Mycolicibacterium nivoides sp. nov isolated from a peat bog.

A fast-growing, non-chromogenic, acid-fast-staining bacterium (DL90T) was isolated from a peat bog in northern Minnesota. On the basis of 16S rRNA gene sequence similarity (99.8 % identity with Mycolicibacterium septicum and 98 % with Mycolicibacterium peregrinum) and chemotaxonomic data (fatty acid content), strain DL90T represents a member of the genus Mycolicibacterium. Physiological tests (growth curves, biofilm formation, antibiotic sensitivity, colony morphologies and heat tolerance) and biochemical analysis (arylsulfatase activity and fatty acid profiles) distinguish DL90T from its closest relative M. septicum. Phylogenomic reconstruction of the 'Fortuitium-Vaccae' clade, digital DNA-DNA hybridization (DDH) values of 61 %, and average nucleotide identity (ANI) values of approximately 95 % indicate that DL90T is likely to be diverged from M. septicum. Thus, we propose that DL90T represents a novel species, given the name Mycolicibacterium nivoides with the type strain being isolate DL90T (=JCM 32796T=NCCB 100660T).

Evidence for hydrogen oxidation and metabolic plasticity in widespread deep-sea sulfur-oxidizing bacteria.

Hydrothermal vents are a well-known source of energy that powers chemosynthesis in the deep sea. Recent work suggests that microbial chemosynthesis is also surprisingly pervasive throughout the dark oceans, serving as a significant CO(2) sink even at sites far removed from vents. Ammonia and sulfur have been identified as potential electron donors for this chemosynthesis, but they do not fully account for measured rates of dark primary production in the pelagic water column. Here we use metagenomic and metatranscriptomic analyses to show that deep-sea populations of the SUP05 group of uncultured sulfur-oxidizing Gammaproteobacteria, which are abundant in widespread and diverse marine environments, contain and highly express genes encoding group 1 Ni, Fe hydrogenase enzymes for H(2) oxidation. Reconstruction of near-complete genomes of two cooccurring SUP05 populations in hydrothermal plumes and deep waters of the Gulf of California enabled detailed population-specific metatranscriptomic analyses, revealing dynamic patterns of gene content and transcript abundance. SUP05 transcripts for genes involved in H(2) and sulfur oxidation are most abundant in hydrothermal plumes where these electron donors are enriched. In contrast, a second hydrogenase has more abundant transcripts in background deep-sea samples. Coupled with results from a bioenergetic model that suggest that H(2) oxidation can contribute significantly to the SUP05 energy budget, these findings reveal the potential importance of H(2) as a key energy source in the deep ocean. This study also highlights the genomic plasticity of SUP05, which enables this widely distributed group to optimize its energy metabolism (electron donor and acceptor) to local geochemical conditions.

Syntrophic growth of Desulfovibrio alaskensis requires genes for H2 and formate metabolism as well as those for flagellum and biofilm formation.

In anaerobic environments, mutually beneficial metabolic interactions between microorganisms (syntrophy) are essential for oxidation of organic matter to carbon dioxide and methane. Syntrophic interactions typically involve a microorganism degrading an organic compound to primary fermentation by-products and sources of electrons (i.e., formate, hydrogen, or nanowires) and a partner producing methane or respiring the electrons via alternative electron accepting processes. Using a transposon gene mutant library of the sulfate-reducing Desulfovibrio alaskensis G20, we screened for mutants incapable of serving as the electron-accepting partner of the butyrate-oxidizing bacterium, Syntrophomonas wolfei. A total of 17 gene mutants of D. alaskensis were identified as incapable of serving as the electron-accepting partner. The genes identified predominantly fell into three categories: membrane surface assembly, flagellum-pilus synthesis, and energy metabolism. Among these genes required to serve as the electron-accepting partner, the glycosyltransferase, pilus assembly protein (tadC), and flagellar biosynthesis protein showed reduced biofilm formation, suggesting that each of these components is involved in cell-to-cell interactions. Energy metabolism genes encoded proteins primarily involved in H2 uptake and electron cycling, including a rhodanese-containing complex that is phylogenetically conserved among sulfate-reducing Deltaproteobacteria. Utilizing an mRNA sequencing approach, analysis of transcript abundance in wild-type axenic and cocultures confirmed that genes identified as important for serving as the electron-accepting partner were more highly expressed under syntrophic conditions. The results imply that sulfate-reducing microorganisms require flagellar and outer membrane components to effectively couple to their syntrophic partners; furthermore, H2 metabolism is essential for syntrophic growth of D. alaskensis G20.

