Evidence for Iron-Dependent Nitrate Respiration in the Dissimilatory Iron-Reducing Bacterium Geobacter metallireducens

The dissimilatory iron-reducing bacterium Geobacter metallireducens was found to require iron at a concentration in excess of 50 μM for continuous cultivation on nitrate. Growth yield (~3-fold), cytochrome c content (~7-fold), and nitrate (~4.5-fold) and nitrite (~70-fold) reductase activities were all increased significantly when the growth medium was amended with 500 μM iron.     

Direct Interspecies Electron Transfer between Geobacter metallireducens and Methanosarcina barkeri

Direct interspecies electron transfer (DIET) is potentially an effective form of syntrophy in methanogenic communities, but little is known about the diversity of methanogens capable of DIET. The ability of Methanosarcina barkeri to participate in DIET was evaluated in coculture with Geobacter metallireducens. Cocultures formed aggregates that shared electrons via DIET during the stoichiometric conversion of ethanol to methane. Cocultures could not be initiated with a pilin-deficient G. metallireducens strain, suggesting that long-range electron transfer along pili was important for DIET. Amendments of granular activated carbon permitted the pilin-deficient G. metallireducens isolates to share electrons with M. barkeri, demonstrating that this conductive material could substitute for pili in promoting DIET. When M. barkeri was grown in coculture with the H₂-producing Pelobacter carbinolicus, incapable of DIET, M. barkeri utilized H₂ as an electron donor but metabolized little of the acetate that P. carbinolicus produced. This suggested that H₂, but not electrons derived from DIET, inhibited acetate metabolism. P. carbinolicus-M. barkeri cocultures did not aggregate, demonstrating that, unlike DIET, close physical contact was not necessary for interspecies H₂ transfer. M. barkeri is the second methanogen found to accept electrons via DIET and the first methanogen known to be capable of using either H₂ or electrons derived from DIET for CO₂ reduction. Furthermore, M. barkeri is genetically tractable, making it a model organism for elucidating mechanisms by which methanogens make biological electrical connections with other cells.     

Anaerobic Benzene Oxidation via Phenol in Geobacter metallireducens

Anaerobic activation of benzene is expected to represent a novel biochemistry of environmental significance. Therefore, benzene metabolism was investigated in Geobacter metallireducens, the only genetically tractable organism known to anaerobically degrade benzene. Trace amounts (<0.5 μM) of phenol accumulated in cultures of Geobacter metallireducens anaerobically oxidizing benzene to carbon dioxide with the reduction of Fe(III). Phenol was not detected in cell-free controls or in Fe(II)- and benzene-containing cultures of Geobacter sulfurreducens, a Geobacter species that cannot metabolize benzene. The phenol produced in G. metallireducens cultures was labeled with ¹⁸O during growth in H₂¹⁸O, as expected for anaerobic conversion of benzene to phenol. Analysis of whole-genome gene expression patterns indicated that genes for phenol metabolism were upregulated during growth on benzene but that genes for benzoate or toluene metabolism were not, further suggesting that phenol was an intermediate in benzene metabolism. Deletion of the genes for PpsA or PpcB, subunits of two enzymes specifically required for the metabolism of phenol, removed the capacity for benzene metabolism. These results demonstrate that benzene hydroxylation to phenol is an alternative to carboxylation for anaerobic benzene activation and suggest that this may be an important metabolic route for benzene removal in petroleum-contaminated groundwaters, in which Geobacter species are considered to play an important role in anaerobic benzene degradation.     

Decolorization of azo dyes by Geobacter metallireducens

Geobacter metallireducens was found to be capable of decolorizing several azo dyes with different structures to various extents. Pyruvate, ethanol, acetate, propionate, and benzoate could support 66.3?±?2.6?93.7?±?2.1 % decolorization of 0.1 mM acid red 27 (AR27) in 40 h. The dependence of the specific decolorization rate on AR27 concentration (25 to 800 μM) followed Michaelis–Menten kinetics (K _m?=?186.9?±?1.4 μΜ, V _max?=?0.65?±?0.02 μmol?mg protein^?1 h^?1). Enhanced AR27 decolorization was observed with the increase of cell concentrations ranging from 7.5 to 45 mgL^?1. AR27 decolorization by G. metallireducens was retarded by the presence of goethite, which competed electrons with AR27 and was reduced to Fe(II). The addition of low concentrations of humic acid (1?100 mgL^?1) or 2-hydroxy–1,4-naphthoquinone (0.5?50 μM) could improve the decolorization performance of G. metallireducens. High-performance liquid chromatography analysis suggested reductive pathway to be responsible for decolorization. This was the first study on azo dye decolorization by Geobacter strain and might improve our understanding of natural attenuation and bioremediation of environments polluted by azo dyes.     

