Freshwater mussels and host fish gut microbe community composition shifts after agricultural contaminant exposure.

AIMS: We examined the effects of a mixture of contaminants found in agricultural watersheds on the gut microbiota and physiology of both the freshwater mussel Lampsilis cardium, and L. cardium host fish Micropterus salmoides. METHODS AND RESULTS: Lampsilis cardium and M. salmoides were exposed to three concentrations of agricultural contaminants for 60 days (observing behaviour daily) before being sampled for gut microbiota analyses. DNA was extracted from the gut samples, amplified via PCR, and sequenced using the Illumina Mi-Seq platform. Only L. cardium guts had differing microbiota across treatments, with an increase in potentially pathogenic Aeromonas. We also provide novel evidence of a core microbiota within L. cardium and M. salmoides. In terms of physiology, female L. cardium exhibited a decrease in movement and marsupial gill display in contaminant exposures. CONCLUSIONS: Exposure to contaminants from agricultural watersheds may affect population recruitment within freshwater mussel communities over time. Specifically, increased pathogenic micro-organisms and altered behaviour can reduce the likelihood of glochidia dispersal. SIGNIFICANCE AND IMPACT OF THE STUDY: This study supports emerging research that contaminants found in agricultural watersheds may be a factor in freshwater mussel population declines. It also provides novel evidence that unionids have a core gut microbiota.

Metagenomics of Antarctic Marine Sediment Reveals Potential for Diverse Chemolithoautotrophy.

The microbial biogeochemical processes occurring in marine sediment in Antarctica remain underexplored due to limited access. Further, these polar habitats are unique, as they are being exposed to significant changes in their climate. To explore how microbes drive biogeochemistry in these sediments, we performed a shotgun metagenomic survey of marine surficial sediment (0 to 3 cm of the seafloor) collected from 13 locations in western Antarctica and assembled 16 high-quality metagenome assembled genomes for focused interrogation of the lifestyles of some abundant lineages. We observe an abundance of genes from pathways for the utilization of reduced carbon, sulfur, and nitrogen sources. Although organotrophy is pervasive, nitrification and sulfide oxidation are the dominant lithotrophic pathways and likely fuel carbon fixation via the reverse tricarboxylic acid and Calvin cycles. Oxygen-dependent terminal oxidases are common, and genes for reduction of oxidized nitrogen are sporadically present in our samples. Our results suggest that the underlying benthic communities are well primed for the utilization of settling organic matter, which is consistent with findings from highly productive surface water. Despite the genetic potential for nitrate reduction, the net catabolic pathway in our samples remains aerobic respiration, likely coupled to the oxidation of sulfur and nitrogen imported from the highly productive Antarctic water column above. IMPORTANCE The impacts of climate change in polar regions, like Antarctica, have the potential to alter numerous ecosystems and biogeochemical cycles. Increasing temperature and freshwater runoff from melting ice can have profound impacts on the cycling of organic and inorganic nutrients between the pelagic and benthic ecosystems. Within the benthos, sediment microbial communities play a critical role in carbon mineralization and the cycles of essential nutrients like nitrogen and sulfur. Metagenomic data collected from sediment samples from the continental shelf of western Antarctica help to examine this unique system and document the metagenomic potential for lithotrophic metabolisms and the cycles of both nitrogen and sulfur, which support not only benthic microbes but also life in the pelagic zone.

Microbial community structure and microbial networks correspond to nutrient gradients within coastal wetlands of the Laurentian Great Lakes.

Microbial communities within the soil of Laurentian Great Lakes coastal wetlands drive biogeochemical cycles and provide several other ecosystem services. However, there exists a lack of understanding of how microbial communities respond to nutrient gradients and human activity in these systems. This research sought to address the lack of understanding through exploration of relationships among nutrient gradients, microbial community diversity, and microbial networks. Significant differences in microbial community structure were found among coastal wetlands within the western basin of Lake Erie and all other wetlands studied (three regions within Saginaw Bay and one region in the Beaver Archipelago). These diversity differences coincided with higher nutrient levels within the Lake Erie region. Site-to-site variability also existed within the majority of the regions studied, suggesting site-scale heterogeneity may impact microbial community structure. Several subnetworks of microbial communities and individual community members were related to chemical gradients among wetland regions, revealing several candidate indicator communities and taxa that may be useful for Great Lakes coastal wetland management. This research provides an initial characterization of microbial communities among Great Lakes coastal wetlands and demonstrates that microbial communities could be negatively impacted by anthropogenic activities.

