Nitrogen Isotope Fractionation During Archaeal Ammonia Oxidation: Coupled Estimates From Measurements of Residual Ammonium and Accumulated Nitrite

The naturally occurring nitrogen (N) isotopes,N-15 and(14)N, exhibit different reaction rates during many microbial N transformation processes, which results in N isotope fractionation. Such isotope effects are critical parameters for interpreting natural stable isotope abundances as proxies for biological process rates in the environment across scales. The kinetic isotope effect of ammonia oxidation (AO) to nitrite (NO2-), performed by ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB), is generally ascribed to the enzyme ammonia monooxygenase (AMO), which catalyzes the first step in this process. However, the kinetic isotope effect of AMO, or epsilon(AMO), has been typically determined based on isotope kinetics during product formation (cumulative product, NO2-) alone, which may have overestimated epsilon(AMO)due to possible accumulation of chemical intermediates and alternative sinks of ammonia/ammonium (NH3/NH4+). Here, we analyzed(15)N isotope fractionation during archaeal ammonia oxidation based on both isotopic changes in residual substrate (RS, NH4+) and cumulative product (CP, NO2-) pools in pure cultures of the soil strainNitrososphaera viennensisEN76 and in highly enriched cultures of the marine strainNitrosopumilus adriaticusNF5, under non-limiting substrate conditions. We obtained epsilon(AMO)values of 31.9-33.1 parts per thousand for both strains based on RS (delta(NH4+)-N-15) and showed that estimates based on CP (delta(NO2-)-N-15) give larger isotope fractionation factors by 6-8 parts per thousand. Complementary analyses showed that, at the end of the growth period, microbial biomass was(15)N-enriched (10.1 parts per thousand), whereas nitrous oxide (N2O) was highly(15)N depleted (-38.1 parts per thousand) relative to the initial substrate. Although we did not determine the isotope effect of NH(4)(+)assimilation (biomass formation) and N2O production by AOA, our results nevertheless show that the discrepancy between epsilon(AMO)estimates based on RS and CP might have derived from the incorporation of(15)N-enriched residual NH(4)(+)after AMO reaction into microbial biomass and that N2O production did not affect isotope fractionation estimates significantly

Microbial community assembly and evolution in subseafloor sediment

Bacterial and archaeal communities inhabiting the subsurface seabed live under strong energy limitation and have growth rates that are orders of magnitude slower than laboratory-grown cultures. It is not understood how subsurface microbial communities are assembled and whether populations undergo adaptive evolution or accumulate mutations as a result of impaired DNA repair under such energy-limited conditions. Here we use amplicon sequencing to explore changes of microbial communities during burial and isolation from the surface to the > 5,000-y-old subsurface of marine sediment and identify a small core set of mostly uncultured bacteria and archaea that is present throughout the sediment column. These persisting populations constitute a small fraction of the entire community at the surface but become predominant in the subsurface. We followed patterns of genome diversity with depth in four dominant lineages of the persisting populations by mapping metagenomic sequence reads onto single-cell genomes. Nucleotide sequence diversity was uniformly low and did not change with age and depth of the sediment. Likewise, therewas no detectable change inmutation rates and efficacy of selection. Our results indicate that subsurface microbial communities predominantly assemble by selective survival of taxa able to persist under extreme energy limitation

Single-Cell Genomics Reveals a Diverse Metabolic Potential of Uncultivated Desulfatiglans-Related Deltaproteobacteria Widely Distributed in Marine Sediment

