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.

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

BACKGROUND: Ferrovum 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. RESULTS: Here 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. CONCLUSIONS: Previously 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.

Efficient Low-pH Iron Removal by a Microbial Iron Oxide Mound Ecosystem at Scalp Level Run.

Acid mine drainage (AMD) is a major environmental problem affecting tens of thousands of kilometers of waterways worldwide. Passive bioremediation of AMD relies on microbial communities to oxidize and remove iron from the system; however, iron oxidation rates in AMD environments are highly variable among sites. At Scalp Level Run (Cambria County, PA), first-order iron oxidation rates are 10 times greater than at other coal-associated iron mounds in the Appalachians. We examined the bacterial community at Scalp Level Run to determine whether a unique community is responsible for the rapid iron oxidation rate. Despite strong geochemical gradients, including a >10-fold change in the concentration of ferrous iron from 57.3 mg/liter at the emergence to 2.5 mg/liter at the base of the coal tailings pile, the bacterial community composition was nearly constant with distance from the spring outflow. Scalp Level Run contains many of the same taxa present in other AMD sites, but the community is dominated by two strains of Ferrovum myxofaciens, a species that is associated with high rates of Fe(II) oxidation in laboratory studies.IMPORTANCE Acid mine drainage pollutes more than 19,300 km of rivers and streams and 72,000 ha of lakes worldwide. Remediation is frequently ineffective and costly, upwards of $100 billion globally and nearly $5 billion in Pennsylvania alone. Microbial Fe(II) oxidation is more efficient than abiotic Fe(II) oxidation at low pH (P. C. Singer and W. Stumm, Science 167:1121-1123, 1970, https://doi.org/10.1126/science.167.3921.1121). Therefore, AMD bioremediation could harness microbial Fe(II) oxidation to fuel more-cost-effective treatments. Advances will require a deeper understanding of the ecology of Fe(II)-oxidizing microbial communities and the factors that control their distribution and rates of Fe(II) oxidation. We investigated bacterial communities that inhabit an AMD site with rapid Fe(II) oxidation and found that they were dominated by two operational taxonomic units (OTUs) of Ferrovum myxofaciens, a taxon associated with high laboratory rates of iron oxidation. This research represents a step forward in identifying taxa that can be used to enhance cost-effective AMD bioremediation.

Physiology, Not Nutrient Availability, May Have Limited Primary Productivity After the Emergence of Oxygenic Photosynthesis.

The evolution of oxygenic photosynthesis in Cyanobacteria was a transformative event in Earth's history. However, the scientific community disagrees over the duration of the delay between the origin of oxygenic photosynthesis and oxygenation of Earth's atmosphere, with estimates ranging from less than a hundred thousand to more than a billion years, depending on assumptions about rates of oxygen production and fluxes of reductants. Here, we propose a novel ecological hypothesis that a geologically significant delay could have been caused by biomolecular inefficiencies within proto-Cyanobacteria-ancestors of modern Cyanobacteria-that limited their maximum rates of oxygen production. Consideration of evolutionary processes and genomic data suggest to us that proto-cyanobacterial primary productivity was initially limited by photosystem instability, oxidative damage, and photoinhibition rather than nutrients or ecological competition. We propose that during the Archean era, cyanobacterial photosystems experienced protracted evolution, with biomolecular inefficiencies initially limiting primary productivity and oxygen production. Natural selection led to increases in efficiency and thus primary productivity through time. Eventually, evolutionary advances produced sufficient biomolecular efficiency that environmental factors, such as nutrient availability, limited primary productivity and shifted controls on oxygen production from physiological to environmental limitations. If correct, our novel hypothesis predicts a geologically significant interval of time between the first local oxygen production and sufficient production for oxygenation of environments. It also predicts that evolutionary rates were likely highly variable due to strong environmental selection pressures and potentially high mutation rates but low competitive interactions.

Limiting factors in the operation of photosystems I and II in cyanobacteria.