Metabolic flexibility of enigmatic SAR324 revealed through metagenomics and metatranscriptomics.

Chemolithotrophy is a pervasive metabolic lifestyle for microorganisms in the dark ocean. The SAR324 group of Deltaproteobacteria is ubiquitous in the ocean and has been implicated in sulfur oxidation and carbon fixation, but also contains genomic signatures of C1 utilization and heterotrophy. Here, we reconstructed the metagenome and metatranscriptome of a population of SAR324 from a hydrothermal plume and surrounding waters in the deep Gulf of California to gain insight into the genetic capability and transcriptional dynamics of this enigmatic group. SAR324's metabolism is signified by genes that encode a novel particulate hydrocarbon monooxygenase (pHMO), degradation pathways for corresponding alcohols and short-chain fatty acids, dissimilatory sulfur oxidation, formate dehydrogenase (FDH) and a nitrite reductase (NirK). Transcripts of the pHMO, NirK, FDH and transporters for exogenous carbon and amino acid uptake were highly abundant in plume waters. Sulfur oxidation genes were also abundant in the plume metatranscriptome, indicating SAR324 may also utilize reduced sulfur species in hydrothermal fluids. These results suggest that aspects of SAR324's versatile metabolism (lithotrophy, heterotrophy and alkane oxidation) operate simultaneously, and may explain SAR324's ubiquity in the deep Gulf of California and in the global marine biosphere.

Sulfur cycling connects microbiomes and biogeochemistry in deep-sea hydrothermal plumes.

In globally distributed deep-sea hydrothermal vent plumes, microbiomes are shaped by the redox energy landscapes created by reduced hydrothermal vent fluids mixing with oxidized seawater. Plumes can disperse over thousands of kilometers and their characteristics are determined by geochemical sources from vents, e.g., hydrothermal inputs, nutrients, and trace metals. However, the impacts of plume biogeochemistry on the oceans are poorly constrained due to a lack of integrated understanding of microbiomes, population genetics, and geochemistry. Here, we use microbial genomes to understand links between biogeography, evolution, and metabolic connectivity, and elucidate their impacts on biogeochemical cycling in the deep sea. Using data from 36 diverse plume samples from seven ocean basins, we show that sulfur metabolism defines the core microbiome of plumes and drives metabolic connectivity in the microbial community. Sulfur-dominated geochemistry influences energy landscapes and promotes microbial growth, while other energy sources influence local energy landscapes. We further demonstrated the consistency of links among geochemistry, function, and taxonomy. Amongst all microbial metabolisms, sulfur transformations had the highest MW-score, a measure of metabolic connectivity in microbial communities. Additionally, plume microbial populations have low diversity, short migration history, and gene-specific sweep patterns after migrating from background seawater. Selected functions include nutrient uptake, aerobic oxidation, sulfur oxidation for higher energy yields, and stress responses for adaptation. Our findings provide the ecological and evolutionary bases of change in sulfur-driven microbial communities and their population genetics in adaptation to changing geochemical gradients in the oceans.

A low-input strategy for chromium removal from industrial stormwater using peat sorbent.

Low-cost and low-input water treatment systems are important for industrial stormwater remediation. Here we examine a flow-through reactor treatment installation where water exceeds the allowable maximum concentration for drinking water in multiple metals (e.g., chromium [Cr], cadmium [Cd], zinc [Zn]) prior to treatment. Specifically, we seek to understand why Cr attenuated in the reactors is not leachable by identifying the specific chemical form of Cr and dominant mechanisms promoting sequestration in the reactors. Total solid-phase Cr concentration in the peat media ranged from 50 to 150 mg/kg after 1 yr of exposure to stormwater to 300 to 900 mg/kg after 3.5 yr. X-ray fluorescence mapping images show Cr, iron (Fe), and Zn spatially correlated over a scale of 10 µm to 5 mm. Chromium rinds form on the edges of peat particles as Cr accumulates. Chromium and Fe K-edge X-ray absorption near edge structure spectroscopy reveal chromium predominately in the 3+ oxidation state with lesser amounts of elemental Cr. We propose the primary means of chromium attenuation in the reactors is precipitation as Cr-Fe hydroxides combined with trivalent Cr adsorption onto peat surfaces.

