Diversity at single nucleotide to pangenome scales among sulfur cycling bacteria in salt marshes.

IMPORTANCE: Salt marshes are known for their significant carbon storage capacity, and sulfur cycling is closely linked with the ecosystem-scale carbon cycling in these ecosystems. Sulfate reducers are key for the decomposition of organic matter, and sulfur oxidizers remove toxic sulfide, supporting the productivity of marsh plants. To date, the complexity of coastal environments, heterogeneity of the rhizosphere, high microbial diversity, and uncultured majority hindered our understanding of the genomic diversity of sulfur-cycling microbes in salt marshes. Here, we use comparative genomics to overcome these challenges and provide an in-depth characterization of sulfur-cycling microbial diversity in salt marshes. We characterize communities across distinct sites and plant species and uncover extensive genomic diversity at the taxon level and specific genomic features present in MAGs affiliated with uncultivated sulfur-cycling lineages. Our work provides insights into the partnerships in salt marshes and a roadmap for multiscale analyses of diversity in complex biological systems.

Metagenomics and metatranscriptomics reveal broadly distributed, active, novel methanotrophs in the Gulf of Mexico hypoxic zone and in the marine water column.

The northern Gulf of Mexico (nGOM) hypoxic zone is a shallow water environment where methane, a potent greenhouse gas, fluxes from sediments to bottom water and remains trapped due to summertime stratification. When the water column is destratified, an active planktonic methanotrophic community could mitigate the efflux of methane, which accumulates to high concentrations, to the atmosphere. To investigate the possibility of such a biofilter in the nGOM hypoxic zone we performed metagenome assembly, and metagenomic and metatranscriptomic read mapping. Methane monooxygenase (pmoA) was an abundant transcript, yet few canonical methanotrophs have been reported in this environment, suggesting a role for non-canonical methanotrophs. To determine the identity of these methanotrophs, we reconstructed six novel metagenome-assembled genomes (MAGs) in the Planctomycetota, Verrucomicrobiota and one putative Latescibacterota, each with at least one pmoA gene copy. Based on ribosomal protein phylogeny, closely related microbes (mostly from Tara Oceans) and isolate genomes were selected and co-analyzed with the nGOM MAGs. Gene annotation and read mapping suggested that there is a large, diverse and unrecognized community of active aerobic methanotrophs in the nGOM hypoxic zone and in the global ocean that could mitigate methane flux to the atmosphere.

Predator dispersal influences predator distribution but not prey diversity in pitcher plant microbial metacommunities.

The spatial distribution of predators can affect both the distribution and diversity of their prey. Therefore, differences in predator dispersal ability that affect their spatial distribution, could also affect prey communities. Here, we use the microbial communities within pitcher plant leaves as a model system to test the relationship between predator (protozoa) dispersal ability and distribution, and its consequences for prey (bacteria) diversity and composition. We hypothesized that limited predator dispersal results in clustered distributions and heterogeneous patches for prey species, whereas wide predator dispersal and distribution could homogenize prey metacommunities. We analyzed the distribution of two prominent bacterivore protozoans from a 2-year survey of an intact field of Sarracenia purpurea pitcher plants, and found a clustered distribution of Tetrahymena and homogeneous distribution of Poterioochromonas. We manipulated the sources of protozoan colonists and recorded protozoan recruitment and bacterial diversity in target leaves in a field experiment. We found the large ciliate, Tetrahymena, was dispersal limited and occupied few leaves, whereas the small flagellate Poterioochromonas was widely dispersed. However, the bacterial communities these protozoans feed on was unaffected by clustering of Tetrahymena, but likely influenced by Poterioochromonas and other bacterivores dispersing in the field. We propose that bacterial communities in this system are structured by a combination of well dispersed bacterivores, bacterial dispersal, and bottom-up mechanisms. Clustered predators could become strong drivers of prey communities if they were specialists or keystone predators, or if they exerted a dominant influence on other predators in top-down controlled systems. Linking dispersal ability within trophic levels and its consequences for trophic dynamics can lead to a more robust perspective on trophic metacommunities.

Bacterial communities vary across populations and tissue type in red mangroves (Rhizophora mangle, Rhizophoraceae) along an expanding front.

