Extracellular Acceptors Research Articles

Enhancing electron transfer in anaerobic process by supercapacitor materials: Polyaniline functionated activated carbon

Strengthening the direct interspecies electron transfer (DIET) is an effective strategy to improve the performance of anaerobic digestion (AD) process. In this study, the polyaniline functionated activated carbon (AC-PANi) was prepared by chemical oxidative polymerization. This material possessed pseudo-capacitance properties as well as excellent charge transfer capability. The experimental results demonstrated that the incorporation of AC-PANi in AD process could efficiently increase the chemical oxygen demand (COD) removal (18.6 %) and daily methane production rate (35.3 %). The AC-PANi can also act as an extracellular acceptor to promote the synthesis of adenosine triphosphate (ATP) and secretion of extracellular enzymes as well as cytochrome C (Cyt-C). The content of coenzyme F420 on methanogens was also shown to be increased by 60.9 % with the addition of AC-PANi in AD reactor. Overall, this work provides an easy but feasible way to enhance AD performance by promoting DIET between acetate-producing bacteria and methanogens.

Different solubilizing ability of cyclodextrin derivatives for cholesterol in Niemann-Pick disease type C treatment.

Niemann-Pick disease type C (NPC) is a fatal neurodegenerative disorder caused by abnormal intracellular cholesterol trafficking. Cyclodextrins (CDs), the most promising therapeutic candidates for NPC, but with concerns about ototoxicity, are cyclic oligosaccharides with dual functions of unesterified cholesterol (UC) shuttle and sink that catalytically enhance the bidirectional flux and net efflux of UC, respectively, between the cell membrane and the extracellular acceptors. However, the properties of CDs that regulate these functions and how they could be used to improve treatments for NPC are unclear. We estimated CD-UC complexation for nine CD derivatives derived from native α-, β-, and γ-CD with different cavity sizes, using solubility and molecular docking analyses. The stoichiometry and complexation ability of the resulting complexes were investigated in relation to the therapeutic effectiveness and toxicity of each CD derivative in NPC experimental models. We found that shuttle and sink activities of CDs are dependent on cavity size-dependent stoichiometry and substituent-associated stability of CD-UC complexation. The ability of CD derivatives to form 1:1 and 2:1 complexes with UC were correlated with their ability to normalize intracellular cholesterol trafficking serving as shuttle and with their cytotoxicity associated with cellular UC efflux acting as sink, respectively, in NPC model cells. Notably, the ability of CD derivatives to form an inclusion complex with UC was responsible for not only efficacy but ototoxicity, while a representative derivative without this ability negligibly affected auditory function, underscoring its preventability. Our findings highlight the importance of strategies for optimizing the molecular structure of CDs to overcome this functional dilemma in the treatment of NPC.

Assessing Thermal Response of Redox Conduction for Anti-Arrhenius Kinetics in a Microbial Cytochrome Nanowire.

A micrometers-long helical homopolymer of the outer-membrane cytochrome type S (OmcS) from Geobacter sulfurreducens is proposed to transport electrons to extracellular acceptors in an ancient respiratory strategy of biogeochemical and technological significance. OmcS surprisingly exhibits higher conductivity upon cooling (anti-Arrhenius kinetics), an effect previously attributed to H-bond restructuring and heme redox potential shifts. Herein, the temperature sensitivity of redox conductivity is more thoroughly examined with conventional and constant-redox and -pH molecular dynamics and quantum mechanics/molecular mechanics. A 30 K drop in temperature constituted a weak perturbation to electron transfer energetics, changing electronic couplings (⟨Hmn⟩), reaction free energies (ΔGmn), reorganization energies (λmn), and activation energies (Ea) by at most |0.002|, |0.050|, |0.120|, and |0.045| eV, respectively. Changes in ΔGmn reflected -0.07 ± 0.03 V shifts in redox potentials that were caused in roughly equal measure by altered electrostatic interactions with the solvent and protein. Changes in intraprotein H-bonding reproduced the earlier observations. Single-particle diffusion and multiparticle steady-state flux models, parametrized with Marcus theory rates, showed that biologically relevant incoherent hopping cannot qualitatively or quantitatively describe electrical conductivity measured by atomic force microscopy in filamentous OmcS. The discrepancy is attributed to differences between solution-phase simulations and solid-state measurements and the need to model intra- and intermolecular vibrations explicitly.

