Acetylenotrophic and Diazotrophic Bradyrhizobium sp. Strain I71 from TCE-Contaminated Soils.

Acetylene (C2H2) is a molecule rarely found in nature, with very few known natural sources, but acetylenotrophic microorganisms can use acetylene as their primary carbon and energy source. As of 2018 there were 15 known strains of aerobic and anaerobic acetylenotrophs; however, we hypothesize there may yet be unrecognized diversity of acetylenotrophs in nature. This study expands the known diversity of acetylenotrophs by isolating the aerobic acetylenotroph, Bradyrhizobium sp. strain I71, from trichloroethylene (TCE)-contaminated soils. Strain I71 is a member of the class Alphaproteobacteria and exhibits acetylenotrophic and diazotrophic activities, the only two enzymatic reactions known to transform acetylene. This unique capability in the isolated strain may increase the genus' economic impact beyond agriculture as acetylenotrophy is closely linked to bioremediation of chlorinated contaminants. Computational analyses indicate that the Bradyrhizobium sp. strain I71 genome contains 522 unique genes compared to close relatives. Moreover, applying a novel hidden Markov model of known acetylene hydratase (AH) enzymes identified a putative AH enzyme. Protein annotation with I-TASSER software predicted the AH from the microbe Syntrophotalea acetylenica as the closest structural and functional analog. Furthermore, the putative AH was flanked by horizontal gene transfer (HGT) elements, like that of AH in anaerobic acetylenotrophs, suggesting an unknown source of acetylene or acetylenic substrate in the environment that is selecting for the presence of AH. IMPORTANCE The isolation of Bradyrhizobium strain I71 expands the distribution of acetylene-consuming microbes to include a group of economically important microorganisms. Members of Bradyrhizobium are well studied for their abilities to improve plant health and increase crop yields by providing bioavailable nitrogen. Additionally, acetylene-consuming microbes have been shown to work in tandem with other microbes to degrade soil contaminants. Based on genome, cultivation, and protein prediction analysis, the ability to consume acetylene is likely not widespread within the genus Bradyrhizobium. These findings suggest that the suite of phenotypic capabilities of strain I71 may be unique and make it a good candidate for further study in several research avenues.

Respiratory Selenite Reductase from Bacillus selenitireducens Strain MLS10.

The putative respiratory selenite [Se(IV)] reductase (Srr) from Bacillus selenitireducens MLS10 has been identified through a polyphasic approach involving genomics, proteomics, and enzymology. Nondenaturing gel assays were used to identify Srr in cell fractions, and the active band was shown to contain a single protein of 80 kDa. The protein was identified through liquid chromatography-tandem mass spectrometry (LC-MS/MS) as a homolog of the catalytic subunit of polysulfide reductase (PsrA). It was found to be encoded as part of an operon that contains six genes that we designated srrE, srrA, srrB, srrC, srrD, and srrF SrrA is the catalytic subunit (80 kDa), with a twin-arginine translocation (TAT) leader sequence indicative of a periplasmic protein and one putative 4Fe-4S binding site. SrrB is a small subunit (17 kDa) with four putative 4Fe-4S binding sites, SrrC (43 kDa) is an anchoring subunit, and SrrD (24 kDa) is a chaperon protein. Both SrrE (38 kDa) and SrrF (45 kDa) were annotated as rhodanese domain-containing proteins. Phylogenetic analysis revealed that SrrA belonged to the PsrA/PhsA clade but that it did not define a distinct subgroup, based on the putative homologs that were subsequently identified from other known selenite-respiring bacteria (e.g., Desulfurispirillum indicum and Pyrobaculum aerophilum). The enzyme appeared to be specific for Se(IV), showing no activity with selenate, arsenate, or thiosulfate, with a Km of 145 ± 53 µM, a Vmax of 23 ± 2.5 µM min-1, and a kcat of 23 ± 2.68 s-1 These results further our understanding of the mechanisms of selenium biotransformation and its biogeochemical cycle.IMPORTANCE Selenium is an essential element for life, with Se(IV) reduction a key step in its biogeochemical cycle. This report identifies for the first time a dissimilatory Se(IV) reductase, Srr, from a known selenite-respiring bacterium, the haloalkalophilic Bacillus selenitireducens strain MLS10. The work extends the versatility of the complex iron-sulfur molybdoenzyme (CISM) superfamily in electron transfer involving chalcogen substrates with different redox potentials. Further, it underscores the importance of biochemical and enzymological approaches in establishing the functionality of these enzymes.

