Project description:We established simple synthetic microbial communities in a microcosm model system to determine the mechanisms that underlay cross-feeding in microbial methane-consuming communities. Co-occurring strains from Lake Washington sediment were used that are involved in methane consumption, a methanotroph and two non-methanotrophic methylotrophs.
Project description:Coastal marine sediments, as locations of substantial fixed nitrogen loss, are very important to the nitrogen budget and to the primary productivity of the oceans. Coastal sediment systems are also highly dynamic and subject to periodic natural and anthropogenic organic substrate additions. The response to organic matter by the microbial community involved in nitrogen loss processes was evaluated using mesocosms of Chesapeake Bay sediments. Over the course of a 50-day incubation, rates of anammox and denitrification were measured weekly using 15N tracer incubations, and samples were collected for genetic analysis. Rates of both nitrogen loss processes and gene abundances associated with them corresponded loosely, probably because heterogeneities in sediments obscured a clear relationship. The rates of denitrification were stimulated more by the higher organic matter addition, and the fraction of nitrogen loss attributed to anammox slightly reduced. Furthermore, the large organic matter pulse drove a significant and rapid shift in the denitrifier community as determined using a nirS microarray, indicating the diversity of these organisms plays an essential role in responding to anthropogenic inputs. We also suggest that the proportion of nitrogen loss due to anammox in these coastal estuarine sediments may be underestimated due to temporal dynamics as well as from methodological artifacts related to conventional sediment slurry incubation approaches.
Project description:Coastal marine sediments, as locations of substantial fixed nitrogen loss, are very important to the nitrogen budget and to the primary productivity of the oceans. Coastal sediment systems are also highly dynamic and subject to periodic natural and anthropogenic organic substrate additions. The response to organic matter by the microbial community involved in nitrogen loss processes was evaluated using mesocosms of Chesapeake Bay sediments. Over the course of a 50-day incubation, rates of anammox and denitrification were measured weekly using 15N tracer incubations, and samples were collected for genetic analysis. Rates of both nitrogen loss processes and gene abundances associated with them corresponded loosely, probably because heterogeneities in sediments obscured a clear relationship. The rates of denitrification were stimulated more by the higher organic matter addition, and the fraction of nitrogen loss attributed to anammox slightly reduced. Furthermore, the large organic matter pulse drove a significant and rapid shift in the denitrifier community as determined using a nirS microarray, indicating the diversity of these organisms plays an essential role in responding to anthropogenic inputs. We also suggest that the proportion of nitrogen loss due to anammox in these coastal estuarine sediments may be underestimated due to temporal dynamics as well as from methodological artifacts related to conventional sediment slurry incubation approaches. Two color array (Cy3 and Cy5): the universal standard 20-mer oligo is printed to the slide with a 70-mer oligo (an archetype). Environmental DNA sequences (fluoresced with Cy3) within 15% of the 70-mer conjugated to a 20-mer oligo (fluoresced with Cy5) complementary to the universal standard will bind to the oligo probes on the array. Signal is the ratio of Cy3 to Cy5. Three replicate probes were printed for each archetype. Two replicate arrays were run on duplicate targets.
Project description:In this study, we investigated Mn3+-cycling microbial populations enriched from Lake Matano, Indonesia using metagenomics and metaproteomics. Lake Matano contains an active Mn cycle that links the oxic-anoxic interface with anoxic deep waters that are enriched in iron and manganese, and depleted in sulfate, phosphate, and oxidized nitrogen (Crowe et al., 2008; Jones et al., 2011). Sediments were incubated with sequential transfers for ~1 year with Mn3+ as the sole electron acceptor and methane as organic carbon until achieving sediment-free conditions. Here we investigate this novel species of Dechloromonas (Betaproteobacteria), “Candidatus Dechloromonas occultata,” which was the dominant population in enrichment cultures with active Mn3+ reduction. “Ca. D. occultata” expressed electron conduits related to those involved in Fe2+ oxidation (Mto-like), as well as a novel cytochrome c-rich gene cluster putatively involved in extracellular electron transfer, and an atypical nitrous oxide reductase. According to ribosomal counts, Dechloromonas outnumber Geobacter. In terms of functional genes, Dechloromonas expresses a wider variety and number of genes. Dechloromonas therefore seems to have a (selective?) advantage over Geobacter. Previous experiments revealed that Dechloromonas express nitrogen regulators, reductases and scavenging genes, as well as many carbon central metabolic pathways, and aromatic carbon degradation pathways. Dechloromonas is a beta proteobacteria, and these are "experts" in nitrogen metabolism. Geobacter, on the other hand, is well known for carbon degradation. Our previous experiments lead to our hypothesis that Dechloromonas is more active because they are more successful at acquiring nitrogen, a limiting nutrient for Geobacter. This would further suggest that carbon is not the limiting nutrient. We will test 2 hypotheses with the next suite of experiments 1) pyrophosphate supports the community, by allowing carbon fixation , 2)Dechloromonas has a (selective?) advantage over Geobacter. To test this hypothesis, bioreactors will be used to grow biotriplicate cultures of (1)- CH4 vs. pyrophosphate and (2)-CH4 vs. Mn(III) pyrophosphate. Here we have analyzed whole cell pellets using gas phase fractionations on the Q Exactive. Are Dechloromonas capable of out-competing Geobacter when grown in media with methane as the only carbon source bioreactors because they are capable of acquiring more nitrogen? Source of inoculum. Lake Matano is a metal-rich, ancient ocean analog (Crowe et al. 2011, Jones et al. 2011). Organic carbon in Lake Matano is mostly mineralized via methanogenesis before reaching the iron-rich sediments, limiting organic matter bioavailability for metal-reducers (Kuntz et al. 2015). A 15-cm sediment core from 200 m water depth in Lake Matano, Sulawesi Island, Indonesia (02°26′27.1′′S, 121°15′12.3′′E; in situ sediment temperature ~27°C) was sampled in November 2014 and sub-sampled at 5 cm increments. Sediments were sealed in gas-tight Mylar bags with no headspace (Hansen et al. 2000) and stored at 4°C until incubations began in December 2015.
