Michael W. W. Adams

Ferredoxin (Fd) from the hyperthermophilic archaeon Pyrococcus furiosus (Pf), Pf-Fd, functions in electron transfer, serving to transfer an electron from various oxidoreductases to an H2-evolving, membrane-bound hydrogenase. The [4Fe-4S] cluster of Pf-Fd is coordinated by three cysteine residues and the carboxylate group of an aspartate residue. Recently, we have found that several such site-differentiated [4Fe-4S]1+ clusters are photoactive, inspiring us to investigate the photoreactivity of the site-differentiated [4Fe-4S]1+ cluster in Pf-Fd. We show here through X-band EPR and 13C/15N Q-band ENDOR studies that visible-light irradiation of the cyanide-bound [4Fe-4S]1+ cluster of Pf-Fd, Pf-Fd-CN, at 12 K results in photodissociation of the cyanide anion, which geminately recombines only upon annealing to 150 K. In addition, this work revisits our previous CW 13C/15N ENDOR studies of Pf-Fd-CN with pulsed-ENDOR techniques. Of particular note, we definitively determine the absolute sign of the 13C hyperfine coupling with the PESTRE pulsed-ENDOR protocol and from this draw inferences about Fe valence localization within the cyanide-bound cluster.

Glycerol is abundant in natural environments and is utilized by many microbes. In aerobes, it is typically converted, via glycerol-3-phosphate (G3P), to dihydroxyacetone phosphate (DHAP) by a quinone-linked dehydrogenase thereby coupling glycolysis and respiration. Under anaerobic conditions, an NAD+-dependent dehydrogenase has been proposed to oxidize G3P to DHAP in several bacteria but this reaction is thermodynamically unfavorable. Herein, we have purified a heterotrimeric electron bifurcating enzyme from the archaeon Pyrococcus furiosus where the simultaneous oxidation of both G3P (Eo′ −190 mV) and reduced ferredoxin (Em ∼ −450 mV) enables reduction of NAD+(Eo′ −320 mV) and production of DHAP. Surprisingly, this ferredoxin-dependent NAD+G3P oxidoreductase (FngABC) is unrelated evolutionarily to the four known families of bifurcating enzyme. Analysis by UV–visible spectroscopy and of its predicted structure suggest FngABC has a nonclassical bifurcation mechanism. Phylogenetic and genome context analyses of 4,273 P. furiosus FngA homologues revealed they are widespread with ∼35% found mainly in Clostridia as FngABC and ∼10% found mainly in Actinomyces as FngAB, most of which utilize polyols in addition to glycerol. The remaining ∼40% are present in Gammaproteobacteria and halophilic archaea but here FngA is associated with homologues of E. coli GlpBC, which are unrelated to FngBC, and these enzymes typically only use glycerol. It is therefore proposed that P. furiosus FngABC and the previously characterized E. coli GlpABC are disparate members of a new electron bifurcating family widespread in the microbial world that are involved in the metabolism of a range of sugars as well as glycerol.

Eubacterium limosum is an abundant gut microbe that efficiently metabolizes lactate to produce the short-chain fatty acids, butyrate and acetate. The key oxidoreductase enzymes, formate dehydrogenase (FDH), carbon monoxide dehydrogenase (CODH) of the Wood-Ljungdahl pathway, pyruvate ferredoxin oxidoreductase (POR), and an electron-bifurcating tungsten-containing oxidoreductase (WOR) that detoxifies acetaldehyde produced as a byproduct of the POR reaction, are all utilized during lactate metabolism. Fermentative anaerobes typically utilize a single ferredoxin as an electron carrier for their primary pathways, which is replaced by flavodoxin under iron-limiting conditions. However, the E. limosum genome encodes two putative ferredoxins (Fd1 and Fd2) and three putative flavodoxins (Fld, Fld-like1, and Fld-like2). All five proteins were heterologously expressed in Escherichia coli , but the UV-visible absorption properties of purified Fld-like proteins 1 and 2 were not typical of canonical flavodoxins. Both POR and CODH reduced Fd1, Fd2, and Fld at comparable rates. Partially purified WOR and FDH had low activity using Fd1, Fd2, and Fld as electron carriers. With WOR, NAD-linked bifurcating activity could not be demonstrated with any of the electron carriers. Deletion mutants of E. limosum lacking Fd1, Fd2, or Fld exhibited similar lag phases during growth on glucose, and this increased on lactate for the Fld mutant but much less so for the Fd mutants. We conclude that E. limosum can utilize either Fd1, Fd2, or Fld as the primary redox protein, but that during growth on lactate, Fld plays a more prominent role than Fd1 or Fd2, even under iron-sufficient conditions. IMPORTANCE: Eubacterium limosum is an abundant gut microbe that is beneficial to human health due to its production of short-chain fatty acids particularly during growth on lactate. It is also of interest due to its ability to metabolize H ₂ /CO ₂ and C1 substrates. It was assumed that E. limosum , like other fermentative anaerobes, utilizes a single ferredoxin as an electron carrier during primary carbon metabolism and only utilizes flavodoxin under iron-limited conditions. However, we show here that this organism utilizes flavodoxin as its main electron carrier. This may be a significant advantage in the gut environment where competition with the host and with other gut microbes for iron is intense. This may have both biotechnological and health implications for this organism.