Corrigendum: "Candidatus Chlorobium masyuteum," a Novel Photoferrotrophic Green Sulfur Bacterium Enriched From a Ferruginous Meromictic Lake.

[This corrects the article DOI: 10.3389/fmicb.2021.695260.].

"Candidatus Chlorobium masyuteum," a Novel Photoferrotrophic Green Sulfur Bacterium Enriched From a Ferruginous Meromictic Lake.

Anoxygenic phototrophic bacteria can be important primary producers in some meromictic lakes. Green sulfur bacteria (GSB) have been detected in ferruginous lakes, with some evidence that they are photosynthesizing using Fe(II) as an electron donor (i.e., photoferrotrophy). However, some photoferrotrophic GSB can also utilize reduced sulfur compounds, complicating the interpretation of Fe-dependent photosynthetic primary productivity. An enrichment (BLA1) from meromictic ferruginous Brownie Lake, Minnesota, United States, contains an Fe(II)-oxidizing GSB and a metabolically flexible putative Fe(III)-reducing anaerobe. "Candidatus Chlorobium masyuteum" grows photoautotrophically with Fe(II) and possesses the putative Fe(II) oxidase-encoding cyc2 gene also known from oxygen-dependent Fe(II)-oxidizing bacteria. It lacks genes for oxidation of reduced sulfur compounds. Its genome encodes for hydrogenases and a reverse TCA cycle that may allow it to utilize H2 and acetate as electron donors, an inference supported by the abundance of this organism when the enrichment was supplied by these substrates and light. The anaerobe "Candidatus Pseudopelobacter ferreus" is in low abundance (∼1%) in BLA1 and is a putative Fe(III)-reducing bacterium from the Geobacterales ord. nov. While "Ca. C. masyuteum" is closely related to the photoferrotrophs C. ferroooxidans strain KoFox and C. phaeoferrooxidans strain KB01, it is unique at the genomic level. The main light-harvesting molecule was identified as bacteriochlorophyll c with accessory carotenoids of the chlorobactene series. BLA1 optimally oxidizes Fe(II) at a pH of 6.8, and the rate of Fe(II) oxidation was 0.63 ± 0.069 mmol day-1, comparable to other photoferrotrophic GSB cultures or enrichments. Investigation of BLA1 expands the genetic basis for phototrophic Fe(II) oxidation by GSB and highlights the role these organisms may play in Fe(II) oxidation and carbon cycling in ferruginous lakes.

Novel Microbial Groups Drive Productivity in an Archean Iron Formation.

Deep subsurface environments are decoupled from Earth's surface processes yet diverse, active, and abundant microbial communities thrive in these isolated environments. Microbes inhabiting the deep biosphere face unique challenges such as electron donor/acceptor limitations, pore space/fracture network limitations, and isolation from other microbes within the formation. Of the few systems that have been characterized, it is apparent that nutrient limitations likely facilitate diverse microbe-microbe interactions (i.e., syntrophic, symbiotic, or parasitic) and that these interactions drive biogeochemical cycling of major elements. Here we describe microbial communities living in low temperature, chemically reduced brines at the Soudan Underground Mine State Park, United States. The Soudan Iron mine intersects a massive hematite formation at the southern extent of the Canadian Shield. Fractured rock aquifer brines continuously flow from exploratory boreholes drilled circa 1960 and are enriched in deuterium compared to the global meteoric values, indicating brines have had little contact with surface derived waters, and continually degas low molecular weight hydrocarbons C1-C4. Microbial enrichments suggest that once brines exit the boreholes, oxidation of the hydrocarbons occur. Amplicon sequencing show these borehole communities are low in diversity and dominated by Firmicute and Proteobacteria phyla. From the metagenome assemblies, we recovered approximately thirty genomes with estimated completion over 50%. Analysis of genome taxonomy generally followed the amplicon data, and highlights that several of the genomes represent novel families and genera. Metabolic reconstruction shows two carbon-fixation pathways were dominant, the Wood-Ljungdahl (acetogenesis) and Calvin-Benson-Bassham (via RuBisCo), indicating that inorganic carbon likely enters into the microbial foodweb with differing carbon fractionation potentials. Interestingly, methanogenesis is likely driven by Methanolobus and suggests cycling of methylated compounds and not H2/CO2 or acetate. Furthermore, the abundance of sulfate in brines suggests cryptic sulfur cycling may occur, as we detect possible sulfate reducing and thiosulfate oxidizing microorganisms. Finally, a majority of the microorganisms identified contain genes that would allow them to participate in several element cycles, highlighting that in these deep isolated systems metabolic flexibility may be an important life history trait.