Lack of Specificity in Geobacter Periplasmic Electron Transfer.

Reduction of extracellular acceptors requires electron transfer across the periplasm. In Geobacter sulfurreducens, three separate cytoplasmic membrane cytochromes are utilized depending on redox potential, and at least five cytochrome conduits span the outer membrane. Because G. sulfurreducens produces 5 structurally similar triheme periplasmic cytochromes (PpcABCDE) that differ in expression level, midpoint potential, and heme biochemistry, many hypotheses propose distinct periplasmic carriers could be used for specific redox potentials, terminal acceptors, or growth conditions. Using a panel of marker-free single, quadruple, and quintuple mutants, little support for these models could be found. Three quadruple mutants containing only one paralog (PpcA, PpcB, and PpcD) reduced Fe(III) citrate and Fe(III) oxide at the same rate and extent, even though PpcB and PpcD were at much lower periplasmic levels than PpcA. Mutants containing only PpcC and PpcE showed defects, but these cytochromes were nearly undetectable in the periplasm. When expressed sufficiently, PpcC and PpcE supported wild-type Fe(III) reduction. PpcA and PpcE from G. metallireducens similarly restored metal respiration in G. sulfurreducens. PgcA, an unrelated extracellular triheme c-type cytochrome, also participated in periplasmic electron transfer. While triheme cytochromes were important for metal reduction, sextuple ΔppcABCDE ΔpgcA mutants grew near wild-type rates with normal cyclic voltammetry profiles when using anodes as electron acceptors. These results reveal broad promiscuity in the periplasmic electron transfer network of metal-reducing Geobacter and suggest that an as-yet-undiscovered periplasmic mechanism supports electron transfer to electrodes. IMPORTANCE Many inner and outer membrane cytochromes used by Geobacter for electron transfer to extracellular acceptors have specific functions. How these are connected by periplasmic carriers remains poorly understood. G. sulfurreducens contains multiple triheme periplasmic cytochromes with unique biochemical properties and expression profiles. It is hypothesized that each could be involved in a different respiratory pathway, depending on redox potential or energy needs. Here, we show that Geobacter periplasmic cytochromes instead show evidence of being highly promiscuous. Any of 6 triheme cytochromes supported similar growth with soluble or insoluble metals, but none were required when cells utilized electrodes. These findings fail to support many models of Geobacter electron transfer, and question why these organisms produce such an array of periplasmic cytochromes.

Neuropeptide Y regulates cholesterol uptake and efflux in macrophages and promotes foam cell formation.

The dysregulation of lipid metabolic pathways (cholesterol uptake and efflux) in macrophages results in the formation of lipid-dense macrophages, named foam cells, that participate in plaque formation. NPY binding to NPY receptors in macrophages can modulate cell functions and affect the process of atherosclerotic plaques. The present study aimed to determine whether NPY affects the formation of macrophage-derived foam cells and its underlying mechanisms in macrophages. THP-1-derived macrophages were incubated with oxidized low-density lipoprotein (ox-LDL) and treated with different concentrations of NPY. We analysed the relative levels of proteins related to cholesterol uptake and efflux. We found that NPY effectively increased cholesterol uptake and intracellular cholesterol content via the Y1 and Y5 receptors, and this effect was blocked by Y1 and Y5 antagonists. Mechanistically, NPY enhanced the expression of SRA and CD36 via the PKC/PPARγ pathways, promoting macrophage cholesterol uptake. Moreover, NPY significantly decreased cholesterol efflux to the extracellular cholesterol acceptors ApoA1 and HDL in macrophages. NPY mediated decreases in ABCA1, ABCG1 and SR-BI expression through the inhibition of the JAK/STAT3 pathways. Our results suggest that NPY binding to the Y1 and Y5 receptors enhances foam cell formation by regulating cholesterol uptake and efflux in macrophages.

