An Alternative Path for the Evolution of Biological Nitrogen Fixation

Nitrogenase catalyzed nitrogen fixation is the process by which life converts dinitrogen gas into fixed nitrogen in the form of bioavailable ammonia. The most common form of nitrogenase today requires a complex metal cluster containing molybdenum (Mo), although alternative forms exist which contain vanadium (V) or only iron (Fe). It has been suggested that Mo-independent forms of nitrogenase (V and Fe) were responsible for N2 fixation on early Earth because oceans were Mo-depleted and Fe-rich. Phylogenetic- and structure-based examinations of multiple nitrogenase proteins suggest that such an evolutionary path is unlikely. Rather, our results indicate an evolutionary path whereby Mo-dependent nitrogenase emerged within the methanogenic archaea and then gave rise to the alternative forms suggesting that they arose later, perhaps in response to local Mo limitation. Structural inferences of nitrogenase proteins and related paralogs suggest that the ancestor of all nitrogenases had an open cavity capable of binding metal clusters which conferred reactivity. The evolution of the nitrogenase ancestor and its associated bound metal cluster was controlled by the availability of fixed nitrogen in combination with local environmental factors that influenced metal availability until a point in Earth’s geologic history where the most desirable metal, Mo, became sufficiently bioavailable to bring about and refine the solution (Mo-nitrogenase) we see perpetuated in extant biology

Characterization of diverse bacteriohopanepolyols in a permanently stratified, hyper-euxinic lake

Bacteriohopanepolyols (BHPs) are a diverse class of bacterial lipids that hold promise as biomarkers of specific microbes, microbial processes, and environmental conditions. BHPs have been characterized in a variety of terrestrial and aquatic environments, but less is known about their distribution and abundance in extreme environmental systems. In the present study, samples taken from the water column and upper sediments of the hyper-euxinic, meromictic Mahoney Lake (Canada) were analyzed for BHPs. Analyses show distinct BHP distributions within the oxic mixolimnion, the chemocline, and the euxinic monimolimnion. Bacteriohopanetetrol (BHT) and unsaturated BHT are the dominant BHPs found in the oxic mixolimnion and at the chemocline, whereas a novel BHP (tentatively identified as diunsaturated aminotriol) dominates the euxinic monimolimnion. Along with the novel BHP structure, composite BHPs (i.e., BHT-cyclitol ether and BHT-glucosamine) were observed in the euxinic monimolimnion and sediments, indicating their production by anaerobic bacteria. Complementary metagenomic analysis of genes involved in BHP biosynthesis (i.e., shc, hpnH, hpnO, hpnP, and hpnR) further revealed that BHPs in Mahoney Lake are most likely produced by bacteria belonging to Deltaproteobacteria, Chloroflexi, Planctomycetia, and Verrucomicrobia. The combined observations of BHP distribution and metagenomic analyses additionally indicate that 2- and 3-methyl BHTs are produced within the euxinic sediments in response to low oxygen and high osmotic concentrations, as opposed to being diagnostic biomarkers of cyanobacteria and aerobic metabolisms

Genomics, Exometabolomics, and Metabolic Probing Reveal Conserved Proteolytic Metabolism of Thermoflexus hugenholtzii and Three Candidate Species From China and Japan

Thermoflexus hugenholtzii JAD2 , the only cultured representative of the Chloroflexota order Thermoflexales, is abundant in Great Boiling Spring (GBS), NV, United States, and close relatives inhabit geothermal systems globally. However, no defined medium exists for T. hugenholtzii JAD2 and no single carbon source is known to support its growth, leaving key knowledge gaps in its metabolism and nutritional needs. Here, we report comparative genomic analysis of the draft genome of T. hugenholtzii JAD2 and eight closely related metagenome-assembled genomes (MAGs) from geothermal sites in China, Japan, and the United States, representing “Candidatus Thermoflexus japonica,” “Candidatus Thermoflexus tengchongensis,” and “Candidatus Thermoflexus sinensis.” Genomics was integrated with targeted exometabolomics and C metabolic probing of T. hugenholtzii. The Thermoflexus genomes each code for complete central carbon metabolic pathways and an unusually high abundance and diversity of peptidases, particularly Metallo- and Serine peptidase families, along with ABC transporters for peptides and some amino acids. The T. hugenholtzii JAD2 exometabolome provided evidence of extracellular proteolytic activity based on the accumulation of free amino acids. However, several neutral and polar amino acids appear not to be utilized, based on their accumulation in the medium and the lack of annotated transporters. Adenine and adenosine were scavenged, and thymine and nicotinic acid were released, suggesting interdependency with other organisms in situ. Metabolic probing of T. hugenholtzii JAD2 using C-labeled compounds provided evidence of oxidation of glucose, pyruvate, cysteine, and citrate, and functioning glycolytic, tricarboxylic acid (TCA), and oxidative pentose-phosphate pathways (PPPs). However, differential use of position-specific C-labeled compounds showed that glycolysis and the TCA cycle were uncoupled. Thus, despite the high abundance of Thermoflexus in sediments of some geothermal systems, they appear to be highly focused on chemoorganotrophy, particularly protein degradation, and may interact extensively with other microorganisms in situ. T T T 13 T T 13 1