Syntrophotalea acetylenivorans sp. nov., a diazotrophic, acetylenotrophic anaerobe isolated from intertidal sediments.

A Gram-stain-negative, strictly anaerobic, non-motile, rod-shaped bacterium, designated SFB93T, was isolated from the intertidal sediments of South San Francisco Bay, located near Palo Alto, CA, USA. SFB93T was capable of acetylenotrophic and diazotrophic growth, grew at 22-37 °C, pH 6.3-8.5 and in the presence of 10-45 g l-1 NaCl. Phylogenetic analyses based on 16S rRNA gene sequencing showed that SFB93T represented a member of the genus Syntrophotalea with highest 16S rRNA gene sequence similarities to Syntrophotalea acetylenica DSM 3246T (96.6 %), Syntrophotalea carbinolica DSM 2380T (96.5 %), and Syntrophotalea venetiana DSM 2394T (96.7 %). Genome sequencing revealed a genome size of 3.22 Mbp and a DNA G+C content of 53.4 %. SFB93T had low genome-wide average nucleotide identity (81-87.5 %) and <70 % digital DNA-DNA hybridization value with other members of the genus Syntrophotalea. The phylogenetic position of SFB93T within the family Syntrophotaleaceae and as a novel member of the genus Syntrophotalea was confirmed via phylogenetic reconstruction based on concatenated alignments of 92 bacterial core genes. On the basis of the results of phenotypic, genotypic and phylogenetic analyses, a novel species, Syntrophotalea acetylenivorans sp. nov., is proposed, with SFB93T (=DSM 106009T=JCM 33327T=ATCC TSD-118T) as the type strain.

Metabolic Capability and Phylogenetic Diversity of Mono Lake during a Bloom of the Eukaryotic Phototroph Picocystis sp. Strain ML.

Algal blooms in lakes are often associated with anthropogenic eutrophication; however, they can occur without the human introduction of nutrients to a lake. A rare bloom of the alga Picocystis sp. strain ML occurred in the spring of 2016 at Mono Lake, a hyperalkaline lake in California, which was also at the apex of a multiyear-long drought. These conditions presented a unique sampling opportunity to investigate microbiological dynamics and potential metabolic function during an intense natural algal bloom. We conducted a comprehensive molecular analysis along a depth transect near the center of the lake from the surface to a depth of 25 m in June 2016. Across sampled depths, rRNA gene sequencing revealed that Picocystis-associated chloroplasts were found at 40 to 50% relative abundance, greater than values recorded previously. Despite high relative abundances of the photosynthetic oxygenic algal genus Picocystis, oxygen declined below detectable limits below a depth of 15 m, corresponding with an increase in microorganisms known to be anaerobic. In contrast to previously sampled years, both metagenomic and metatranscriptomic data suggested a depletion of anaerobic sulfate-reducing microorganisms throughout the lake's water column. Transcripts associated with photosystem I and II were expressed at both 2 m and 25 m, suggesting that limited oxygen production could occur at extremely low light levels at depth within the lake. Blooms of Picocystis appear to correspond with a loss of microbial activity such as sulfate reduction within Mono Lake, yet microorganisms may survive within the sediment to repopulate the lake water column as the bloom subsides.IMPORTANCE Mono Lake, California, provides a habitat to a unique ecological community that is heavily stressed due to recent human water diversions and a period of extended drought. To date, no baseline information exists from Mono Lake to understand how the microbial community responds to human-influenced drought or algal bloom or what metabolisms are lost in the water column as a consequence of such environmental pressures. While previously identified anaerobic members of the microbial community disappear from the water column during drought and bloom, sediment samples suggest that these microorganisms survive at the lake bottom or in the subsurface. Thus, the sediments may represent a type of seed bank that could restore the microbial community as a bloom subsides. Our work sheds light on the potential photosynthetic activity of the halotolerant alga Picocystis sp. strain ML and how the function and activity of the remainder of the microbial community responds during a bloom at Mono Lake.

Improved ZnS nanoparticle properties through sequential NanoFermentation.

