Evidence that OmcB and OmpB of Geobacter sulfurreducens are outer membrane surface proteins

The c-type cytochrome (OmcB) and the multicopper protein (OmpB) required for Fe(III) oxide reduction by Geobacter sulfurreducens were predicted previously to be outer membrane proteins, but it is not clear whether they are positioned in a manner that permits the interaction with Fe(III). Treatment of whole cells with proteinase K inhibited Fe(III) reduction, but had no impact on the inner membrane-associated fumarate reduction. OmcB was digested by protease, resulting in a smaller peptide. However, immunogold labeling coupled with transmission electron microscopy did not detect OmcB, suggesting that it is only partially exposed on the cell surface. In contrast, OmpB was completely digested with protease. OmpB was loosely associated with the cell surface as a substantial portion of it was recovered in the culture supernatant. Immunogold labeling demonstrated that OmpB associated with the cell was evenly distributed on the cell surface rather than localized to one side of the cell like the conductive pili. Although several proteins required for Fe(III) oxide reduction are shown to be exposed on the outer surface of G. sulfurreducens, the finding that OmcB is also surface exposed is the first report of a protein required for optimal Fe(III) citrate reduction at least partially accessible on the cell surface.     

pH, redox potential and local biofilm potential microenvironments within Geobacter sulfurreducens biofilms and their roles in electron transfer

The limitation of pH inside electrode‐respiring biofilms is a well‐known concept. However, little is known about how pH and redox potential are affected by increasing current inside biofilms respiring on electrodes. Quantifying the variations in pH and redox potential with increasing current is needed to determine how electron transfer is tied to proton transfer within the biofilm. In this research, we quantified pH and redox potential variations in electrode‐respiring Geobacter sulfurreducens biofilms as a function of respiration rates, measured as current. We also characterized pH and redox potential at the counter electrode. We concluded that (1) pH continued to decrease in the biofilm through different growth phases, showing that the pH is not always a limiting factor in a biofilm and (2) decreasing pH and increasing redox potential at the biofilm electrode were associated only with the biofilm, demonstrating that G. sulfurreducens biofilms respire in a unique internal environment. Redox potential inside the biofilm was also compared to the local biofilm potential measured by a graphite microelectrode, where the tip of the microelectrode was allowed to acclimatize inside the biofilm. Biotechnol. Bioeng. 2012; 109: 2651–2662. © 2012 Wiley Periodicals, Inc.     

Fluorescent properties of c-type cytochromes reveal their potential role as an extracytoplasmic electron sink in Geobacter sulfurreducens

A novel fluorescence technique for monitoring the redox status of c-type cytochromes in Geobacter sulfurreducens was developed in order to evaluate the capacity of these extracytoplasmic cytochromes to store electrons during periods in which an external electron acceptor is not available. When intact cells in which the cytochromes were in a reduced state were excited at a wavelength of 350 nm, they fluoresced with maxima at 402 and 437 nm. Oxidation of the cytochromes resulted in a loss of fluorescence. This method was much more sensitive than the traditional approach of detecting c-type cytochromes via visible light absorbance. Furthermore, fluorescence of reduced cytochromes in individual cells could be detected via fluorescence microscopy, and the cytochromes in a G. sulfurreducens biofilm, remotely excited with an optical fibre, could be detected at distances as far as 5 cm. Fluorescence analysis of cytochrome oxidation and reduction of the external electron acceptor, anthraquinone-2,6-disulfonate, suggested that the extracytoplasmic cytochromes of G. sulfurreducens could store approximately 10(7) electrons per cell. Independent analysis of the haem content of the cells determined from analysis of incorporation of (55)Fe into cytochromes provided a similar estimate of cytochrome electron-storage capacity. This electron-storage capacity could, in the absence of an external electron acceptor, permit continued electron transfer across the inner membrane sufficient to supply the maintenance energy requirements for G. sulfurreducens for up to 8 min or enough proton motive force to power flagella motors for G. sulfurreducens motility. The fluorescence approach described here provides a sensitive method for evaluating the redox status of Geobacter species in culture and/or its environments. Furthermore, these results suggest that the periplasmic and outer-membrane cytochromes of Geobacter species act as capacitors, allowing continued electron transport, and thus viability and motility, for Geobacter species as they move between heterogeneously dispersed Fe(III) oxides during growth in the subsurface.     