Exploration of bifurcated electron transfer mechanism in Bacillus cereus for enhanced power generation in double-chambered microbial fuel cells

Extracellular Electron Transfer (EET) is considered as the communication bridge between bacterial cells and outer electron acceptors. Apart from a few model microorganisms conventionally studied, the literature regarding the EET mechanism in Gram-positive bacteria and its conductive interaction is sparse. In the present study, proteolytic Gram-positive bacteria - Bacillus cereus is explored for enhancement of power generation in Microbial Fuel Cells (MFC). A low zeta potential (−0.1 V) of B. cereus, enables it to release electrons and protons much faster. We achieve a power density as high as 400 mW m−2 with a current density of ~5.8 mA cm−2 by inoculating an MFC with B. cereus. A genome wide transcriptome analysis by RNA-seq is used to categorize the genes and the significant pathways potentially involved in EET in B. cereus. The expression of gene cluster encodes for PTS (Phosphotransferase system) that catalyzes and transport phosphorylation of disaccharide present in the medium. This plays a regulatory role in biofilm formation. Simultaneously the secretion and assembly of extracellular flavin molecules by B. cereus, shuttle and mediate electron to the extracellular acceptor. In short, B. cereus uses bifurcated pathway i.e., directly by aligning the cytochrome complex and indirectly by secreting flavins molecules to promote electron transfer. These findings reveal its significant functional role in MFCs’ electrode community for sustainable bioenergy production.

Structure and transport mechanism of the human cholesterol transporter ABCG1.

The reverse cholesterol transport pathway is responsible for the maintenance of human cholesterol homeostasis, an imbalance of which usually leads to atherosclerosis. As a key component of this pathway, the ATP-binding cassette transporter ABCG1 forwards cellular cholesterol to the extracellular acceptor nascent high-density lipoprotein (HDL). Here, we report a 3.26-Å cryo-electron microscopy structure of cholesterol-bound ABCG1 in an inward-facing conformation, which represents a turnover condition upon ATP binding. Structural analyses combined with functional assays reveals that a cluster of conserved hydrophobic residues, in addition to two sphingomyelins, constitute a well-defined cholesterol-binding cavity. The exit of this cavity is closed by three pairs of conserved Phe residues, which constitute a hydrophobic path for the release of cholesterol in an acceptor concentration-dependent manner. Overall, we propose an ABCG1-driven cholesterol transport cycle initiated by sphingomyelin-assisted cholesterol recruitment and accomplished by the release of cholesterol to HDL.

Making protons tag along with electrons

Every living cell needs to get rid of leftover electrons when metabolism extracts energy through the oxidation of nutrients. Common soil microbes such as Geobacter sulfurreducens live in harsh environments that do not afford the luxury of soluble, ingestible electron acceptors like oxygen. Instead of resorting to fermentation, which requires the export of reduced compounds (e.g. ethanol or lactate derived from pyruvate) from the cell, these organisms have evolved a means to anaerobically respire by using nanowires to export electrons to extracellular acceptors in a process called extracellular electron transfer (EET) [ 1]. Since 2005, these nanowires were thought to be pili filaments [ 2]. But recent studies have revealed that nanowires are composed of multiheme cytochromes OmcS [ 3, 4] and OmcZ [ 5] whereas pili remain inside the cell during EET and are required for the secretion of nanowires [ 6]. However, how electrons are passed to these nanowires remains a mystery ( Figure 1A). Periplasmic cytochromes (Ppc) called PpcA-E could be doing the job, but only two of them (PpcA and PpcD) can couple electron/proton transfer - a necessary condition for energy generation. In a recent study, Salgueiro and co-workers selectively replaced an aromatic with an aliphatic residue to couple electron/proton transfer in PpcB and PpcE (Biochem. J. 2021, 478 (14): 2871-2887). This significant in vitro success of their protein engineering strategy may enable the optimization of bioenergetic machinery for bioenergy, biofuels, and bioelectronics applications.

Engineering Wired Life: Synthetic Biology for Electroactive Bacteria.

Electroactive bacteria produce or consume electrical current by moving electrons to and from extracellular acceptors and donors. This specialized process, known as extracellular electron transfer, relies on pathways composed of redox active proteins and biomolecules and has enabled technologies ranging from harvesting energy on the sea floor, to chemical sensing, to carbon capture. Harnessing and controlling extracellular electron transfer pathways using bioengineering and synthetic biology promises to heighten the limits of established technologies and open doors to new possibilities. In this review, we provide an overview of recent advancements in genetic tools for manipulating native electroactive bacteria to control extracellular electron transfer. After reviewing electron transfer pathways in natively electroactive organisms, we examine lessons learned from the introduction of extracellular electron transfer pathways into Escherichia coli. We conclude by presenting challenges to future efforts and give examples of opportunities to bioengineer microbes for electrochemical applications.