Project description:Epigenetic variation has the potential to control environmentally dependent development and contribute to phenotypic responses to local environments. Environmental epigenetic studies of sexual organisms confirm the responsiveness of epigenetic variation, which should be even more important when genetic variation is lacking. A previous study of an asexual snail, Potamopyrgus antipodarum, demonstrated that different populations derived from a single clonal lineage differed in both shell phenotype and methylation signature when comparing lake versus river populations. Here, we examine methylation variation among lakes that differ in environmental disturbance and pollution histories. The differential DNA methylation regions (DMRs) identified among the different lake comparisons suggested a higher number of DMRs and variation between rural Lake 1 and one urban Lake 2 and between the two urban Lakes 2 and 3, but limited variation between the rural Lake 1 and urban Lake 3. DMR genomic characteristics and gene associations were investigated. Observations suggest there is no effect of geographic distance or any consistent pattern of DMRs between urban and rural lakes. Environmental factors may influence epigenetic response.
Project description:Chemosynthetic symbioses occur worldwide in marine habitats, but comprehensive physiological studies of chemoautotrophic bacteria thriving on animals are scarce. Stilbonematinae are coated by monocultures of thiotrophic Gammaproteobacteria. As these nematodes migrate through the redox zone, their ectosymbionts experience varying oxygen concentrations. Here, by applying omics, Raman microspectroscopy and stable isotope labeling, we investigated the effect of oxygen on the metabolism of Candidatus Thiosymbion oneisti. Unexpectedly, sulfur oxidation genes were upregulated in anoxic relative to oxic conditions, but carbon fixation genes and incorporation of 13C-labeled bicarbonate were not. Instead, several genes involved in carbon fixation, organic carbon assimilation and polyhydroxyalkanoate (PHA) biosynthesis, as well as nitrogen fixation and urea utilization were upregulated in oxic conditions. Furthermore, in the presence of oxygen, stress-related genes were upregulated together with vitamin biosynthesis genes likely necessary to withstand its deleterious effects, and fewer symbionts were detected to divide. Based on this first global physiological study of an uncultured chemosynthetic ectosymbiont, we propose that, in anoxic sediment, its proliferation is powered by anaerobic sulfur oxidation coupled to denitrification, whereas in upper layers it makes use of aerobic respiration to facilitate assimilation of carbon and nitrogen, and to survive oxidative stress. The ectosymbiont’s versatile metabolism is thus well-adapted to exploiting a highly changeable environment.
Project description:Denitrification, a crucial biochemical pathway prevalent among haloarchaea in hypersaline ecosystems, has garnered considerable attention in recent years due to its ecological implications. Nevertheless, the underlying molecular mechanisms and genetic regulation governing this respiration/detoxification process in haloarchaea remain largely unexplored. In this study, RNA-sequencing was used to compare the transcriptomes of the haloarchaeon Haloferax mediterranei under oxic and denitrifying conditions, shedding light on the intricate metabolic alterations occurring within the cell such as the accurate control of the metal homeostasis. Furthermore, the investigation identifies several genes encoding transcriptional regulators and potential accessory proteins with putative roles in denitrification. Among these are bacterioopsin transcriptional activators, proteins harbouring a domain of unknown function (DUF2249), and a cyanoglobin. Additionally, the study delves into the genetic regulation of denitrification, finding a regulatory motif within promoter regions that activates numerous denitrification-related genes. This research serves as a starting point for future molecular biology studies in haloarchaea, offering a promising avenue to unravel the intricate mechanisms governing haloarchaeal denitrification, a pathway of paramount ecological importance.