Molecular evidence for the adaptive evolution of Geobacter sulfurreducens to perform dissimilatory iron reduction in natural environments

The electrically conductive pili (e-pili) of Geobacter species enable extracellular electron transfer to insoluble metallic minerals, electrodes and other microbial species, which confers biogeochemical significance and global prevalence on Geobacter in diverse anaerobic environments. E-pili are constructed by truncated PilA which is considered to have evolved from full-length pilin by gene fission under positive evolutionary selection. However, this hypothesis is based on phylogenetic analysis and has not yet been experimentally confirmed. Here, we reconstructed an ancestral strain of G. sulfurreducens (designated COMB) carrying full-length PilA by combining genes GSU1496 and GSU1497. The results demonstrated that strain COMB expressed and assembled the full-length fused PilA and exhibited an outer membrane c-type cytochrome profile similar to the wild-type strain. Surprisingly, the generated COMB-pili were also conductive, indicating the evolution of truncated PilA did not occur for conductivity. Moreover, strain COMB minimally reduced Fe(III) oxides but maintained its ability to respire electrodes, demonstrating the truncation of pilin enables iron respiration. This study provides the first experimental evidence that the truncation of pilin in Geobacter species confers adaption to Fe(III)-mineral-mediated selective pressures, and suggests an evolutionary event during which the separation of the GSU1497 gene helped Geobacter survive and thrive in natural environments.     

Potential regulates metabolism and extracellular respiration of electroactive Geobacter biofilm

Dissimilatory metal reducer Geobacter sulfurreducens can mediate redox processes through extracellular electron transfer and exhibit potential-dependent electrochemical activity in biofilm. Understanding the microbial acclimation to potential is of critical importance for developing robust electrochemically active biofilms and facilitating their environmental, geochemical, and energy applications. In this study, the metabolism and redox conduction behaviors of G. sulfurreducens biofilms developed at different potentials were explored. We found that electrochemical acclimation occurred at the initial hours of polarizing G. sulfurreducens cells to the potentials. Two mechanisms of acclimation were found, depending on the polarizing potential. In the mature biofilms, a low level of biosynthesis and a high level of catabolism were maintained at +0.2 V versus standard hydrogen electrode (SHE). The opposite results were observed at potentials higher than or equal to +0.4 V versus SHE. The potential also regulated the constitution of the electron transfer network by synthesizing more extracellular cytochrome c such as OmcS at 0.0 and +0.2 V and exhibited a better conductivity. These findings provide reasonable explanations for the mechanism governing the electrochemical respiration and activity in G. sulfurreducens biofilms.     

Efficient and precise control of gene expression in Geobacter sulfurreducens through new genetic elements and tools for pollutant conversion

Geobacter species, exhibiting exceptional extracellular electron transfer aptitude, hold great potential for applications in pollution remediation, bioenergy production, and natural elemental cycles. Nonetheless, a scarcity of well-characterized genetic elements and gene expression tools constrains the effective and precise fine-tuning of gene expression in Geobacter species, thereby limiting their applications. Here, we examined a suite of genetic elements and developed a new genetic editing tool in Geobacter sulfurreducens to enhance their pollutant conversion capacity. First, the performances of the widely used inducible promoters, constitutive promoters, and ribosomal binding sites (RBSs) elements in G. sulfurreducens were quantitatively evaluated. Also, six native promoters with superior expression levels than constitutive promoters were identified on the genome of G. sulfurreducens. Employing the characterized genetic elements, the clustered regularly interspaced short palindromic repeats interference (CRISPRi) system was constructed in G. sulfurreducens to achieve the repression of an essential gene-aroK and morphogenic genes-ftsZ and mreB. Finally, applying the engineered strain to the reduction of tungsten trioxide (WO₃), methyl orange (MO), and Cr(VI), We found that morphological elongation through ftsZ repression amplified the extracellular electron transfer proficiency of G. sulfurreducens and facilitated its contaminant transformation efficiency. These new systems provide rapid, versatile, and scalable tools poised to expedite advancements in Geobacter genomic engineering to favor environmental and other biotechnological applications.     