Cyanobacteria are important targets for biotechnological applications due to their ability to grow in a wide variety of environments, rapid growth rates, and tractable genetic systems. They and their bioproducts can be used as bioplastics, biofertilizers, and in carbon capture and produce important secondary metabolites that can be used as pharmaceuticals. However, the photosynthetic process in cyanobacteria can be limited by a wide variety of environmental factors such as light intensity and wavelength, exposure to UV light, nutrient limitation, temperature, and salinity. Carefully considering these limitations, modifying the environment, and/or selecting cyanobacterial species will allow cyanobacteria to be used in biotechnological applications.

Biogeographic distribution of five Antarctic cyanobacteria using large-scale k-mer searching with sourmash branchwater.

Cyanobacteria form diverse communities and are important primary producers in Antarctic freshwater environments, but their geographic distribution patterns in Antarctica and globally are still unresolved. There are however few genomes of cultured cyanobacteria from Antarctica available and therefore metagenome-assembled genomes (MAGs) from Antarctic cyanobacteria microbial mats provide an opportunity to explore distribution of uncultured taxa. These MAGs also allow comparison with metagenomes of cyanobacteria enriched communities from a range of habitats, geographic locations, and climates. However, most MAGs do not contain 16S rRNA gene sequences, making a 16S rRNA gene-based biogeography comparison difficult. An alternative technique is to use large-scale k-mer searching to find genomes of interest in public metagenomes. This paper presents the results of k-mer based searches for 5 Antarctic cyanobacteria MAGs from Lake Fryxell and Lake Vanda, assigned the names Phormidium pseudopriestleyi FRX01, Microcoleus sp. MP8IB2.171, Leptolyngbya sp. BulkMat.35, Pseudanabaenaceae cyanobacterium MP8IB2.15, and Leptolyngbyaceae cyanobacterium MP9P1.79 in 498,942 unassembled metagenomes from the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA). The Microcoleus sp. MP8IB2.171 MAG was found in a wide variety of environments, the P. pseudopriestleyi MAG was found in environments with challenging conditions, the Leptolyngbyaceae cyanobacterium MP9P1.79 MAG was only found in Antarctica, and the Leptolyngbya sp. BulkMat.35 and Pseudanabaenaceae cyanobacterium MP8IB2.15 MAGs were found in Antarctic and other cold environments. The findings based on metagenome matches and global comparisons suggest that these Antarctic cyanobacteria have distinct distribution patterns ranging from locally restricted to global distribution across the cold biosphere and other climatic zones.

Novel Gloeobacterales spp. from Diverse Environments across the Globe.

Photosynthetic Cyanobacteria and their descendants are the only known organisms capable of oxygenic photosynthesis. Their metabolism permanently changed the Earth's surface and the evolutionary trajectory of life, but little is known about their evolutionary history. Genomes of the Gloeobacterales, an order of deeply divergent photosynthetic Cyanobacteria, may hold clues about the evolutionary process. However, there are only three published genomes within this order, and it is difficult to make broad inferences based on such little data. Here, I describe five species within the Gloeobacterales retrieved from publicly available databases and examine their photosynthetic gene content and the environments in which Gloeobacterales genomes and 16S rRNA gene sequences are found. The Gloeobacterales contain reduced photosystems and inhabit cold, wet-rock, and low-light environments. They are likely present in low abundances due to their low growth rate. Future searches for Gloeobacterales should target these environments, and samples should be deeply sequenced to capture the low-abundance taxa. Publicly available databases contain undescribed taxa within the Gloeobacterales. However, searching through all available data with current methods is computationally expensive. Therefore, new methods must be developed to search for these and other evolutionarily important taxa. Once identified, these novel photosynthetic Cyanobacteria will help illuminate the origin and evolution of oxygenic photosynthesis. IMPORTANCE Early branching photosynthetic Cyanobacteria such as the Gloeobacterales may provide clues into the evolutionary history of oxygenic photosynthesis, but there are few genomes or cultured taxa from this order. Five new metagenome-assembled genomes suggest that members of the Gloeobacterales all contain reduced photosystems and lack genes associated with thylakoids and circadian rhythms. Their distribution suggests that they may thrive in environments that are marginal for other species, including wet-rock and cold environments. These traits may aid in the discovery and cultivation of novel species in this clade.