Evidence for iron-dependent nitrate respiration in the dissimilatory iron-reducing bacterium Geobacter metallireducens

The dissimilatory iron-reducing bacterium Geobacter metallireducens was found to require iron at a concentration in excess of 50 microM for continuous cultivation on nitrate. Growth yield (approximately 3-fold), cytochrome c content (approximately 7-fold), and nitrate (approximately 4.5-fold) and nitrite (approximately 70-fold) reductase activities were all increased significantly when the growth medium was amended with 500 microM iron.     

Reduction of Prussian Blue by the two iron-reducing microorganisms Geobacter metallireducens and Shewanella alga

Cyanide or cyanide-metal complexes are frequent contaminants of soil or aquifers at industrial sites, which can be released from such sites by outgassing or transport with the groundwater. They form very stable complexes with iron, which may occur in the subsurface as an insoluble blue mineral, the so-called Prussian Blue (Fe(4)[Fe(CN)(6)](3)). In this study, we show that the insoluble and colloidal Fe(III)-cyanide complex Prussian Blue can be reduced and utilized as electron acceptor by the dissimilatory iron-reducing bacteria Geobacter metallireducens and Shewanella alga strain BrY. The microbial reduction of the dark blue pigment Prussian Blue leads to the formation of a completely colourless solid mineral, presumably Prussian White (Fe(2)[Fe(CN)(6)]), which could be reoxidized through exposure to air, regaining the dark blue colour. In addition, the microorganisms were able to grow with Prussian Blue, using it as the sole electron acceptor. Geobacter metallireducens could also reduce Prussian Blue coatings on sand, which was sampled from a contaminated site.     

How Geobacteraceae may dominate subsurface biodegradation: physiology of Geobacter metallireducens in slow-growth habitat-simulating retentostats

Geobacteraceae dominate many iron-reducing subsurface environments and are associated with biodegradation of organic pollutants. In order to enhance the understanding of the environmental role played by Geobacteraceae , the physiology of Geobacter metallireducens was investigated at the low growth rates found in its subsurface habitat. Cultivation in retentostats (a continuous culturing device with biomass retention) under electron acceptor and electron donor limitation enabled growth rates as low as 0.0008 h⁻¹. The maximum growth yield was between 0.05 and 0.09 C-mol biomass per C-mol acetate and comparable to that observed in batch experiments. Maintenance energy demand is among the lowest reported for heterotrophic bacteria, under both acetate and AQDS limitation. The cells were able to use alternative electron acceptors directly, without requiring de novo protein synthesis. We discuss how the extremely low maintenance energy demand and the ability to readily use alternative electron acceptors may help Geobacter species to become ubiquitous and dominant microorganisms in many iron-reducing subsurface settings.     

Heat Production by the Denitrifying Bacterium Pseudomonas fluorescens and the Dissimilatory Ammonium-Producing Bacterium Pseudomonas putrefaciens during Anaerobic Growth with Nitrate as the Electron Acceptor

The heat production rate and the simultaneous nitrate consumption and production and consumption of nitrite and nitrous oxide were monitored during the anaerobic growth of two types of dissimilatory nitrate reducers. Pseudomonas fluorescens, a denitrifier, consumed nitrate and accumulated small amounts of nitrite or nitrous oxide. The heat production rate increased steadily during the course of nitrate consumption and decreased rapidly concomitant with the depletion of the electron acceptors. A mean experimental enthalpy change value of −800 kJ/mol of nitrate and a mean growth yield value of 33 g (dry weight)/mol of nitrate consumed were obtained for different concentrations of nitrate. For Pseudomonas putrefaciens, a dissimilatory ammonium producer, the nitrate consumption resulted in an accumulation of nitrite and nitrous oxide. Nitrite consumption commenced after depletion of the nitrate; consequently, two phases were noted in the heat production rate curve during growth. A mean experimental enthalpy change value of −810 kJ/mol of nitrate was obtained for different concentrations of nitrate.     