Plant-associated microbial communities may be important sources of functional diversity and genetic variation that influence host evolution. Bacteria provide benefits for their hosts, yet in most plant systems we know little about their taxonomic composition or variation across tissues and host range. Red Mangrove (Rhizophora mangle L.) is a vital coastal plant species that is currently expanding poleward and with it, perhaps, its microbiome. We explored variability in bacterial communities across tissues, individuals, and populations. We collected samples from six sample types from 5 to 10 individuals at each of three populations and used 16S rRNA gene (iTag) sequencing to describe their bacterial communities. Core community members and dominant bacterial classes were determined for each sample type. Pairwise PERMANOVA of Bray-Curtis dissimilarity and Indicator Species Analysis revealed significant differences in bacterial communities between sample types and populations. We described the previously unexplored microbiome of the reproductive tissues of R. mangle. Populations and most sample types were associated with distinct communities. Bacterial communities associated with R. mangle are influenced by host geography and sample type. Our study provides a foundation for future work exploring the functional roles of these microbes and their relevance to biogeochemical cycling.

Effects of Community Richness and Competitive Asymmetry on Protozoa Evolution in Sarracenia purpurea Leaves.

AbstractPredicting evolution in natural systems will require understanding how selection operates in multispecies communities. We predicted that the amount that traits evolve in multispecies mixtures would be less than the amount that would be predicted from the additive contributions of the pairwise interactions and that subordinate species will be more likely to evolve in competitive systems than dominant species. We conducted an experimental test of these predictions using a guild of protozoans found in the water-filled leaves of the pitcher plant Sarracenia purpurea. The response to selection did not significantly change as we increased richness from monocultures to two- and four-species mixtures. In accordance with our second prediction, subordinate species demonstrated greater growth in competition after selection than before, while dominant species generally showed no response to selection. Monod-type experiments to determine minimum resource levels found that the dominant species had much higher resource requirements than the subordinate species and that the minimum resource requirements evolved to be higher in the subordinate species. Importantly, these results suggest that subordinate species evolve to become more similar to dominant species, which may involve resource use convergence. Our findings and other recent works suggest that community diversity can affect evolution in surprising ways that warrant further investigation.

Metagenomic analysis of microbial communities across a transect from low to highly hydrocarbon-contaminated soils in King George Island, Maritime Antarctica.

Soil samples from a transect from low to highly hydrocarbon-contaminated soils were collected around the Brazilian Antarctic Station Comandante Ferraz (EACF), located at King George Island, Antarctica. Quantitative PCR (qPCR) analysis of bacterial 16S rRNA genes, 16S rRNA gene (iTag), and shotgun metagenomic sequencing were used to characterize microbial community structure and the potential for petroleum degradation by indigenous microbes. Hydrocarbon contamination did not affect bacterial abundance in EACF soils (bacterial 16S rRNA gene qPCR). However, analysis of 16S rRNA gene sequences revealed a successive change in the microbial community along the pollution gradient. Microbial richness and diversity decreased with the increase of hydrocarbon concentration in EACF soils. The abundance of Cytophaga, Methyloversatilis, Polaromonas, and Williamsia was positively correlated (p-value = <.05) with the concentration of total petroleum hydrocarbons (TPH) and/or polycyclic aromatic hydrocarbons (PAH). Annotation of metagenomic data revealed that the most abundant hydrocarbon degradation pathway in EACF soils was related to alkyl derivative-PAH degradation (mainly methylnaphthalenes) via the CYP450 enzyme family. The abundance of genes related to nitrogen fixation increased in EACF soils as the concentration of hydrocarbons increased. The results obtained here are valuable for the future of bioremediation of petroleum hydrocarbon-contaminated soils in polar environments.

Correction: Host and environmental determinants of microbial community structure in the marine phyllosphere.

[This corrects the article DOI: 10.1371/journal.pone.0235441.].

Composition of seagrass phyllosphere microbial communities suggests rapid environmental regulation of community structure.