Sequential NanoFermentation (SNF) is a novel process which entails sparging microbially produced gas containing H2S from a primary reactor through a concentrated metal-acetate solution contained in a secondary reactor, thereby precipitating metallic sulfide nanoparticles (e.g., ZnS, CuS, or SnS). SNF holds an advantage over single reactor nanoparticle synthesis strategies, because it avoids exposing the microorganisms to high concentrations of toxic metal and sulfide ions. Also, by segregating the nanoparticle products from biological materials, SNF avoids coating nanoparticles with bioproducts that alter their desired properties. Herein, we report the properties of ZnS nanoparticles formed from SNF as compared with ones produced directly in a primary reactor (i.e., conventional NanoFermentation, or "CNF"), commercially available ZnS, and ZnS chemically synthesized by bubbling H2S gas through a Zn-acetate solution. The ZnS nanoparticles produced by SNF provided improved optical properties due to their smaller crystallite size, smaller overall particle sizes, reduced biotic surface coatings, and reduced structural defects. SNF still maintained the advantages of NanoFermentation technology over chemical synthesis including scalability, reproducibility, and lower hazardous waste burden.

Arsenate-dependent growth is independent of an ArrA mechanism of arsenate respiration in the termite hindgut isolate Citrobacter sp. strain TSA-1.

Citrobacter sp. strain TSA-1 is an enteric bacterium isolated from the hindgut of the termite. Strain TSA-1 displays anaerobic growth with selenite, fumarate, tetrathionate, nitrate, or arsenate serving as electron acceptors, and it also grows aerobically. In regards to arsenate, genome sequencing revealed that strain TSA-1 lacks a homolog for respiratory arsenate reductase, arrAB, and we were unable to obtain amplicons of arrA. This raises the question as to how strain TSA-1 achieves As(V)-dependent growth. We show that growth of strain TSA-1 on glycerol, which it cannot ferment, is linked to the electron acceptor arsenate. A series of transcriptomic experiments were conducted to discern which genes were upregulated during growth on arsenate, as opposed to those on fumarate or oxygen. For As(V), upregulation was noted for 1 of the 2 annotated arsC genes, while there was no clear upregulation for tetrathionate reductase (ttr), suggesting that this enzyme is not an alternative to arrAB as occurs in certain hyperthermophilic archaea. A gene-deletion mutant strain of TSA-1 deficient in arsC could not achieve anaerobic respiratory growth on As(V). Our results suggest that Citrobacter sp. strain TSA-1 has an unusual and as yet undefined means of achieving arsenate respiration, perhaps involving its ArsC as a respiratory reductase as well as a detoxifying agent.

The genetic basis of anoxygenic photosynthetic arsenite oxidation.

'Photoarsenotrophy', the use of arsenite as an electron donor for anoxygenic photosynthesis, is thought to be an ancient form of phototrophy along with the photosynthetic oxidation of Fe(II), H2 S, H2 and NO2-. Photoarsenotrophy was recently identified from Paoha Island's (Mono Lake, CA) arsenic-rich hot springs. The genomes of several photoarsenotrophs revealed a gene cluster, arxB2AB1CD, where arxA is predicted to encode for the sole arsenite oxidase. The role of arxA in photosynthetic arsenite oxidation was confirmed by disrupting the gene in a representative photoarsenotrophic bacterium, resulting in the loss of light-dependent arsenite oxidation. In situ evidence of active photoarsenotrophic microbes was supported by arxA mRNA detection for the first time, in red-pigmented microbial mats within the hot springs of Paoha Island. This work expands on the genetics for photosynthesis coupled to new electron donors and elaborates on known mechanisms for arsenic metabolism, thereby highlighting the complexities of arsenic biogeochemical cycling.

Detection of Diazotrophy in the Acetylene-Fermenting Anaerobe Pelobacter sp. Strain SFB93.

