David A. Stahl

Ammonia oxidation kinetics determine niche separation of nitrifying Archaea and Bacteria

The discovery of ammonia oxidation by mesophilic and thermophilic Crenarchaeota and the widespread distribution of these organisms in marine and terrestrial environments indicated an important role for them in the global nitrogen cycle. However, very little is known about their physiology or their contribution to nitrification. Here we report oligotrophic ammonia oxidation kinetics and cellular characteristics of the mesophilic crenarchaeon ‘Candidatus Nitrosopumilus maritimus’ strain SCM1. Unlike characterized ammonia-oxidizing bacteria, SCM1 is adapted to life under extreme nutrient limitation, sustaining high specific oxidation rates at ammonium concentrations found in open oceans. Its half-saturation constant (Km = 133 nM total ammonium) and substrate threshold (≤10 nM) closely resemble kinetics of in situ nitrification in marine systems and directly link ammonia-oxidizing Archaea to oligotrophic nitrification. The remarkably high specific affinity for reduced nitrogen (68,700 l per g cells per h) of SCM1 suggests that Nitrosopumilus-like ammonia-oxidizing Archaea could successfully compete with heterotrophic bacterioplankton and phytoplankton. Together these findings support the hypothesis that nitrification is more prevalent in the marine nitrogen cycle than accounted for in current biogeochemical models.

Nitrosopumilus maritimus genome reveals unique mechanisms for nitrification and autotrophy in globally distributed marine crenarchaea

Ammonia-oxidizing archaea are ubiquitous in marine and terrestrial environments and now thought to be significant contributors to carbon and nitrogen cycling. The isolation of Candidatus “Nitrosopumilus maritimus” strain SCM1 provided the opportunity for linking its chemolithotrophic physiology with a genomic inventory of the globally distributed archaea. Here we report the 1,645,259-bp closed genome of strain SCM1, revealing highly copper-dependent systems for ammonia oxidation and electron transport that are distinctly different from known ammonia-oxidizing bacteria. Consistent with in situ isotopic studies of marine archaea, the genome sequence indicates N. maritimus grows autotrophically using a variant of the 3-hydroxypropionate/4-hydroxybutryrate pathway for carbon assimilation, while maintaining limited capacity for assimilation of organic carbon. This unique instance of archaeal biosynthesis of the osmoprotectant ectoine and an unprecedented enrichment of multicopper oxidases, thioredoxin-like proteins, and transcriptional regulators points to an organism responsive to environmental cues and adapted to handling reactive copper and nitrogen species that likely derive from its distinctive biochemistry. The conservation of N. maritimus gene content and organization within marine metagenomes indicates that the unique physiology of these specialized oligophiles may play a significant role in the biogeochemical cycles of carbon and nitrogen.

Distinct gene set in two different lineages of ammonia-oxidizing archaea supports the phylum Thaumarchaeota

Globally distributed archaea comprising ammonia oxidizers of moderate terrestrial and marine environments are considered the most abundant archaeal organisms on Earth. Based on 16S rRNA phylogeny, initial assignment of these archaea was to the Crenarchaeota. By contrast, features of the first genome sequence from a member of this group suggested that they belong to a novel phylum, the Thaumarchaeota. Here, we re-investigate the Thaumarchaeota hypothesis by including two newly available genomes, that of the marine ammonia oxidizer Nitrosopumilus maritimus and that of Nitrososphaera gargensis, a representative of another evolutionary lineage within this group predominantly detected in terrestrial environments. Phylogenetic studies based on r-proteins and other core genes, as well as comparative genomics, confirm the assignment of these organisms to a separate phylum and reveal a Thaumarchaeota-specific set of core informational processing genes, as well as potentially ancestral features of the archaea.

