Dissimilatory Fe(III) reduction by Clostridium beijerinckii isolated from freshwater sediment using Fe(III) maltol enrichment

A microorganism which reduces Fe(III) during the fermentation of glucose was isolated from freshwater sediment. The Fe(III) was supplied to enrichment cultures as a soluble complex with the bidentate ligand maltol (3-hydroxy-2-methyl-4-pyrone). Advantages that were afforded by the use of Fe(III)(maltol)3 over previously published methods included negation of the requirement for assays of Fe(II) formation. Because Fe(III)(maltol)3 has a characteristic deep red colour, Fe(III) reduction could be quantified spectrophotometrically by monitoring the disappearance of the complex in liquid cultures. Furthermore, Fe(III) reduction on agar plates containing the complex was apparent by zones of decolourisation around the bacterial colonies. 16S rRNA gene sequencing indicated the isolate to be a strain of Clostridium beijerinckii. Growth experiments were performed on the isolate in batch cultures with varying concentrations of Fe(III) citrate and 50 mM glucose. Increasing the level of Fe(III) citrate present was found to alter the fermentation balance, with less acidic products being formed. The presence of Fe(III) led to increases in the growth rate and growth yield, which were both approximately doubled when the supply of the cation reached 25 mM. A NAD(P)H-dependent Fe(III) reductase activity was localised to the bacterial membrane and found not to be sensitive to respiratory inhibitors. Taken together, these data suggest that dissimilatory Fe(III) reduction by the isolate provides a means of utilising the cation as an electron sink, thus facilitating pyridine nucleotide to be recycled during fermentative metabolism.     

Fe(III)的微生物异化还原

异化Fe(III)还原微生物是厌氧环境中广泛存在的一类主要微生物类群,它们的共同特征是可以利用Fe(III)作为末端电子受体而获能。异化Fe(III)还原微生物具有强大的代谢功能,可还原许多有毒重金属包括一些放射性核素,还可降解利用许多有机污染物,在污染环境的生物修复中具有重要的应用价值。本文对异化Fe(III)还原微生物的分布、分类,代谢功能多样性以及异化Fe(III)还原的意义做了评述,旨在加强相关领域的研究人员对此的了解和重视,通过学科的交叉和合作加快我国在这一领域的研究。     

Adhesion of Dissimilatory Fe(III)-Reducing Bacteria to Fe(III) Minerals

The metabolism of dissimilatory iron-reducing bacteria (DIRB) may provide a means of remediating contaminated subsurface soils. The factors controlling the rate and extent of bacterial F(III) mineral reduction are poorly understood. Recent research suggests that molecular-scale interactions between DIRB cells and Fe(III) mineral particles play an important role in this process. One of these interactions, cell adhesion to Fe(III) mineral particles, appears to be a complex process that is, at least in part, mediated by a variety of surface proteins. This study examined the hypothesis that the flagellum serves as an adhesin to different Fe(III) minerals that range in their surface area and degree of crystallinity. Deflagellated cells of the DIRB Shewanella algae BrY showed a reduced ability to adhere to hydrous ferric oxide (HFO) relative to flagellated cells. Flagellated cells were also more hydrophobic than deflagellated cells. This was significant because hydrophobic interactions have been previously shown to dominate S. algae cell adhesion to Fe(III) minerals. Pre-incubating HFO, goethite, or hematite with purified flagella inhibited the adhesion of S. algae BrY cells to these minerals. Transposon mutagenesis was used to generate a flagellum-deficient mutant designated S. algae strain NF. There was a significant difference in the rate and extent of S. algae NF adhesion to HFO, goethite, and hematite relative to that of S. algae BrY. Amiloride, a specific inhibitor of Na + -driven flagellar motors, inhibited S. algae BrY motility but did not affect the adhesion of S. algae BrY to HFO. S.algae NF reduced HFO at the same rate as S. algae BrY. Collectively, the results of this study support the hypothesis that the flagellum of S. algae functions as a specific Fe(III) mineral adhesin. However, these results suggest that flagellum-mediated adhesion is not requisite for Fe(III) mineral reduction.     