Carbon and nitrogen recycling during cyanoHABs in dreissenid-invaded and non-invaded US midwestern lakes and reservoirs

Lakes and reservoirs play key roles in global carbon cycling, especially as a carbon sink. Enrichment of nutrients in lakes and reservoirs (eutrophication) and rising global temperatures favors the proliferation of bloom-forming cyanobacteria. Harmful blooms of cyanobacteria (cyanoHABs) alter carbon and nutrient cycling in freshwater ecosystems. Some evidence suggests the introduction or establishment of invasive mussel species (i.e., Dreissena spp.) also favor cyanoHAB formation through selective filter feeding, a process through which they may also impact biogeochemical processes including carbon cycling and sequestration. However, few studies have considered the combined effects of invasive mussels and cyanoHABs on carbon and nitrogen cycling in freshwater ecosystems. Here, we examined microbial community composition and biogeochemical attributes (including carbon and nitrogen stable isotopes) in eutrophic lakes, reservoirs, and rivers in western Ohio, eastern Indiana, and northern Kentucky during the cyanobacterial bloom period of the summer of 2015. Our samples include both sites impacted by invasive mussels and those where invasive mussels have not yet been observed. Based on 16S and 18S rRNA gene sequence analysis, we found that cyanobacterial and algal communities varied across sites and were most closely related to habitat (sediment or water column sample) and site, regardless of the presence of invasive mussels or other environmental factors. However, we did find evidence that invasive mussels may influence both carbon and nitrogen cycling. While the results are based on a single time point sampling, they highlight the interactions of multiple environmental stressors in aquatic ecosystems and the critical need for more temporally intensive studies of carbon and nutrient cycling in bloom- and mussel-impacted waters

Metagenome-Assembled Genomes of Novel Taxa from an Acid Mine Drainage Environment

Acid mine drainage (AMD) is a global problem in which iron sulfide minerals oxidize and generate acidic, metal-rich water. Bioremediation relies on understanding how microbial communities inhabiting an AMD site contribute to biogeochemical cycling. A number of studies have reported community composition in AMD sites from 16S rRNA gene amplicons, but it remains difficult to link taxa to function, especially in the absence of closely related cultured species or those with published genomes. Unfortunately, there is a paucity of genomes and cultured taxa from AMD environments. Here, we report 29 novel metagenome-assembled genomes from Cabin Branch, an AMD site in the Daniel Boone National Forest, Kentucky, USA. The genomes span 11 bacterial phyla and one archaeal phylum and include taxa that contribute to carbon, nitrogen, sulfur, and iron cycling. These data reveal overlooked taxa that contribute to carbon fixation in AMD sites as well as uncharacterized Fe(II)-oxidizing bacteria. These data provide additional context for 16S rRNA gene studies, add to our understanding of the taxa involved in biogeochemical cycling in AMD environments, and can inform bioremediation strategies. IMPORTANCE Bioremediating acid mine drainage requires understanding how microbial communities influence geochemical cycling of iron and sulfur and biologically important elements such as carbon and nitrogen. Research in this area has provided an abundance of 16S rRNA gene amplicon data. However, linking these data to metabolisms is difficult because many AMD taxa are uncultured or lack published genomes. Here, we present metagenome-assembled genomes from 29 novel AMD taxa and detail their metabolic potential. These data provide information on AMD taxa that could be important for bioremediation strategies, including taxa that are involved in cycling iron, sulfur, carbon, and nitrogen

Productivity and Community Composition of Low Biomass/High Silica Precipitation Hot Springs: A Possible Window to Earth’s Early Biosphere?

Terrestrial hot springs have provided a niche space for microbial communities throughout much of Earth’s history, and evidence for hydrothermal deposits on the Martian surface suggest this could have also been the case for the red planet. Prior to the evolution of photosynthesis, life in hot springs on early Earth would have been supported though chemoautotrophy. Today, hot spring geochemical and physical parameters can preclude the occurrence of oxygenic phototrophs, providing an opportunity to characterize the geochemical and microbial components. In the absence of the photo-oxidation of water, chemoautotrophy in these hot springs (and throughout Earth’s history) relies on the delivery of exogenous electron acceptors and donors such as H2, H2S, and Fe2+. Thus, systems fueled by chemoautotrophy are likely energy substrate-limited and support low biomass communities compared to those where oxygenic phototrophs are prevalent. Low biomass silica-precipitating systems have implications for preservation, especially over geologic time. Here, we examine and compare the productivity and composition of low biomass chemoautotrophic versus photoautotrophic communities in silica-saturated hot springs. Our results indicate low biomass chemoautotrophic microbial communities in Yellowstone National Park are supported primarily by sulfur redox reactions and, while similar in total biomass, show higher diversity in anoxygenic phototrophic communities compared to chemoautotrophs. Our data suggest productivity in Archean terrestrial hot springs may be directly linked to redox substrate availability, and there may be high potential for geochemical and physical biosignature preservation from these communities

