Bacterium Geobacter Sulfurreducens Research Articles

Periplasmic electron transfer network in Geobacter sulfurreducens revealed by biomolecular interaction studies

AbstractMultiheme cytochromes located in different compartments are crucial for extracellular electron transfer in the bacterium Geobacter sulfurreducens to drive important environmental processes and biotechnological applications. Recent studies have unveiled that for particular sets of electron terminal acceptors, discrete respiratory pathways selectively recruit specific cytochromes from both the inner and outer membranes. However, such specificity was not observed for the abundant periplasmic cytochromes, namely the triheme cytochrome family PpcA‐E. In this work, the distinctive NMR spectroscopic signatures of these proteins in different redox states were explored to monitor pairwise interactions and electron transfer reactions between each pair of cytochromes. The results showed that the five proteins interact transiently and can exchange electrons between each other revealing intra‐promiscuity within the members of this family. This discovery is discussed in the light of the establishment of an effective electron transfer network by this pool of cytochromes. This network is advantageous to the bacteria as it enables the maintenance of the functional working potential redox range within the cells.

Characterization of melanin from Exophiala mesophila with the prospect of potential biotechnological applications.

Fungal melanin is an underexplored natural biomaterial of great biotechnological interest in different areas. This study investigated the physical, chemical, electrochemical, and metal-binding properties of melanin extracted from the metallotolerant black fungus Exophiala mesophila strain IRTA-M2-F10. Specific inhibitory studies with tricyclazole and biochemical profiling of whole cells by synchrotron radiation-based Fourier-transform infrared spectral microscopy (SR-FTIRM) were performed. An optimized extraction protocol was implemented, and purified fungal melanin was characterized using an array of spectrophotometric techniques (UV-Vis, FTIR, and EPR) and by cyclic voltammetry (CV) experiments. The metal-binding capacity of melanin extracts was also assessed by using Cr(VI) as a model heavy metal. Inhibitory studies indicated that 1,8-dihydroxynaphthalene may be the main precursor molecule of E. mesophila melanin (DHN-melanin). The biochemical characterization of fungal melanin extracts were benchmarked against those from two melanins comprising the precursor molecule L-3,4-dihydroxiphenylalanine (DOPA-melanin): extracts from the ink of the cephalopod Sepia officinalis and DOPA-melanin synthesized in the laboratory. The CV results of melanin extracts incubated with and without cell suspensions of the electroconductive bacterium Geobacter sulfurreducens were indicative of novel semiquinone/hydroquinone redox transformations specific for each melanin type. These interactions may play an important role in cation exchange for the adsorption of metals and in microbial interspecies electron transfer processes. The obtained results provided further evidence for the DHN-nature of E. mesophila melanin. The FTIR profiling of melanin extracts exposed to Cr(VI), compared to unexposed melanin, resulted in useful information on the distinct surface-binding properties of fungal melanin. The parameters of the Langmuir and Freundlicht isotherms for the adsorption of Cr(VI) were determined and compared to bibliographic data. Altogether, the inherent properties of fungal melanin suggest its promising potential as a biomaterial for environmental applications.

Bioproduction of cerium-bearing magnetite and application to improve carbon-black supported platinum catalysts

BackgroundBiogeochemical processing of metals including the fabrication of novel nanomaterials from metal contaminated waste streams by microbial cells is an area of intense interest in the environmental sciences.ResultsHere we focus on the fate of Ce during the microbial reduction of a suite of Ce-bearing ferrihydrites with between 0.2 and 4.2 mol% Ce. Cerium K-edge X-ray absorption near edge structure (XANES) analyses showed that trivalent and tetravalent cerium co-existed, with a higher proportion of tetravalent cerium observed with increasing Ce-bearing of the ferrihydrite. The subsurface metal-reducing bacterium Geobacter sulfurreducens was used to bioreduce Ce-bearing ferrihydrite, and with 0.2 mol% and 0.5 mol% Ce, an Fe(II)-bearing mineral, magnetite (Fe(II)(III)2O4), formed alongside a small amount of goethite (FeOOH). At higher Ce-doping (1.4 mol% and 4.2 mol%) Fe(III) bioreduction was inhibited and goethite dominated the final products. During microbial Fe(III) reduction Ce was not released to solution, suggesting Ce remained associated with the Fe minerals during redox cycling, even at high Ce loadings. In addition, Fe L2,3 X-ray magnetic circular dichroism (XMCD) analyses suggested that Ce partially incorporated into the Fe(III) crystallographic sites in the magnetite. The use of Ce-bearing biomagnetite prepared in this study was tested for hydrogen fuel cell catalyst applications. Platinum/carbon black electrodes were fabricated, containing 10% biomagnetite with 0.2 mol% Ce in the catalyst. The addition of bioreduced Ce-magnetite improved the electrode durability when compared to a normal Pt/CB catalyst.ConclusionDifferent concentrations of Ce can inhibit the bioreduction of Fe(III) minerals, resulting in the formation of different bioreduction products. Bioprocessing of Fe-minerals to form Ce-containing magnetite (potentially from waste sources) offers a sustainable route to the production of fuel cell catalysts with improved performance.Graphical

