Biology subjects

Stewart, F. J.

Publications and source records attributed to Stewart, F. J..

8 recordsLinked to original sources

bioRxiv2023

Active and abundant alphaproteobacterial nitric oxide transforming enzymes in a marine oxygen deficient zone

Marine oxygen deficient zones (ODZs) are portions of the ocean where intense nitrogen loss occurs primarily via denitrification and anammox. Despite many decades of study, the identity of the microbes that catalyze nitrogen loss in ODZs are still being elucidated. Intriguingly, high transcription of genes in the same family as the nitric oxide dismutase (nod) gene from Methylomirabilota has been reported in the anoxic core of ODZs. Here, we show that the most abundantly transcribed nod genes in the Eastern Tropical North Pacific ODZ belong to a new order (UBA11136) of Alphaproteobacteria, rather than Methylomirabilota as previously assumed. Gammaproteobacteria and Planctomycetia also transcribe nod, but at lower relative abundance than UBA11136 in the upper ODZ. The nod-transcribing Alphaproteobacteria likely use formaldehyde and formate as a source of electrons for aerobic respiration, with additional electrons possibly from sulfide oxidation. They also transcribe multiheme cytochrome (here named ptd) genes for a putative porin-cytochrome protein complex of unknown function, potentially involved in extracellular electron transfer. Molecular oxygen for aerobic respiration may originate from nitric oxide dismutation via cryptic oxygen cycling. Our results implicate Alphaproteobacteria order UBA11136 as a significant player in marine nitrogen loss and highlight their potential in one-carbon, nitrogen, and sulfur metabolism in ODZs. Significance statementIn marine oxygen deficient zones, microbes transform bioavailable nitrogen to gaseous nitrogen, with nitric oxide as a key intermediate. The Eastern Tropical North Pacific contains the worlds largest oxygen deficient zone, but the identity of the microbes transforming nitric oxide remain unknown. Here, we show that highly transcribed nitric oxide dismutase (nod) genes belong to Alphaproteobacteria of the novel order UBA11136, which lacks cultivated isolates. These Alphaproteobacteria show evidence for aerobic respiration, using oxygen potentially sourced from nitric oxide dismutase, and possess a novel porin-cytochrome protein complex with unknown function. Gammaproteobacteria and Planctomycetia transcribe nod at lower levels. Our results pinpoint the microbes mediating a key step in marine nitrogen loss and reveal an unexpected predicted metabolism for marine Alphaproteobacteria.

microbiology↗

bioRxiv2022

Microbial and Viral Genome and Proteome Nitrogen Demand Varies Across Multiple Spatial Scales Within a Marine Oxygen Minimum Zone

Nutrient availability can significantly influence microbial genomic and proteomic streamlining, for example by selecting for lower nitrogen to carbon ratios. Oligotrophic open ocean microbes have streamlined genomic nitrogen requirements relative to their counterparts in nutrient-rich coastal waters. However, steep gradients in nutrient availability occur at meter- and even micron-level spatial scales. It is unclear if such gradients also structure genomic and proteomic stoichiometry. Focusing on the eastern tropical North Pacific oxygen minimum zone (OMZ), we use comparative metagenomics to examine how nitrogen availability shapes microbial and viral genome properties along the vertical gradient across the OMZ and between two size fractions distinguishing free-living versus particle-associated microbes. We find a substantial increase in nitrogen content of encoded proteins in particle-associated over free-living bacteria and archaea across nitrogen availability regimes over depth. Within each size-fraction, we find that bacterial and viral genomic nitrogen tends to increase with increasing nitrate concentrations with depth. In contrast to cellular genes, the nitrogen content of virus proteins does not differ between size fractions. We identified arginine as a key amino acid in modulating the C:N ratio of core genes for bacteria, archaea, and viruses. Functional analysis reveals that particle-associated bacterial metagenomes are enriched for genes involved in arginine metabolism and organic nitrogen compound catabolism. Our results are consistent with nitrogen streamlining in both cellular and viral genomes on spatial scales of meters to microns. These effects are similar in magnitude to those previously reported across scales of thousands of kilometers. IMPORTANCEThe genomes of marine microbes can be shaped by nutrient cycles, with ocean-scale gradients in nitrogen availability known to influence microbial amino acid usage. It is unclear, however, how genomic properties are shaped by nutrient changes over much smaller spatial scales, for example along the vertical transition into oxygen minimum zones (OMZs) or from the exterior to interior of detrital particles. Here, we measure protein nitrogen usage by marine bacteria, archaea, and viruses using metagenomes from the nitracline of the eastern tropical North Pacific OMZ including both particle-associated and non-associated biomass. Our results show higher genomic and proteomic nitrogen content in particle-associated microbes and at depths with higher nitrogen availability for cellular and viral genomes. This discovery suggests that stoichiometry influences microbial and viral evolution across multiple scales, including the micro- to millimeter scale associated with particle-associated versus free-living lifestyles.