Metagenomics of Antarctic Marine Sediment Reveals Potential for Diverse Chemolithoautotrophy.

The microbial biogeochemical processes occurring in marine sediment in Antarctica remain underexplored due to limited access. Further, these polar habitats are unique, as they are being exposed to significant changes in their climate. To explore how microbes drive biogeochemistry in these sediments, we performed a shotgun metagenomic survey of marine surficial sediment (0 to 3 cm of the seafloor) collected from 13 locations in western Antarctica and assembled 16 high-quality metagenome assembled genomes for focused interrogation of the lifestyles of some abundant lineages. We observe an abundance of genes from pathways for the utilization of reduced carbon, sulfur, and nitrogen sources. Although organotrophy is pervasive, nitrification and sulfide oxidation are the dominant lithotrophic pathways and likely fuel carbon fixation via the reverse tricarboxylic acid and Calvin cycles. Oxygen-dependent terminal oxidases are common, and genes for reduction of oxidized nitrogen are sporadically present in our samples. Our results suggest that the underlying benthic communities are well primed for the utilization of settling organic matter, which is consistent with findings from highly productive surface water. Despite the genetic potential for nitrate reduction, the net catabolic pathway in our samples remains aerobic respiration, likely coupled to the oxidation of sulfur and nitrogen imported from the highly productive Antarctic water column above. IMPORTANCE The impacts of climate change in polar regions, like Antarctica, have the potential to alter numerous ecosystems and biogeochemical cycles. Increasing temperature and freshwater runoff from melting ice can have profound impacts on the cycling of organic and inorganic nutrients between the pelagic and benthic ecosystems. Within the benthos, sediment microbial communities play a critical role in carbon mineralization and the cycles of essential nutrients like nitrogen and sulfur. Metagenomic data collected from sediment samples from the continental shelf of western Antarctica help to examine this unique system and document the metagenomic potential for lithotrophic metabolisms and the cycles of both nitrogen and sulfur, which support not only benthic microbes but also life in the pelagic zone.

Biogeochemical and physical controls on methane fluxes from two ferruginous meromictic lakes.

Meromictic lakes with anoxic bottom waters often have active methane cycles whereby methane is generally produced biogenically under anoxic conditions and oxidized in oxic surface waters prior to reaching the atmosphere. Lakes that contain dissolved ferrous iron in their deep waters (i.e., ferruginous) are rare, but valuable, as geochemical analogues of the conditions that dominated the Earth's oceans during the Precambrian when interactions between the iron and methane cycles could have shaped the greenhouse regulation of the planet's climate. Here, we explored controls on the methane fluxes from Brownie Lake and Canyon Lake, two ferruginous meromictic lakes that contain similar concentrations (max. >1 mM) of dissolved methane in their bottom waters. The order Methanobacteriales was the dominant methanogen detected in both lakes. At Brownie Lake, methanogen abundance, an increase in methane concentration with respect to depths closer to the sediment, and isotopic data suggest methanogenesis is an active process in the anoxic water column. At Canyon Lake, methanogenesis occurred primarily in the sediment. The most abundant aerobic methane-oxidizing bacteria present in both water columns were associated with the Gammaproteobacteria, with little evidence of anaerobic methane oxidizing organisms being present or active. Direct measurements at the surface revealed a methane flux from Brownie Lake that was two orders of magnitude greater than the flux from Canyon Lake. Comparison of measured versus calculated turbulent diffusive fluxes indicates that most of the methane flux at Brownie Lake was non-diffusive. Although the turbulent diffusive methane flux at Canyon Lake was attenuated by methane oxidizing bacteria, dissolved methane was detected in the epilimnion, suggestive of lateral transport of methane from littoral sediments. These results highlight the importance of direct measurements in estimating the total methane flux from water columns, and that non-diffusive transport of methane may be important to consider from other ferruginous systems.

Comparison of species richness estimates obtained using nearly complete fragments and simulated pyrosequencing-generated fragments in 16S rRNA gene-based environmental surveys.