Tungsten-containing oxidoreductases (WORs) are a diverse family of enzymes with over 4,000 known members that can be subdivided into 92 clades based on the phylogeny of the large pyranopterin cofactor-containing large subunit (WorL). Despite being widespread in Bacteria and Archaea, particularly in members of the human microbiome, only five of the 92 WOR clades contain a WOR with a defined physiological role in cellular metabolism, primarily—but not exclusively—involved in oxidation of various aldehydes. However, this phylogenetic-based organizational system lacks a perspective on the diversity and complexity of WOR enzymes. Herein, we propose a non-phylogenetic classification system for WORs based on predicted subunit composition and electron carrier specificity that provides insight into potential physiological roles. WORs can be divided into five classes that range in complexity and predicted function. The simpler cytoplasmic Class I-III WORs are involved in aldehyde detoxification, a modified glycolysis pathway and cold adaptation. More complex multimeric WORs are proposed to use multiple electron carriers in bifurcating reactions (Class IV) or interact with various respiratory systems via associations with the cell membrane (Class V). We characterized two new WORs, one Class I and one Class V, from the human gut bacterium Cetobacterium somerae , and showed that the former enzyme had aldehyde oxidation activity but the latter did not. By combining phylogenetic information with the new WOR classification system, we can predict structural and functional characteristics of as-yet uncharaterized WORs and identify unique and novel enzymes for future studies.

The chemical and biochemical engineering, microbiology, and biotechnology communities deeply mourn the passing of Dr. Robert M. Kelly, the Alcoa Professor of Chemical and Biomolecular Engineering and Director of the Biotechnology Program at NC State University. A towering pioneer in the biology of extremophiles, an esteemed educator and mentor, and a dedicated editor for Applied and Environmental Microbiology (AEM), Bob leaves behind an extraordinary legacy that reshaped our understanding of life at high temperatures.

Groundwater acidification co-occurring with nitrate pollution is a common, global environmental health hazard. Denitrifying bacteria have been leveraged for the in situ removal of nitrate in groundwater. However, co-existing stressors—such as low pH—reduce the efficacy of biological removal processes. Castellaniella sp. str. MT123 is a complete denitrifier that was isolated from acidic, nitrate-contaminated groundwater. The strain grows robustly by nitrate respiration at pH < 6.0, completely reducing nitrate to dinitrogen gas. Genomic analyses of MT123 revealed few previously characterized acid tolerance genes. Thus, we utilized a combination of proteomics, metabolomics, and competitive mutant fitness to characterize the genetic mechanisms of MT123 acclimation to growth under mildly acidic conditions. We found that glutamate accumulation is critical in the acid acclimation of MT123, possibly through consumption of intracellular protons via glutamate decarboxylation to GABA. This is despite the fact that MT123 lacks the canonical glutamate decarboxylase-glutamate/GABA antiporter system implicated in acid tolerance in other bacteria. In contrast, branched-chain amino acid (BCAA) accumulation was detrimental to cell growth at lower pHs, possibly through indirect mechanisms impacting the cellular glutamate pool. Genetic analysis previously linked MT123 to a population of Castellaniella that bloomed—concurrent to nitrate removal—during a biostimulation effort to reduce groundwater nitrate concentrations at MT123’s location of origin. Thus, our analyses provide novel insight into mechanisms of acclimation to acidic conditions in a strain with significant potential for nitrate bioremediation. IMPORTANCE: Nitrate pollution in groundwater is a major threat to both environmental and human health. This nitrate pollution can come from a variety of sources, including farm fertilizers, sewage, animal waste, septic systems, and industrial discharge. Bacteria known as “denitrifiers” can convert this nitrate into harmless nitrogen gas, a process known as “denitrification.” Denitrifiers can be used to clean up nitrate-contaminated groundwater. However, their ability to do this can be disrupted by changing environmental conditions. For example, groundwater that is polluted with nitrate is often acidic. Acidic conditions make it challenging for denitrifiers to survive, which results in less conversion of nitrate to nitrogen gas. In this study, we investigated how one denitrifying bacterium—originating from acidic, nitrate-contaminated groundwater—can cope with acidic conditions.