Acetylene (C2H2) is a trace constituent of the present Earth's oxidizing atmosphere, reflecting a mixture of terrestrial and marine emissions from anthropogenic, biomass-burning, and unidentified biogenic sources. Fermentation of acetylene was serendipitously discovered during C2H2 block assays of N2O reductase, and Pelobacter acetylenicus was shown to grow on C2H2 via acetylene hydratase (AH). AH is a W-containing, catabolic, low-redox-potential enzyme that, unlike nitrogenase (N2ase), is specific for acetylene. Acetylene fermentation is a rare metabolic process that is well characterized only in P. acetylenicus DSM3246 and DSM3247 and Pelobacter sp. strain SFB93. To better understand the genetic controls for AH activity, we sequenced the genomes of the three acetylene-fermenting Pelobacter strains. Genome assembly and annotation produced three novel genomes containing gene sequences for AH, with two copies being present in SFB93. In addition, gene sequences for all five compulsory genes for iron-molybdenum N2ase were also present in the three genomes, indicating the cooccurrence of two acetylene transformation pathways. Nitrogen fixation growth assays showed that DSM3426 could ferment acetylene in the absence of ammonium, but no ethylene was produced. However, SFB93 degraded acetylene and, in the absence of ammonium, produced ethylene, indicating an active N2ase. Diazotrophic growth was observed under N2 but not in experimental controls incubated under argon. SFB93 exhibits acetylene fermentation and nitrogen fixation, the only known biochemical mechanisms for acetylene transformation. Our results indicate complex interactions between N2ase and AH and suggest novel evolutionary pathways for these relic enzymes from early Earth to modern days.IMPORTANCE Here we show that a single Pelobacter strain can grow via acetylene fermentation and carry out nitrogen fixation, using the only two enzymes known to transform acetylene. These findings provide new insights into acetylene transformations and adaptations for nutrient (C and N) and energy acquisition by microorganisms. Enhanced understanding of acetylene transformations (i.e., extent, occurrence, and rates) in modern environments is important for the use of acetylene as a potential biomarker for extraterrestrial life and for degradation of anthropogenic contaminants.

Acetylene Fuels TCE Reductive Dechlorination by Defined Dehalococcoides/Pelobacter Consortia.

Acetylene (C2H2) can be generated in contaminated groundwater sites as a consequence of chemical degradation of trichloroethene (TCE) by in situ minerals, and C2H2 is known to inhibit bacterial dechlorination. In this study, we show that while high C2H2 (1.3 mM) concentrations reversibly inhibit reductive dechlorination of TCE by Dehalococcoides mccartyi isolates as well as enrichment cultures containing D. mccartyi sp., low C2H2 (0.4 mM) concentrations do not inhibit growth or metabolism of D. mccartyi. Cocultures of Pelobacter SFB93, a C2H2-fermenting bacterium, with D. mccartyi strain 195 or with D. mccartyi strain BAV1 were actively sustained by providing acetylene as the electron donor and carbon source while TCE or cis-DCE served as the electron acceptor. Inhibition by acetylene of reductive dechlorination and methanogenesis in the enrichment culture ANAS was observed, and the inhibition was removed by adding Pelobacter SFB93 into the consortium. Transcriptomic analysis of D. mccartyi strain 195 showed genes encoding for reductive dehalogenases (e.g., tceA) were not affected during the C2H2-inhibition, while genes encoding for ATP synthase, biosynthesis, and Hym hydrogenase were down-regulated during C2H2 inhibition, consistent with the physiological observation of lower cell yields and reduced dechlorination rates in strain 195. These results will help facilitate the optimization of TCE-bioremediation at contaminated sites containing both TCE and C2H2.

Functional metagenomic selection of ribulose 1, 5-bisphosphate carboxylase/oxygenase from uncultivated bacteria.

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) is a critical yet severely inefficient enzyme that catalyses the fixation of virtually all of the carbon found on Earth. Here, we report a functional metagenomic selection that recovers physiologically active RubisCO molecules directly from uncultivated and largely unknown members of natural microbial communities. Selection is based on CO2 -dependent growth in a host strain capable of expressing environmental deoxyribonucleic acid (DNA), precluding the need for pure cultures or screening of recombinant clones for enzymatic activity. Seventeen functional RubisCO-encoded sequences were selected using DNA extracted from soil and river autotrophic enrichments, a photosynthetic biofilm and a subsurface groundwater aquifer. Notably, three related form II RubisCOs were recovered which share high sequence similarity with metagenomic scaffolds from uncultivated members of the Gallionellaceae family. One of the Gallionellaceae RubisCOs was purified and shown to possess CO2 /O2 specificity typical of form II enzymes. X-ray crystallography determined that this enzyme is a hexamer, only the second form II multimer ever solved and the first RubisCO structure obtained from an uncultivated bacterium. Functional metagenomic selection leverages natural biological diversity and billions of years of evolution inherent in environmental communities, providing a new window into the discovery of CO2 -fixing enzymes not previously characterized.