Recent studies have revealed that seagrass blade surfaces, also known as the phyllosphere, are rich habitats for microbes; however, the primary drivers of composition and structure in these microbial communities are largely unknown. This study utilized a reciprocal transplant approach between two sites with different environmental conditions combined with 16S rRNA gene sequencing (iTag) to examine the relative influence of environmental conditions and host plant on phyllosphere community composition of the seagrass Thalassia testudinum. After 30 days, identity of phyllosphere microbial community members was more similar within the transplant sites than between despite differences in the source of host plant. Additionally, the diversity and evenness of these communities was significantly different between the two sites. These results indicated that local environmental conditions can be a primary driver in structuring seagrass phyllosphere microbial communities over relatively short time scales. Composition of microbial community members in this study also deviated from those in previous seagrass phyllosphere studies with a higher representation of candidate bacterial phyla and archaea than previously observed. The capacity for seagrass phyllosphere microbial communities to shift dramatically with environmental conditions, including ecosystem perturbations, could significantly affect seagrass-microbe interactions in ways that may influence the health of the seagrass host.

Enrichment of potential pathogens in marine microbiomes with different degrees of anthropogenic activity.

Anthropogenic activities in coastal marine ecosystems can lead to an increase in the abundance of potentially harmful microorganisms in the marine environment. To understand anthropogenic impacts on the marine microbiome, we first used publicly available microbial phylogenetic and functional data to establish a dataset of bacterial genera potentially related to pathogens that cause diseases (BGPRD) in marine organisms. Representatives of low-, medium- and highly impacted marine coastal environments were selected, and the abundance and composition of their microbial communities were determined by quantitative PCR and 16 S rRNA gene sequencing. In total, 72 BGPRD were cataloged, and 11, 36 and 37 BGPRD were found in low-, medium- and highly human-impacted ecosystems, respectively. The absolute abundance of BGPRD and the co-occurrence of antibiotic resistance genes (AGR) increased with the degree of anthropogenic perturbation in these ecosystems. Anthropogenically impacted coastal microbiomes were compositionally and functionally distinct from those of less impacted sites, presenting features that may contribute to adverse outcomes for marine macrobiota in the Anthropocene era.

Host and environmental determinants of microbial community structure in the marine phyllosphere.

Although seagrasses are economically and ecologically critical species, little is known about their blade surface microbial communities and how these communities relate to the plant host. To determine microbial community composition and diversity on seagrass blade surfaces and in the surrounding seawater,16S rRNA gene sequencing (iTag) was used for samples collected at five sites along a gradient of freshwater input in the northern Gulf of Mexico on three separate sampling dates. Additionally, seagrass surveys were performed and environmental parameters were measured to characterize host characteristics and the abiotic conditions at each site. Results showed that Thalassia testudinum (turtle grass) blades hosted unique microbial communities that were distinct in composition and diversity from the water column. Environmental conditions, including water depth, salinity, and temperature, influenced community structure as blade surface microbial communities varied among sites and sampling dates in correlation with changes in environmental parameters. Microbial community composition also correlated with seagrass host characteristics, including growth rates and blade nutrient composition. There is some evidence for a core community for T. testudinum as 21 microorganisms from five phyla (Cyanobacteria, Proteobacteria, Planctomycetes, Chloroflexi, and Bacteroidetes) were present in all blade surface samples. This study provides new insights and understanding of the processes that influence the structure of marine phyllosphere communities, how these microbial communities relate to their host, and their role as a part of the seagrass holobiont, which is an important contribution given the current decline of seagrass coverage worldwide.

Investigating promising substrates for promoting 1,4-dioxane biodegradation: effects of ethane and tetrahydrofuran on microbial consortia.

Cometabolic biodegradation of 1,4-dioxane (dioxane) in the presence of primary substrates is a promising strategy for treating dioxane at environmentally relevant concentrations. Seven aqueous amendments (i.e., tetrahydrofuran (THF), butanone, acetone, 1-butanol, 2-butanol, phenol and acetate) and five gaseous amendments (i.e., C1-C4 alkanes and ethylene) were evaluated as the primary substrates for dioxane degradation by mixed microbial consortia. The aqueous amendments were tested in microcosm bottles and the gaseous amendments were tested in a continuous-flow membrane biofilm reactor with hollow fibers pressurized by the gaseous amendments. Ethane was found to be the most effective gaseous substrate and THF was the only aqueous substrate that promoted dioxane degradation. A diverse microbial community consisting of several putative dioxane degraders-Mycobacterium, Flavobacterium and Bradyrhizobiaceae-were enriched in the presence of ethane. This is the first study showing that ethane was the most effective substrate among the short-chain alkanes and it promoted dioxane degradation by enriching dioxane-degraders that did not harbor the well-known dioxane/tetrahydrofuran monooxygenase.