The primary quinone acceptor and the membrane-bound c-type cytochromes of the halophilic purple nonsulfur bacterium Rhodospirillum salinarum: A spectroscopic and thermodynamic study

The mid-point potential (Em_7.0) of the primary quinone acceptor (Qa) and the biochemical features (Em_7.0 and apparent molecular mass, MM) of the membrane bound c-type cytochromes (cyt) involved in photosynthetic electron transfer of the halophilic phototrophic bacterium Rhodospirillum (Rs.) salinarum were determined. A tetrahemic RC bound cytochrome was found (MM of 39.8 kDa) with Em_7.0 of the hemes equal to +304, +98, +21, –134 (± 8) mV as determined by dark equilibrium redox titrations in the isolated purified form. The highest potential heme (Em_7.0 = +304 mV, band at 556 nm) was able to reduce the photo-oxidized reaction center (P⁺) in a sub-millisecond ( 20 s) time scale reaction, acting most likely as the direct electron donor to P⁺). The midpoint potential of the primary electron donor (Em_7.0 = + 455 mV) was found to be close to that reported for the primary donor of the non-halophilic Rhodospirillum species Rs. rubrum, whereas the quinone primary electron acceptor (Qa) was different showing the spectral features of a menaquinone molecule with Em_7.0 at –128 (± 5) mV. A membrane bound c-type heme with Em_7.0 of 259 (± 1) and MM of 40 kDa was also isolated and referred to an orthodox cytochrome c₁). The present data on the photosynthetic apparatus, along with the previous results on the respiratory system [Moschettini et al. (1997) Arch Microbiol 168: 302-309], suggest that Rs. salinarum is biochemically distinct from Rs. rubrum, the most representative specie of the genus.     

生物地球化学锰循环中的微生物胞外电子传递机制

微生物是生物地球化学元素循环的重要驱动者,在锰等变价金属元素的氧化还原过程中起着至关重要的作用。近年来,Mn(Ⅲ)的发现以及在一些环境中的广泛存在,丰富了人们对Mn(Ⅲ)以及自然界锰循环过程的认识。研究发现,锰的生物地球化学循环,尤其是锰还原过程,与微生物胞外电子传递紧密相关,且目前已知的5种胞外电子传递机制均与锰还原有关联。因此,本文综述了锰的生物地球化学循环及其意义,并从微生物胞外电子传递的机制、微生物介导锰氧化、微生物介导锰还原等3个方面来介绍参与锰循环的微生物多样性;以及微生物地球化学锰循环的环境意义。对微生物参与锰循环过程的研究不仅可以进一步丰富相关理论,同时也能推动生物除锰、污染物原位修复及生物冶金等应用领域的发展。     

Structures and solution properties of two novel periplasmic sensor domains with c-type heme from chemotaxis proteins of Geobacter sulfurreducens: implications for signal transduction

Periplasmic sensor domains from two methyl-accepting chemotaxis proteins from Geobacter sulfurreducens (encoded by genes GSU0935 and GSU0582) were expressed in Escherichia coli. The sensor domains were isolated, purified, characterized in solution, and their crystal structures were determined. In the crystal, both sensor domains form swapped dimers and show a PAS-type fold. The swapped segment consists of two helices of about 45 residues at the N terminus with the hemes located between the two monomers. In the case of the GSU0582 sensor, the dimer contains a crystallographic 2-fold symmetry and the heme is coordinated by an axial His and a water molecule. In the case of the GSU0935 sensor, the crystals contain a non-crystallographic dimer, and surprisingly, the coordination of the heme in each monomer is different; monomer A heme has His-Met ligation and monomer B heme has His-water ligation as found in the GSU0582 sensor. The structures of these sensor domains are the first structures of PAS domains containing covalently bound heme. Optical absorption, electron paramagnetic resonance and NMR spectroscopy have revealed that the heme groups of both sensor domains are high-spin and low-spin in the oxidized and reduced forms, respectively, and that the spin-state interconversion involves a heme axial ligand replacement. Both sensor domains bind NO in their ferric and ferrous forms but bind CO only in the reduced form. The binding of both NO and CO occurs via an axial ligand exchange process, and is fully reversible. The reduction potentials of the sensor domains differ by 95 mV (− 156 mV and − 251 mV for sensors GSU0582 and GSU0935, respectively). The swapped dimerization of these sensor domains and redox-linked ligand switch might be related to the mechanism of signal transduction by these chemotaxis proteins.     

Role of Met58 in the regulation of electron/proton transfer in trihaem cytochrome PpcA from Geobacter sulfurreducens

The bacterium Gs (Geobacter sulfurreducens) is capable of oxidizing a large variety of compounds relaying electrons out of the cytoplasm and across the membranes in a process designated as extracellular electron transfer. The trihaem cytochrome PpcA is highly abundant in Gs and is most probably the reservoir of electrons destined for the outer surface. In addition to its role in electron transfer pathways, we have previously shown that this protein could perform e⁻/H⁺ energy transduction. This mechanism is achieved by selecting the specific redox states that the protein can access during the redox cycle and might be related to the formation of proton electrochemical potential gradient across the periplasmic membrane. The regulatory role of haem III in the functional mechanism of PpcA was probed by replacing Met⁵⁸, a residue that controls the solvent accessibility of haem III, with serine, aspartic acid, asparagine or lysine. The data obtained from the mutants showed that the preferred e⁻/H⁺ transfer pathway observed for PpcA is strongly dependent on the reduction potential of haem III. It is striking to note that one residue can fine tune the redox states that can be accessed by the trihaem cytochrome enough to alter the functional pathways.     