Biosynthesis Parameters Control the Physicochemical and Catalytic Properties of Microbially Supported Pd Nanoparticles.

The biosynthesis of Pd nanoparticles supported on microorganisms (bio-Pd) is achieved via the enzymatic reduction of Pd(II) to Pd(0) under ambient conditions using inexpensive buffers and electron donors, like organic acids or hydrogen. Sustainable bio-Pd catalysts are effective for C-C coupling and hydrogenation reactions, but their industrial application is limited by challenges in controlling nanoparticle properties. Here, using the metal-reducing bacterium Geobacter sulfurreducens, it is demonstrated that synthesizing bio-Pd under different Pd loadings and utilizing different electron donors (acetate, formate, hydrogen, no e- donor) influences key properties such as nanoparticle size, Pd(II):Pd(0) ratio, and cellular location. Controlling nanoparticle size and location controls the activity of bio-Pd for the reduction of 4-nitrophenol, whereas high Pd loading on cells synthesizes bio-Pd with high activity, comparable to commercial Pd/C, for Suzuki-Miyaura coupling reactions. Additionally, the study demonstrates the novel synthesis of microbially-supported ≈2nm PdO nanoparticles due to the hydrolysis of biosorbed Pd(II) in bicarbonate buffer. Bio-PdO nanoparticles show superior activity in 4-nitrophenol reduction compared to commercial Pd/C catalysts. Overall, controlling biosynthesis parameters, such as electron donor, metal loading, and solution chemistry, enables tailoring of bio-Pd physicochemical and catalytic properties.

Global transcriptional analysis of Geobacter sulfurreducens gsu1771 mutant biofilm grown on two different support structures.

Electroactive biofilms formation by the metal-reducing bacterium Geobacter sulfurreducens is a step crucial for bioelectricity generation and bioremediation. The transcriptional regulator GSU1771 controls the expression of essential genes involved in electron transfer and biofilm formation in G. sulfurreducens, with GSU1771-deficient producing thicker and more electroactive biofilms. Here, RNA-seq analyses were conducted to compare the global gene expression patterns of wild-type and Δgsu1771 mutant biofilms grown on non-conductive (glass) and conductive (graphite electrode) materials. The Δgsu1771 biofilm grown on the glass surface exhibited 467 differentially expressed (DE) genes (167 upregulated and 300 downregulated) versus the wild-type biofilm. In contrast, the Δgsu1771 biofilm grown on the graphite electrode exhibited 119 DE genes (79 upregulated and 40 downregulated) versus the wild-type biofilm. Among these DE genes, 67 were also differentially expressed in the Δgsu1771 biofilm grown on glass (56 with the same regulation and 11 exhibiting counter-regulation). Among the upregulated genes in the Δgsu1771 biofilms, we identified potential target genes involved in exopolysaccharide synthesis (gsu1961-63, gsu1959, gsu1972-73, gsu1976-77). RT-qPCR analyses were then conducted to confirm the differential expression of a selection of genes of interest. DNA-protein binding assays demonstrated the direct binding of the GSU1771 regulator to the promoter region of pgcA, pulF, relA, and gsu3356. Furthermore, heme-staining and western blotting revealed an increase in c-type cytochromes including OmcS and OmcZ in Δgsu1771 biofilms. Collectively, our findings demonstrated that GSU1771 is a global regulator that controls extracellular electron transfer and exopolysaccharide synthesis in G. sulfurreducens, which is crucial for electroconductive biofilm development.