Metabolic diversity and co-occurrence of multiple Ferrovum species at an acid mine drainage site

BackgroundFerrovum spp. are abundant in acid mine drainage sites globally where they play an important role in biogeochemical cycling. All known taxa in this genus are Fe(II) oxidizers. Thus, co-occurring members of the genus could be competitors within the same environment. However, we found multiple, co-occurring Ferrovum spp. in Cabin Branch, an acid mine drainage site in the Daniel Boone National Forest, KY.ResultsHere we describe the distribution of Ferrovum spp. within the Cabin Branch communities and metagenome assembled genomes (MAGs) of two new Ferrovum spp. In contrast to previous studies, we recovered multiple 16S rRNA gene sequence variants suggesting the commonly used 97% cutoff may not be appropriate to differentiate Ferrovum spp. We also retrieved two nearly-complete Ferrovum spp. genomes from metagenomic data. The genomes of these taxa differ in several key ways relating to nutrient cycling, motility, and chemotaxis.ConclusionsPreviously reported Ferrovum genomes are also diverse with respect to these categories suggesting that the genus Ferrovum contains substantial metabolic diversity. This diversity likely explains how the members of this genus successfully co-occur in Cabin Branch and why Ferrovum spp. are abundant across geochemical gradients

Hot Spring Microbial Community Composition, Morphology, and Carbon Fixation: Implications for Interpreting the Ancient Rock Record

Microbial communities in hydrothermal systems exist in a range of macroscopic morphologies including stromatolites, mats, and filaments. The architects of these structures are typically autotrophic, serving as primary producers. Structures attributed to microbial life have been documented in the rock record dating back to the Archean including recent reports of microbially-related structures in terrestrial hot springs that date back as far as 3.5 Ga. Microbial structures exhibit a range of complexity from filaments to more complex mats and stromatolites and the complexity impacts preservation potential. As a result, interpretation of these structures in the rock record relies on isotopic signatures in combination with overall morphology and paleoenvironmental setting. However, the relationships between morphology, microbial community composition, and primary productivity remain poorly constrained. To begin to address this gap, we examined community composition and carbon fixation in filaments, mats, and stromatolites from the Greater Obsidian Pool Area (GOPA) of the Mud Volcano Area, Yellowstone National Park, WY. We targeted morphologies dominated by bacterial phototrophs located in close proximity within the same pool which are exposed to similar geochemistry as well as bacterial mat, algal filament and chemotrophic filaments from nearby springs. Our results indicate (i) natural abundance δ13C values of biomass from these features (−11.0 to −24.3‰) are similar to those found in the rock record; (ii) carbon uptake rates of photoautotrophic communities is greater than chemoautotrophic; (iii) oxygenic photosynthesis, anoxygenic photosynthesis, and chemoautotrophy often contribute to carbon fixation within the same morphology; and (iv) increasing phototrophic biofilm complexity corresponds to a significant decrease in rates of carbon fixation—filaments had the highest uptake rates whereas carbon fixation by stromatolites was significantly lower. Our data highlight important differences in primary productivity between structures despite indistinguishable δ13C values of the biomass. Furthermore, low primary productivity by stromatolites compared to other structures underscores the need to consider a larger role for microbial mats and filaments in carbon fixation and O2 generation during the Archean and Proterozoic

Metabolic diversity and ecological niches of Achromatium populations revealed with single-cell genomic sequencing

Large, sulfur-cycling, calcite-precipitating bacteria in the genus Achromatium represent a significant proportion of bacterial communities near sediment-water interfaces throughout the world. Our understanding of their potentially crucial roles in calcium, carbon, sulfur, nitrogen, and iron cycling is limited because they have not been cultured or sequenced using environmental genomics approaches to date. We utilized single-cell genomic sequencing to obtain one incomplete and two nearly complete draft genomes for Achromatium collected at Warm Mineral Springs, FL. Based on 16S rRNA gene sequences, the three cells represent distinct and relatively distant Achromatium populations (91-92% identity). The draft genomes encode key genes involved in sulfur and hydrogen oxidation; oxygen, nitrogen and polysulfide respiration; carbon and nitrogen fixation; organic carbon assimilation and storage; chemotaxis; twitching motility; antibiotic resistance; and membrane transport. Known genes for iron and manganese energy metabolism were not detected. The presence of pyrophosphatase and vacuolar (V)-type ATPases, which are generally rare in bacterial genomes, suggests a role for these enzymes in calcium transport, proton pumping, and/or energy generation in the membranes of calcite-containing inclusions