Effects of protein-protein interactions on electron transfer: docking and electron transfer calculations for complexes between flavodoxin and c-type cytochromes

 Theoretical studies of protein-protein association and electron transfer were performed on the binary systems formed by Desulfovibrio vulgaris Hildenborough (D. v. H.) flavodoxin and D. v. H. cytochrome c ₅₅₃ and by flavodoxin and horse heart cytochrome c. Initial structures for the complexes were obtained by rigid-body docking and were refined by MD to allow for molecular flexibility. The structures thus obtained were analysed in terms of their relative stability through the calculation of excess energies. Electrostatic, van der Waals and solvation energy terms showed all to have significant contributions to the stability of complexes. In the best association solutions found for both cytochromes, these bind to different zones of flavodoxin. The binding site of flavodoxin observed for cytochrome c is in accordance with earlier works [27]. The various association modes found were characterised in terms of electron transfer using the Pathways model. For complexes between flavodoxin and horse heart cytochrome c, some correlation was observed between electron tunnelling coupling factors and conformation energy; the best conformation found for electron transfer corresponded also to the best one in terms of energy. For complexes between flavodoxin and cytochrome c ₅₅₃ this was not the case and a lower correlation was observed between electron tunnelling coupling factors and excess energies. These results are in accordance with the differences in the experimental dependence of electron transfer rates with ionic strength observed between these two cases. Received: 29 December 1998 / Accepted: 22 March 1999     

The role of the membrane bound cytochromes of b- and c-type in the electron transport chain of Rhodobacter capsulatus

(1) The electron transport system of heterotrophically dark-grown Rhodobacter capsulatus was investigated using the wild-type strain MT1131 and the phototrophic non-competent (Ps^-) mutant MT-GS18 carrying deletions of the genes for cytochrome c ₁ and b of the bc ₁ complex and for cytochrome c ₂. (2) Spectroscopic and thermodynamic data demonstrate that deletion of both bc ₁ complex and cyt. c ₂ still leaves several haems of c- and b-type with Em_7.0 of +265 mV and +354 mV at 551–542 nm, and +415 mV and +275 mV at 561–575 nm, respectively. (3) Analysis of the oxidoreduction kinetic patterns of cytochromes indicated that cyt. b ₄₁₅ and cyt. b ₂₇₅ are reduced by either ascorbate-diaminodurene or NADH, respectively. (4) Growth on different carbon and nitrogen sources revealed that the membrane-bound electron transport chain of both MT1131 and MT-GS18 strains undergoes functional modifications in response to the composition of the growth medium used. (5) Excitation of membrane fragments from cells grown in malate minimal medium by a train of single turnover flashes of light led to a rapid oxidation of 32% of the membrane-bound c-type haem complement. Conversely, membranes prepared from peptone/yeast extract grown cells did not show cyt. c photooxidation. These results are discussed within the framework of an electron transport chain in which alternative pathways bypassing both the cyt. c ₂ and bc ₁ complex might involve high-potential membrane bound haems of b- and c-type.Abbreviations AA antimycin A - CCCP carbonylcyanide m-chlorophenyl hydrazone - CN^- cyanide - DAD diaminodurene - Q₂H₂ ubiquinol-2 - Q-pool ubiquinone-10 pool - RC photochemical reaction center     

Effect of isotope substitution and controlled dehydration on the photoinduced electron transport reactions of quinone acceptors and multiheme cytochrome C in bacterial photosynthetic reaction center