Extent of the annual Gulf of Mexico hypoxic zone influences microbial community structure.

Rich geochemical datasets generated over the past 30 years have provided fine-scale resolution on the northern Gulf of Mexico (nGOM) coastal hypoxic (≤ 2 mg of O2 L-1) zone. In contrast, little is known about microbial community structure and activity in the hypoxic zone despite the implication that microbial respiration is responsible for forming low dissolved oxygen (DO) conditions. Here, we hypothesized that the extent of the hypoxic zone is a driver in determining microbial community structure, and in particular, the abundance of ammonia-oxidizing archaea (AOA). Samples collected across the shelf for two consecutive hypoxic seasons in July 2013 and 2014 were analyzed using 16S rRNA gene sequencing, oligotyping, microbial co-occurrence analysis, and quantification of thaumarchaeal 16S rRNA and archaeal ammonia-monooxygenase (amoA) genes. In 2014 Thaumarchaeota were enriched and inversely correlated with DO while Cyanobacteria, Acidimicrobiia, and Proteobacteria where more abundant in oxic samples compared to hypoxic. Oligotyping analysis of Nitrosopumilus 16S rRNA gene sequences revealed that one oligotype was significantly inversely correlated with DO in both years. Oligotyping analysis revealed single nucleotide variation among all Nitrosopumilaceae, including Nitrosopumilus 16S rRNA gene sequences, with one oligotype possibly being better adapted to hypoxia. We further provide evidence that in the hypoxic zone of both year 2013 and 2014, low DO concentrations and high Thaumarchaeota abundances influenced microbial co-occurrence patterns. Taken together, the data demonstrated that the extent of hypoxic conditions could potentially drive patterns in microbial community structure, with two years of data revealing the annual nGOM hypoxic zone to be emerging as a low DO adapted AOA hotspot.

Carbon fixation and energy metabolisms of a subseafloor olivine biofilm.

Earth's largest aquifer ecosystem resides in igneous oceanic crust, where chemosynthesis and water-rock reactions provide the carbon and energy that support an active deep biosphere. The Calvin Cycle is the predominant carbon fixation pathway in cool, oxic, crust; however, the energy and carbon metabolisms in the deep thermal basaltic aquifer are poorly understood. Anaerobic carbon fixation pathways such as the Wood-Ljungdahl pathway, which uses hydrogen (H2) and CO2, may be common in thermal aquifers since water-rock reactions can produce H2 in hydrothermal environments and bicarbonate is abundant in seawater. To test this, we reconstructed the metabolisms of eleven bacterial and archaeal metagenome-assembled genomes from an olivine biofilm obtained from a Juan de Fuca Ridge basaltic aquifer. We found that the dominant carbon fixation pathway was the Wood-Ljungdahl pathway, which was present in seven of the eight bacterial genomes. Anaerobic respiration appears to be driven by sulfate reduction, and one bacterial genome contained a complete nitrogen fixation pathway. This study reveals the potential pathways for carbon and energy flux in the deep anoxic thermal aquifer ecosystem, and suggests that ancient H2-based chemolithoautotrophy, which once dominated Earth's early biosphere, may thus remain one of the dominant metabolisms in the suboceanic aquifer today.

Microbial Community Analysis Provides Insights into the Effects of Tetrahydrofuran on 1,4-Dioxane Biodegradation.