(Invited) Using the Model Bacterium Geobacter Sulfurreducens to Understand Microbial Electrochemical Conversion of Nitrogen Gas into Ammonium

There is a growing need to decrease reliance on the Haber-Bosch process. To convert nitrogen gas (N2) into ammonia (NH3) for fertilizers, this temperature- and pressure-intensive process consumes roughly 2–3% of global energy and releases over 740 million tons of CO2 each year. Many microorganisms naturally convert or “fix” N2 into ammonium (NH4 +) at room temperature and pressure using an enzyme called the nitrogenase. Attempts to increase NH4 + yields from microorganisms through genetic engineering are seeing signs of success, but we lack methods to increase NH4 + generation rates and overcome one of the largest challenges: O2 gas. Increasing O2 gas concentrations typically increases metabolic rates of aerobic microorganisms; however, in the case of microorganisms that fix N2 (called diazotrophs), O2 can shut down the nitrogenase and stop cell growth. To overcome these challenges, we are using electricity to drive N2 fixation in exoelectrogenic bacteria. These bacteria have the unique ability to naturally transfer electrons to anode electrodes in microbial electrochemical technologies (METs). Increasing the voltage applied to METs increases the metabolic rates of exoelectrogens. The anode chamber is kept anaerobic, which eliminates O2-driven inhibition of the nitrogenase. We previously showed that the N2 fixation rates of a mixed microbial community in a MET increased more than three times when the applied whole-cell potential increased from 0.7 V to 1.0 V. By adding a chemical inhibitor that prevented the incorporation of NH4 + into larger biomolecules, NH4 + was excreted by the cells and into the medium. Based on the acetylene reduction assay (a proxy for N2 fixation), we estimated that we recovered about 10% of the theoretical NH4 +generated by the cells. If close to 100% of the theoretical NH4 + could be excreted from the cells and recovered, we predicted an energy demand of around 3 MJ/mol-NH4 +, which is close to the range of value reported for the Haber-Bosch process. To develop METs that generate NH4 + at competitive production rates and energy demands, we are now focusing on the model exoelectrogenic diazotroph Geobacter sulfurreducens. Using a single organism with a fully sequenced genome allows us to understand how the N2 fixation process responds to electrochemical variables. Towards these efforts, we conducted a transcriptomic analysis of G. sulfurreducens at two fixed anode potentials: +0.15 V and −0.15 V. This approach revealed which genes are turned on and off in response to these potentials. The presence of NH4 + decreased the expression of genes associated with N2 fixation which was expected due to the known sensitivity of nitrogenases to NH4 +. On the other hand, the two anode potentials had a dramatic and unexpected impact on the expression levels of N2 fixation genes. At −0.15 V, nitrogenase genes were significantly up-regulated, as were genes associated with NH4 + uptake and transport, such as glutamine and glutamate synthetases. Considering that −0.15 V provides less energy to the cells relative to +0.15 V (based on thermodynamic predictions), the results suggest that the cells responded to this highly energy-constrained environment by increasing expression of N2 fixation pathways. Based on our new knowledge of how G. sulfurredcuens regulates N2 fixation and NH4 + production, we have developed new strains of G. sulfurreducens that can excrete NH4 + during N2 fixation. The new engineering toolkit that we have created in this bacterium can now be leveraged to maximize NH4 + production in METs.

Extracellular cytochrome nanowires appear to be ubiquitous in prokaryotes

Electrically conductive appendages from the anaerobic bacterium Geobacter sulfurreducens, recently identified as extracellular cytochrome nanowires (ECNs), have received wide attention due to numerous potential applications. However, whether other organisms employ similar ECNs for electron transfer remains unknown. Here, using cryoelectron microscopy, we describe the atomic structures of two ECNs from two major orders of hyperthermophilic archaea present in deep-sea hydrothermal vents and terrestrial hot springs. Homologs of Archaeoglobus veneficus ECN are widespread among mesophilic methane-oxidizing Methanoperedenaceae, alkane-degrading Syntrophoarchaeales archaea, and in the recently described megaplasmids called Borgs. The ECN protein subunits lack similarities in their folds; however, they share a common heme arrangement, suggesting an evolutionarily optimized heme packing for efficient electron transfer. The detection of ECNs in archaea suggests that filaments containing closely stacked hemes may be a common and widespread mechanism for long-range electron transfer in both prokaryotic domains of life.