An Elegant Four-Helical Fold in NOX and STEAP Enzymes Facilitates Electron Transport across Biomembranes-Similar Vehicle, Different Destination.

The ferric reductase superfamily comprises several oxidoreductases that use an intracellular electron source to reduce an extracellular acceptor substrate. NADPH oxidases (NOXs) and six-transmembrane epithelial antigen of the prostate enzymes (STEAPs) are iconic members of the superfamily. NOXs produce extracellular reactive oxygen species that exert potent bactericidal activities and trigger redox-signaling cascades that regulate cell division and differentiation. STEAPs catalyze the reduction of extracellular iron and copper which is necessary for the bioavailability of these essential elements. Both NOXs and STEAPs are present as multiple isozymes with distinct regulatory properties and physiological roles. Despite the important roles of NOXs and STEAPs in human physiology and despite their wide involvement in diseases like cancer, their mode of action at the molecular level remained incompletely understood for a long time, in part due to the absence of high-resolution models of the complete enzymes. Our two laboratories have elucidated the three-dimensional structures of NOXs and STEAPs, providing key insight into their mechanisms and evolution. The enzymes share a conserved transmembrane helical domain with an eye-catching hourglass shape. On the extracellular side, a heme prosthetic group is at the bottom of a pocket where the substrate (O2 in NOX, chelated iron or copper in STEAP) is reduced. On the intracellular side, the inner heme of NOX and the FAD of STEAP are bound to topological equivalent sites. This is a rare case where critical amino acid substitutions and local conformational changes enable a cofactor (heme vs FAD) swap between two structurally and functionally conserved scaffolds. The catalytic core of these enzymes is completed by distinct cytosolic NADPH-binding domains that are topologically unrelated (a ferredoxin reductase-like flavoprotein domain in NOX and a F420H2:NADP+-like domain in STEAP), feature different quaternary structures, and underlie specific regulatory mechanisms. Despite their differences, these domains all establish electron-transfer chains that direct the electrons from NADPH to the transmembrane domain. The multistep nature of the process and the chemical nature of the products pose considerable problems in the enzymatic assays. We learned that great care must be exerted in the validation of a candidate inhibitor. Multiple orthogonal assays are required to rule out off-target effects such as ROS-scavenging activities or nonspecific interference with the enzyme redox chain. The structural analysis of STEAP/NOX enzymes led us to further notice that their transmembrane heme-binding topology is shared by other enzymes. We found that the core domain of the cytochrome b subunits of the mitochondrial complex III and photosynthetic cytochrome b6f are closely related to NOXs and STEAPs and likely arose from the same ancestor protein. This observation expands the substrate portfolio of the superfamily since cytochromes b act on ubiquinone. The rigidly packed helices of the NOX/STEAP/cytochrome b domain contrast with the more malleable membrane proteins like ion channels or amino-acid transporters, which undergo large conformational changes to allow passage of relatively large metabolites. This notion of a rigid hourglass scaffold found an unexpected confirmation in the observation, revealed by structural comparisons, that an helical bundle identical to the NOX/STEAP/cytochrome b enzymes is featured by a de novo designed heme-binding protein, PS1. Apparently, nature and protein designers have independently converged to this fold as a versatile scaffold for heme-mediated reactions. The challenge is now to uncover the molecular mechanisms that implement the isozyme-specific regulation of the enzyme functions and develop much needed inhibitors and modulators for chemical biology and drug design studies.

Thermodynamic properties of triheme cytochrome PpcF from Geobacter metallireducens reveal unprecedented functional mechanism

The bacterium Geobacter metallireducens is highly efficient in long-range extracellular electron transfer, a process that relies on an efficient bridging between the cytoplasmic electron donors and the extracellular acceptors. The periplasmic triheme cytochromes are crucial players in these processes and thus the understanding of their functional mechanism is crucial to elucidate the extracellular electron transfer processes in this microorganism. The triheme cytochrome PpcF from G. metallireducens has the lowest amino acid sequence identity with the remaining cytochromes from the PpcA-family of G. sulfurreducens and G. metallireducens, making it an interesting target for structural and functional studies. In this work, we performed a detailed functional and thermodynamic characterization of cytochrome PpcF by the complementary usage of NMR and visible spectroscopic techniques. The results obtained show that the heme reduction potentials are negative, different from each other and are also modulated by the redox and redox-Bohr interactions that assure unprecedented mechanistic features to the protein. The results showed that the order of oxidation of the hemes in cytochrome PpcF is maintained in the entire physiological pH range. The considerable separation of the hemes' redox potential values facilitates a sequential transfer within the chain of redox centers in PpcF, thus assuring electron transfer directionality to the electron acceptors.