Isolation and characterization of a heterologously expressed bacterial laccase from the anaerobe Geobacter metallireducens

Applied Microbiology and Biotechnology - Bioinformatics has revealed the presence of putative laccase genes in diverse bacteria, including extremophiles, autotrophs, and, interestingly, anaerobes....     

Constraint-Based Modeling of Carbon Fixation and the Energetics of Electron Transfer in Geobacter metallireducens

Geobacter species are of great interest for environmental and biotechnology applications as they can carry out direct electron transfer to insoluble metals or other microorganisms and have the ability to assimilate inorganic carbon. Here, we report on the capability and key enabling metabolic machinery of Geobacter metallireducens GS-15 to carry out CO₂ fixation and direct electron transfer to iron. An updated metabolic reconstruction was generated, growth screens on targeted conditions of interest were performed, and constraint-based analysis was utilized to characterize and evaluate critical pathways and reactions in G. metallireducens. The novel capability of G. metallireducens to grow autotrophically with formate and Fe(III) was predicted and subsequently validated in vivo. Additionally, the energetic cost of transferring electrons to an external electron acceptor was determined through analysis of growth experiments carried out using three different electron acceptors (Fe(III), nitrate, and fumarate) by systematically isolating and examining different parts of the electron transport chain. The updated reconstruction will serve as a knowledgebase for understanding and engineering Geobacter and similar species.     

Biochemical and genetic evidence of benzylsuccinate synthase in toluene-degrading,ferric iron-reducing Geobacter metallireducens

In vitro assays demonstrated that toluene-grown cells of Geobacter metallireducens catalyzed the addition of toluene to fumarate to form benzylsuccinate under anaerobic conditions. The specific in vitro rate of benzylsuccinate formationwas ca. 45% of the specific in vivo rate of toluene consumption. In addition, bssA and bssB, which code for the and subunits of benzylsuccinate synthase (BSS), respectively, were found to have sequences in G. etallireducens similar to the only sequences heretofore available (for three denitrifying strains). This is the first report of the presence of BSS in a ferriciron-reducing bacterium; BSS activity has previously been reported in denitrifying, sulfate-reducing, and anoxygenic phototrophic toluene degraders, as well as in a highly enriched methanogenic, toluene-degrading culture.     

The genome sequence of Geobacter metallireducens: features of metabolism, physiology and regulation common and dissimilar to Geobacter sulfurreducens

Background  

The genome sequence of Geobacter metallireducens is the second to be completed from the metal-respiring genus Geobacter, and is compared in this report to that of Geobacter sulfurreducens in order to understand their metabolic, physiological and regulatory similarities and differences.     

Purification and characterization of active-site components of the putative p-cresol methylhydroxylase membrane complex from Geobacter metallireducens

p-Cresol methylhydroxylases (PCMH) from aerobic and facultatively anaerobic bacteria are soluble, periplasmic flavocytochromes that catalyze the first step in biological p-cresol degradation, the hydroxylation of the substrate with water. Recent results suggested that p-cresol degradation in the strictly anaerobic Geobacter metallireducens involves a tightly membrane-bound PCMH complex. In this work, the soluble components of this complex were purified and characterized. The data obtained suggest a molecular mass of 124 ± 15 kDa and a unique αα′β₂ subunit composition, with α and α′ representing isoforms of the flavin adenine dinucleotide (FAD)-containing subunit and β representing a c-type cytochrome. Fluorescence and mass spectrometric analysis suggested that one FAD was covalently linked to Tyr³⁹⁴ of the α subunit. In contrast, the α′ subunit did not contain any FAD cofactor and is therefore considered to be catalytically inactive. The UV/visible spectrum was typical for a flavocytochrome with two heme c cofactors and one FAD cofactor. p-Cresol reduced the FAD but only one of the two heme cofactors. PCMH catalyzed both the hydroxylation of p-cresol to p-hydroxybenzyl alcohol and the subsequent oxidation of the latter to p-hydroxybenzaldehyde in the presence of artificial electron acceptors. The very low K_m values (1.7 and 2.7 μM, respectively) suggest that the in vivo function of PCMH is to oxidize both p-cresol and p-hydroxybenzyl alcohol. The latter was a mixed inhibitor of p-cresol oxidation, with inhibition constants of a K_ic (competitive inhibition) value of 18 ± 9 μM and a K_iu (uncompetitive inhibition) value of 235 ± 20 μM. A putative functional model for an unusual PCMH enzyme is presented.     

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.