Comparative genomics of 16 Microbacterium spp. that tolerate multiple heavy metals and antibiotics.

A total of 16 different strains of Microbacterium spp. were isolated from contaminated soil and enriched on the carcinogen, hexavalent chromium [Cr(VI)]. The majority of the isolates (11 of the 16) were able to tolerate concentrations (0.1 mM) of cobalt, cadmium, and nickel, in addition to Cr(VI) (0.5-20 mM). Interestingly, these bacteria were also able to tolerate three different antibiotics (ranges: ampicillin 0-16 µg ml-1, chloramphenicol 0-24 µg ml-1, and vancomycin 0-24 µg ml-1). To gain genetic insight into these tolerance pathways, the genomes of these isolates were assembled and annotated. The genomes of these isolates not only have some shared genes (core genome) but also have a large amount of variability. The genomes also contained an annotated Cr(VI) reductase (chrR) that could be related to Cr(VI) reduction. Further, various heavy metal tolerance (e.g., Co/Zn/Cd efflux system) and antibiotic resistance genes were identified, which provide insight into the isolates' ability to tolerate metals and antibiotics. Overall, these isolates showed a wide range of tolerances to heavy metals and antibiotics and genetic diversity, which was likely required of this population to thrive in a contaminated environment.

Microbial subnetworks related to short-term diel O2 fluxes within geochemically distinct freshwater wetlands.

Oxygen (O2) concentrations often fluctuate over diel timescales within wetlands, driven by temperature, sunlight, photosynthesis and respiration. These daily fluxes have been shown to impact biogeochemical transformations (e.g. denitrification), which are mediated by the residing microbial community. However, little is known about how resident microbial communities respond to diel physical and chemical fluxes in freshwater wetland ecosystems. In this study, total microbial (bacterial and archaeal) community structure was significantly related to diel time points in just one out of four distinct freshwater wetlands sampled. This suggests that daily environmental shifts may influence wetlands differentially based upon the resident microbial community and specific physical and chemical conditions of a freshwater wetland. When exploring the microbial communities within each wetland at finer resolutions, subcommunities of taxa within two wetlands were found to correspond to fluctuating O2 levels. Microbial taxa that were found to be susceptible to fluctuating O2 levels within these subnetworks may have intimate ties to metabolism and/or diel redox cycles. This study highlights that freshwater wetland microbial communities are often stable in community structure when confronted with short-term O2 fluxes; however, specialist taxa may be sensitive to these same fluxes.

Investigating diversity of pathogenic microbes in commercial bait trade water.

The recreational bait trade is a potential pathway for pathogen introduction and spread when anglers dump bait shop sourced water into aquatic systems. Despite this possibility, and previous recognition of the importance of the bait trade in the spread of aquatic invasive species (AIS), to date there has been no region wide survey documenting pathogens in retail bait shops. In this study, we analyzed 96 environmental DNA samples from retail bait shops around the Great Lakes region to identify pathogens, targeting the V4 hypervariable region of the 16S rRNA gene. Additionally, we used samples from one site in Lake Michigan as a comparison to pathogen diversity and abundance in natural aquatic systems. Our results identified nine different groups of pathogens in the bait shop samples, including those that pose risks to both humans and fish species. Compared to wild sourced samples, the bait shops had higher relative abundance and greater taxonomic diversity. These findings suggest that the bait trade represents a potentially important pathway that could introduce and spread pathogens throughout the Great Lakes region. Improving pathogen screening and angler outreach should be used in combination to aid in preventing the future spread of high risk pathogens.

Potential for gulls to transport bacteria from human waste sites to beaches.

Contamination of recreational beaches due to fecal waste from gulls complicates beach monitoring and may pose a risk to public health. Gulls that feed at human waste sites may ingest human fecal microorganisms associated with that waste. If these gulls also visit beaches, they may serve as vectors, transporting fecal microorganisms to the beach where they may subsequently contaminate sand and water. In this study, samples collected from landfills, treated wastewater storage lagoons, and public beaches demonstrated a spatial and temporal overlap of markers for gull and human-associated microorganisms. In addition, markers for gull, fecal indicator bacteria, and the human-associated marker, HF183, were detected in gull feces and cloacae samples. Further, HF183 was detected in cloacae samples from gulls that were documented by radio-telemetry traveling between human waste sites and public beaches. This study highlights the potential for gulls that visit human waste sites to disperse human-associated microorganisms in the beach landscape.