Global transcription factors (TFs) control metabolic processes in bacteria to efficiently utilize available carbon. The order Caldicellulosiruptorales has drawn interest due to the ability of its members to degrade components of lignocellulosic biomass. Regulatory reconstruction of Anaerocellum (f. Caldicellulosiruptor) bescii identified two major global transcription factors for xylan utilization, XynR and XylR, and the corresponding putative transcription factor binding sites. Recombinant versions of XynR (LacI family) and XylR (ROK family) were subjected to fluorescence polarization (FP) and biolayer interferometry (BLI) analysis to confirm the predicted binding sites. Four XynR sites and two XylR sites were validated, accounting for 20 of 26 genes regulated by XynR and six of seven genes regulated by XylR. Bioinformatic analysis of the individual genes controlled by the two regulators showed an inter-dependent scheme for xylan conversion; the transport of xylooligosaccharides (XOS) is dependent on XylR, while enzymes responsible for hydrolysis are controlled by both regulators. For xylose catabolism by the xylose isomerase-xylulose kinase pathway, regulation is also split, with XylR controlling xylose isomerase and XynR controlling xylokinase. The XynR/XylR regulator pair within A. bescii is conserved in all sequenced species of Caldicellulosiruptorales , suggesting similarities in regulating linear xylan conversion. In other xylanolytic thermophiles, XylR homologs control xylan degradation, compared to just 6 out of 26 genes for A. bescii . These results show that two separate regulatory schemes (dual repression) are coordinated by A. bescii to effectively regulate the hemicellulose inventory and xylan catabolism. IMPORTANCE: To take full advantage of extreme thermophiles as platform metabolic engineering microorganisms, the tools for genetic manipulation must be further developed, and strategies that exploit a better understanding of metabolic regulation need to be discerned. Anaerocellum bescii , the most studied of the extremely thermophilic fermentative anaerobic bacteria that can utilize microcrystalline cellulose, can degrade microcrystalline cellulose and hemicellulose and has been metabolically engineered to convert the resulting sugars to products such as ethanol and acetone. For xylan, in particular, two major global transcription factors (TFs), XynR and XylR, play a role in sugar metabolism, although their predicted regulatory interdependence from bioinformatics analysis has not been elucidated experimentally. Here, fluorescence polarization (FP) and biolayer interferometry (BLI) were used to explore this issue to support metabolic engineering efforts aimed at improving carbohydrate processing to industrial chemicals.

Members of the tungsten-containing oxidoreductase (WOR) family, which contain a tungstopyranopterin (Tuco) cofactor, are typically either monomeric (WorL) or heterodimeric (WorLS). These enzymes oxidize aldehydes to the corresponding acids while reducing the redox protein ferredoxin. They have been structurally characterized mainly using WORs from hyperthermophilic archaea. The WORs of some bacteria contain three additional subunits of the BfuABC family and these chimeric WorABCSL enzymes catalyze an electron-bifurcating reaction in which aldehyde oxidation is coupled to the simultaneous reduction of ferredoxin and nicotinamide adenine dinucleotide. In human gut microbes, electron bifurcation by WorABSL is proposed to enable the detoxification of aldehydes generated from cooked foods and in the tungstocentric production of beneficial short chain fatty acids from lactate, potentially impacting health. Herein we present the high-resolution cryogenic electron microscopy (cryo-EM) structure of the WorABCSL purified from the bacterium Acetomicrobium mobile. The structure reveals a surprising 1:3 stoichiometry between WorABC and WorSL, with the WorSL units forming a nanowire-like architecture leading from three Tuco-containing catalytic sites in WorL via strings of multiple iron-sulfur clusters in WorS to a single bifurcating WorABC core. Our structure uncovers a distinct domain arrangement that links three Tuco-dependent aldehyde oxidation sites with the bifurcation process and potentially facilitates environmental aldehyde oxidation.

Cetobacterium somerae ATCC BAA-474 was first isolated from human feces and is emerging as an important species for animal and human health. Here, we report the complete genome sequence of this strain of C. somerae to improve the currently available genome sequence.