Nitrogen Fixation and Ammonium Assimilation Pathway Expression of Geobacter sulfurreducens Changes in Response to the Anode Potential in Microbial Electrochemical Cells.

Nitrogen gas (N2) fixation in the anode-respiring bacterium Geobacter sulfurreducens occurs through complex, multistep processes. Optimizing ammonium (NH4+) production from this bacterium in microbial electrochemical technologies (METs) requires an understanding of how those processes are regulated in response to electrical driving forces. In this study, we quantified gene expression levels (via RNA sequencing) of G. sulfurreducens growing on anodes fixed at two different potentials (-0.15 V and +0.15 V versus standard hydrogen electrode). The anode potential had a significant impact on the expression levels of N2 fixation genes. At -0.15 V, the expression of nitrogenase genes, such as nifH, nifD, and nifK, significantly increased relative to that at +0.15 V, as well as genes associated with NH4+ uptake and transformation, such as glutamine and glutamate synthetases. Metabolite analysis confirmed that both of these organic compounds were present in significantly higher intracellular concentrations at -0.15 V. N2 fixation rates (estimated using the acetylene reduction assay and normalized to total protein) were significantly larger at -0.15 V. Genes expressing flavin-based electron bifurcation complexes, such as electron-transferring flavoproteins (EtfAB) and the NADH-dependent ferredoxin:NADP reductase (NfnAB), were also significantly upregulated at -0.15 V, suggesting that these mechanisms may be involved in N2 fixation at that potential. Our results show that in energy-constrained situations (i.e., low anode potential), the cells increase per-cell respiration and N2 fixation rates. We hypothesize that at -0.15 V, they increase N2 fixation activity to help maintain redox homeostasis, and they leverage electron bifurcation as a strategy to optimize energy generation and use. IMPORTANCE Biological nitrogen fixation coupled with ammonium recovery provides a sustainable alternative to the carbon-, water-, and energy-intensive Haber-Bosch process. Aerobic biological nitrogen fixation technologies are hindered by oxygen gas inhibition of the nitrogenase enzyme. Electrically driving biological nitrogen fixation in anaerobic microbial electrochemical technologies overcomes this challenge. Using Geobacter sulfurreducens as a model exoelectrogenic diazotroph, we show that the anode potential in microbial electrochemical technologies has a significant impact on nitrogen gas fixation rates, ammonium assimilation pathways, and expression of genes associated with nitrogen gas fixation. These findings have important implications for understanding regulatory pathways of nitrogen gas fixation and will help identify target genes and operational strategies to enhance ammonium production in microbial electrochemical technologies.

Microbial Reduction of Antimony(V)-Bearing Ferrihydrite by Geobacter sulfurreducens.

The reduction of Sb(V)-bearing ferrihydrite by Geobacter sulfurreducens was studied to determine the fate of the metalloid in Fe-rich systems undergoing redox transformations. Sb(V) added at a range of concentrations adsorbed readily to ferrihydrite, and the loadings had a pronounced impact on the rate and extent of Fe(III) reduction and the products formed. Magnetite dominated at low (0.5 and 1 mol%) Sb(V) concentrations, with crystallite sizes decreasing at higher Sb loadings: 37-, 25-, and 17-nm particles for no-Sb, 0.5% Sb, and 1% Sb samples, respectively. In contrast, goethite was the dominant end product for samples with higher antimony loadings (2 and 5 mol%), with increased goethite grain size in the 5% Sb sample. Inductively coupled mass spectrometry (ICP-MS) analysis confirmed that Sb was not released to solution during the bioreduction process, and X-ray photoelectron spectroscopy (XPS) analyses showed that no Sb(III) was formed throughout the experiments, confirming that the Fe(III)-reducing bacterium Geobacter sulfurreducens cannot reduce Sb(V) enzymatically or via biogenic Fe(II). These findings suggest that Fe (bio)minerals have a potential role in limiting antimony pollution in the environment, even when undergoing redox transformations. IMPORTANCE Antimony is an emerging contaminant that shares chemical characteristics with arsenic. Metal-reducing bacteria (such as Geobacter sulfurreducens) can cause the mobilization of arsenic from Fe(III) minerals under anaerobic conditions, causing widespread contamination of aquifers worldwide. This research explores whether metal-reducing bacteria can drive the mobilization of antimony under similar conditions. In this study, we show that G. sulfurreducens cannot reduce Sb(V) directly or cause Sb release during the bioreduction of the Fe(III) mineral ferrihydrite [although the sorbed Sb(V) did alter the Fe(II) mineral end products formed]. Overall, this study highlights the tight associations between Fe and Sb in environmental systems, suggesting that the microbial reduction of Fe(III)/Sb mineral assemblages may not lead to Sb release (in stark contrast to the mobilization of As in iron-rich systems) and offers potential Fe-based remediation options for Sb-contaminated environments.