microbiology↗

bioRxiv2020

Gulf of Mexico blue hole harbors high levels of novel microbial lineages

Exploration of oxygen-depleted marine environments has consistently revealed novel microbial taxa and metabolic capabilities that expand our understanding of microbial evolution and ecology. Marine blue holes are shallow karst formations characterized by low oxygen and high organic matter content. They are logistically challenging to sample, and thus our understanding of their biogeochemistry and microbial ecology is limited. We present a metagenomic characterization of Amberjack Hole on the Florida continental shelf (Gulf of Mexico). Dissolved oxygen became depleted at the holes rim (32 m water depth), remained low but detectable in an intermediate hypoxic zone (40-75 m), and then increased to a secondary peak before falling below detection in the bottom layer (80-110 m), concomitant with increases in nutrients, dissolved iron, and a series of sequentially more reduced sulfur species. Microbial communities in the bottom layer contained heretofore undocumented levels of the recently discovered phylum Woesearchaeota (up to 58% of the community), along with lineages in the bacterial Candidate Phyla Radiation (CPR). Thirty-one high-quality metagenome-assembled genomes (MAGs) showed extensive biochemical capabilities for sulfur and nitrogen cycling, as well as for resisting and respiring arsenic. One uncharacterized gene associated with a CPR lineage differentiated hypoxic from anoxic zone communities. Overall, microbial communities and geochemical profiles were stable across two sampling dates in the spring and fall of 2019. The blue hole habitat is a natural marine laboratory that provides opportunities for sampling taxa with under-characterized but potentially important roles in redox-stratified microbial processes.

microbiology↗

bioRxiv2019

Expression of extracellular multiheme cytochromes discovered in a betaproteobacterium during Mn(III) reduction

Soluble ligand-bound Mn(III) can support anaerobic microbial respiration in diverse aquatic environments. Thus far, Mn(III) reduction has only been associated with certain Gammaproteobacteria. Here, we characterized microbial communities enriched from Mn-replete sediments of Lake Matano, Indonesia. Our results provide the first evidence for biological reduction of soluble Mn(III) outside the Gammaproteobacteria. Metagenome assembly and binning revealed a novel betaproteobacterium, which we designate "Candidatus Dechloromonas occultata." This organism dominated the enrichment and expressed a porin-cytochrome c complex typically associated with iron-oxidizing Betaproteobacteria and a novel cytochrome c-rich protein cluster (Occ), including an undecaheme putatively involved in extracellular electron transfer. This occ gene cluster was also detected in diverse aquatic bacteria, including uncultivated Betaproteobacteria from the deep subsurface. These observations provide new insight into the taxonomic and functional diversity of microbially-driven Mn(III) reduction in natural environments. Originality-Significance StatementRecent observations suggest that Mn(III)-ligand complexes are geochemically important in diverse aquatic environments. Thus far, microbially-driven Mn(III) reduction has only been associated with Gammaproteobacteria encoding three-component outer-membrane porin-cytochrome c conduits. Here, we demonstrate that Betaproteobacteria dominate in abundance and with respect to protein expression during biologically-mediated Mn(III) reduction in an enrichment culture from an anoxic lacustrine system. Using metaproteomics, we detect for the first time that Betaproteobacteria express a two-component porin-cytochrome c conduit, and an uncharacterized extracellular undecaheme (11-heme) c-type cytochrome. Although this is the first definitive report of an undecaheme within the Betaproteobacteria, we find evidence that they are widespread in uncultivated strains. These results widen the phylogenetic diversity of Mn(III)-reducing bacteria, and provide new insights into potential molecular mechanisms for soluble Mn(III) reduction

microbiology↗

bioRxiv2019

Phylogenetic and structural diversity of aromatically dense pili from environmental metagenomes