Isotope substitution of H₂O by ²H₂O causes an increase in the rate of dark recombination between photooxidized bacteriochlorophyll (P⁺) and reduced primary quinone acceptor in Rhodobacter sphaeroides reaction centers (RC) at room temperature. The isotopic effect declines upon decreasing the temperature. Dehydration of RC complexes of Ectothiorhodospira shaposhnikovii chromatophores containing multiheme cytochrome c causes a decrease in the efficiency of transfer of a photomobilized electron between the primary and secondary quinone acceptors and from cytochrome to P⁺. In the case of H₂O medium these effects are observed at a lower hydration than in ²H₂O-containing medium. In the E. shaposhnikovii chromatophores subjected to dehydration in H₂O, the rate of electron transfer from the nearest high-potential cytochrome heme to P⁺ is virtually independent of hydration within the P/P₀ range from 0.1 to 0.5. In samples hydrated in ²H₂O this rate is approximately 1.5 times lower than in H₂O. However, the isotopic effect of this reaction disappears upon dehydration. The intramolecular electron transfer between two high-potential hemes of cytochrome c in samples with ²H₂O is inhibited within this range of P/P₀, whereas in RC samples with H₂O there is a trend toward gradual inhibition of the interheme electron transfer with dehydration. The experimental results are discussed in terms of the effects of isotope substitution and dehydration on relaxation processes and charge state of RC on implementation of the reactive states of RC providing electron transfer control.     

Regulation of proton-to-electron stoichiometry in photosynthetic electron transport: physiological function in photoprotection

The primary stable products of photosynthetic electron flow are NADPH and ATP. Stoichiometry of their production depends on the ratio of protons pumped across the thylakoid membrane to electrons passed through the electron transport pathway (H⁺/e⁻ ratio). Flexible requirements of the ATP/NADPH ratio by various assimilatory reactions in chloroplasts must be fulfilled by the H⁺/e⁻ ratio during the electron flow. In addition to the well-known role of ΔpH during ATP synthesis, ΔpH also functions as a trigger of the down-regulation of photosystem II (PSII) photochemistry. Excessive light energy is safely dissipated as heat by this regulatory process to suppress the generation of toxic reactive oxygen species. Thus, regulation of the H⁺/e⁻ ratio may function in the photoprotection, as well as in the regulation of the ATP/NADPH production ratio. It has long been the consensus that the H⁺/e⁻ ratio can be controlled by regulating the proton-transporting Q-cycle in the cytochrome b ₆ f complex and by the cyclic electron flow around photosystem I (PSI). Despite the possible physiological importance and the long history of interest, the molecular identity of Q-cycle regulation and the cyclic electron flow around PSI have been remained unclear. The recent improvements in research tools, including the genetic approach using chlorophyll fluorescence imaging and establishment of the chloroplast transformation technique, are providing new insights into classical topics. In this review, we focus on regulation of the H⁺/e⁻ ratio especially from the view of photosynthetic regulation. Received: August 2, 2001 / Accepted: October 1, 2001     

Cleavage of the iron-methionine bond in c-type cytochromes: Crystal structure of oxidized and reduced cytochrome c2 from Rhodopseudomonas palustris and its ammonia complex

The three-dimensional structures of the native cytochrome c(2) from Rhodopseudomonas palustris and of its ammonia complex have been obtained at pH 4.4 and pH 8.5, respectively. The structure of the native form has been refined in the oxidized state at 1.70 A and in the reduced state at 1.95 A resolution. These are the first high-resolution crystal structures in both oxidation states of a cytochrome c(2) with relatively high redox potential (+350 mV). The differences between the two oxidation states of the native form, including the position of internal water molecules, are small. The unusual six-residue insertion Gly82-Ala87, which precedes the heme binding Met93, forms an isolated 3(10)-helix secondary structural element not previously observed in other c-type cytochromes. Furthermore, this cytochrome shows an external methionine residue involved in a strained folding near the exposed edge of the heme. The structural comparison of the present cytochrome c(2) with other c-type cytochromes has revealed that the presence of such a residue, with torsion angles phi and psi of approximately -140 and -130 degrees, respectively, is a typical feature of this family of proteins. The refined crystal structure of the ammonia complex, obtained at 1.15 A resolution, shows that the sulphur atom of the Met93 axial ligand does not coordinate the heme iron atom, but is replaced by an exogenous ammonia molecule. This is the only example so far reported of an X-ray structure with the heme iron coordinated by an ammonia molecule. The detachment of Met93 is accompanied by a very localized change in backbone conformation, involving mainly the residues Lys92, Met93, and Thr94. Previous studies under typical denaturing conditions, including high-pH values and the presence of exogenous ligands, have shown that the detachment of the Met axial ligand is a basic step in the folding/unfolding process of c-type cytochromes. The ammonia adduct represents a structural model for this important step of the unfolding pathway. Factors proposed to be important for the methionine dissociation are the strength of the H-bond between the Met93 and Tyr66 residues that stabilizes the native form, and the presence in this bacterial cytochrome c(2) of the rare six-residue insertion in the helix 3(10) conformation that increases Met loop flexibility.