Microbial Antimony Biogeochemistry: Enzymes, Regulation, and Related Metabolic Pathways.

Antimony (Sb) is a toxic metalloid that occurs widely at trace concentrations in soil, aquatic systems, and the atmosphere. Nowadays, with the development of its new industrial applications and the corresponding expansion of antimony mining activities, the phenomenon of antimony pollution has become an increasingly serious concern. In recent years, research interest in Sb has been growing and reflects a fundamental scientific concern regarding Sb in the environment. In this review, we summarize the recent research on bacterial antimony transformations, especially those regarding antimony uptake, efflux, antimonite oxidation, and antimonate reduction. We conclude that our current understanding of antimony biochemistry and biogeochemistry is roughly equivalent to where that of arsenic was some 20 years ago. This portends the possibility of future discoveries with regard to the ability of microorganisms to conserve energy for their growth from antimony redox reactions and the isolation of new species of "antimonotrophs."

Microbiological oxidation of antimony(III) with oxygen or nitrate by bacteria isolated from contaminated mine sediments.

Bacterial oxidation of arsenite [As(III)] is a well-studied and important biogeochemical pathway that directly influences the mobility and toxicity of arsenic in the environment. In contrast, little is known about microbiological oxidation of the chemically similar anion antimonite [Sb(III)]. In this study, two bacterial strains, designated IDSBO-1 and IDSBO-4, which grow on tartrate compounds and oxidize Sb(III) using either oxygen or nitrate, respectively, as a terminal electron acceptor, were isolated from contaminated mine sediments. Both isolates belonged to the Comamonadaceae family and were 99% similar to previously described species. We identify these novel strains as Hydrogenophaga taeniospiralis strain IDSBO-1 and Variovorax paradoxus strain IDSBO-4. Both strains possess a gene with homology to the aioA gene, which encodes an As(III)-oxidase, and both oxidize As(III) aerobically, but only IDSBO-4 oxidized Sb(III) in the presence of air, while strain IDSBO-1 could achieve this via nitrate respiration. Our results suggest that expression of aioA is not induced by Sb(III) but may be involved in Sb(III) oxidation along with an Sb(III)-specific pathway. Phylogenetic analysis of proteins encoded by the aioA genes revealed a close sequence similarity (90%) among the two isolates and other known As(III)-oxidizing bacteria, particularly Acidovorax sp. strain NO1. Both isolates were capable of chemolithoautotrophic growth using As(III) as a primary electron donor, and strain IDSBO-4 exhibited incorporation of radiolabeled [(14)C]bicarbonate while oxidizing Sb(III) from Sb(III)-tartrate, suggesting possible Sb(III)-dependent autotrophy. Enrichment cultures produced the Sb(V) oxide mineral mopungite and lesser amounts of Sb(III)-bearing senarmontite as precipitates.

Microbiological reduction of Sb(V) in anoxic freshwater sediments.

Microbiological reduction of millimolar concentrations of Sb(V) to Sb(III) was observed in anoxic sediments from two freshwater settings: (1) a Sb- and As-contaminated mine site (Stibnite Mine) in central Idaho and 2) an uncontaminated suburban lake (Searsville Lake) in the San Francisco Bay Area. Rates of Sb(V) reduction in anoxic sediment microcosms and enrichment cultures were enhanced by amendment with lactate or acetate as electron donors but not by H2, and no reduction occurred in sterilized controls. Addition of 2-(14)C-acetate to Stibnite Mine microcosms resulted in the production of (14)CO2 coupled to Sb(V) reduction, suggesting that this process proceeds by a dissimilatory respiratory pathway in those sediments. Antimony(V) reduction in Searsville Lake sediments was not coupled to acetate mineralization and may be associated with Sb-resistance. The microcosms and enrichment cultures also reduced sulfate, and the precipitation of insoluble Sb(III)-sulfide complexes was a major sink for reduced Sb. The reduction of Sb(V) by Stibnite Mine sediments was inhibited by As(V), suggesting that As(V) is a preferred electron acceptor for the indigenous community. These findings indicate a novel pathway for anaerobic microbiological respiration and suggest that communities capable of reducing high concentrations of Sb(V) commonly occur naturally in the environment.