Methylmercury formation in biofilms of Geobacter sulfurreducens.

Mercury (Hg) is a major environmental pollutant that accumulates in biota predominantly in the form of methylmercury (MeHg). Surface-associated microbial communities (biofilms) represent an important source of MeHg in natural aquatic systems. In this work, we report MeHg formation in biofilms of the iron-reducing bacterium Geobacter sulfurreducens. Biofilms were prepared in media with varied nutrient load for 3, 5, or 7 days, and their structural properties were characterized using confocal laser scanning microscopy, cryo-scanning electron microscopy and Fourier-transform infrared spectroscopy. Biofilms cultivated for 3 days with vitamins in the medium had the highest surface coverage, and they also contained abundant extracellular matrix. Using 3 and 7-days-old biofilms, we demonstrate that G. sulfurreducens biofilms prepared in media with various nutrient load produce MeHg, of which a significant portion is released to the surrounding medium. The Hg methylation rate constant determined in 6-h assays in a low-nutrient assay medium with 3-days-old biofilms was 3.9 ± 2.0 ∙ 10-14 L ∙ cell-1 ∙ h-1, which is three to five times lower than the rates found in assays with planktonic cultures of G. sulfurreducens in this and previous studies. The fraction of MeHg of total Hg within the biofilms was, however, remarkably high (close to 50%), and medium/biofilm partitioning of inorganic Hg (Hg(II)) indicated low accumulation of Hg(II) in biofilms. These findings suggest a high Hg(II) methylation capacity of G. sulfurreducens biofilms and that Hg(II) transfer to the biofilm is the rate-limiting step for MeHg formation in this systems.

Metabolic turnover of cysteine-related thiol compounds at environmentally relevant concentrations by Geobacter sulfurreducens.

Low-molecular-mass (LMM) thiol compounds are known to be important for many biological processes in various organisms but LMM thiols are understudied in anaerobic bacteria. In this work, we examined the production and turnover of nanomolar concentrations of LMM thiols with a chemical structure related to cysteine by the model iron-reducing bacterium Geobacter sulfurreducens. Our results show that G. sulfurreducens tightly controls the production, excretion and intracellular concentration of thiols depending on cellular growth state and external conditions. The production and cellular export of endogenous cysteine was coupled to the extracellular supply of Fe(II), suggesting that cysteine excretion may play a role in cellular trafficking to iron proteins. Addition of excess exogenous cysteine resulted in a rapid and extensive conversion of cysteine to penicillamine by the cells. Experiments with added isotopically labeled cysteine confirmed that penicillamine was formed by a dimethylation of the C-3 atom of cysteine and not via indirect metabolic responses to cysteine exposure. This is the first report of de novo metabolic synthesis of this compound. Penicillamine formation increased with external exposure to cysteine but the compound did not accumulate intracellularly, which may suggest that it is part of G. sulfurreducens' metabolic strategy to maintain cysteine homeostasis. Our findings highlight and expand on processes mediating homeostasis of cysteine-like LMM thiols in strict anaerobic bacteria. The formation of penicillamine is particularly noteworthy and this compound warrants more attention in microbial metabolism studies.

Electrochemical Bioremediation of Uranium VI Using Geobacter Sulfurreducens on Boron-Doped Diamond Electrode Surface