Dissimilatory reduction of Fe(III) and other electron acceptors by a Thermus isolate

A thermophilic bacterium that can use O2, NO3-, Fe(III), and S0 as terminal electron acceptors for growth was isolated from groundwater sampled at a 3.2-km depth in a South African gold mine. This organism, designated SA-01, clustered most closely with members of the genus Thermus, as determined by 16S rRNA gene (rDNA) sequence analysis. The 16S rDNA sequence of SA-01 was >98% similar to that of Thermus strain NMX2 A.1, which was previously isolated by other investigators from a thermal spring in New Mexico. Strain NMX2 A.1 was also able to reduce Fe(III) and other electron acceptors. Neither SA-01 nor NMX2 A.1 grew fermentatively, i.e., addition of an external electron acceptor was required for anaerobic growth. Thermus strain SA-01 reduced soluble Fe(III) complexed with citrate or nitrilotriacetic acid (NTA); however, it could reduce only relatively small quantities (0.5 mM) of hydrous ferric oxide except when the humic acid analog 2,6-anthraquinone disulfonate was added as an electron shuttle, in which case 10 mM Fe(III) was reduced. Fe(III)-NTA was reduced quantitatively to Fe(II); reduction of Fe(III)-NTA was coupled to the oxidation of lactate and supported growth through three consecutive transfers. Suspensions of Thermus strain SA-01 cells also reduced Mn(IV), Co(III)-EDTA, Cr(VI), and U(VI). Mn(IV)-oxide was reduced in the presence of either lactate or H2. Both strains were also able to mineralize NTA to CO2 and to couple its oxidation to Fe(III) reduction and growth. The optimum temperature for growth and Fe(III) reduction by Thermus strains SA-01 and NMX2 A.1 is approximately 65 degrees C; their optimum pH is 6.5 to 7.0. This is the first report of a Thermus sp. being able to couple the oxidation of organic compounds to the reduction of Fe, Mn, or S.     

Dissimilatory reduction of Cr(VI), Fe(III), and U(VI) by Cellulomonas isolates

The reduction of Cr(VI), Fe(III), and U(VI) was studied using three recently isolated environmental Cellulomonas sp. (WS01, WS18, and ES5) and a known Cellulomonas strain ( Cellulomonas flavigena ATCC 482) under anaerobic, non-growth conditions. In all cases, these cultures were observed to reduce Cr(VI), Fe(III), and U(VI). In 100 h, with lactate as electron donor, the Cellulomonas isolates (500 mg/l total cell protein) reduced nitrilotriacetic acid chelated Fe(III) [Fe(III)-NTA] from 5 mM to less than 2.2 mM, Cr(VI) from 0.2 mM to less than 0.001 mM, and U(VI) from 0.2 mM to less than 0.12 mM. All Cellulomonas isolates also reduced Cr(VI), Fe(III), and U(VI) in the absence of lactate, while no metal reduction was observed in either the cell-free or heat-killed cell controls. This is the first report of Cellulomonas sp. reducing Fe(III) and U(VI). Further, this is the first report of Cellulomonas spp. coupling the oxidation of lactate, or other unknown electron donors in the absence of lactate, to the reduction of Cr(VI), Fe(III), and U(VI).     

Dissimilatory Fe(III) and Mn(IV) Reduction by Shewanella putrefaciens Requires ferE, a Homolog of the pulE (gspE) Type II Protein Secretion Gene

Shewanella putrefaciens strain 200 respires anaerobically on a wide range of compounds as the sole terminal electron acceptor, including ferric iron [Fe(III)] and manganese oxide [Mn(IV)]. Previous studies demonstrated that a 23.3-kb S. putrefaciens wild-type DNA fragment conferred metal reduction capability to a set of respiratory mutants with impaired Fe(III) and Mn(IV) reduction activities (T. DiChristina and E. DeLong, J. Bacteriol. 176:1468-1474, 1994). In the present study, the smallest complementing fragment was found to contain one open reading frame (ORF) (ferE) whose translated product displayed 87% sequence similarity to Aeromonas hydrophila ExeE, a member of the PulE (GspE) family of proteins found in type II protein secretion systems. Insertional mutants E726 and E912, constructed by targeted replacement of wild-type ferE with an insertionally inactivated ferE construct, were unable to respire anaerobically on Fe(III) or Mn(IV) yet retained the ability to grow on all other terminal electron acceptors. Nucleotide sequence analysis of regions flanking ferE revealed the presence of one partial and two complete ORFs whose translated products displayed 55 to 70% sequence similarity to the PulD, -F, and -G homologs of type II secretion systems. A contiguous cluster of 12 type II secretion genes (pulC to -N homologs) was found in the unannotated genome sequence of Shewanella oneidensis (formerly S. putrefaciens) MR-1. A 91-kDa heme-containing protein involved in Fe(III) reduction was present in the peripheral proteins loosely attached to the outside face of the outer membrane of the wild-type and complemented (Fer+) B31 transconjugates yet was missing from this location in Fer mutants E912 and B31 and in uncomplemented (Fer-) B31 transconjugates. Membrane fractionation studies with the wild-type strain supported this finding: the 91-kDa heme-containing protein was detected with the outer membrane fraction and not with the inner membrane or soluble fraction. These findings provide the first genetic evidence linking dissimilatory metal reduction to type II protein secretion and provide additional biochemical evidence supporting outer membrane localization of S. putrefaciens proteins involved in anaerobic respiration on Fe(III) and Mn(IV).     

