Jennifer B. Glass

The importance of abiotic reactions for nitrous oxide production

The continuous rise of atmospheric nitrous oxide (N2O) is an environmental issue of global concern. In biogeochemical studies, N2O production is commonly assumed to arise solely from enzymatic reactions in microbes and fungi. However, iron, manganese and organic compounds readily undergo redox reactions with intermediates in the nitrogen cycle that produce N2O abiotically under relevant environmental conditions at circumneutral pH. Although these abiotic N2O production pathways have been known to occur for close to a century, they are often neglected in modern ecological studies. In this Synthesis and Emerging Ideas paper, we highlight the defining characteristics, environmental controls, and isotopic signatures of abiotic reactions between nitrogen cycle intermediates (hydroxylamine, nitric oxide, and nitrite), redox-active metals (iron and manganese) and organic matter (humic and fulvic acids) that can lead to N2O production. We also discuss the emerging idea that abiotic reactions coupled to biotic processes have widespread ecological relevance and encourage consideration of abiotic production mechanisms in future biogeochemical investigations of N2O cycling.

SAR11 bacteria linked to ocean anoxia and nitrogen loss

Bacteria of the SAR11 clade constitute up to one half of all microbial cells in the oxygen-rich surface ocean. SAR11 bacteria are also abundant in oxygen minimum zones (OMZs), where oxygen falls below detection and anaerobic microbes have vital roles in converting bioavailable nitrogen to N2 gas. Anaerobic metabolism has not yet been observed in SAR11, and it remains unknown how these bacteria contribute to OMZ biogeochemical cycling. Here, genomic analysis of single cells from the world’s largest OMZ revealed previously uncharacterized SAR11 lineages with adaptations for life without oxygen, including genes for respiratory nitrate reductases (Nar). SAR11 nar genes were experimentally verified to encode proteins catalysing the nitrite-producing first step of denitrification and constituted ~40% of OMZ nar transcripts, with transcription peaking in the anoxic zone of maximum nitrate reduction activity. These results link SAR11 to pathways of ocean nitrogen loss, redefining the ecological niche of Earth’s most abundant organismal group.

The Sphagnum microbiome: new insights from an ancient plant lineage

57 I. 57 II. 58 III. 59 IV. 59 V. 61 VI. 62 63 References 63 SUMMARY: Peat mosses of the genus Sphagnum play a major role in global carbon storage and dominate many northern peatland ecosystems, which are currently being subjected to some of the most rapid climate changes on Earth. A rapidly expanding database indicates that a diverse community of microorganisms is intimately associated with Sphagnum, inhabiting the tissues and surface of the plant. Here we summarize the current state of knowledge regarding the Sphagnum microbiome and provide a perspective for future research directions. Although the majority of the microbiome remains uncultivated and its metabolic capabilities uncharacterized, prokaryotes and fungi have the potential to act as mutualists, symbionts, or antagonists of Sphagnum. For example, methanotrophic and nitrogen-fixing bacteria may benefit the plant host by providing up to 20-30% of Sphagnum carbon and nitrogen, respectively. Next-generation sequencing approaches have enabled the detailed characterization of microbiome community composition in peat mosses. However, as with other ecologically or economically important plants, our knowledge of Sphagnum-microbiome associations is in its infancy. In order to attain a predictive understanding of the role of the microbiome in Sphagnum productivity and ecosystem function, the mechanisms of plant-microbiome interactions and the metabolic potential of constituent microbial populations must be revealed.