ArxA, a new clade of arsenite oxidase within the DMSO reductase family of molybdenum oxidoreductases.

Arsenotrophy, growth coupled to autotrophic arsenite oxidation or arsenate respiratory reduction, occurs only in the prokaryotic domain of life. The enzymes responsible for arsenotrophy belong to distinct clades within the DMSO reductase family of molybdenum-containing oxidoreductases: specifically arsenate respiratory reductase, ArrA, and arsenite oxidase, AioA (formerly referred to as AroA and AoxB). A new arsenite oxidase clade, ArxA, represented by the haloalkaliphilic bacterium Alkalilimnicola ehrlichii strain MLHE-1 was also identified in the photosynthetic purple sulfur bacterium Ectothiorhodospira sp. strain PHS-1. A draft genome sequence of PHS-1 was completed and an arx operon similar to MLHE-1 was identified. Gene expression studies showed that arxA was strongly induced with arsenite. Microbial ecology investigation led to the identification of additional arxA-like sequences in Mono Lake and Hot Creek sediments, both arsenic-rich environments in California. Phylogenetic analyses placed these sequences as distinct members of the ArxA clade of arsenite oxidases. ArxA-like sequences were also identified in metagenome sequences of several alkaline microbial mat environments of Yellowstone National Park hot springs. These results suggest that ArxA-type arsenite oxidases appear to be widely distributed in the environment presenting an opportunity for further investigations of the contribution of Arx-dependent arsenotrophy to the arsenic biogeochemical cycle.

Desulfohalophilus alkaliarsenatis gen. nov., sp. nov., an extremely halophilic sulfate- and arsenate-respiring bacterium from Searles Lake, California.

A haloalkaliphilic sulfate-respiring bacterium, strain SLSR-1, was isolated from a lactate-fed stable enrichment culture originally obtained from the extreme environment of Searles Lake, California. The isolate proved capable of growth via sulfate-reduction over a broad range of salinities (125-330 g/L), although growth was slowest at salt-saturation. Strain SLSR-1 was also capable of growth via dissimilatory arsenate-reduction and displayed an even broader range of salinity tolerance (50-330 g/L) when grown under these conditions. Strain SLSR-1 could also grow via dissimilatory nitrate reduction to ammonia. Growth experiments in the presence of high borate concentrations indicated a greater sensitivity of sulfate-reduction than arsenate-respiration to this naturally abundant anion in Searles Lake. Strain SLSR-1 contained genes involved in both sulfate-reduction (dsrAB) and arsenate respiration (arrA). Amplicons of 16S rRNA gene sequences obtained from DNA extracted from Searles Lake sediment revealed the presence of close relatives of strain SLSR-1 as part of the flora of this ecosystem despite the fact that sulfate-reduction activity could not be detected in situ. We conclude that strain SLSR-1 can only achieve growth via arsenate-reduction under the current chemical conditions prevalent at Searles Lake. Strain SLSR-1 is a deltaproteobacterium in the family Desulfohalobiacea of anaerobic, haloalkaliphilic bacteria, for which we propose the name Desulfohalophilus alkaliarsenatis gen. nov., sp. nov.

Got acetylene: a personal research retrospective.

In research, sometimes sheer happenstance and serendipity make for an unexpected discovery. Once revealed and if interesting enough, such a finding and its follow-up investigations can lead to advances by others that leave its originators 'scooped' and mulling about what next to do with their unpublished data, specifically what journals could it still be published in and be perceived as original. This is what occurred with us nearly 40 years ago with regard to our follow-up observations of acetylene fermentation and led us to concoct a 'cock-and-bull' story. We hypothesized about a plausible role for acetylene metabolism in the primordial biogeochemistry of Earth and the possibility of acetylene serving as a key life-sustaining substrate for alien microbes dwelling in the orbs of the outer solar system. With the passage of time, advances were made in whole-genome sequencing coupled with major in silico progress in bioinformatics. In parallel came the results of explorations of the outer solar system (i.e. the Cassini mission to Saturn and its moons). It now appears that these somewhat harebrained ideas of ours, arisen at first out of a sense of desperation, actually ring true in fact, and particularly well in song: 'Tell a tale of cock and bull, Of convincing detail full Tale tremendous, Heav'n defend us! What a tale of cock and bull!' From 'The Yeoman of the Guard' by Gilbert & Sullivan.