Pyrosequencing-based 16S rRNA gene surveys are increasingly utilized to study highly diverse bacterial communities, with special emphasis on utilizing the large number of sequences obtained (tens to hundreds of thousands) for species richness estimation. However, it is not yet clear how the number of operational taxonomic units (OTUs) and, hence, species richness estimates determined using shorter fragments at different taxonomic cutoffs correlates with the number of OTUs assigned using longer, nearly complete 16S rRNA gene fragments. We constructed a 16S rRNA clone library from an undisturbed tallgrass prairie soil (1,132 clones) and used it to compare species richness estimates obtained using eight pyrosequencing candidate fragments (99 to 361 bp in length) and the nearly full-length fragment. Fragments encompassing the V1 and V2 (V1+V2) region and the V6 region (generated using primer pairs 8F-338R and 967F-1046R) overestimated species richness; fragments encompassing the V3, V7, and V7+V8 hypervariable regions (generated using primer pairs 338F-530R, 1046F-1220R, and 1046F-1392R) underestimated species richness; and fragments encompassing the V4, V5+V6, and V6+V7 regions (generated using primer pairs 530F-805R, 805F-1046R, and 967F-1220R) provided estimates comparable to those obtained with the nearly full-length fragment. These patterns were observed regardless of the alignment method utilized or the parameter used to gauge comparative levels of species richness (number of OTUs observed, slope of scatter plots of pairwise distance values for short and nearly complete fragments, and nonparametric and parametric species richness estimates). Similar results were obtained when analyzing three other datasets derived from soil, adult Zebrafish gut, and basaltic formations in the East Pacific Rise. Regression analysis indicated that these observed discrepancies in species richness estimates within various regions could readily be explained by the proportions of hypervariable, variable, and conserved base pairs within an examined fragment.

Novelty and uniqueness patterns of rare members of the soil biosphere.

Soil bacterial communities typically exhibit a distribution pattern in which most bacterial species are present in low abundance. Due to the relatively small size of most culture-independent sequencing surveys, a detailed phylogenetic analysis of rare members of the community is lacking. To gain access to the rarely sampled soil biosphere, we analyzed a data set of 13,001 near-full-length 16S rRNA gene clones derived from an undisturbed tall grass prairie soil in central Oklahoma. Rare members of the soil bacterial community (empirically defined at two different abundance cutoffs) represented 18.1 to 37.1% of the total number of clones in the data set and were, on average, less similar to their closest relatives in public databases when compared to more abundant members of the community. Detailed phylogenetic analyses indicated that members of the soil rare biosphere either belonged to novel bacterial lineages (members of five novel bacterial phyla identified in the data set, as well as members of multiple novel lineages within previously described phyla or candidate phyla), to lineages that are prevalent in other environments but rarely encountered in soil, or were close relatives to more abundant taxa in the data set. While a fraction of the rare community was closely related to more abundant taxonomic groups in the data set, a significant portion of the rare biosphere represented evolutionarily distinct lineages at various taxonomic cutoffs. We reason that these novelty and uniqueness patterns provide clues regarding the origins and potential ecological roles of members of the soil's rare biosphere.

Identification and Removal of Contaminant Sequences From Ribosomal Gene Databases: Lessons From the Census of Deep Life.

Earth's subsurface environment is one of the largest, yet least studied, biomes on Earth, and many questions remain regarding what microorganisms are indigenous to the subsurface. Through the activity of the Census of Deep Life (CoDL) and the Deep Carbon Observatory, an open access 16S ribosomal RNA gene sequence database from diverse subsurface environments has been compiled. However, due to low quantities of biomass in the deep subsurface, the potential for incorporation of contaminants from reagents used during sample collection, processing, and/or sequencing is high. Thus, to understand the ecology of subsurface microorganisms (i.e., the distribution, richness, or survival), it is necessary to minimize, identify, and remove contaminant sequences that will skew the relative abundances of all taxa in the sample. In this meta-analysis, we identify putative contaminants associated with the CoDL dataset, recommend best practices for removing contaminants from samples, and propose a series of best practices for subsurface microbiology sampling. The most abundant putative contaminant genera observed, independent of evenness across samples, were Propionibacterium, Aquabacterium, Ralstonia, and Acinetobacter. While the top five most frequently observed genera were Pseudomonas, Propionibacterium, Acinetobacter, Ralstonia, and Sphingomonas. The majority of the most frequently observed genera (high evenness) were associated with reagent or potential human contamination. Additionally, in DNA extraction blanks, we observed potential archaeal contaminants, including methanogens, which have not been discussed in previous contamination studies. Such contaminants would directly affect the interpretation of subsurface molecular studies, as methanogenesis is an important subsurface biogeochemical process. Utilizing previously identified contaminant genera, we found that ∼27% of the total dataset were identified as contaminant sequences that likely originate from DNA extraction and DNA cleanup methods. Thus, controls must be taken at every step of the collection and processing procedure when working with low biomass environments such as, but not limited to, portions of Earth's deep subsurface. Taken together, we stress that the CoDL dataset is an incredible resource for the broader research community interested in subsurface life, and steps to remove contamination derived sequences must be taken prior to using this dataset.