Electroactive type IV pili, or e-pili, are used by some microbial species for extracellular electron transfer. Recent studies suggest that e-pili may be more phylogenetically and structurally diverse than previously assumed. Here, we used updated aromatic density thresholds ([≥]9.8% aromatic amino acids, [≤]22-aa aromatic gaps, and aromatic amino acids at residues 1, 24, 27, 50 and/or 51, and 32 and/or 57) to search for putative e-pilin genes in metagenomes from diverse ecosystems with active microbial metal cycling. Environmental putative e-pilins were diverse in length and phylogeny, and included truncated e-pilins in Geobacter spp., as well as longer putative e-pilins in Fe(II)-oxidizing Betaproteobacteria and Zetaproteobacteria.\n\nOriginality and SignificanceElectroactive pili (e-pili) are used by microorganisms to respire solid metals in their environment through extracellular electron transfer. Thus, e-pili enable microbes to occupy specific environmental niches. Additionally, e-pili have important potential for biotechnological applications. Currently the repertoire of known e-pili is small, and their environmental distribution is largely unknown. Using sequence analysis, we identified numerous genes encoding putative e-pili from diverse anoxic, metal-rich ecosystems. Our results expand the diversity of putative e-pili in environments where metal oxides may be important electron acceptors for microbial respiration.

microbiology↗

bioRxiv2019

Adaptations of Atribacteria to life in methane hydrates: hot traits for cold life

Gas hydrates harbor gigatons of natural gas, yet their microbiomes remain understudied. We bioprospected 16S rRNA amplicons, metagenomes, and metaproteomes from methane hydrate-bearing sediments under Hydrate Ridge (offshore Oregon, USA, ODP Site 1244, 2-69 mbsf) for novel microbial metabolic and biosynthetic potential. Atribacteria sequences generally increased in relative sequence abundance with increasing sediment depth. Most Atribacteria ASVs belonged to JS-1-Genus 1 and clustered with other sequences from gas hydrate-bearing sediments. We recovered 21 metagenome-assembled genomic bins spanning three geochemical zones in the sediment core: the sulfate-methane transition zone, metal (iron/manganese) reduction zone, and gas hydrate stability zone. We found evidence for bacterial fermentation as a source of acetate for aceticlastic methanogenesis and as a driver of iron reduction in the metal reduction zone. In multiple zones, we identified a Ni-Fe hydrogenase-Na+/H+ antiporter supercomplex (Hun) in Atribacteria and Firmicutes bins and in other deep subsurface bacteria and cultured hyperthermophiles from the Thermotogae phylum. Atribacteria expressed tripartite ATP-independent (TRAP) transporters downstream from a novel regulator (AtiR). Atribacteria also possessed adaptations to survive extreme conditions (e.g., high salt brines, high pressure, and cold temperatures) including the ability to synthesize the osmolyte di-myo-inositol-phosphate as well as expression of K+-stimulated pyrophosphatase and capsule proteins. Originality-Significance StatementThis work provides insights into the metabolism and adaptations of microbes that are ubiquitous and abundant in methane-rich ecosystems. Our findings suggest that bacterial fermentation is a source of acetate for aceticlastic methanogenesis and a driver of iron reduction in the metal reduction zone. Atribacteria, the most abundant phylum in gas hydrate-bearing sediments, possess multiple strategies to cope with environmental stress.

microbiology↗

bioRxiv2018

Feeding rapidly alters microbiome composition and gene transcription in the clownfish gut