Organic Microbial Electrochemical Transistor Monitoring Extracellular Electron Transfer.

Extracellular electron transfer (EET) denotes the process of microbial respiration with electron transfer to extracellular acceptors and has been exploited in a range of microbial electrochemical systems (MESs). To further understand EET and to optimize the performance of MESs, a better understanding of the dynamics at the microscale is needed. However, the real‐time monitoring of EET at high spatiotemporal resolution would require sophisticated signal amplification. To amplify local EET signals, a miniaturized bioelectronic device, the so‐called organic microbial electrochemical transistor (OMECT), is developed, which includes Shewanella oneidensis MR‐1 integrated onto organic electrochemical transistors comprising poly(3,4‐ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) combined with poly(vinyl alcohol) (PVA). Bacteria are attached to the gate of the transistor by a chronoamperometric method and the successful attachment is confirmed by fluorescence microscopy. Monitoring EET with the OMECT configuration is achieved due to the inherent amplification of the transistor, revealing fast time‐responses to lactate. The limits of detection when using microfabricated gates as charge collectors are also investigated. The work is a first step toward understanding and monitoring EET in highly confined spaces via microfabricated organic electronic devices, and it can be of importance to study exoelectrogens in microenvironments, such as those of the human microbiome.

Two Routes for Extracellular Electron Transfer in Enterococcus faecalis.

Enterococcus faecalis cells are known to have ferric reductase activity and the ability to transfer electrons generated in metabolism to the external environment. We have isolated mutants defective in ferric reductase activity and studied their electron transfer properties to electrodes mediated by ferric ions and an osmium complex-modified redox polymer (OsRP). Electron transfer mediated with ferric ions and ferric reductase activity were both found to be dependent on the membrane-associated Ndh3 and EetA proteins, consistent with findings in Listeria monocytogenes In contrast, electron transfer mediated with OsRP was independent of these two proteins. Quinone in the cell membrane was required for the electron transfer with both mediators. The combined results demonstrate that extracellular electron transfer from reduced quinone to ferric ions and to OsRP occurs via different routes in the cell envelope of E. faecalisIMPORTANCE The transfer of reducing power in the form of electrons, generated in the catabolism of nutrients, from a bacterium to an extracellular acceptor appears to be common in nature. The electron acceptor can be another cell or abiotic material. Such extracellular electron transfer contributes to syntrophic metabolism and is of wide environmental, industrial, and medical importance. Electron transfer between microorganisms and electrodes is fundamental in microbial fuel cells for energy production and for electricity-driven synthesis of chemical compounds in cells. In contrast to the much-studied extracellular electron transfer mediated by cell surface exposed cytochromes, little is known about components and mechanisms for such electron transfer in organisms without these cytochromes and in Gram-positive bacteria such as E. faecalis, which is a commensal gut lactic acid bacterium and opportunistic pathogen.

Monitoring the itinerary of lysosomal cholesterol in Niemann-Pick Type C1-deficient cells after cyclodextrin treatment

Niemann-Pick disease type C (NPC) disease is a lipid-storage disorder that is caused by mutations in the genes encoding NPC proteins and results in lysosomal cholesterol accumulation. 2-Hydroxypropyl-β-cyclodextrin (CD) has been shown to reduce lysosomal cholesterol levels and enhance sterol homeostatic responses, but CD's mechanism of action remains unknown. Recent work provides evidence that CD stimulates lysosomal exocytosis, raising the possibility that lysosomal cholesterol is released in exosomes. However, therapeutic concentrations of CD do not alter total cellular cholesterol, and cholesterol homeostatic responses at the ER are most consistent with increased ER membrane cholesterol. To address these disparate findings, here we used stable isotope labeling to track the movement of lipoprotein cholesterol cargo in response to CD in NPC1-deficient U2OS cells. Although released cholesterol was detectable, it was not associated with extracellular vesicles. Rather, we demonstrate that lysosomal cholesterol trafficks to the plasma membrane (PM), where it exchanges with lipoprotein-bound cholesterol in a CD-dependent manner. We found that in the absence of suitable extracellular cholesterol acceptors, cholesterol exchange is abrogated, cholesterol accumulates in the PM, and reesterification at the ER is increased. These results support a model in which CD promotes intracellular redistribution of lysosomal cholesterol, but not cholesterol exocytosis or efflux, during the restoration of cholesterol homeostatic responses.

