Microbial Transformation of 3-Nitro-4-Hydroxybenzene Arsonic Acid (Roxarsone)

Land application of chicken litter from production houses that use the organoarsenical 3-nitro-4-hydroxybenzenearsonic acid (roxarsone) results in deposition of significant quantities of arsenic into United States soil each year. Used widely as a feed additive in the poultry industry, roxarsone was originally thought to be stable in the environment. However, recent evidence from our lab, as well as others, has established that this benign compound is readily transformed via several intermediates into inorganic arsenate. While microbial activity has been implicated in this process, there is no direct evidence suggesting the specific processes or the organisms involved. Using a physiological and proteomic approach, this study demonstrates increased anaerobic growth of the arsenate respiring Clostridium species strain OhILAs in the presence of roxarsone using two different carbon sources, as well as provides evidence to suggest the respiratory arsenate reductase (Arr) system may be constitutively expressed. Increased growth of Clostridium OhILAs was coupled to a loss of roxarsone as determined by spectrophotometric assays. Similar results have been obtained with the structurally similar ortho-nitrophenol. Additionally, arsenate reductase activity assays of OhILAs grown in the presence of lactate alone or in the presence of lactate and either roxarsone, ortho-nitrophenol or sodium arsenate have suggested that the respiratory arsenate reductase is constitutively expressed

Enzyme systems involved in interspecies hydrogen and formate transfer between syntrophic fatty and aromatic acid degraders and Methanospirillum hungatei

The oxidation of either the fatty acid butyrate or the aromatic acid benzoate is essential for the efficient degradation of complex organic material to methane because they are central intermediates. The oxidation of butyrate to acetate and hydrogen is energy requiring under physiological conditions. Likewise, the oxidation of benzoate to acetate, carbon dioxide and hydrogen is energy requiring under physiological conditions. However, when hydrogen or formate are maintained at very low levels by a partner organism, through metabolic cooperation known as syntrophy, the oxidation of both butyrate and benzoate becomes energetically favorable. An essential feature of both the syntrophic oxidation of butyrate and benzoate is the need to produce hydrogen (E’ = -260 mV at 1 Pa hydrogen) or formate (E’ = -290 mV at 1 µM formate) from butyryl-CoA (butyrate oxidation by S. wolfei) or from glutaryl-CoA (benzoate oxidation by S. aciditrophicus). Ion gradients, and consequently membrane bound protein complexes, are known to be important for butyrate oxidation by S. wolfei and benzoate oxidation by bacteria related to S. aciditrophicus – Syntrophus gentianae and Syntrophus buswellii. The main goal of this research was to investigate the mechanisms of reverse electron transfer in S. wolfei and S. aciditrophicus. I used proteomic and enzymological approaches and coupled these approaches with mRNA expression analyses. Here, I showed that an FeS oxidoreductase, the gene of which is linked on the chromosome to genes coding for electron transferring flavoprotein subunits, and components of a cytochrome b –linked hydrogenase (hydIIABC gene product) are codetected in a complex unique to syntrophic growth on butyrate. Expression analyses of the genes coding for the electron transferring flavoprotein subunits, FeS oxidoreductase and hydIIABC gene product argue for the importance of these systems for syntrophic growth on butyrate. In S. aciditrophicus, I showed that peptides derived from an Rnf-like complex were detected in membrane complexes from S. aciditrophicus cells. I used the low potential acceptors, benzyl viologen (E0’ = -360 mV) and methyl viologen (E0’ = -460 mV) to test for the ability of S. aciditrophicus cells to catalyze an Rnf-like activity - reduction of the viologen dyes with NADH (E0’ = -320 mV). I showed that membrane fractions of S. wolfei catalyze the reduction of both benzyl and methyl viologen with electrons derived from NADH and that specific activity for this direction is higher than for the more thermodynamically favorable oxidation of benzyl or methyl viologen with concomitant reduction of NAD+. Moreover, I showed that the Rnf-like activity is highest in cells grown syntrophically. I used size exclusion chromatography to partially purify the Rnf-like activity and I showed that peptides derived from an Rnf-like complex are present in these fractions. Finally, I investigated the genome of Methanospirillum hungatei strain JF1 and used whole cell shotgun proteomics to interrogate the response of M. hungatei to growth in syntrophic partnership with S. wolfei. M. hungatei is a hydrogenotrophic methanogen which is capable of utilizing formate or hydrogen for methane production. M. hungatei is a partner organism for several syntrophic systems and members of the genus Methanospirillum have been found in many environments where syntrophy is important. Proteomic analysis showed that M. hungatei uses both hydrogenases and formate dehydrogenases and increases the relative abundance of the core methanogenic machinery during syntrophic growth relative to pure culture growth on hydrogen and formate. The relative abundance of peptides associated with energy production and cofactor synthesis increased while those involved in translation decreased in syntrophically grown cells compared to axenically-grown cells. The above data are consistent with a strategy to maximize energy production efficiency and curtail biosynthesis during syntrophic growth