BackgroundDiet is a major determinant of intestinal microbiome composition. While studies have evaluated microbiome responses to diet variation, less is understood of how the act of feeding influences the microbiome, independent of diet type. Here, we use the clownfish Premnas biaculeatus, a species reared commonly in ornamental marine aquaculture, to test how the diversity, predicted gene content, and gene transcription of the microbiome vary over a two-day diurnal period with a single daily feeding event. This study used fish fed four times daily, once daily, or every three days prior to the diurnal period, allowing us also to test how feeding frequency affected microbiome diversity. The amount of time between feedings had no affect on baseline diversity of the microbiome. In contrast, the act of feeding itself caused a significant short term change in the microbiome, with microbiome diversity, predicted gene content, and gene transcription varying significantly between time points immediately before and 1.5 hours post feeding. Variation was driven by abundance shifts involving exact sequence variants (ESVs), with one ESV identified as Photobacterium sp. increasing from <0.5% of sequences immediately pre-feeding to 34% at 1.5 hours post-feeding. Other ESVs from a range of microbial groups also increased dramatically after feeding, with the majority also detected in the food. One ESV identified as Clostridium perfringens represented up to 55% of sequences but did not vary significantly over the diurnal period and was not detected in the food. Post-feeding samples were enriched in transcripts and predicted genes for social interactions, cell motility, and coping with foreign DNA, whereas time points farther from feeding were enriched in genes of diverse catabolic and biosynthetic functions. These results confirm feeding as a significant destabilizing force in clownfish intestinal microbiomes, likely due to both input of cells attached to food and stimulation of resident microbes. Microbes such as Photobacterium may episodically transition from environmental reservoirs to growth in the gut, likely in association with food particles. This transition may be facilitated by functions for navigating a new environment and interacting with neighboring microbes and host cells. Other taxa, such as Clostridium, are comparatively stable intestinal members and less likely to be affected by passing food. Conclusions about microbiome ecology may therefore differ based on when samples were collected relative to the last feeding.\n\nImportanceDespite extensive study of intestinal microbiome diversity and the role of diet type in structuring gut microbial communities, we know very little about short-term changes in the intestinal microbiome as a result of feeding alone. Sampling microbiomes over a feeding cycle will allow us to differentiate opportunistic, feeding-responsive microbes from resident, potentially commensal members of the gut community. Also, since feeding has the potential to alter microbiome structure, sampling at different points relative to the last feeding event will likely yield different conclusions about microbiome composition and function. This variation should be addressed in comparative microbiome studies. Our study contributes to knowledge of short-term changes in the gut microbiome associated with feeding events.

microbiology↗

bioRxiv2016

Shifting microbial communities sustain multi-year iron reduction and methanogenesis in ferruginous sediment incubations

Reactive Fe(III) minerals can influence methane (CH4) emissions by inhibiting microbial methanogenesis or by stimulating anaerobic CH4 oxidation. The balance between Fe(III) reduction, methanogenesis, and methane oxidation in ferruginous Archean and Paleoproterozoic oceans would have controlled CH4 fluxes to the atmosphere, thereby regulating the capacity for CH4 to warm the early Earth under the Faint Young Sun. We studied CH4 and Fe cycling in anoxic incubations of ferruginous sediment from the ancient ocean analogue Lake Matano, Indonesia over three successive transfers (500 days total). Iron reduction, methanogenesis, methane oxidation, and microbial taxonomy were monitored in treatments amended with ferrihydrite or goethite. After three dilutions, Fe(III) reduction persisted only in bottles with ferrihydrite. Enhanced CH4 production was observed in the presence of goethite, highlighting the potential for reactive Fe(III)-oxides to inhibit methanogenesis. Supplementing the media with hydrogen, nickel and selenium did not stimulate methanogenesis. There was limited evidence for Fe(III)-dependent CH4 oxidation, although some incubations displayed CH4-stimulated Fe(III)-reduction. 16S rRNA profiles continuously changed over the course of enrichment, with ultimate dominance of unclassified members of the order Desulfuromonadales in all treatments. Microbial diversity decreased markedly over the course of incubation, with subtle differences between ferrihydrite and goethite amendments. These results suggest that Fe(III)-oxide mineralogy and availability of electron donors could have led to spatial separation of Fe(III)-reducing and methanogenic microbial communities in ferruginous marine sediments, potentially explaining the persistence of CH4 as a greenhouse gas throughout the first half of Earth history.

microbiology↗