Organohalide respiration in Dehalococcoides strains represents a novel mode of proton motive force generation.

Desulfitobacterium hafniense was isolated for its ability to use organohalogens as terminal electron acceptors via organohalide respiration (OHR). In contrast to obligate OHR bacteria, Desulfitobacterium spp. show a highly versatile energy metabolism with the capacity to use different electron donors and acceptors and to grow fermentatively. Desulfitobacterium genomes display numerous and apparently redundant members of redox enzyme families which confirm their metabolic potential. Nonetheless, the enzymes responsible for many metabolic traits are not yet identified. In the present work, we conducted an extended proteomic study by comparing the proteomes of Desulfitobacterium hafniense strain DCB-2 cultivated in combinations of electron donors and acceptors, triggering five alternative respiratory metabolisms that include OHR, as well as fermentation. Tandem Mass Tag labelling proteomics allowed us to identify and quantify almost 60% of the predicted proteome of strain DCB-2 (2,796 proteins) in all six growth conditions. Raw data are available via ProteomeXchange with identifier PXD030393. This dataset was analyzed in order to highlight the proteins that were significantly up-regulated in one or a subset of growth conditions and to identify possible key players in the different energy metabolisms. The addition of sodium sulfide as reducing agent in the medium - a very widespread practice in the cultivation of strictly anaerobic bacteria - triggered the expression of the dissimilatory sulfite reduction pathway in relatively less favorable conditions such as fermentative growth on pyruvate, respiration with H2 as electron donor and OHR conditions. The presence of H2, CO2 and acetate in the medium induced several metabolic pathways involved in carbon metabolism including the Wood-Ljungdahl pathway and two pathways related to the fermentation of butyrate that rely on electron-bifurcating enzymes. While the predicted fumarate reductase appears to be constitutively expressed, a new lactate dehydrogenase and lactate transporters were identified. Finally, the OHR metabolism with 3-chloro-4-hydroxyphenylacetate as electron acceptor strongly induced proteins encoded in several reductive dehalogenase gene clusters, as well as four new proteins related to corrinoid metabolism. We believe that this extended proteomic database represents a new landmark in understanding the metabolic versatility of Desulfitobacterium spp. and provides a solid basis for addressing future research questions.

Microbial fuel cells (MFC) have recently received increasing attention due to their promising potential in sustainable wastewater treatment and contaminant removal. In general, contaminants can be removed either as an electron donor via microbial catalyzed oxidization at the anode or removed at the cathode as electron acceptors through reduction. Some contaminants can also function as electron mediators at the anode or cathode. While previous studies have done a thorough assessment of electron donors, cathodic electron acceptors and mediators have not been as well described. Oxygen is widely used as an electron acceptor due to its high oxidation potential and ready availability. Recent studies, however, have begun to assess the use of different electron acceptors because of the (1) diversity of redox potential, (2) needs of alternative and more efficient cathode reaction, and (3) expanding of MFC based technologies in different areas. The aim of this review was to evaluate the performance and applicability of various electron acceptors and mediators used in MFCs. This review also evaluated the corresponding performance, advantages and disadvantages, and future potential applications of select electron acceptors (e.g., nitrate, iron, copper, perchlorate) and mediators.

The genus Desulfoluna comprises two anaerobic sulfate-reducing strains, D. spongiiphila AA1T and D. butyratoxydans MSL71T, of which only the former was shown to perform organohalide respiration (OHR). Here we isolated a third strain, designated D. spongiiphila strain DBB, from marine intertidal sediment using 1,4-dibromobenzene and sulfate as the electron acceptors and lactate as the electron donor. Each strain harbors three reductive dehalogenase gene clusters (rdhABC) and corrinoid biosynthesis genes in their genomes, and dehalogenated brominated but not chlorinated organohalogens. The Desulfoluna strains maintained OHR in the presence of 20 mM sulfate or 20 mM sulfide, which often negatively affect other organohalide-respiring bacteria. Strain DBB sustained OHR with 2% oxygen in the gas phase, in line with its genetic potential for reactive oxygen species detoxification. Reverse transcription-quantitative PCR revealed differential induction of rdhA genes in strain DBB in response to 1,4-dibromobenzene or 2,6-dibromophenol. Proteomic analysis confirmed expression of rdhA1 with 1,4-dibromobenzene, and revealed a partially shared electron transport chain from lactate to 1,4-dibromobenzene and sulfate, which may explain accelerated OHR during concurrent sulfate reduction. Versatility in using electron donors, de novo corrinoid biosynthesis, resistance to sulfate, sulfide and oxygen, and concurrent sulfate reduction and OHR may confer an advantage to marine Desulfoluna strains.

