Deep-Sea Archaea Fix and Share Nitrogen in Methane-Consuming Microbial Consortia

Nitrogen-fixing (diazotrophic) microorganisms regulate productivity in diverse ecosystems; however, the identities of diazotrophs are unknown in many oceanic environments. Using single-cell–resolution nanometer secondary ion mass spectrometry images of ^(15)N incorporation, we showed that deep-sea anaerobic methane-oxidizing archaea fix N_2, as well as structurally similar CN^–, and share the products with sulfate-reducing bacterial symbionts. These archaeal/bacterial consortia are already recognized as the major sink of methane in benthic ecosystems, and we now identify them as a source of bioavailable nitrogen as well. The archaea maintain their methane oxidation rates while fixing N_2 but reduce their growth, probably in compensation for the energetic burden of diazotrophy. This finding extends the demonstrated lower limits of respiratory energy capable of fueling N_2 fixation and reveals a link between the global carbon, nitrogen, and sulfur cycles

Quantifying microbial utilization of petroleum hydrocarbons in salt-marsh sediments using the ^(13)C content of bacterial rRNA

Natural remediation of oil spills is catalyzed by complex microbial consortia. Here we take a whole-community approach to investigate bacterial incorporation of petroleum hydrocarbons from a simulated oil spill. We utilized the natural difference in carbon-isotopic abundance between a salt marsh ecosystem supported by the ^(13)C-enriched C4 grass, Spartina alterniflora, and the ^(13)C-depleted composition of petroleum to monitor changes in the ^(13)C content of biomass. Magnetic-bead capture methods for the selective recovery of bacterial RNA were used to monitor the ^(13)C content of bacterial biomass during a two-week experiment. The data show that by the end of the experiment, up to 26% of bacterial biomass derived from consumption of the freshly-spilled oil. The results contrast with the inertness of a nearby relict spill, which occurred in 1969 in West Falmouth, MA. Sequences of 16S rRNA genes from our experimental samples also were consistent with previous reports suggesting the importance of {gamma}- and {delta}-Proteobacteria and Firmicutes in the remineralization of hydrocarbons. The magnetic-bead capture approach makes it possible to quantify uptake of petroleum hydrocarbons by microbes in-situ. Although employed here at the Domain level, RNA-capture procedures can be highly specific. The same strategy could be used with genus-level specificity, something which is not currently possible using the ^(13)C content of biomarker lipids

Activity and interactions of methane seep microorganisms assessed by parallel transcription and FISH-NanoSIMS analyses

To characterize the activity and interactions of methanotrophic archaea (ANME) and Deltaproteobacteria at a methane-seeping mud volcano, we used two complimentary measures of microbial activity: a community-level analysis of the transcription of four genes (16S rRNA, methyl coenzyme M reductase A (mcrA), adenosine-5′-phosphosulfate reductase α-subunit (aprA), dinitrogenase reductase (nifH)), and a single-cell-level analysis of anabolic activity using fluorescence in situ hybridization coupled to nanoscale secondary ion mass spectrometry (FISH-NanoSIMS). Transcript analysis revealed that members of the deltaproteobacterial groups Desulfosarcina/Desulfococcus (DSS) and Desulfobulbaceae (DSB) exhibit increased rRNA expression in incubations with methane, suggestive of ANME-coupled activity. Direct analysis of anabolic activity in DSS cells in consortia with ANME by FISH-NanoSIMS confirmed their dependence on methanotrophy, with no ^(15)NH^+_4 assimilation detected without methane. In contrast, DSS and DSB cells found physically independent of ANME (i.e., single cells) were anabolically active in incubations both with and without methane. These single cells therefore comprise an active ‘free-living’ population, and are not dependent on methane or ANME activity. We investigated the possibility of N_2 fixation by seep Deltaproteobacteria and detected nifH transcripts closely related to those of cultured diazotrophic Deltaproteobacteria. However, nifH expression was methane-dependent. ^(15)N_2 incorporation was not observed in single DSS cells, but was detected in single DSB cells. Interestingly, ^(15)N_2 incorporation in single DSB cells was methane-dependent, raising the possibility that DSB cells acquired reduced ^(15)N products from diazotrophic ANME while spatially coupled, and then subsequently dissociated. With this combined data set we address several outstanding questions in methane seep microbial ecosystems and highlight the benefit of measuring microbial activity in the context of spatial associations

Polyphosphate Storage during Sporulation in the Gram-Negative Bacterium Acetonema longum

Using electron cryotomography, we show that the Gram-negative sporulating bacterium Acetonema longum synthesizes high-density storage granules at the leading edges of engulfing membranes. The granules appear in the prespore and increase in size and number as engulfment proceeds. Typically, a cluster of 8 to 12 storage granules closely associates with the inner spore membrane and ultimately accounts for ∼7% of the total volume in mature spores. Energy-dispersive X-ray spectroscopy (EDX) analyses show that the granules contain high levels of phosphorus, oxygen, and magnesium and therefore are likely composed of polyphosphate (poly-P). Unlike the Gram-positive Bacilli and Clostridia, A. longum spores retain their outer spore membrane upon germination. To explore the possibility that the granules in A. longum may be involved in this unique process, we imaged purified Bacillus cereus, Bacillus thuringiensis, Bacillus subtilis, and Clostridium sporogenes spores. Even though B. cereus and B. thuringiensis contain the ppk and ppx genes, none of the spores from Gram-positive bacteria had granules. We speculate that poly-P in A. longum may provide either the energy or phosphate metabolites needed for outgrowth while retaining an outer membrane