Complete genome analysis of a Siphoviridae phage TSK1 showing biofilm removal potential against Klebsiella pneumoniae.

Multidrug-resistant Klebsiella pneumoniae is a nosocomial pathogen, produces septicemia, pneumonia and UTI. Excessive use of antibiotics contributes towards emergence of multidrug-resistance. Bacteriophage-therapy is a potential substitute of antibiotics with many advantages. In this investigation, microbiological and genome characterization of TSK1 bacteriophage and its biofilm elimination capability are presented. TSK1 showed narrow host range and highest stability at pH 7 and 37 °C. TSK1 reduced the growth of K. pneumoniae during the initial 14 hours of infection. Post-treatment with TSK1 against different age K. pneumoniae biofilms reduced 85-100% biomass. Pre-treatment of TSK1 bacteriophage against the biofilm of Klebsiella pneumoniae reduced > 99% biomass in initial 24 hr of incubation. The genome of TSK1 phage comprised 49,836 base pairs with GC composition of 50.44%. Total seventy-five open reading frames (ORFs) were predicted, 25 showed homology with known functional proteins, while 50 were called hypothetical, as no homologs with proved function exists in the genome databases. Blast and phylogenetic analysis put it in the Kp36 virus genus of family Siphoviridae. Proposed packaging strategy of TSK1 bacteriophage genome is headful packaging using the pac sites. The potential of TSK1 bacteriophage could be used to reduce the bacterial load and biofilm in clinical and non-clinical settings.

Complete Genome Sequence of Desulfovibrio desulfuricans Strain G11, a Model Sulfate-Reducing, Hydrogenotrophic, and Syntrophic Partner Organism.

Here, we report the draft genome of the Gram-negative, sulfate-reducing bacterium Desulfovibrio desulfuricans strain G11. Isolated from a rumen fluid enrichment, this culture has been a model syntrophic partner due to its metabolic flexibility. The assembly yielded a single circular chromosome of 3,414,943 bp and a 57% G+C content.

Pyrophosphate-Dependent ATP Formation from Acetyl Coenzyme A in Syntrophus aciditrophicus, a New Twist on ATP Formation.

UNLABELLED: Syntrophus aciditrophicus is a model syntrophic bacterium that degrades key intermediates in anaerobic decomposition, such as benzoate, cyclohexane-1-carboxylate, and certain fatty acids, to acetate when grown with hydrogen-/formate-consuming microorganisms. ATP formation coupled to acetate production is the main source for energy conservation by S. aciditrophicus However, the absence of homologs for phosphate acetyltransferase and acetate kinase in the genome of S. aciditrophicus leaves it unclear as to how ATP is formed, as most fermentative bacteria rely on these two enzymes to synthesize ATP from acetyl coenzyme A (CoA) and phosphate. Here, we combine transcriptomic, proteomic, metabolite, and enzymatic approaches to show that S. aciditrophicus uses AMP-forming, acetyl-CoA synthetase (Acs1) for ATP synthesis from acetyl-CoA. acs1 mRNA and Acs1 were abundant in transcriptomes and proteomes, respectively, of S. aciditrophicus grown in pure culture and coculture. Cell extracts of S. aciditrophicus had low or undetectable acetate kinase and phosphate acetyltransferase activities but had high acetyl-CoA synthetase activity under all growth conditions tested. Both Acs1 purified from S. aciditrophicus and recombinantly produced Acs1 catalyzed ATP and acetate formation from acetyl-CoA, AMP, and pyrophosphate. High pyrophosphate levels and a high AMP-to-ATP ratio (5.9 ± 1.4) in S. aciditrophicus cells support the operation of Acs1 in the acetate-forming direction. Thus, S. aciditrophicus has a unique approach to conserve energy involving pyrophosphate, AMP, acetyl-CoA, and an AMP-forming, acetyl-CoA synthetase. IMPORTANCE: Bacteria use two enzymes, phosphate acetyltransferase and acetate kinase, to make ATP from acetyl-CoA, while acetate-forming archaea use a single enzyme, an ADP-forming, acetyl-CoA synthetase, to synthesize ATP and acetate from acetyl-CoA. Syntrophus aciditrophicus apparently relies on a different approach to conserve energy during acetyl-CoA metabolism, as its genome does not have homologs to the genes for phosphate acetyltransferase and acetate kinase. Here, we show that S. aciditrophicus uses an alternative approach, an AMP-forming, acetyl-CoA synthetase, to make ATP from acetyl-CoA. AMP-forming, acetyl-CoA synthetases were previously thought to function only in the activation of acetate to acetyl-CoA.