The development of nuclear science and technology has led to increasing nuclear waste containing uranium. The contamination associated with nuclear activity, especially uranium, is a significant concern for human and environmental health. Due to the high toxicity, radioactivity, and extensive half-life of uranium, it is necessary to find a way to remove it from the environment. One alternative to solve this problem is bioremediation. With this technique, microorganisms can be used to reduce, eliminate, contain, or transform hazardous waste like radionuclides. This research proposes using the bacteria Geobacter sulfurreducens to reduce uranium (VI) (U(VI)) to uranium (IV) (U(IV)). The experiment consists in the G. sulfurreducens' immobilization using the boron-doped diamond electrodes (BDD) as an electron donor by electrochemical processes. Taking advantage of the bacteria's microbial metabolism process and using U (VI) as an electron acceptor will reduce the ion to U (IV). First, a potential of -500mV (vs. Ag/AgCl) will be applied to immobilize the G. sulfurreducens’ to the BDD electrode surface. Second, with the application of a potential between -500mV to -700mV (vs. Ag/AgCl), it will expect to reduce the uranium ion and remove it from the media. Preliminary studies of the behavior of BDD electrodes in the presence of U(VI) had been carried out to determine the electrochemical conditions at which the substrate works with this ion. The results found with these studies will show how G. sulfurreducens’ will improve U(VI) bioremediation through electrochemical processes.This research has the financial support from the DOE- Minority Serving Institutions Partnership Program, National Nuclear Security Administration, "Scholarly Partnership in Nuclear Security (SPINS)", Grant Number DE NA0003980. Figure 1

Cryo-EM structure of an extracellular Geobacter OmcE cytochrome filament reveals tetrahaem packing.

Electrically conductive appendages from the anaerobic bacterium Geobacter sulfurreducens were first observed two decades ago, with genetic and biochemical data suggesting that conductive fibres were type IV pili. Recently, an extracellular conductive filament of G. sulfurreducens was found to contain polymerized c-type cytochrome OmcS subunits, not pilin subunits. Here we report that G. sulfurreducens also produces a second, thinner appendage comprised of cytochrome OmcE subunits and solve its structure using cryo-electron microscopy at ~4.3 Å resolution. Although OmcE and OmcS subunits have no overall sequence or structural similarities, upon polymerization both form filaments that share a conserved haem packing arrangement in which haems are coordinated by histidines in adjacent subunits. Unlike OmcS filaments, OmcE filaments are highly glycosylated. In extracellular fractions from G. sulfurreducens, we detected type IV pili comprising PilA-N and -C chains, along with abundant B-DNA. OmcE is the second cytochrome filament to be characterized using structural and biophysical methods. We propose that there is a broad class of conductive bacterial appendages with conserved haem packing (rather than sequence homology) that enable long-distance electron transport to chemicals or other microbial cells.

Fumarate disproportionation by Geobacter sulfurreducens and its involvement in biocorrosion and interspecies electron transfer

The model electroactive bacterium Geobacter sulfurreducens can acquire electrons directly from solid donors including metals and other species. Reports on this physiology concluding that solid donors are the only electron sources were conducted with fumarate believed to serve exclusively as the terminal electron acceptor (TEA). Here, G. sulfurreducens was repeatedly transferred for adaptation within a growth medium containing only fumarate and no other solid or soluble substrate. The resulting evolved strain grew efficiently with either the C4-dicarboxylate fumarate or malate acting simultaneously as electron donor, carbon source, and electron acceptor via disproportionation. Whole-genome sequencing identified 38 mutations including one in the regulator PilR known to repress the expression of the C4-dicarboxylate antiporter DcuB essential to G. sulfurreducens when growing with fumarate. Futhermore, the PilR mutation was identical to the sole mutation previously reported in an evolved G. sulfurreducens grown in a co-culture assumed to derive energy solely from direct interspecies electron transfer, but cultivated with fumarate as the TEA. When cultivating the fumarate-adapted strain in the presence of stainless steel and fumarate, biocorrosion was observed and bacterial growth was accelerated 2.3 times. These results suggest that G. sulfurreducens can conserve energy concomitantly from C4-dicarboxylate disproportionation and the oxidation of a solid electron donor. This co-metabolic capacity confers an advantage to Geobacter for survival and colonization and explains in part why these microbes are omnipresent in different anaerobic ecosystems.

Elucidation of complex respiratory chains: a straightforward strategy to monitor electron transfer between cytochromes.