Tetrahydrofuran (THF) is known to induce the biodegradation of 1,4-dioxane (dioxane), an emerging contaminant, but the mechanisms by which THF affects dioxane biodegradation in microbial communities are not well understood. To fill this knowledge gap, changes in the microbial community structure in microcosm experiments with synthetic medium and landfill leachate were examined over time using 16S rRNA gene amplicon sequencing and functional gene quantitative PCR assays. The overarching hypothesis being tested was that THF promoted dioxane biodegradation by increasing the abundance of dioxane-degrading bacteria in the consortium. The data revealed that in experiments with synthetic medium, the addition of THF significantly increased the abundance of Pseudonocardia, a genus with several representatives that can grow on both dioxane and THF, and of Rhodococcus ruber, a species that can use THF as the primary growth substrate while cometabolizing dioxane. However, in similar experiments with landfill leachate, only R. ruber was significantly enriched. When the THF concentration was higher than the dioxane concentration, THF competitively inhibited dioxane degradation since dioxane degradation was negligible, while the dioxane-degrading bacteria and the corresponding THF/dioxane monooxygenase gene copies increased by a few orders of magnitude.IMPORTANCE Widespread in groundwater and carcinogenic to humans, 1,4-dioxane (dioxane) is attracting significant attention in recent years. Advanced oxidation processes can effectively remove dioxane but require high energy consumption and operation costs. Biological removal of dioxane is of particular interest due to the ability of some bacteria to mineralize dioxane at a low energy cost. Although dioxane is generally considered recalcitrant to biodegradation, more than 20 types of bacteria can degrade dioxane as the sole electron donor substrate or the secondary electron donor substrate. In the latter case, tetrahydrofuran (THF) is commonly studied as the primary electron donor substrate. Previous work has shown that THF promotes dioxane degradation at a low THF concentration but inhibits dioxane degradation at a high THF concentration. Our work expanded on the previous work by mechanically examining the effects of THF on dioxane degradation in a microbial community context.

Metagenomic Assembly and Prokaryotic Metagenome-Assembled Genome Sequences from the Northern Gulf of Mexico "Dead Zone".

Coastal regions experiencing declining dissolved oxygen are increasing in number and severity around the world. However, despite the importance of microbial metabolism in coastal hypoxia, few metagenomic surveys exist. Our data set from within the second largest human-caused hypoxic region provides opportunities to more deeply explore the microbiology of these systems.

Predator identity more than predator richness structures aquatic microbial assemblages in Sarracenia purpurea leaves.

The importance of predators in influencing community structure is a well-studied area of ecology. However, few studies test ecological hypotheses of predation in multi-predator microbial communities. The phytotelmic community found within the water-filled leaves of the pitcher plant, Sarracenia purpurea, exhibits a simple trophic structure that includes multiple protozoan predators and microbial prey. Using this system, we sought to determine whether different predators target distinct microorganisms, how interactions among protozoans affect resource (microorganism) use, and how predator diversity affects prey community diversity. In particular, we endeavored to determine if protozoa followed known ecological patterns such as keystone predation or generalist predation. For these experiments, replicate inquiline microbial communities were maintained for seven days with five protozoan species. Microbial community structure was determined by 16S rRNA gene amplicon sequencing (iTag) and analysis. Compared to the control (no protozoa), two ciliates followed patterns of keystone predation by increasing microbial evenness. In pairwise competition treatments with a generalist flagellate, prey communities resembled the microbial communities of the respective keystone predator in monoculture. The relative abundance of the most common bacterial Operational Taxonomic Unit (OTU) in our system decreased compared to the control in the presence of these ciliates. This OTU was 98% similar to a known chitin degrader and nitrate reducer, important functions for the microbial community and the plant host. Collectively, the data demonstrated that predator identity had a greater effect on prey diversity and composition than overall predator diversity.

Metabolic Roles of Uncultivated Bacterioplankton Lineages in the Northern Gulf of Mexico "Dead Zone".