Microbial community diversity patterns are related to physical and chemical differences among temperate lakes near Beaver Island, MI.

Lakes are dynamic and complex ecosystems that can be influenced by physical, chemical, and biological processes. Additionally, individual lakes are often chemically and physically distinct, even within the same geographic region. Here we show that differences in physicochemical conditions among freshwater lakes located on (and around) the same island, as well as within the water column of each lake, are significantly related to aquatic microbial community diversity. Water samples were collected over time from the surface and bottom-water within four freshwater lakes located around Beaver Island, MI within the Laurentian Great Lakes region. Three of the sampled lakes experienced seasonal lake mixing events, impacting either O2, pH, temperature, or a combination of the three. Microbial community alpha and beta diversity were assessed and individual microbial taxa were identified via high-throughput sequencing of the 16S rRNA gene. Results demonstrated that physical and chemical variability (temperature, dissolved oxygen, and pH) were significantly related to divergence in the beta diversity of surface and bottom-water microbial communities. Despite its correlation to microbial community structure in unconstrained analyses, constrained analyses demonstrated that dissolved organic carbon (DOC) concentration was not strongly related to microbial community structure among or within lakes. Additionally, several taxa were correlated (either positively or negatively) to environmental variables, which could be related to aerobic and anaerobic metabolisms. This study highlights the measurable relationships between environmental conditions and microbial communities within freshwater temperate lakes around the same island.

Biogeochemical and Microbial Variation across 5500 km of Antarctic Surface Sediment Implicates Organic Matter as a Driver of Benthic Community Structure.

Western Antarctica, one of the fastest warming locations on Earth, is a unique environment that is underexplored with regards to biodiversity. Although pelagic microbial communities in the Southern Ocean and coastal Antarctic waters have been well-studied, there are fewer investigations of benthic communities and most have a focused geographic range. We sampled surface sediment from 24 sites across a 5500 km region of Western Antarctica (covering the Ross Sea to the Weddell Sea) to examine relationships between microbial communities and sediment geochemistry. Sequencing of the 16S and 18S rRNA genes showed microbial communities in sediments from the Antarctic Peninsula (AP) and Western Antarctica (WA), including the Ross, Amundsen, and Bellingshausen Seas, could be distinguished by correlations with organic matter concentrations and stable isotope fractionation (total organic carbon; TOC, total nitrogen; TN, and δ(13)C). Overall, samples from the AP were higher in nutrient content (TOC, TN, and NH4 (+)) and communities in these samples had higher relative abundances of operational taxonomic units (OTUs) classified as the diatom, Chaetoceros, a marine cercozoan, and four OTUs classified as Flammeovirgaceae or Flavobacteria. As these OTUs were strongly correlated with TOC, the data suggests the diatoms could be a source of organic matter and the Bacteroidetes and cercozoan are grazers that consume the organic matter. Additionally, samples from WA have lower nutrients and were dominated by Thaumarchaeota, which could be related to their known ability to thrive as lithotrophs. This study documents the largest analysis of benthic microbial communities to date in the Southern Ocean, representing almost half the continental shoreline of Antarctica, and documents trophic interactions and coupling of pelagic and benthic communities. Our results indicate potential modifications in carbon sequestration processes related to change in community composition, identifying a prospective mechanism that links climate change to carbon availability.

Metabolic and genomic analysis elucidates strain-level variation in Microbacterium spp. isolated from chromate contaminated sediment.

Hexavalent chromium [Cr(VI)] is a soluble carcinogen that has caused widespread contamination of soil and water in many industrial nations. Bacteria have the potential to aid remediation as certain strains can catalyze the reduction of Cr(VI) to insoluble and less toxic Cr(III). Here, we examine Cr(VI) reducing Microbacterium spp. (Cr-K1W, Cr-K20, Cr-K29, and Cr-K32) isolated from contaminated sediment (Seymore, Indiana) and show varying chromate responses despite the isolates' phylogenetic similarity (i.e., identical 16S rRNA gene sequences). Detailed analysis identified differences based on genomic metabolic potential, growth and general metabolic capabilities, and capacity to resist and reduce Cr(VI). Taken together, the discrepancies between the isolates demonstrate the complexity inter-strain variation can have on microbial physiology and related biogeochemical processes.