Cytochromes are electron transfer (ET) proteins essential in various biological systems, playing crucial roles in the respiratory chains of bacteria. These proteins are particularly abundant in electrogenic microorganisms and are responsible for the efficient delivery of electrons to the cells' exterior. The capability of sending electrons outside the cells open new avenues to be explored for emerging biotechnological applications in bioremediation, microbial electrosynthesis, and bioenergy fields. To develop these applications, it is critical to identify the different redox partners and to elucidate the stepwise ET along the respiratory paths. However, investigating direct ET events between proteins with identical features in nearly all spectroscopic techniques is extremely challenging. Nuclear magnetic resonance (NMR) spectroscopy offers the possibility to overcome this difficulty by analysing the alterations of the spectral signatures of each protein caused by electron exchange events. The uncrowded NMR spectral regions containing the heme resonances of the cytochromes display unique and distinct signatures in the reduced and oxidized states, which can be explored to monitor ET within the redox complex. In this study, we present a strategy for a fast and straightforward monitorization of ET between c-type cytochromes, using as model a triheme periplasmic cytochrome and a membrane-associated monoheme cytochrome from the electrogenic bacterium Geobacter sulfurreducens. The comparison between the 1D 1H NMR spectra obtained for samples containing the two cytochromes and for samples containing the individual proteins clearly demonstrated a unidirectional ET within the redox complex. This strategy provides a simple and straightforward means to elucidate complex biologic respiratory ET chains.

Mechanisms underlying Clostridium pasteurianum's metabolic shift when grown with Geobacter sulfurreducens.

Recently, a study showed that glycerol fermentation by Clostridium pasteurianum could be metabolically redirected when the electroactive bacterium Geobacter sulfurreducens was added in the culture. It was assumed that this metabolic shift of the fermentative species resulted from an interspecies electron transfer. The aim of this study was to find out the mechanisms used for this interaction and how they affect the metabolism of C. pasteurianum. To get insights into the mechanisms involved, several coculture setups and RNA sequencing with differential expression analysis were performed. As a result, a putative interaction model was proposed: G. sulfurreducens produces cobamide molecules that possibly modify C. pasteurianum metabolic pathway at the key enzyme glycerol dehydratase, and affect its vanadium nitrogenase expression. In addition, the results suggested that G. sulfurreducens' electrons could enter C. pasteurianum through its transmembrane flavin-bound polyferredoxin and cellular cytochrome b5-rubredoxin interplay, putatively reinforcing the metabolic shift. Unravelling the mechanisms behind the interaction between fermentative and electroactive bacteria helps to better understand the role of bacterial interactions in fermentation setups. KEY POINTS: • C. pasteurianum-G. sulfurreducens interaction inducing a metabolic shift is mediated • C. pasteurianum's metabolic shift in coculture might be induced by cobamides • Electrons possibly enter C. pasteurianum through a multiflavin polyferredoxin.

Rational design of electron/proton transfer mechanisms in the exoelectrogenic bacteria Geobacter sulfurreducens.

The redox potential values of cytochromes can be modulated by the protonation/deprotonation of neighbor groups (redox-Bohr effect), a mechanism that permits the proteins to couple electron/proton transfer. In the respiratory chains, this effect is particularly relevant if observed in the physiological pH range, as it may contribute to the electrochemical gradient for ATP synthesis. A constitutively produced family of five triheme cytochromes (PpcA-E) from the bacterium Geobacter sulfurreducens plays a crucial role in extracellular electron transfer, a hallmark that permits this bacterium to be explored for several biotechnological applications. Two members of this family (PpcA and PpcD) couple electron/proton transfer in the physiological pH range, a feature not shared with PpcB and PpcE. That ability is crucial for G. sulfurreducens' growth in Fe(III)-reducing habitats since extra contributors to the electrochemical gradient are needed. It was postulated that the redox-Bohr effect is determined by the nature of residue 6, a leucine in PpcA/PpcD and a phenylalanine in PpcB/PpcE. To confirm this hypothesis, Phe6 was replaced by leucine in PpcB and PpcE. The functional properties of these mutants were investigated by NMR and UV-visible spectroscopy to assess their capability to couple electron/proton transfer in the physiological pH range. The results obtained showed that the mutants have an increased redox-Bohr effect and are now capable of coupling electron/proton transfer. This confirms the determinant role of the nature of residue 6 in the modulation of the redox-Bohr effect in this family of cytochromes, opening routes to engineer Geobacter cells with improved biomass production.

Cobalt Resistance via Detoxification and Mineralization in the Iron-Reducing Bacterium Geobacter sulfurreducens.