Formate Formation and Formate Conversion in Biological Fuels Production

Biomethanation is a mature technology for fuel production. Fourth generation biofuels research will focus on sequestering CO2 and providing carbon-neutral or carbon-negative strategies to cope with dwindling fossil fuel supplies and environmental impact. Formate is an important intermediate in the methanogenic breakdown of complex organic material and serves as an important precursor for biological fuels production in the form of methane, hydrogen, and potentially methanol. Formate is produced by either CoA-dependent cleavage of pyruvate or enzymatic reduction of CO2 in an NADH- or ferredoxin-dependent manner. Formate is consumed through oxidation to CO2 and H2 or can be further reduced via the Wood-Ljungdahl pathway for carbon fixation or industrially for the production of methanol. Here, we review the enzymes involved in the interconversion of formate and discuss potential applications for biofuels production

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

Complete genome sequence of Syntrophobacter fumaroxidans strain (MPOB(T)).

Syntrophobacter fumaroxidans strain MPOB(T) is the best-studied species of the genus Syntrophobacter. The species is of interest because of its anaerobic syntrophic lifestyle, its involvement in the conversion of propionate to acetate, H2 and CO2 during the overall degradation of organic matter, and its release of products that serve as substrates for other microorganisms. The strain is able to ferment fumarate in pure culture to CO2 and succinate, and is also able to grow as a sulfate reducer with propionate as an electron donor. This is the first complete genome sequence of a member of the genus Syntrophobacter and a member genus in the family Syntrophobacteraceae. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 4,990,251 bp long genome with its 4,098 protein-coding and 81 RNA genes is a part of the Microbial Genome Program (MGP) and the Genomes to Life (GTL) Program project

A phenomenology of motives: An existential-dramatistic approach

The present dissertation adds another voice to the small but growing trend in communication scholarship examining the relationship between Kenneth Burke\u27s dramatism and existential phenomenology, represented here by Jean-Paul Sartre, Martin Heidegger, and Maurice Merleau-Ponty. While these two theoretical traditions might, in some ways, be seen as an unlikely pair, the dissertation demonstrates many points of compatibility between them. In addition, the two approaches to human existence and interaction were applied to a concrete aspect of social life: motives. Beginning with a puzzling phrase found in Burke\u27s A Grammar of Motives one essentially suggesting that motives are created through action, this study investigates the phenomenon of motives through a detailed exploration of human action. Each chapter unfolds a different aspect of human action, but does so in a holistic way, keeping the project as a whole always in sight. Therefore, the early chapters, dealing with action versus knowledge, human embodiment, and the lived world, find their full clarification only in the final substantive chapter, which studies the relationship between temporality, human finitude, and action. In the conclusion, it is suggested why these discussions of human action, far from reaching a conclusion, are but an initial framework for further inquiry into both the phenomenon of motive and the interconnection of dramatism and existential philosophy

Proteomic analysis reveals metabolic and regulatory systems involved 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 proteomic 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 macromolecular stability and energy metabolism to S. wolfei and presence of regulatory mechanisms responsive to external stimuli and cellular physiological status

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