Widespread nitrogen fixation in sediments from diverse deep-sea sites of elevated carbon loading

Nitrogen fixation, the biological conversion of N_2 to NH_3, is critical to alleviating nitrogen limitation in many marine ecosystems. To date, few measurements exist of N_2 fixation in deep‐sea sediments. Here, we conducted > 400 bottle incubations with sediments from methane seeps, whale falls and background sites off the western coast of the United States from 600 to 2893 m water depth to investigate the potential rates, spatial distribution and biological mediators of benthic N_2 fixation. We found that N2 fixation was widespread, yet heterogeneously distributed with sediment depth at all sites. In some locations, rates exceeded previous measurements by > 10×, and provided up to 30% of the community anabolic growth requirement for nitrogen. Diazotrophic activity appeared to be inhibited by pore water ammonium: N_2 fixation was only observed if incubation ammonium concentrations were ≤ 25 μM, and experimental additions of ammonium reduced diazotrophy. In seep sediments, N_2 fixation was dependent on CH_4 and coincident with sulphate reduction, consistent with previous work showing diazotrophy by microorganisms mediating sulphate‐coupled methane oxidation. However, the pattern of diazotrophy was different in whale‐fall and associated reference sediments, where it was largely unaffected by CH_4, suggesting catabolically different diazotrophs at these sites

High Mass Triple Systems: The Classical Cepheid Y Car

We have obtained an HST STIS ultraviolet high dispersion Echelle mode spectrum the binary companion of the double mode classical Cepheid Y Car. The velocity measured for the hot companion from this spectrum is very different from reasonable predictions for binary motion, implying that the companion is itself a short period binary. The measured velocity changed by 7 km/ s during the 4 days between two segments of the observation confirming this interpretation. We summarize "binary" Cepheids which are in fact members of triple system and find at least 44% are triples. The summary of information on Cepheids with orbits makes it likely that the fraction is under-estimated.Comment: accepted by A

Widespread nitrogen fixation in sediments from diverse deep-sea sites of elevated carbon loading

Nitrogen fixation, the biological conversion of N_2 to NH_3, is critical to alleviating nitrogen limitation in many marine ecosystems. To date, few measurements exist of N_2 fixation in deep‐sea sediments. Here, we conducted > 400 bottle incubations with sediments from methane seeps, whale falls and background sites off the western coast of the United States from 600 to 2893 m water depth to investigate the potential rates, spatial distribution and biological mediators of benthic N_2 fixation. We found that N2 fixation was widespread, yet heterogeneously distributed with sediment depth at all sites. In some locations, rates exceeded previous measurements by > 10×, and provided up to 30% of the community anabolic growth requirement for nitrogen. Diazotrophic activity appeared to be inhibited by pore water ammonium: N_2 fixation was only observed if incubation ammonium concentrations were ≤ 25 μM, and experimental additions of ammonium reduced diazotrophy. In seep sediments, N_2 fixation was dependent on CH_4 and coincident with sulphate reduction, consistent with previous work showing diazotrophy by microorganisms mediating sulphate‐coupled methane oxidation. However, the pattern of diazotrophy was different in whale‐fall and associated reference sediments, where it was largely unaffected by CH_4, suggesting catabolically different diazotrophs at these sites

Sediment C isotope ratios from incubations amended with 13C-labeled substrates from samples collected on cruise OC1703A aboard R/V Oceanus and cruise AT36 aboard R/V Atlantis

Dataset: OC1703A and AT36 sediment C isotope ratiosThis dataset includes sediment C isotope ratios from incubations amended with 13C-labeled substrates. Sediments were collected on cruise OC1703A aboard R/V Oceanus in March 2017 and on cruise AT36 aboard R/V Atlantis in July-August 2016. Cores were collected using an MC-800 multicore and an MC-400 multicore. The generation of these data was completed on April 9, 2020. For a complete list of measurements, refer to the full dataset description in the supplemental file 'Dataset_description.pdf'. The most current version of this dataset is available at: https://www.bco-dmo.org/dataset/863144NSF Division of Ocean Sciences (NSF OCE) OCE-163429

Pore water geochemistry from sediments collected on cruise OC1703A aboard R/V Oceanus and cruise AT36 aboard R/V Atlantis

Dataset: OC1703A and AT36 sediment geochemistryThis dataset includes pore water geochemistry from sediments collected on cruise OC1703A aboard R/V Oceanus in March 2017 and on cruise AT36 aboard R/V Atlantis in July-August 2016. Sediment cores were collected using an MC-800 multicore and an MC-400 multicore. For a complete list of measurements, refer to the full dataset description in the supplemental file 'Dataset_description.pdf'. The most current version of this dataset is available at: https://www.bco-dmo.org/dataset/862499NSF Division of Ocean Sciences (NSF OCE) OCE-163429

NCBI accession numbers describing 16S rRNA and 16S rRNA gene amplicon sequences from sediment samples collected offshore of San Francisco, Califronia, USA in March 2017 on R/V Oceanus cruise OC1703A

Dataset: OC1703A Sediment 16S rRNA and 16S rRNA gene amplicon sequencesThis dataset includes metadata and NCBI accession numbers describing 16S rRNA and 16S rRNA gene amplicon sequences from sediment samples collected offshore of San Francisco, Califronia, USA in March 2017 on R/V Oceanus cruise OC1703A. For a complete list of measurements, refer to the full dataset description in the supplemental file 'Dataset_description.pdf'. The most current version of this dataset is available at: https://www.bco-dmo.org/dataset/862690NSF Division of Ocean Sciences (NSF OCE) OCE-163429