Proteomic analysis reveals metabolic and regulatory systems involved in the syntrophic and axenic lifestyle of Syntrophomonas wolfei.

Microbial syntrophy is a vital metabolic interaction necessary for the complete oxidation of organic biomass to methane in all-anaerobic ecosystems. However, this process is thermodynamically constrained and represents an ecosystem-level metabolic bottleneck. To gain insight into the physiology of this process, a shotgun proteomics approach was used to quantify the protein landscape of the model syntrophic metabolizer, Syntrophomonas wolfei, grown axenically and syntrophically with Methanospirillum hungatei. Remarkably, the abundance of most proteins as represented by normalized spectral abundance factor (NSAF) value changed very little between the pure and coculture growth conditions. Among the most abundant proteins detected were GroEL and GroES chaperonins, a small heat shock protein, and proteins involved in electron transfer, beta-oxidation, and ATP synthesis. Several putative energy conservation enzyme systems that utilize NADH and ferredoxin were present. The abundance of an EtfAB2 and the membrane-bound iron-sulfur oxidoreductase (Swol_0698 gene product) delineated a potential conduit for electron transfer between acyl-CoA dehydrogenases and membrane redox carriers. Proteins detected only when S. wolfei was grown with M. hungatei included a zinc-dependent dehydrogenase with a GroES domain, whose gene is present in genomes in many organisms capable of syntrophy, and transcriptional regulators responsive to environmental stimuli or the physiological status of the cell. The proteomic analysis revealed an emphasis on macromolecular stability and energy metabolism by S. wolfei and presence of regulatory mechanisms responsive to external stimuli and cellular physiological status.

Microbial communities of the Lemon Creek Glacier show subtle structural variation yet stable phylogenetic composition over space and time.

Glaciers are geologically important yet transient ecosystems that support diverse, biogeochemically significant microbial communities. During the melt season glaciers undergo dramatic physical, geochemical, and biological changes that exert great influence on downstream biogeochemical cycles. Thus, we sought to understand the temporal melt-season dynamics of microbial communities and associated geochemistry at the terminus of Lemon Creek Glacier (LCG) in coastal southern Alaska. Due to late season snowfall, sampling of LCG occurred in three interconnected areas: proglacial Lake Thomas, the lower glacial outflow stream, and the glacier's terminus. LCG associated microbial communities were phylogenetically diverse and varied by sampling location. However, Betaproteobacteria, Alphaproteobacteria, and Bacteroidetes dominated communities at all sampling locations. Strict anaerobic groups such as methanogens, SR1, and OP11 were also recovered from glacier outflows, indicating anoxic conditions in at least some portions of the LCG subglacial environment. Microbial community structure was significantly correlated with sampling location and sodium concentrations. Microbial communities sampled from terminus outflow waters exhibited day-to-day fluctuation in taxonomy and phylogenetic similarity. However, these communities were not significantly different from randomly constructed communities from all three sites. These results indicate that glacial outflows share a large proportion of phylogenetic overlap with downstream environments and that the observed significant shifts in community structure are driven by changes in relative abundance of different taxa, and not complete restructuring of communities. We conclude that LCG glacial discharge hosts a diverse and relatively stable microbiome that shifts at fine taxonomic scales in response to geochemistry and likely water residence time.