Desulfatiglans-related organisms comprise one of the most abundant deltaproteobacterial lineages in marine sediments where they occur throughout the sediment column in a gradient of increasing sulfate and organic carbon limitation with depth. Characterized Desulfatiglans isolates are dissimilatory sulfate reducers able to grow by degrading aromatic hydrocarbons. The ecophysiology of environmental Desulfatiglans-populations is poorly understood, however, possibly utilization of aromatic compounds may explain their predominance in marine subsurface sediments. We sequenced and analyzed seven Desulfatiglans-related single-cell genomes (SAGs) from Aarhus Bay sediments to characterize their metabolic potential with regard to aromatic compound degradation and energy metabolism. The average genome assembly size was 1.3 Mbp and completeness estimates ranged between 20 and 50%. Five of the SAGs (group 1) originated from the sulfate-rich surface part of the sediment while two (group 2) originated from sulfate-depleted subsurface sediment. Based on 16S rRNA gene amplicon sequencing group 2 SAGs represent the more frequent types of Deaufatig/ans-populations in Aarhus Bay sediments. Genes indicative of aromatic compound degradation could be identified in both groups, but the two groups were metabolically distinct with regard to energy conservation. Group 1 SAGs carry a full set of genes for dissimilatory sulfate reduction, whereas the group 2 SAGs lacked any genetic evidence for sulfate reduction. The latter may be due to incompleteness of the SAGs, but as alternative energy metabolisms group 2 SAGs carry the genetic potential for growth by acetogenesis and fermentation. Group 1 SAGs encoded reductive dehalogenase genes, allowing them to access organohalides and possibly conserve energy by their reduction. Both groups possess sulfatases unlike their cultured relatives allowing them to utilize sulfate esters as source of organic carbon and sulfate. In conclusion, the uncultivated marine Desulfatiglans populations are metabolically diverse, likely reflecting different strategies for coping with energy and sulfate limitation in the subsurface seabed

Marine Deep Biosphere Microbial Communities Assemble in Near-Surface Sediments in Aarhus Bay

Analyses of microbial diversity in marine sediments have identified a core set of taxa unique to the marine deep biosphere. Previous studies have suggested that these specialized communities are shaped by processes in the surface seabed, in particular that their assembly is associated with the transition from the bioturbated upper zone to the nonbioturbated zone below. To test this hypothesis, we performed a fine-scale analysis of the distribution and activity of microbial populations within the upper 50 cm of sediment from Aarhus Bay (Denmark). Sequencing and qPCR were combined to determine the depth distributions of bacterial and archaeal taxa (16S rRNA genes) and sulfate-reducing microorganisms (SRM) (dsrB gene). Mapping of radionuclides throughout the sediment revealed a region of intense bioturbation at 0–6 cm depth. The transition from bioturbated sediment to the subsurface below (7 cm depth) was marked by a shift from dominant surface populations to common deep biosphere taxa (e.g., Chloroflexi and Atribacteria). Changes in community composition occurred in parallel to drops in microbial activity and abundance caused by reduced energy availability below the mixed sediment surface. These results offer direct evidence for the hypothesis that deep subsurface microbial communities present in Aarhus Bay mainly assemble already centimeters below the sediment surface, below the bioturbation zone

In vitro interaction network of a synthetic gut bacterial community

A key challenge in microbiome research is to predict the functionality of microbial communities based on community membership and (meta)-genomic data. As central microbiota functions are determined by bacterial community networks, it is important to gain insight into the principles that govern bacteria-bacteria interactions. Here, we focused on the growth and metabolic interactions of the Oligo-Mouse-Microbiota (OMM12) synthetic bacterial community, which is increasingly used as a model system in gut microbiome research. Using a bottom-up approach, we uncovered the directionality of strain-strain interactions in mono- and pairwise co-culture experiments as well as in community batch culture. Metabolic network reconstruction in combination with metabolomics analysis of bacterial culture supernatants provided insights into the metabolic potential and activity of the individual community members. Thereby, we could show that the OMM12 interaction network is shaped by both exploitative and interference competition in vitro in nutrient-rich culture media and demonstrate how community structure can be shifted by changing the nutritional environment. In particular, Enterococcus faecalis KB1 was identified as an important driver of community composition by affecting the abundance of several other consortium members in vitro. As a result, this study gives fundamental insight into key drivers and mechanistic basis of the OMM12 interaction network in vitro, which serves as a knowledge base for future mechanistic in vivo studies

Helicobacter hepaticus is required for immune targeting of bacterial heat shock protein 60 and fatal colitis in mice

