 shared between them and nearly than half deriving from candidate divisions. The abundance and composition of predicted photosynthetic primary producers (Cyanobacteria) was markedly different between sites: Devils Hole planktonic and sediment communities were dominated by Oscillatoria spp. (13.2% mean relative abundance), which proved virtually undetectable in AMFCF. Conversely, AMFCF was dominated by a predicted heterotroph from the Verrucomicrobiaceae family (31.7%); which was comparatively rare (<2.4%) in Devils Hole. We propose that the paucity of bioavailable nitrogen in AMFCF, perhaps resulting from physical isolation from allochthonous environmental inputs, is reflected in the microbial assemblage disparity, influences biogeochemical cycling of other dissolved constituents, and may ultimately impact survivorship and recruitment of refuge populations of the Devils Hole pupfish.

Microbial community characterization of ozone-biofiltration systems in drinking water and potable reuse applications.

Microbial community structure in the ozone-biofiltration systems of two drinking water and two wastewater treatment facilities was characterized using 16S rRNA gene sequencing. Collectively, these datasets enabled comparisons by facility, water type (drinking water, wastewater), pre-oxidation (ozonation, chlorination), media type (anthracite, activated carbon), media depth, and backwash dynamics. Proteobacteria was the most abundant phylum in drinking water filters, whereas Bacteroidetes, Chloroflexi, Firmicutes, and Planctomycetes were differentially abundant in wastewater filters. A positive correlation was observed between media depth and relative abundance of Cyanobacteria in drinking water filters, but there was only a slight increase in one alpha diversity metric with depth in the wastewater filters. Media type had a significant effect on beta but not alpha diversity in drinking water and wastewater filters. Pre-ozonation caused a significant decrease in alpha diversity in the wastewater filters, but the effect on beta diversity was not statistically significant. An evaluation of backwash dynamics resulted in two notable observations: (1) endosymbionts such as Neochlamydia and Legionella increased in relative abundance following backwashing and (2) nitrogen-fixing Bradyrhizobium dominated the microbial community in wastewater filters operated with infrequent backwashing. Bradyrhizobium is known to generate extracellular polymeric substances (EPS), which may adversely impact biofilter performance and effluent water quality. These findings have important implications for public health and the operation and resiliency of biofiltration systems.

Association between degradation of pharmaceuticals and endocrine-disrupting compounds and microbial communities along a treated wastewater effluent gradient in Lake Mead.

The role of microbial communities in the degradation of trace organic contaminants in the environment is little understood. In this study, the biotransformation potential of 27 pharmaceuticals and endocrine-disrupting compounds was examined in parallel with a characterization of the native microbial community in water samples from four sites variously impacted by urban run-off and wastewater discharge in Lake Mead, Nevada and Arizona, USA. Samples included relatively pristine Colorado River water at the upper end of the lake, nearly pure tertiary-treated municipal wastewater entering via the Las Vegas Wash, and waters of mixed influence (Las Vegas Bay and Boulder Basin), which represented a gradient of treated wastewater effluent impact. Microbial diversity analysis based on 16S rRNA gene censuses revealed the community at this site to be distinct from the less urban-impacted locations, although all sites were similar in overall diversity and richness. Similarly, Biolog EcoPlate assays demonstrated that the microbial community at Las Vegas Wash was the most metabolically versatile and active. Organic contaminants added as a mixture to laboratory microcosms were more rapidly and completely degraded in the most wastewater-impacted sites (Las Vegas Wash and Las Vegas Bay), with the majority exhibiting shorter half-lives than at the other sites or in a bacteriostatic control. Although the reasons for enhanced degradation capacity in the wastewater-impacted sites remain to be established, these data are consistent with the acclimatization of native microorganisms (either through changes in community structure or metabolic regulation) to effluent-derived trace contaminants. This study suggests that in urban, wastewater-impacted watersheds, prior exposure to organic contaminants fundamentally alters the structure and function of microbial communities, which in turn translates into greater potential for the natural attenuation of these compounds compared to more pristine sites.