Meta-omic signatures of microbial metal and nitrogen cycling in marine oxygen minimum zones

Iron (Fe) and copper (Cu) are essential cofactors for microbial metalloenzymes, but little is known about the metalloenyzme inventory of anaerobic marine microbial communities despite their importance to the nitrogen cycle. We compared dissolved O2, NO3−, NO2−, Fe and Cu concentrations with nucleic acid sequences encoding Fe and Cu-binding proteins in 21 metagenomes and 9 metatranscriptomes from Eastern Tropical North and South Pacific oxygen minimum zones and 7 metagenomes from the Bermuda Atlantic Time-series Station. Dissolved Fe concentrations increased sharply at upper oxic-anoxic transition zones, with the highest Fe:Cu molar ratio (1.8) occurring at the anoxic core of the Eastern Tropical North Pacific oxygen minimum zone and matching the predicted maximum ratio based on data from diverse ocean sites. The relative abundance of genes encoding Fe-binding proteins was negatively correlated with O2, driven by significant increases in genes encoding Fe-proteins involved in dissimilatory nitrogen metabolisms under anoxia. Transcripts encoding cytochrome c oxidase, the Fe- and Cu-containing terminal reductase in aerobic respiration, were positively correlated with O2 content. A comparison of the taxonomy of genes encoding Fe- and Cu-binding vs. bulk proteins in OMZs revealed that Planctomycetes represented a higher percentage of Fe genes while Thaumarchaeota represented a higher percentage of Cu genes, particularly at oxyclines. These results are broadly consistent with higher relative abundance of genes encoding Fe-proteins in the genome of a marine planctomycete vs. higher relative abundance of genes encoding Cu-proteins in the genome of a marine thaumarchaeote. These findings highlight the importance of metalloenzymes for microbial processes in oxygen minimum zones and suggest preferential Cu use in oxic habitats with Cu > Fe vs. preferential Fe use in anoxic niches with Fe > Cu.

Metagenomic Binning Recovers a Transcriptionally Active Gammaproteobacterium Linking Methanotrophy to Partial Denitrification in an Anoxic Oxygen Minimum Zone

Diverse planktonic microorganisms play a crucial role in mediating methane flux from the ocean to the atmosphere. The distribution and composition of the marine methanotroph community is determined partly by oxygen availability. The low oxygen conditions of oxygen minimum zones (OMZs) may select for methanotrophs that oxidize methane using inorganic nitrogen compounds (e.g., nitrate, nitrite) in place of oxygen. However, environmental evidence for methane-nitrogen linkages in OMZs remains sparse, as does our knowledge of the genomic content and metabolic capacity of organisms catalyzing OMZ methane oxidation. Here, binning of metagenome sequences from a coastal anoxic OMZ recovered the first near complete (95%) draft genome representing the methanotroph clade OPU3. Phylogenetic reconstruction of concatenated single copy marker genes confirmed the OPU3-like bacterium as a divergent member of the type Ia methanotrophs, with an estimated genome size half that of other sequenced taxa in this group. The proportional abundance of this bacterium peaked at 4% of the total microbial community at the top of the anoxic zone in areas of nitrite and nitrate availability but declining methane concentrations. Genes mediating dissimilatory nitrate and nitrite reduction were identified in the OPU3 genome, and transcribed in conjunction with key enzymes catalyzing methane oxidation to formaldehyde and the ribulose monophosphate (RuMP) pathway for formaldehyde assimilation, suggesting partial denitrification linked to methane oxidation. Together, these data provide the first field-based evidence for methanotrophic partial denitrification by the OPU3 cluster under anoxic conditions, supporting a role for OMZs as key sites in pelagic methane turnover.