Shuttle/sink model composed of β-cyclodextrin and simvastatin-loaded discoidal reconstituted high-density lipoprotein for enhanced cholesterol efflux and drug uptake in macrophage/foam cells.

Targeting drug delivery to macrophage/foam cells is challenged owing to the poor cell permeability and fluidity resulting from the massive accumulation of intracellular cholesterol in atherosclerosis (AS). Discoidal reconstituted high-density lipoprotein (d-rHDL) has been well regarded as a potential drug delivery system for AS by virtue of its plaque-targeting and cholesterol removal abilities, while the latter is compromised by the high activation energy of cholesterol efflux. It is reported that a low concentration of β-cyclodextrin (β-CD) can function as a cholesterol shuttle to promote cholesterol efflux from cells to the extracellular acceptors (cholesterol sink, such as HDL particles), but it is still unknown whether the combination of β-CD with a drug-loaded d-rHDL can function as a shuttle/sink model to promote the remodeling and drug release of the d-rHDL carrier after accelerating the cholesterol efflux. Furthermore, it is interesting to investigate whether enhanced cholesterol efflux can improve the cellular drug uptake by restoring the permeability and fluidity of the cell membrane. Here, simvastatin-loaded d-rHDL (ST-d-rHDL) was combined with different concentrations of β-CD. Compared with ST-d-rHDL alone, the cholesterol removal ability of ST-d-rHDL combined with 0.5 mM of β-CD increased by 31-fold after incubation for 6 h and the cumulative drug release of ST-d-rHDL increased by two-fold during the initial 1 h in an acellular mimetic system. In macrophage/foam cells, 0.5 mM of β-CD showed superior promoting effects in the cholesterol removal ability and remodeling of ST-d-rHDL compared to 0.1 mM of β-CD. The high concentration of β-CD at 2 mM displayed a low efficiency for accelerating cholesterol efflux, which might function as a cholesterol sink rather than a cholesterol shuttle. Moreover, the permeability and fluidity of the cell membrane were improved by combining 0.5 mM of β-CD with ST-d-rHDL, which exhibited an enhanced cellular drug uptake and inhibiting effect on the intracellular lipid deposition and secretion of inflammatory cytokine. Collectively, combination of β-CD and ST-d-rHDL as a shuttle/sink model could enhance cholesterol efflux and drug uptake to suppress inflammation in macrophage/foam cells.

Direct Extracellular Electron Transfer of the Geobacter sulfurreducens Pili Relevant to Interaromatic Distances.

Microorganisms can transfer electrons directly to extracellular acceptors, during which organic compounds are oxidized to carbon dioxide. One of these microbes, Geobacter sulfurreducens, is well known for the “metallic-like” conductivity of its type IV pili. However, there is no consensus on what the mechanism for electron transfer along these conductive pili is. Based on the aromatic distances and orientations of our predicted models, the mechanism of electron transfer in the Geobacter sulfurreducens (GS) pili was explored by quantum chemical calculations with Marcus theory of electron transfer reactions. Three aromatic residues from the N-terminal α-helix of the GS pilin subunit are packed together, resulting in a continuous pi-pi interaction chain. The theoretical conductance (4.69 μS/3.85 μS) of the predicted models is very similar to that in the experiments reported recently (3.40 μS). These findings offer a new concept that the GS pili belongs to a new class of proteins that can transport electrons through pi-pi interaction between aromatic residues and also provide a valuable tool for guiding further researches of these conductive pili, to investigate their roles in biogeochemical cycling, and potential applications in biomaterials, bioelectronics, and bioenergy.

Backbone, side chain and heme resonance assignment of the triheme cytochrome PpcA from Geobacter metallireducens in the oxidized state.