Bacteria in the genus Geobacter thrive in iron- and manganese-rich environments where the divalent cobalt cation (CoII) accumulates to potentially toxic concentrations. Consistent with selective pressure from environmental exposure, the model laboratory representative Geobacter sulfurreducens grew with CoCl2 concentrations (1 mM) typically used to enrich for metal-resistant bacteria from contaminated sites. We reconstructed from genomic data canonical pathways for CoII import and assimilation into cofactors (cobamides) that support the growth of numerous syntrophic partners. We also identified several metal efflux pumps, including one that was specifically upregulated by CoII. Cells acclimated to metal stress by downregulating non-essential proteins with metals and thiol groups that CoII preferentially targets. They also activated sensory and regulatory proteins involved in detoxification as well as pathways for protein and DNA repair. In addition, G. sulfurreducens upregulated respiratory chains that could have contributed to the reductive mineralization of the metal on the cell surface. Transcriptomic evidence also revealed pathways for cell envelope modification that increased metal resistance and promoted cell-cell aggregation and biofilm formation in stationary phase. These complex adaptive responses confer on Geobacter a competitive advantage for growth in metal-rich environments that are essential to the sustainability of cobamide-dependent microbiomes and the sequestration of the metal in hitherto unknown biomineralization reactions.

Isotope exchange between mercuric [Hg(II)] chloride and Hg(II) bound to minerals and thiolate ligands: Implications for enriched isotope tracer studies

Enriched mercury (Hg) stable isotopes have been widely used as tracers in field and laboratory investigations of Hg(II) biogeochemical transformations such as methylation and demethylation. Few studies, however, have considered concurrent isotope exchange reactions between newly spiked and pre-existing Hg(II) in environmental matrices, which may alter redistribution and thus transformation of the spiked and pre-existing Hg(II). Using enriched 198Hg [as mercuric Hg(II) or HgCln species], this study investigated isotope exchange between 198Hg and pre-existing Hg(II) bound to metacinnabar (β-HgS), sediments, low-molecular-weight (LMW) thiols, and dissolved organic matter (DOM). The impact of isotope exchange on methylmercury production in the presence of organic ligands was also evaluated with an iron-reducing bacterium Geobacter sulfurreducens PCA in a phosphate buffered solution (pH 7.4). We found that spiked 198Hg readily exchanged with mineral-bound ambient Hg(II) despite concurrent Hg(II) adsorption and immobilization on the solids. Rapid exchange (<3 min) was also observed between spiked 198Hg and 200Hg pre-equilibrated with LMW thiols and DOM in solution. While the exchange did not cause net changes in Hg(II) chemical speciation, it resulted in redistribution of Hg(II) isotopes bound to the ligands and thus an apparently similar methylation rate and magnitude of the spiked 198Hg and pre-existing 200Hg by PCA cells when 198Hg and 200Hg were added at 1:1 ratio. These observations underscore the importance of isotope exchange when an enriched Hg isotope is applied in environmental matrices, as the exchange could potentially lead to biased rate calculations of Hg(II) transformation and bioaccumulation and thus risk assessments of new Hg(II) input to the natural ecosystems.

Fast removal of toxic hexavalent chromium from an aqueous solution by high-density Geobacter sulfurreducens

Hexavalent chromium (Cr(VI)) is a carcinogenic compound that can be removed from contaminated sites by the activity of metal-reducing bacteria. The model bacterium Geobacter sulfurreducens reduces Cr(VI) to less toxic Cr(III) and accumulates Cr ions intracellularly. However, this process is usually slow with small concentrations of Cr(VI) removed in a matter of days. Here, high-density G. sulfurreducens cultures were tested for the capacity to remove Cr(VI) readily. With an initial G. sulfurreducens density of 5.8 × 108 cells ml−1, 99.0 ± 0.8% of 100 mg l−1 Cr(VI) was removed after 20 min. With a higher starting Cr(VI) concentration of 200 mg l−1, G. sulfurreducens with a density of 11.4 × 108 cells ml−1 removed 99.0 ± 0.4% Cr(VI) after 2 h. Experiments performed with cell-free spent medium indicate that extracellular proteins are major contributors for the reduction of Cr(VI) to Cr(III). Furthermore, results show that most Cr(III) ions ultimately end up inside the bacterial cells where they are less susceptible to re-oxidation. The fast Cr(VI) removal rates observed with high-density G. sulfurreducens demonstrate the potential of this bacterium for bioremediation applications such as the cleaning of industrial wastewaters.