Acetylene-Fueled Trichloroethene Reductive Dechlorination in a Groundwater Enrichment Culture.

In aquifers, acetylene (C2H2) is a product of abiotic degradation of trichloroethene (TCE) catalyzed by in situ minerals. C2H2 can, in turn, inhibit multiple microbial processes including TCE dechlorination and metabolisms that commonly support dechlorination, in addition to supporting the growth of acetylenotrophic microorganisms. Previously, C2H2 was shown to support TCE reductive dechlorination in synthetic, laboratory-constructed cocultures containing the acetylenotroph Pelobacter sp. strain SFB93 and Dehalococcoides mccartyi strain 195 or strain BAV1. In this study, we demonstrate TCE and perchloroethene (PCE) reductive dechlorination by a microbial community enriched from contaminated groundwater and amended with C2H2 as the sole electron donor and organic carbon source. The metagenome of the stable, enriched community was analyzed to elucidate putative community functions. A novel anaerobic acetylenotroph in the phylum Actinobacteria was identified using metagenomic analysis. These results demonstrate that the coupling of acetylenotrophy and reductive dechlorination can occur in the environment with native bacteria and broaden our understanding of biotransformation at contaminated sites containing both TCE and C2H2IMPORTANCE Understanding the complex metabolisms of microbial communities in contaminated groundwaters is a challenge. PCE and TCE are among the most common groundwater contaminants in the United States that, when exposed to certain minerals, exhibit a unique abiotic degradation pathway in which C2H2 is a product. C2H2 can act as both an inhibitor of TCE dechlorination and of supporting metabolisms and an energy source for acetylenotrophic bacteria. Here, we combine laboratory microcosm studies with computational approaches to enrich and characterize an environmental microbial community that couples two uncommon metabolisms, demonstrating unique metabolic interactions only yet reported in synthetic, laboratory-constructed settings. Using this comprehensive approach, we have identified the first reported anaerobic acetylenotroph in the phylum Actinobacteria, demonstrating the yet-undescribed diversity of this metabolism that is widely considered to be uncommon.

Identification of a novel arsenite oxidase gene, arxA, in the haloalkaliphilic, arsenite-oxidizing bacterium Alkalilimnicola ehrlichii strain MLHE-1.

Although arsenic is highly toxic to most organisms, certain prokaryotes are known to grow on and respire toxic metalloids of arsenic (i.e., arsenate and arsenite). Two enzymes are known to be required for this arsenic-based metabolism: (i) the arsenate respiratory reductase (ArrA) and (ii) arsenite oxidase (AoxB). Both catalytic enzymes contain molybdopterin cofactors and form distinct phylogenetic clades (ArrA and AoxB) within the dimethyl sulfoxide (DMSO) reductase family of enzymes. Here we report on the genetic identification of a "new" type of arsenite oxidase that fills a phylogenetic gap between the ArrA and AoxB clades of arsenic metabolic enzymes. This "new" arsenite oxidase is referred to as ArxA and was identified in the genome sequence of the Mono Lake isolate Alkalilimnicola ehrlichii MLHE-1, a chemolithoautotroph that can couple arsenite oxidation to nitrate reduction. A genetic system was developed for MLHE-1 and used to show that arxA (gene locus ID mlg_0216) was required for chemoautotrophic arsenite oxidation. Transcription analysis also showed that mlg_0216 was only expressed under anaerobic conditions in the presence of arsenite. The mlg_0216 gene is referred to as arxA because of its greater homology to arrA relative to aoxB and previous reports that implicated Mlg_0216 (ArxA) of MLHE-1 in reversible arsenite oxidation and arsenate reduction in vitro. Our results and past observations support the position that ArxA is a distinct clade within the DMSO reductase family of proteins. These results raise further questions about the evolutionary relationships between arsenite oxidases (AoxB) and arsenate respiratory reductases (ArrA).