Molybdenum-based diazotrophy in a Sphagnum peatland in northern Minnesota

ABSTRACT Microbial N2 fixation (diazotrophy) represents an important nitrogen source to oligotrophic peatland ecosystems, which are important sinks for atmospheric CO2 and are susceptible to the changing climate. The objectives of this study were (i) to determine the active microbial group and type of nitrogenase mediating diazotrophy in an ombrotrophic Sphagnum-dominated peat bog (the S1 peat bog, Marcell Experimental Forest, Minnesota, USA); and (ii) to determine the effect of environmental parameters (light, O2, CO2, and CH4) on potential rates of diazotrophy measured by acetylene (C2H2) reduction and 15N2 incorporation. A molecular analysis of metabolically active microbial communities suggested that diazotrophy in surface peat was primarily mediated by Alphaproteobacteria (Bradyrhizobiaceae and Beijerinckiaceae). Despite higher concentrations of dissolved vanadium ([V] 11 nM) than molybdenum ([Mo] 3 nM) in surface peat, a combination of metagenomic, amplicon sequencing, and activity measurements indicated that Mo-containing nitrogenases dominate over the V-containing form. Acetylene reduction was only detected in surface peat exposed to light, with the highest rates observed in peat collected from hollows with the highest water contents. Incorporation of 15N2 was suppressed 90% by O2 and 55% by C2H2 and was unaffected by CH4 and CO2 amendments. These results suggest that peatland diazotrophy is mediated by a combination of C2H2-sensitive and C2H2-insensitive microbes that are more active at low concentrations of O2 and show similar activity at high and low concentrations of CH4. IMPORTANCE Previous studies indicate that diazotrophy provides an important nitrogen source and is linked to methanotrophy in Sphagnum-dominated peatlands. However, the environmental controls and enzymatic pathways of peatland diazotrophy, as well as the metabolically active microbial populations that catalyze this process, remain in question. Our findings indicate that oxygen levels and photosynthetic activity override low nutrient availability in limiting diazotrophy and that members of the Alphaproteobacteria (Rhizobiales) catalyze this process at the bog surface using the molybdenum-based form of the nitrogenase enzyme.

Microbial Manganese(III) Reduction Fueled by Anaerobic Acetate Oxidation

Soluble manganese in the intermediate +III oxidation state (Mn3+ ) is a newly identified oxidant in anoxic environments, whereas acetate is a naturally abundant substrate that fuels microbial activity. Microbial populations coupling anaerobic acetate oxidation to Mn3+ reduction, however, have yet to be identified. We isolated a Shewanella strain capable of oxidizing acetate anaerobically with Mn3+ as the electron acceptor, and confirmed this phenotype in other strains. This metabolic connection between acetate and soluble Mn3+ represents a new biogeochemical link between carbon and manganese cycles. Genomic analyses uncovered four distinct genes that allow for pathway variations in the complete dehydrogenase-driven TCA cycle that could support anaerobic acetate oxidation coupled to metal reduction in Shewanella and other Gammaproteobacteria. An oxygen-tolerant TCA cycle supporting anaerobic manganese reduction is thus a new connection in the manganese-driven carbon cycle, and a new variable for models that use manganese as a proxy to infer oxygenation events on early Earth.

Trace Metal Imaging of Sulfate-Reducing Bacteria and Methanogenic Archaea at Single-Cell Resolution by Synchrotron X-Ray Fluorescence Imaging

ABSTRACT Metal cofactors are required for many enzymes in anaerobic microbial respiration. This study examined iron, cobalt, nickel, copper, and zinc in cellular and abiotic phases at the single-cell scale for a sulfate-reducing bacterium (Desulfococcus multivorans) and a methanogenic archaeon (Methanosarcina acetivorans) using synchrotron X-ray fluorescence microscopy. Relative abundances of cellular metals were also measured by inductively coupled plasma mass spectrometry. For both species, zinc and iron were consistently the most abundant cellular metals. M. acetivorans contained higher nickel and cobalt content than D. multivorans, likely due to elevated metal requirements for methylotrophic methanogenesis. Cocultures contained spheroid zinc sulfides and cobalt/copper sulfides.