The bacterium Geobacter metallireducens is capable of transferring electrons to the cell exterior, a process designated extracellular electron transfer. This mechanism allows the microorganism to reduce extracellular acceptors such as Fe(III) (hydr)oxides and water toxic and/or radioactive contaminants including Cr(VI) and U(VI). It is also capable of oxidizing waste water aromatic organic compounds being an important microorganism for bioremediation of polluted waters. Extracellular electron transfer also allows electricity harvesting from microbial fuel cells, a promising sustainable form of energy production. However, extracellular electron transfer processes in this microorganism are still poorly characterized. The triheme c-type cytochrome PpcA from G. metallireducens is abundant in the periplasm and is crucial for electron transfer between the cytoplasm and the cell's exterior. In this work, we report near complete assignment of backbone, side chain and heme resonances for PpcA in the oxidized state that will permit its structure determination and identification of interactions with physiological redox partners.

Determination of the magnetic properties and orientation of the heme axial ligands of PpcA from Geobacter metallireducens by paramagnetic NMR

The rising interest in the use of Geobacter bacteria for biotechnological applications demands a deep understanding of how these bacteria are able to thrive in a variety of environments and perform extracellular electron transfer. The Geobacter metallireducens bacterium can couple the oxidation of a wide range of compounds to the reduction of several extracellular acceptors, including heavy metals, toxic organic compounds or electrode surfaces. The periplasmic c-type cytochrome PpcA from this bacterium is a member of a family composed of five periplasmic triheme cytochromes, which are important to bridge the electron transfer between the cytoplasm and the extracellular environment. To better understand the functional mechanism of PpcA it is essential to obtain structural data for this cytochrome. In this work, the geometry of the heme axial ligands, as well as the magnetic properties of the hemes were determined for the oxidized form of the cytochrome, using the 13C NMR chemical shifts of the heme α-substituents. The results were further compared with those previously obtained for the homologous cytochrome from Geobacter sulfurreducens. The orientations of the axial histidine planes and the magnetic properties of the hemes are conserved in both proteins. Overall, the results obtained allowed the definition of the orientation of the magnetic axes of PpcA from G. metallireducens, which will be used as constraints to assist the solution structure determination of the cytochrome in the oxidized form.

Electrochemiluminescence for the identification of electrochemically active bacteria

Electrochemically active bacteria (EAB) use extracellular electron transfer (EET) to exchange electron with extracellular acceptors. Previous studies regarding the measurement of EAB were based on either extracellular reduction or oxidation. In this work, we developed a simple electrochemiluminescence (ECL) assay for the identification and detection of EAB. The results of this proposed method revealed that EET of EAB influenced the content of dissolved oxygen and the formation of Ru(bpy)32+• thus leading to qualitative changes of the ECL signal. EAB with the ability of extracellular reduction (such as Shewanella oneidensis MR-1) gave enhanced signal on ECL emission while those displaying the ability of extracellular oxidation (i.e., Sulfobacillus acidophilus) showed the opposite effect on ECL emission, but non-EAB (i.e., Escherichia coli) did not. These changes in ECL intensity were also proportional to the cell density that could be quantitatively detected in the concentration range of (1.1 ± 1) × 105–212 ± 2 CFU/mL (i.e. Shewanella oneidensis MR-1). Moreover, the measurement of the ability of EAB using this approach was in agreement with measurements using the dissimilatory Fe(III) reduction method. Compared to previous reports, this method displayed a continual and steady ECL signal that allowed accurate measurements of EAB. Most important, only a low cell density was needed in this Ru(bpy)32+ - based ECL method, which is beneficial for cell detection.

High density lipoprotein promotes nascent apolipoprotein A-V secretion from mRNA transfected cells

Despite its exceptionally low circulating concentration, apolipoprotein (apo) A-V is a potent modulator of plasma triacylglycerol levels. The secretion efficiency of nascent apoA-V was investigated in cultured cells transfected with mRNA. Following transfection of HepG2 cells with wild type apoA-V mRNA, apoA-V protein was detectable in cell lysates by 6 h. At 24 h post transfection, evidence of apoA-V secretion into media was obtained, although most apoA-V was recovered in the cell lysate fraction. By contrast, apoA-I was efficiently secreted into the culture medium. A positive correlation between culture medium fetal bovine serum content and the percentage of apoA-V recovered in conditioned media was observed. When transfected cells were cultured in serum-free media supplemented with increasing amounts of high density lipoprotein, a positive correlation with apoA-V secretion was observed. The data indicate that, following signal sequence cleavage, the bulk of nascent apoA-V remains cell associated. Transit of nascent apoA-V out of cultured cells is enhanced by the availability of extracellular lipid particle acceptors.