Multiple Lateral Transfers of Dissimilatory Sulfite Reductase Genes between Major Lineages of Sulfate-Reducing Prokaryotes

A large fragment of the dissimilatory sulfite reductase genes (dsrAB) was PCR amplified and fully sequenced from 30 reference strains representing all recognized lineages of sulfate-reducing bacteria. In addition, the sequence of the dsrAB gene homologs of the sulfite reducer Desulfitobacterium dehalogenans was determined. In contrast to previous reports, comparative analysis of all available DsrAB sequences produced a tree topology partially inconsistent with the corresponding 16S rRNA phylogeny. For example, the DsrAB sequences of several Desulfotomaculum species (low G+C gram-positive division) and two members of the genus Thermodesulfobacterium (a separate bacterial division) were monophyletic with delta-proteobacterial DsrAB sequences. The most parsimonious interpretation of these data is that dsrAB genes from ancestors of as-yet-unrecognized sulfate reducers within the delta-Proteobacteria were laterally transferred across divisions. A number of insertions and deletions in the DsrAB alignment independently support these inferred lateral acquisitions of dsrAB genes. Evidence for a dsrAB lateral gene transfer event also was found within the delta-Proteobacteria, affecting Desulfobacula toluolica. The root of the dsr tree was inferred to be within the Thermodesulfovibrio lineage by paralogous rooting of the alpha and beta subunits. This rooting suggests that the dsrAB genes in Archaeoglobus species also are the result of an ancient lateral transfer from a bacterial donor. Although these findings complicate the use of dsrAB genes to infer phylogenetic relationships among sulfate reducers in molecular diversity studies, they establish a framework to resolve the origins and diversification of this ancient respiratory lifestyle among organisms mediating a key step in the biogeochemical cycling of sulfur.

Metabolic modeling of a mutualistic microbial community

The rate of production of methane in many environments depends upon mutualistic interactions between sulfate‐reducing bacteria and methanogens. To enhance our understanding of these relationships, we took advantage of the fully sequenced genomes of Desulfovibrio vulgaris and Methanococcus maripaludis to produce and analyze the first multispecies stoichiometric metabolic model. Model results were compared to data on growth of the co‐culture on lactate in the absence of sulfate. The model accurately predicted several ecologically relevant characteristics, including the flux of metabolites and the ratio of D. vulgaris to M. maripaludis cells during growth. In addition, the model and our data suggested that it was possible to eliminate formate as an interspecies electron shuttle, but hydrogen transfer was essential for syntrophic growth. Our work demonstrated that reconstructed metabolic networks and stoichiometric models can serve not only to predict metabolic fluxes and growth phenotypes of single organisms, but also to capture growth parameters and community composition of simple bacterial communities.

Intact Membrane Lipids of "Candidatus Nitrosopumilus maritimus," a Cultivated Representative of the Cosmopolitan Mesophilic Group I Crenarchaeota

ABSTRACT In this study we analyzed the membrane lipid composition of “Candidatus Nitrosopumilus maritimus,” the only cultivated representative of the cosmopolitan group I crenarchaeota and the only mesophilic isolate of the phylum Crenarchaeota. The core lipids of “Ca. Nitrosopumilus maritimus” consisted of glycerol dialkyl glycerol tetraethers (GDGTs) with zero to four cyclopentyl moieties. Crenarchaeol, a unique GDGT containing a cyclohexyl moiety in addition to four cyclopentyl moieties, was the most abundant GDGT. This confirms unambiguously that crenarchaeol is synthesized by species belonging to the group I.1a crenarchaeota. Intact polar lipid analysis revealed that the GDGTs have hexose, dihexose, and/or phosphohexose head groups. Similar polar lipids were previously found in deeply buried sediments from the Peru margin, suggesting that they were in part synthesized by group I crenarchaeota.

Optimization of Single-Base-Pair Mismatch Discrimination in Oligonucleotide Microarrays

ABSTRACT The discrimination between perfect-match and single-base-pair-mismatched nucleic acid duplexes was investigated by using oligonucleotide DNA microarrays and nonequilibrium dissociation rates (melting profiles). DNA and RNA versions of two synthetic targets corresponding to the 16S rRNA sequences of Staphylococcus epidermidis (38 nucleotides) and Nitrosomonas eutropha (39 nucleotides) were hybridized to perfect-match probes (18-mer and 19-mer) and to a set of probes having all possible single-base-pair mismatches. The melting profiles of all probe-target duplexes were determined in parallel by using an imposed temperature step gradient. We derived an optimum wash temperature for each probe and target by using a simple formula to calculate a discrimination index for each temperature of the step gradient. This optimum corresponded to the output of an independent analysis using a customized neural network program. These results together provide an experimental and analytical framework for optimizing mismatch discrimination among all probes on a DNA microarray.