: Bacteria of the group Dehalococcoides display the ability to respire recalcitrant chlorinated organic compounds. Dehalococcoides strains' respiratory pathways and function of most genome-encoded enzymes responsible for dechlorination, reductive dehalogenases (RDases), remain incompletely annotated. To further the description of the biological organization of Dehalococcoides, this study monitored the trancriptomic response of Dehalococcoides ethenogenes stain 195 using two-color microarrays. This study analyzed the transcriptome of 23 varied continuous feed (or pseudo-steady state (PSS)) conditions and two distinct batch fed conditions. The continuous feed experiments were comprised of 57 cultures with varying electron acceptor feed rates (0-504 micro-eeq/(L-hr)), electron acceptor types (tetrachloroethene (PCE), trichloroethene (TCE), dichloroethene (DCE), 2,3-dichlorophenol (DCP), and no electron acceptor), electron donor to acceptor ratios (0.7 to 17 on an electron equivalence (eeq) basis), and electron donor type (butyrate, lactate, yeast extract, fermented yeast, pure hydrogen, or endogenous biomass decay). When similarly respiring (120 micro-eeq PCE/(L-hr)) batch and PSS cultures were contrasted, the RDases DET1545 and DET0180 were up-regulated in the PSS cultures indicating activity at lower overall electron acceptor concentration. For all continuous fed chloroethene cultures, members of the RDase family and electron transport chain displayed unique clusters of transcripts responding either positively, negatively, or indifferently to the respiration rate. An RDase within the indifferent group, DET1171, was highly (31 + or - 15fold) up regulated in response to DCP being fed as the electron acceptor. DET1171 could potentially play a role with DET1559 (27 + or - 4.1 fold up-regulated during DCP growth) and DET0318 pceA in the dechlorination of chlorophenols.

Researchers have long been interested in replicating the reactivity that occurs in photosynthetic organisms. To mimic the long-lived charge separations characteristic of the reaction center in photosynthesis, researchers have applied the Marcus theory to design synthetic multistep electron-transfer (ET) systems. In this Account, we describe our recent research on the rational design of ET control systems, based on models of the photosynthetic reaction center that rely on the Marcus theory of ET. The key to obtaining a long-lived charge separation is the careful choice of electron donors and acceptors that have small reorganization energies of ET. In these cases, the driving force of back ET is located in the Marcus inverted region, where the lifetime of the charge-separated state lengthens as the driving force of back ET increases. We chose porphyrins as electron donors and fullerenes as electron acceptors, both of which have small ET reorganization energies. By linking electron donor porphyrins and electron acceptor fullerenes at appropriate distances, we achieved charge-separated states with long lifetimes. We could further lengthen the lifetimes of charge-separated states by mixing a variety of components, such as a terminal electron donor, an electron mediator, and an electron acceptor, mimicking both the photosynthetic reaction center and the multistep photoinduced ET that occurs there. However, each step in multistep ET loses a fraction of the initial excitation energy during the long-distance charge separation. To overcome this drawback in multistep ET systems, we used designed new systems where we could finely control the redox potentials and the geometry of simple donor-acceptor dyads. These modifications resulted in a small ET reorganization energy and a high-lying triplet excited state. Our most successful example, 9-mesityl-10-methylacridinium ion (Acr(+)-Mes), can undergo a fast photoinduced ET from the mesityl (Mes) moiety to the singlet excited state of the acridinium ion moiety (Acr(+)) with extremely slow back ET. The high-energy triplet charge-separated state is located deep in the Marcus inverted region, and we have detected the structural changes during the photoinduced ET in this system using X-ray crystallography. To increase the efficiency of both the light-harvesting and photoinduced ET, we assembled the Acr(+)-Mes dyads on gold nanoparticles to bring them in closer proximity to one another. We can also incorporate Acr(+)-Mes molecules within nanosized mesoporous silica-alumina. In contrast to the densely assembled dyads on gold nanoparticles, each Acr(+)-Mes molecule in silica-alumina is isolated in the mesopore, which inhibits the bimolecular back ET and leads to longer lifetimes in solution at room temperature than the natural photosynthetic reaction center. Acr(+)-Mes and related compounds act as excellent organic photocatalysts and facilitate a variety of reactions such as oxygenation, bromination, carbon-carbon bond formation, and hydrogen evolution reactions.