Extracellular haem peroxidases mediate Mn(II) oxidation in a marine Roseobacter bacterium via superoxide production.

Manganese (Mn) oxides are among the strongest sorbents and oxidants in environmental systems. A number of biotic and abiotic pathways induce the oxidation of Mn(II) to Mn oxides. Here, we use a combination of proteomic analyses and activity assays, to identify the enzyme(s) responsible for extracellular superoxide-mediated Mn oxide formation by a bacterium within the ubiquitous Roseobacter clade. We show that animal haem peroxidases (AHPs) located on the outer membrane and within the secretome are responsible for Mn(II) oxidation. These novel peroxidases have previously been implicated in direct Mn(II) oxidation by phylogenetically diverse bacteria. Yet, we show that in this Roseobacter species, AHPs mediate Mn(II) oxidation not through a direct reaction but by producing superoxide and likely also by degrading hydrogen peroxide. These findings point to a eukaryotic-like oscillatory oxidative-peroxidative enzymatic cycle by these AHPs that leads to Mn oxide formation by this organism. AHP expression appears unaffected by Mn(II), yet the large energetic investment required to produce and secrete these enzymes points to an as yet unknown physiological function. These findings are further evidence that bacterial peroxidases and secreted enzymes, in general, are unappreciated controls on the cycling of metals and reactive oxygen species (ROS), and by extension carbon, in natural systems.

Constraints on superoxide mediated formation of manganese oxides.

Manganese (Mn) oxides are among the most reactive sorbents and oxidants within the environment, where they play a central role in the cycling of nutrients, metals, and carbon. Recent discoveries have identified superoxide ([Formula: see text]) both of biogenic and abiogenic origin as an effective oxidant of Mn(II) leading to the formation of Mn oxides. Here we examined the conditions under which abiotically produced superoxide led to oxidative precipitation of Mn and the solid-phases produced. Oxidized Mn, as both aqueous Mn(III) and Mn(III/IV) oxides, was only observed in the presence of active catalase, indicating that hydrogen peroxide (H2O2), a product of the reaction of [Formula: see text] with Mn(II), inhibits the oxidation process presumably through the reduction of Mn(III). Citrate and pyrophosphate increased the yield of oxidized Mn but decreased the amount of Mn oxide produced via formation of Mn(III)-ligand complexes. While complexing ligands played a role in stabilizing Mn(III), they did not eliminate the inhibition of net Mn(III) formation by H2O2. The Mn oxides precipitated were highly disordered colloidal hexagonal birnessite, similar to those produced by biotically generated superoxide. Yet, in contrast to the large particulate Mn oxides formed by biogenic superoxide, abiotic Mn oxides did not ripen to larger, more crystalline phases. This suggests that the deposition of crystalline Mn oxides within the environment requires a biological, or at least organic, influence. This work provides the first direct evidence that, under conditions relevant to natural waters, oxidation of Mn(II) by superoxide can occur and lead to formation of Mn oxides. For organisms that oxidize Mn(II) by producing superoxide, these findings may also point to other microbially mediated processes, in particular enzymatic hydrogen peroxide degradation and/or production of organic ligand metabolites, that allow for Mn oxide formation.

Involvement of Shewanella oneidensis MR-1 LuxS in biofilm development and sulfur metabolism.

The role of LuxS in Shewanella oneidensis MR-1 has been examined by transcriptomic profiling, biochemical, and physiological experiments. The results indicate that a mutation in luxS alters biofilm development, not by altering quorum-sensing abilities but by disrupting the activated methyl cycle (AMC). The S. oneidensis wild type can produce a luminescence response in the AI-2 reporter strain Vibrio harveyi MM32. This luminescence response is abolished upon the deletion of luxS. The deletion of luxS also alters biofilm formations in static and flowthrough conditions. Genetic complementation restores the mutant biofilm defect, but the addition of synthetic AI-2 has no effect. These results suggest that AI-2 is not used as a quorum-sensing signal to regulate biofilm development in S. oneidensis. Growth on various sulfur sources was examined because of the involvement of LuxS in the AMC. A mutation in luxS produced a reduced ability to grow with methionine as the sole sulfur source. Methionine is a key metabolite used in the AMC to produce a methyl source in the cell and to recycle homocysteine. These data suggest that LuxS is important to metabolizing methionine and the AMC in S. oneidensis.