Stochasticity, succession, and environmental perturbations in a fluidic ecosystem

Significance The study of ecological succession remains at the core of ecology. Understanding the trajectories and mechanisms controlling ecological succession is crucial to predicting the responses of ecosystems to environmental change and projecting their future states. By definition, deterministic succession is expected under homogeneous abiotic and biotic starting conditions. This study, however, shows that the succession of groundwater microbial communities in response to nutrient amendment is primarily stochastic, but that the drivers controlling biodiversity and succession are dynamic rather than static. By identifying the mechanisms controlling microbial community assembly and succession, this study makes fundamental contribution to the mechanistic understanding essential for a predictive microbial ecology of many systems ranging from microbiomes of humans and plants to natural and managed ecosystems. Unraveling the drivers of community structure and succession in response to environmental change is a central goal in ecology. Although the mechanisms shaping community structure have been intensively examined, those controlling ecological succession remain elusive. To understand the relative importance of stochastic and deterministic processes in mediating microbial community succession, a unique framework composed of four different cases was developed for fluidic and nonfluidic ecosystems. The framework was then tested for one fluidic ecosystem: a groundwater system perturbed by adding emulsified vegetable oil (EVO) for uranium immobilization. Our results revealed that groundwater microbial community diverged substantially away from the initial community after EVO amendment and eventually converged to a new community state, which was closely clustered with its initial state. However, their composition and structure were significantly different from each other. Null model analysis indicated that both deterministic and stochastic processes played important roles in controlling the assembly and succession of the groundwater microbial community, but their relative importance was time dependent. Additionally, consistent with the proposed conceptual framework but contradictory to conventional wisdom, the community succession responding to EVO amendment was primarily controlled by stochastic rather than deterministic processes. During the middle phase of the succession, the roles of stochastic processes in controlling community composition increased substantially, ranging from 81.3% to 92.0%. Finally, there are limited successional studies available to support different cases in the conceptual framework, but further well-replicated explicit time-series experiments are needed to understand the relative importance of deterministic and stochastic processes in controlling community succession.

Synthesis of methylphosphonic acid by marine microbes: A source for methane in the aerobic ocean

Ocean Methane Is methylphosphonate a natural product? If it is, then its catabolism by microrganisms seeking phosphorus might explain the ubiquity of methane in the oceans. Anaerobic Archaea, so far, are the only significant source of methane known. Thus, Metcalf et al. (p. 1104) looked for evidence of methylphosphonate biochemistry in the widespread archaeon Nitrosopumilus maritimus. The organism can synthesize C-P bonds using phosphoenolpyruvate mutase. By using its gene, ppm, as a marker, a possible phosphonate biosynthesis gene cluster was identified, and adjacent to this a gene for a cupin was found. Together with Fe(II) and O2 the cupin catalyzed the production of methylphosphonate and formate. Homologs of the cupin gene are found in a wide range of common marine microbes, including Pelagibacter ubique and Prochlorococcus. The archaeon Nitrosopumilus maritimus makes what may be a major source of ocean methane. Relative to the atmosphere, much of the aerobic ocean is supersaturated with methane; however, the source of this important greenhouse gas remains enigmatic. Catabolism of methylphosphonic acid by phosphorus-starved marine microbes, with concomitant release of methane, has been suggested to explain this phenomenon, yet methylphosphonate is not a known natural product, nor has it been detected in natural systems. Further, its synthesis from known natural products would require unknown biochemistry. Here we show that the marine archaeon Nitrosopumilus maritimus encodes a pathway for methylphosphonate biosynthesis and that it produces cell-associated methylphosphonate esters. The abundance of a key gene in this pathway in metagenomic data sets suggests that methylphosphonate biosynthesis is relatively common in marine microbes, providing a plausible explanation for the methane paradox.

Abundance of Ammonia-Oxidizing Archaea and Bacteria along an Estuarine Salinity Gradient in Relation to Potential Nitrification Rates

ABSTRACT Abundance of ammonia-oxidizing Archaea (AOA) was found to be always greater than that of ammonia-oxidizing Bacteria along an estuarine salinity gradient, and AOA abundance was highest at intermediate salinity. However, AOA abundance did not correlate with potential nitrification rates. This lack of correlation may be due to methodological limitations or alternative energy sources.