Marine regions that have seasonal to long-term low dissolved oxygen (DO) concentrations, sometimes called "dead zones," are increasing in number and severity around the globe with deleterious effects on ecology and economics. One of the largest of these coastal dead zones occurs on the continental shelf of the northern Gulf of Mexico (nGOM), which results from eutrophication-enhanced bacterioplankton respiration and strong seasonal stratification. Previous research in this dead zone revealed the presence of multiple cosmopolitan bacterioplankton lineages that have eluded cultivation, and thus their metabolic roles in this ecosystem remain unknown. We used a coupled shotgun metagenomic and metatranscriptomic approach to determine the metabolic potential of Marine Group II Euryarchaeota, SAR406, and SAR202. We recovered multiple high-quality, nearly complete genomes from all three groups as well as candidate phyla usually associated with anoxic environments-Parcubacteria (OD1) and Peregrinibacteria Two additional groups with putative assignments to ACD39 and PAUC34f supplement the metabolic contributions by uncultivated taxa. Our results indicate active metabolism in all groups, including prevalent aerobic respiration, with concurrent expression of genes for nitrate reduction in SAR406 and SAR202, and dissimilatory nitrite reduction to ammonia and sulfur reduction by SAR406. We also report a variety of active heterotrophic carbon processing mechanisms, including degradation of complex carbohydrate compounds by SAR406, SAR202, ACD39, and PAUC34f. Together, these data help constrain the metabolic contributions from uncultivated groups in the nGOM during periods of low DO and suggest roles for these organisms in the breakdown of complex organic matter.IMPORTANCE Dead zones receive their name primarily from the reduction of eukaryotic macrobiota (demersal fish, shrimp, etc.) that are also key coastal fisheries. Excess nutrients contributed from anthropogenic activity such as fertilizer runoff result in algal blooms and therefore ample new carbon for aerobic microbial metabolism. Combined with strong stratification, microbial respiration reduces oxygen in shelf bottom waters to levels unfit for many animals (termed hypoxia). The nGOM shelf remains one of the largest eutrophication-driven hypoxic zones in the world, yet despite its potential as a model study system, the microbial metabolisms underlying and resulting from this phenomenon-many of which occur in bacterioplankton from poorly understood lineages-have received only preliminary study. Our work details the metabolic potential and gene expression activity for uncultivated lineages across several low DO sites in the nGOM, improving our understanding of the active biogeochemical cycling mediated by these "microbial dark matter" taxa during hypoxia.

Mississippi River Plume Enriches Microbial Diversity in the Northern Gulf of Mexico.

The Mississippi River (MR) serves as the primary source of freshwater and nutrients to the northern Gulf of Mexico (nGOM). Whether this input of freshwater also enriches microbial diversity as the MR plume migrates and mixes with the nGOM serves as the central question addressed herein. Specifically, in this study physicochemical properties and planktonic microbial community composition and diversity was determined using iTag sequencing of 16S rRNA genes in 23 samples collected along a salinity (and nutrient) gradient from the mouth of the MR, in the MR plume, in the canyon, at the Deepwater Horizon wellhead and out to the loop current. Analysis of these datasets revealed that the MR influenced microbial diversity as far offshore as the Deepwater Horizon wellhead. The MR had the highest microbial diversity, which decreased with increasing salinity. MR bacterioplankton communities were distinct compared to the nGOM, particularly in the surface where Actinobacteria and Proteobacteria dominated, while the deeper MR was also enriched in Thaumarchaeota. Statistical analyses revealed that nutrients input by the MR, along with salinity and depth, were the primary drivers in structuring the microbial communities. These results suggested that the reduced salinity, nutrient enriched MR plume could act as a seed bank for microbial diversity as it mixes with the nGOM. Whether introduced microorganisms are active at higher salinities than freshwater would determine if this seed bank for microbial diversity is ecologically significant. Alternatively, microorganisms that are physiologically restricted to freshwater habitats that are entrained in the plume could be used as tracers for freshwater input to the marine environment.

Comparison of Archaeal and Bacterial Diversity in Methane Seep Carbonate Nodules and Host Sediments, Eel River Basin and Hydrate Ridge, USA.

Anaerobic oxidation of methane (AOM) impacts carbon cycling by acting as a methane sink and by sequestering inorganic carbon via AOM-induced carbonate precipitation. These precipitates commonly take the form of carbonate nodules that form within methane seep sediments. The timing and sequence of nodule formation within methane seep sediments are not well understood. Further, the microbial diversity associated with sediment-hosted nodules has not been well characterized and the degree to which nodules reflect the microbial assemblage in surrounding sediments is unknown. Here, we conducted a comparative study of microbial assemblages in methane-derived authigenic carbonate nodules and their host sediments using molecular, mineralogical, and geochemical methods. Analysis of 16S rRNA gene diversity from paired carbonate nodules and sediments revealed that both sample types contained methanotrophic archaea (ANME-1 and ANME-2) and syntrophic sulfate-reducing bacteria (Desulfobacteraceae and Desulfobulbaceae), as well as other microbial community members. The combination of geochemical and molecular data from Eel River Basin and Hydrate Ridge suggested that some nodules formed in situ and captured the local sediment-hosted microbial community, while other nodules may have been translocated or may represent a record of conditions prior to the contemporary environment. Taken together, this comparative analysis offers clues to the formation regimes and mechanisms of sediment-hosted carbonate nodules.