Multiple prebiotic metals mediate translation

ABSTRACT Today, Mg 2+ is an essential cofactor with diverse structural and functional roles in life’s oldest macromolecular machine, the translation system. We tested whether ancient Earth conditions (low O 2 ,high Fe 2+ , high Mn 2+ ) can revert the ribosome to a functional ancestral state. First, SHAPE (Selective 2’-Hydroxyl Acylation analyzed by Primer Extension) was used to compare the effect of Mg 2+ vs. Fe 2+ on the tertiary structure of rRNA. Then, we used in vitro translation reactions to test whether Fe 2+ or Mn 2+ could mediate protein production, and quantified ribosomal metal content. We found that: (i) Fe 2+ and Mg 2+ had strikingly similar effects on rRNA folding; (ii) Fe 2+ and Mn 2+ can replace Mg 2+ as the dominant divalent cation during translation of mRNA to functional protein; (iii) Fe 2+ and Mn 2+ associated extensively with the ribosome. Given that the translation system originated and matured when Fe 2+ and Mn 2+ were abundant, these findings suggest that Fe 2+ and Mn 2+ played a role in early ribosomal evolution. SIGNIFICANCE Ribosomes are found in every living organism where they are responsible for the translation of messenger RNA into protein. The ribosome’s centrality to cell function is underscored by its evolutionary conservation; the core structure has changed little since its inception ~4 billion years ago when ecosystems were anoxic and metal-rich. The ribosome is a model system for the study of bioinorganic chemistry, owing to the many highly coordinated divalent metal cations that are essential to its function. We studied the structure, function, and cation content of the ribosome under early Earth conditions (low O 2 , high Fe 2+ , high Mn 2+ ). Our results expand the roles of Fe 2+ and Mn 2+ in ancient and extant biochemistry as a cofactor for ribosomal structure and function.The ubiquity of Fe2+ in life, despite its insolubility in the presence of oxygen, appears to stem from conditions of the ancient Earth. Today, Mg2+ is an essential cofactor with diverse structural and functional roles in life’s oldest macromolecular machine, the translation system. We tested whether anoxia and Fe2+ can revert the ribosome to a functional ancestral state. First, SHAPE (Selective 2‘-Hydroxyl Acylation analyzed by Primer Extension) was used to compare the effect of Mg2+ vs. Fe2+ on the tertiary structure of rRNA. Then, we used in vitro translation reactions to test whether Fe2+ could mediate protein production, and quantified ribosomal iron content. We found that: (i) Fe2+ and Mg2+ had strikingly similar effects on rRNA folding; (ii) Fe2+ can replace Mg2+ as the dominant divalent cation during translation of mRNA to functional protein; (iii) Fe2+ associated extensively with the ribosome. Given that the translation system originated and matured when Fe2+ was abundant, these findings suggest that Fe2+ played a role in early ribosomal evolution. SIGNIFICANCE Ribosomes are found in every living organisms where they are responsible for the translation of messenger RNA into protein. The ribosome’s centrality to cell function is underscored by its evolutionary conservation; the core structure has changed little since its inception ∼4 billion years ago when ecosystems were anoxic and Fe2+-rich. The ribosome is a model system for the study of bioinorganic chemistry, owing to the many highly coordinated divalent metal cations that are essential to its function. We studied the structure, function, and cation content of the ribosome under early Earth conditions, (high-Fe2+, low-O2). Our results expand the role of Fe2+ in ancient and extant biochemistry as a cofactor for ribosomal structure and function.The ubiquity of Fe 2+ in life, despite its toxicity and insolubility, appears to stem from conditions of the ancient Earth. Today, Mg 2+ is an essential cofactor with diverse structural and functional roles in life9s oldest macromolecular machine, the translation system. We tested whether anoxia and Fe 2+ can revert the ribosome to a functional ancestral state. We find that Fe 2+ associates extensively with the ribosome and replaces Mg 2+ as the dominant divalent cation during translation of mRNA to functional protein. Given that the translation system originated and matured when Fe 2+ was abundant, these findings suggest that Fe 2+ played a role in early ribosomal evolution.