Use of γ-hexachlorocyclohexane as a terminal electron acceptor by an anaerobic enrichment culture

Microorganisms capable of direct or mediated extracellular electron transfer (EET) have garnered significant attention for their various biotechnological applications, such as bioremediation, metal recovery, wastewater treatment, energy generation in microbial fuel cells, and microbial or enzymatic electrosynthesis. One microorganism of particular interest is the organohalide-respiring bacterium Dehalococcoides mccartyi strain CBDB1, known for its ability to reductively dehalogenate toxic and persistent halogenated organic compounds through organohalide respiration (OHR), using halogenated organics as terminal electron acceptors. A membrane-bound OHR protein complex couples electron transfer to proton translocation across the membrane, generating a proton motive force, which enables metabolism and proliferation. In this study we show that the halogenated compounds can be replaced with redox mediators that can putatively shuttle electrons between the OHR complex and the anode, coupling D. mccartyi cells to an electrode via mediated EET. We identified cobalt-containing metal complexes, referred to as cobalt chelates, as promising mediators using a photometric high throughput methyl viologen-based enzyme activity assay. Through various biochemical approaches, we show that cobalt chelates are specifically reduced by CBDB1 cells, putatively by the reductive dehalogenase subunit (RdhA) of the OHR complex. Using cyclic voltammetry, we also demonstrate that cobalt chelates exchange electrons with a gold electrode, making them promising candidates for bioelectrochemical cultivation. Furthermore, using the AlphaFold 2-calculated RdhA structure and molecular docking, we found that one of the identified cobalt chelates exhibits favorable binding to RdhA, with a binding energy of approximately -28 kJ mol-1. Taken together, our results indicate that bioelectrochemical cultivation of D. mccartyi with cobalt chelates as anode mediators, instead of toxic halogenated compounds, is feasible, which opens new perspectives for bioremediation and other biotechnological applications of strain CBDB1.

Tetra- and trichloroethene (PCE, TCE) are organohalides polluting the environment as a result of inappropriate use, storage, and disposal by various industries. Anthropogenic pollution by organohalides is a major source of concern because of their undesirable effects on human health. Remediation of contaminated sites by the use of microorganisms is a promising approach, especially under anaerobic conditions. Dehalobacter restrictus represents the paradigmatic member of the genus Dehalobacter, which in recent years has proven to be a major player in the biodegradation of a growing number of organohalides, both in situ and in the laboratory. D. restrictus grows only through anaerobic respiration of PCE and TCE with hydrogen as electron donor by a process known as organohalide respiration (OHR). To this day, only a single reductive dehalogenase (PceA/RdhA), the key enzyme in the OHR process, has been characterized on genetic and biochemical levels. However, recent genome analysis of D. restrictus has revealed the presence of 25 rdhA genes. Chapter 2 of this thesis describes a functional genomics and proteomics approach on D. restrictus with a focus on the diversity, composition and expression of rdh gene clusters. Genome analysis also revealed a complete corrinoid biosynthetic pathway, WL pathway for CO2 fixation and hydrogenases. Some of these were identified in proteomic analysis along with main PceABCT, RdhA14 and a few RdhK. OHR bacteria (OHRB) have developed different strategies to satisfy their need of corrinoid (Cobalamin/Vitamin B12 derivatives), as it is an essential cofactor of RdhAs forming the basis for Chapter 3. Obligate OHRB such as Dehalococcoides spp. and D. restrictus cannot de novo synthesize corrinoid. However. genome analysis revealed that in contrast to Dehalococcoides mccartyi, the genome of D. restrictus surprisingly has the complete series of genes for biosynthesis of corrinoid, however a single non-functional gene could account for the corrinoid auxotrophy. Comparative genomics within Dehalobacter spp. revealed that one of the five operons associated with the biosynthesis of corrinoid is unique to D. restrictus, which encoded enzymes corrinoid- salvaging and transport proteins. Omics during corrinoid starvation highlighted the importance of operon-2 in corrinoid homeostasis in D. restrictus along with indicating its augmented corrinoid salvaging strategy. Chapter 4 finally analyses the diversity of RdhK proteins in D. restrictus belonging to the CRP-FNR family of transcriptional regulators. Earlier studies in Desulfitobacterium spp. have allowed the identification and characterization of a transcriptional regulator, CprK known to be involved in the regulation of cpr gene cluster involved in OHR. Moreover recent genome analysis in D. restrictus, revealed the presence of 25 cprK-like rdhK genes found to be located in the direct neighbourhood of the rdh gene clusters strongly suggesting they could be implicated in regulating OHR in D. restrictus. A combination of in silico, in vivo and in vitro analyses have been attempted to characterize the role of a few RdhK proteins and understand the tri-partite interaction of the RdhK with the putative organohalide along with the putative-DNA binding regions (dehaloboxes). However, further efforts are still needed to elucidate the network regulating the OHR metabolism in D. restrictus.