Metabolic Capacity of the Antarctic Cyanobacterium Phormidium pseudopriestleyi That Sustains Oxygenic Photosynthesis in the Presence of Hydrogen Sulfide.

Sulfide inhibits oxygenic photosynthesis by blocking electron transfer between H2O and the oxygen-evolving complex in the D1 protein of Photosystem II. The ability of cyanobacteria to counter this effect has implications for understanding the productivity of benthic microbial mats in sulfidic environments throughout Earth history. In Lake Fryxell, Antarctica, the benthic, filamentous cyanobacterium Phormidium pseudopriestleyi creates a 1-2 mm thick layer of 50 µmol L-1 O2 in otherwise sulfidic water, demonstrating that it sustains oxygenic photosynthesis in the presence of sulfide. A metagenome-assembled genome of P. pseudopriestleyi indicates a genetic capacity for oxygenic photosynthesis, including multiple copies of psbA (encoding the D1 protein of Photosystem II), and anoxygenic photosynthesis with a copy of sqr (encoding the sulfide quinone reductase protein that oxidizes sulfide). The genomic content of P. pseudopriestleyi is consistent with sulfide tolerance mechanisms including increasing psbA expression or directly oxidizing sulfide with sulfide quinone reductase. However, the ability of the organism to reduce Photosystem I via sulfide quinone reductase while Photosystem II is sulfide-inhibited, thereby performing anoxygenic photosynthesis in the presence of sulfide, has yet to be demonstrated.

A phylogenetically novel cyanobacterium most closely related to Gloeobacter.

Clues to the evolutionary steps producing innovations in oxygenic photosynthesis may be preserved in the genomes of organisms phylogenetically placed between non-photosynthetic Vampirovibrionia (formerly Melainabacteria) and the thylakoid-containing Cyanobacteria. However, only two species with published genomes are known to occupy this phylogenetic space, both within the genus Gloeobacter. Here, we describe nearly complete, metagenome-assembled genomes (MAGs) of an uncultured organism phylogenetically placed near Gloeobacter, for which we propose the name Candidatus Aurora vandensis {Au'ro.ra. L. fem. n. aurora, the goddess of the dawn in Roman mythology; van.de'nsis. N.L. fem. adj. vandensis of Lake Vanda, Antarctica}. The MAG of A. vandensis contains homologs of most genes necessary for oxygenic photosynthesis including key reaction center proteins. Many accessory subunits associated with the photosystems in other species either are missing from the MAG or are poorly conserved. The MAG also lacks homologs of genes associated with the pigments phycocyanoerethrin, phycoeretherin and several structural parts of the phycobilisome. Additional characterization of this organism is expected to inform models of the evolution of oxygenic photosynthesis.

Energy, ecology and the distribution of microbial life.

Mechanisms that govern the coexistence of multiple biological species have been studied intensively by ecologists since the turn of the nineteenth century. Microbial ecologists in the meantime have faced many fundamental challenges, such as the lack of an ecologically coherent species definition, lack of adequate methods for evaluating population sizes and community composition in nature, and enormous taxonomic and functional diversity. The accessibility of powerful, culture-independent molecular microbiology methods offers an opportunity to close the gap between microbial science and the main stream of ecological theory, with the promise of new insights and tools needed to meet the grand challenges humans face as planetary engineers and galactic explorers. We focus specifically on resources related to energy metabolism because of their direct links to elemental cycling in the Earth's history, engineering applications and astrobiology. To what extent does the availability of energy resources structure microbial communities in nature? Our recent work on sulfur- and iron-oxidizing autotrophs suggests that apparently subtle variations in the concentration ratios of external electron donors and acceptors select for different microbial populations. We show that quantitative knowledge of microbial energy niches (population-specific patterns of energy resource use) can be used to predict variations in the abundance of specific taxa in microbial communities. Furthermore, we propose that resource ratio theory applied to micro-organisms will provide a useful framework for identifying how environmental communities are organized in space and time.