Ferrous iron folds rRNA and mediates translation

ABSTRACT Today, Mg 2+ is an essential cofactor with diverse structural and functional roles in life’s oldest macromolecular machine, the translation system. We tested whether ancient Earth conditions (low O 2 ,high Fe 2+ , high Mn 2+ ) can revert the ribosome to a functional ancestral state. First, SHAPE (Selective 2’-Hydroxyl Acylation analyzed by Primer Extension) was used to compare the effect of Mg 2+ vs. Fe 2+ on the tertiary structure of rRNA. Then, we used in vitro translation reactions to test whether Fe 2+ or Mn 2+ could mediate protein production, and quantified ribosomal metal content. We found that: (i) Fe 2+ and Mg 2+ had strikingly similar effects on rRNA folding; (ii) Fe 2+ and Mn 2+ can replace Mg 2+ as the dominant divalent cation during translation of mRNA to functional protein; (iii) Fe 2+ and Mn 2+ associated extensively with the ribosome. Given that the translation system originated and matured when Fe 2+ and Mn 2+ were abundant, these findings suggest that Fe 2+ and Mn 2+ played a role in early ribosomal evolution. SIGNIFICANCE Ribosomes are found in every living organism where they are responsible for the translation of messenger RNA into protein. The ribosome’s centrality to cell function is underscored by its evolutionary conservation; the core structure has changed little since its inception ~4 billion years ago when ecosystems were anoxic and metal-rich. The ribosome is a model system for the study of bioinorganic chemistry, owing to the many highly coordinated divalent metal cations that are essential to its function. We studied the structure, function, and cation content of the ribosome under early Earth conditions (low O 2 , high Fe 2+ , high Mn 2+ ). Our results expand the roles of Fe 2+ and Mn 2+ in ancient and extant biochemistry as a cofactor for ribosomal structure and function.The ubiquity of Fe2+ in life, despite its insolubility in the presence of oxygen, appears to stem from conditions of the ancient Earth. Today, Mg2+ is an essential cofactor with diverse structural and functional roles in life’s oldest macromolecular machine, the translation system. We tested whether anoxia and Fe2+ can revert the ribosome to a functional ancestral state. First, SHAPE (Selective 2‘-Hydroxyl Acylation analyzed by Primer Extension) was used to compare the effect of Mg2+ vs. Fe2+ on the tertiary structure of rRNA. Then, we used in vitro translation reactions to test whether Fe2+ could mediate protein production, and quantified ribosomal iron content. We found that: (i) Fe2+ and Mg2+ had strikingly similar effects on rRNA folding; (ii) Fe2+ can replace Mg2+ as the dominant divalent cation during translation of mRNA to functional protein; (iii) Fe2+ associated extensively with the ribosome. Given that the translation system originated and matured when Fe2+ was abundant, these findings suggest that Fe2+ played a role in early ribosomal evolution. SIGNIFICANCE Ribosomes are found in every living organisms where they are responsible for the translation of messenger RNA into protein. The ribosome’s centrality to cell function is underscored by its evolutionary conservation; the core structure has changed little since its inception ∼4 billion years ago when ecosystems were anoxic and Fe2+-rich. The ribosome is a model system for the study of bioinorganic chemistry, owing to the many highly coordinated divalent metal cations that are essential to its function. We studied the structure, function, and cation content of the ribosome under early Earth conditions, (high-Fe2+, low-O2). Our results expand the role of Fe2+ in ancient and extant biochemistry as a cofactor for ribosomal structure and function.The ubiquity of Fe 2+ in life, despite its toxicity and insolubility, appears to stem from conditions of the ancient Earth. Today, Mg 2+ is an essential cofactor with diverse structural and functional roles in life9s oldest macromolecular machine, the translation system. We tested whether anoxia and Fe 2+ can revert the ribosome to a functional ancestral state. We find that Fe 2+ associates extensively with the ribosome and replaces Mg 2+ as the dominant divalent cation during translation of mRNA to functional protein. Given that the translation system originated and matured when Fe 2+ was abundant, these findings suggest that Fe 2+ played a role in early ribosomal evolution.