Sulfurospirillum multivorans is a free-living, physiologically versatile Epsilonproteobacterium able to couple the reductive dehalogenation of chlorinated and brominated ethenes to growth (organohalide respiration). We present proteomic data of S. multivorans grown with different electron donors (formate or pyruvate) and electron acceptors (fumarate, nitrate, or tetrachloroethene [PCE]). To obtain information on the cellular localization of proteins, membrane extracts and soluble fractions were separated before data collection from both fractions. The proteome analysis of S. multivorans was performed by mass spectrometry (nanoLC-MS/MS). Raw data have been deposited at ProteomeXchange, “ProteomeXchange provides globally coordinated proteomics data submission and dissemination” [1], via the PRIDE partner repository with the dataset identifier PRIDE: PXD004011. The data might support further research in organohalide respiration and in the general metabolism of free-living Epsilonproteobacteria. The dataset is associated with a previously published study “Proteomics of the organohalide-respiring Epsilonproteobacterium S. multivorans adapted to tetrachloroethene and other energy substrates” [2].

Assessing the Bacterial Ecology of Organohalide Respiration for the Design of Bioremediation Strategies

Neutral red (NR) was utilized as an electron mediator in microbial fuel cells consuming glucose to study both its efficiency during electricity generation and its role in altering anaerobic growth and metabolism of Escherichia coli and Actinobacillus succinogenes. A study of chemical fuel cells in which NADH, NR, and ferricyanide were the electron donor, the electronophore, and the electron acceptor, respectively, showed that electrical current produced from NADH was proportional to the concentration of NADH. Fourfold more current was produced from NADH in chemical fuel cells when NR was the electron mediator than when thionin was the electron mediator. In microbial fuel cells in which E. coli resting cells were used the amount of current produced from glucose when NR was the electron mediator (3.5 mA) was 10-fold more than the amount produced when thionin was the electron mediator (0.4 mA). The amount of electrical energy generated (expressed in joules per mole of substrate) and the amount of current produced from glucose (expressed in milliamperes) in NR-mediated microbial fuel cells containing either E. coli or A. succinogenes were about 10- and 2-fold greater, respectively, when resting cells were used than when growing cells were used. Cell growth was inhibited substantially when these microbial fuel cells were making current, and more oxidized end products were formed under these conditions. When sewage sludge (i.e., a mixed culture of anaerobic bacteria) was used in the fuel cell, stable (for 120 h) and equivalent levels of current were obtained with glucose, as observed in the pure-culture experiments. These results suggest that NR is better than other electron mediators used in microbial fuel cells and that sludge production can be decreased while electricity is produced in fuel cells. Our results are discussed in relation to factors that may improve the relatively low electrical efficiencies (1.2 kJ/mol) obtained with microbial fuel cells.