Gut microbiota and the immune system are in constant exchange shaping both host immunity and microbial communities. Here, improper immune regulation can cause inflammatory bowel disease (IBD) and colitis. Antibody therapies blocking signaling through the CD40–CD40L axis showed promising results as these molecules are deregulated in certain IBD patients. To better understand the mechanism, we used transgenic DC-LMP1/CD40 animals with a constitutive CD40-signal in CD11c+ cells, causing a lack of intestinal CD103+ dendritic cells (DCs) and failure to induce regulatory T (iTreg) cells. These mice rapidly develop spontaneous fatal colitis, accompanied by dysbiosis and increased inflammatory IL-17+IFN-γ+ Th17/Th1 and IFN-γ + Th1 cells. In the present study, we analyzed the impact of the microbiota on disease development and detected elevated IgA- and IgG-levels in sera from DC-LMP1/CD40 animals. Their serum antibodies specifically bound intestinal bacteria, and by proteome analysis, we identified a 60 kDa chaperonin GroEL (Hsp60) from Helicobacter hepaticus (Hh) as the main specific antigen targeted in the absence of iTregs. When re-derived to a different Hh-free specific-pathogen-free (SPF) microbiota, mice showed few signs of disease, normal microbiota, and no fatality. Upon recolonization of mice with Hh, the disease developed rapidly. Thus, the present work identifies GroEL/Hsp60 as a major Hh-antigen and its role in disease onset, progression, and outcome in this colitis model. Our results highlight the importance of CD103+ DC- and iTreg-mediated immune tolerance to specific pathobionts to maintain healthy intestinal balance

Nitrogen Isotope Fractionation During Archaeal Ammonia Oxidation: Coupled Estimates From Measurements of Residual Ammonium and Accumulated Nitrite.

The naturally occurring nitrogen (N) isotopes, 15N and 14N, exhibit different reaction rates during many microbial N transformation processes, which results in N isotope fractionation. Such isotope effects are critical parameters for interpreting natural stable isotope abundances as proxies for biological process rates in the environment across scales. The kinetic isotope effect of ammonia oxidation (AO) to nitrite (NO2 -), performed by ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB), is generally ascribed to the enzyme ammonia monooxygenase (AMO), which catalyzes the first step in this process. However, the kinetic isotope effect of AMO, or ε A M O , has been typically determined based on isotope kinetics during product formation (cumulative product, NO2 -) alone, which may have overestimated ε A M O due to possible accumulation of chemical intermediates and alternative sinks of ammonia/ammonium (NH3/NH4 +). Here, we analyzed 15N isotope fractionation during archaeal ammonia oxidation based on both isotopic changes in residual substrate (RS, NH4 +) and cumulative product (CP, NO2 -) pools in pure cultures of the soil strain Nitrososphaera viennensis EN76 and in highly enriched cultures of the marine strain Nitrosopumilus adriaticus NF5, under non-limiting substrate conditions. We obtained ε A M O values of 31.9-33.1‰ for both strains based on RS (δ15NH4 +) and showed that estimates based on CP (δ15NO2 -) give larger isotope fractionation factors by 6-8‰. Complementary analyses showed that, at the end of the growth period, microbial biomass was 15N-enriched (10.1‰), whereas nitrous oxide (N2O) was highly 15N depleted (-38.1‰) relative to the initial substrate. Although we did not determine the isotope effect of NH4 + assimilation (biomass formation) and N2O production by AOA, our results nevertheless show that the discrepancy between ε A M O estimates based on RS and CP might have derived from the incorporation of 15N-enriched residual NH4 + after AMO reaction into microbial biomass and that N2O production did not affect isotope fractionation estimates significantly

In vitro interaction network of a synthetic gut bacterial community.

A key challenge in microbiome research is to predict the functionality of microbial communities based on community membership and (meta)-genomic data. As central microbiota functions are determined by bacterial community networks, it is important to gain insight into the principles that govern bacteria-bacteria interactions. Here, we focused on the growth and metabolic interactions of the Oligo-Mouse-Microbiota (OMM12) synthetic bacterial community, which is increasingly used as a model system in gut microbiome research. Using a bottom-up approach, we uncovered the directionality of strain-strain interactions in mono- and pairwise co-culture experiments as well as in community batch culture. Metabolic network reconstruction in combination with metabolomics analysis of bacterial culture supernatants provided insights into the metabolic potential and activity of the individual community members. Thereby, we could show that the OMM12 interaction network is shaped by both exploitative and interference competition in vitro in nutrient-rich culture media and demonstrate how community structure can be shifted by changing the nutritional environment. In particular, Enterococcus faecalis KB1 was identified as an important driver of community composition by affecting the abundance of several other consortium members in vitro. As a result, this study gives fundamental insight into key drivers and mechanistic basis of the OMM12 interaction network in vitro, which serves as a knowledge base for future mechanistic in vivo studies