Vertical variations of pentachlorophenol (PCP) dissipation and microbial community were investigated in a paddy soil with the addition of electron acceptors (NO3(-), SO4(2-)) and donors (crop residues). Crop residues enhanced PCP dissipation by supplying dissolved organic carbon (DOC) as an electron donor, whereas NO3(-) and SO4(2-) inhibited it. The dissipation of PCP in electron donor treatments resulted in the accumulation of 3,4,5-trichlorophenol (3,4,5-TCP) except for wheat residues. The abundance and diversity of phospholipid fatty acids (PLFAs) decreased with increasing soil depth. The succession of predominant PLFAs shifted from aerobic bacteria to anaerobic bacteria when electron acceptors were changed to electron donors. The saturated/monounsaturated fatty acids (S/M) ratio increased with soil depth, which probably implied that nutrient turnover rate declined after the accumulation of 3,4,5-TCP. The results showed that the addition of electron donors and acceptors modified the microbial communities, which then further influenced the degradation pathway of PCP.

The only organohalide-respiring Epsilonproteobacteria (e-proteobacteria) described so far are found in the genus Sulfurospirillum . This genus consists of versatile, often microaerophilic bacteria, growing with many different growth substrates. Only a few of these organisms use halogenated compounds, mainly chlorinated ethenes, as electron acceptors. Organohalide respiration was extensively studied in Sulfurospirillum multivorans, but seems to be similar in other reductively dehalogenating Sulfurospirilla like Sulfurospirillum halorespirans. While most Sulfurospirillum species are unable to utilize organohalides as electron acceptors, many of them grow with other toxic substrates such as arsenate or selenate. Other typical electron acceptors are nitrate and sulfur compounds. Electron donors used are pyruvate, hydrogen and formate. The anaerobic respiratory chains of Sulfurospirillum spp. involve most likely menaquinones and cytochromes for most electron donor/acceptor combinations. The growth substrate range which includes many toxic compounds enables many Sulfurospirillum species to thrive in polluted habitats, which is reflected by the presence of these bacteria in many contaminated sites. The genomes of Sulfurospirillum spp. are small to average in size (about 2.5–3 Mbp) and the genes necessary for organohalide respiration, if present, are clustered in one area, including corrinoid biosynthesis genes responsible for production of the unique norpseudovitamin B12. The gene inventory in this area differs from that of other organohalide-respiring bacterial classes in that a putative quinol dehydrogenase and other accessory proteins are encoded. This points to a respiratory chain differing from other organohalide-respiring bacteria.

In oxidative phosphorylation, respiratory complex I serves as an entry point in the electron transport chain for electrons generated in catabolic processes in the form of NADH. An ancestral version of the complex, lacking the NADH-oxidising module, is encoded in a significant number of bacterial genomes. Amongst them is Desulfitobacterium hafniense, a strict anaerobe capable of conserving energy via organohalide respiration. This study investigates the role of the complex I-like enzyme in D. hafniense energy metabolism using rotenone as a specific complex I inhibitor under different growth conditions. The investigation revealed that the complex I-like enzyme was essential for growth with lactate and pyruvate but not in conditions involving H2 as an electron donor. In addition, a previously published proteomic dataset of strain DCB-2 was analysed to reveal the predominance of the complex under different growth conditions and to identify potential redox partners. This approach revealed seven candidates with expression patterns similar to Nuo homologues, suggesting the use of diverse electron sources. Based on these results, we propose a model where the complex I-like enzyme serves as an electron entry point into the respiratory chain for substrates delivering electrons within the cytoplasm, such as lactate or pyruvate, with ferredoxins shuttling electrons to the complex.

Organohalide respiration is an environmentally important but poorly characterized type of anaerobic respiration. We compared the global proteome of the versatile organohalide-respiring Epsilonproteobacterium Sulfurospirillum multivorans grown with different electron acceptors (fumarate, nitrate, or tetrachloroethene [PCE]). The most significant differences in protein abundance were found for gene products of the organohalide respiration region. This genomic region encodes the corrinoid and FeS cluster containing PCE reductive dehalogenase PceA and other proteins putatively involved in PCE metabolism such as those involved in corrinoid biosynthesis. The latter gene products as well as PceA and a putative quinol dehydrogenase were almost exclusively detected in cells grown with PCE. This finding suggests an electron flow from the electron donor such as formate or pyruvate via the quinone pool and a quinol dehydrogenase to PceA and the terminal electron acceptor PCE. Two putative accessory proteins, an IscU-like protein and a peroxidase-like protein, were detected with PCE only and might be involved in PceA maturation. The proteome of cells grown with pyruvate instead of formate as electron donor indicates a route of electrons from reduced ferredoxin via an Epsilonproteobacterial complex I and the quinone pool to PCE.