Mechanisms for Accessing Insoluble Fe(III) Oxide during Dissimilatory Fe(III) Reduction by Geothrix fermentans

Mechanisms for Fe(III) oxide reduction were investigated in Geothrix fermentans, a dissimilatory Fe(III)-reducing microorganism found within the Fe(III) reduction zone of subsurface environments. Culture filtrates of G. fermentans stimulated the reduction of poorly crystalline Fe(III) oxide by washed cell suspensions, suggesting that G. fermentans released one or more extracellular compounds that promoted Fe(III) oxide reduction. In order to determine if G. fermentans released electron-shuttling compounds, poorly crystalline Fe(III) oxide was incorporated into microporous alginate beads, which prevented contact between G. fermentans and the Fe(III) oxide. G. fermentans reduced the Fe(III) within the beads, suggesting that one of the compounds that G. fermentans releases is an electron-shuttling compound that can transfer electrons from the cell to Fe(III) oxide that is not in contact with the organism. Analysis of culture filtrates by thin-layer chromatography suggested that the electron shuttle has characteristics similar to those of a water-soluble quinone. Analysis of filtrates by ion chromatography demonstrated that there was as much as 250 μM dissolved Fe(III) in cultures of G. fermentans growing with Fe(III) oxide as the electron acceptor, suggesting that G. fermentans released one or more compounds capable of chelating and solubilizing Fe(III). Solubilizing Fe(III) is another strategy for alleviating the need for contact between cells and Fe(III) oxide for Fe(III) reduction. This is the first demonstration of a microorganism that, in defined medium without added electron shuttles or chelators, can reduce Fe(III) derived from Fe(III) oxide without directly contacting the Fe(III) oxide. These results are in marked contrast to those with Geobacter metallireducens, which does not produce electron shuttles or Fe(III) chelators. These results demonstrate that phylogenetically distinct Fe(III)-reducing microorganisms may use significantly different strategies for Fe(III) reduction. Thus, it is important to know which Fe(III)-reducing microorganisms predominate in a given environment in order to understand the mechanisms for Fe(III) reduction in the environment of interest.     

Regulation of Dissimilatory Fe(III) Reduction Activity in Shewanella putrefaciens

Under anaerobic conditions, Shewanella putrefaciens is capable of respiratory-chain-linked, high-rate dissimilatory iron reduction via both a constitutive and inducible Fe(III)-reducing system. In the presence of low levels of dissolved oxygen, however, iron reduction by this microorganism is extremely slow. Fe(II)-trapping experiments in which Fe(III) and O₂ were presented simultaneously to batch cultures of S. putrefaciens indicated that autoxidation of Fe(II) was not responsible for the absence of Fe(III) reduction. Inhibition of cytochrome oxidase with CN⁻ resulted in a high rate of Fe(III) reduction in the presence of dissolved O₂, which suggested that respiratory control mechanisms did not involve inhibition of Fe(III) reductase activities or Fe(III) transport by molecular oxygen. Decreasing the intracellular ATP concentrations by using an uncoupler, 2,4-dinitrophenol, did not increase Fe(III) reduction, indicating that the reduction rate was not controlled by the energy status of the cell. Control of electron transport at branch points could account for the observed pattern of respiration in the presence of the competing electron acceptors Fe(III) and O₂.     

Dissimilatory Fe(III) Reduction by the Marine Microorganism Desulfuromonas acetoxidans

The ability of the marine microorganism Desulfuromonas acetoxidans to reduce Fe(III) was investigated because of its close phylogenetic relationship with the freshwater dissimilatory Fe(III) reducer Geobacter metallireducens. Washed cell suspensions of the type strain of D. acetoxidans reduced soluble Fe(III)-citrate and Fe(III) complexed with nitriloacetic acid. The c-type cytochrome(s) of D. acetoxidans was oxidized by Fe(III)-citrate and Mn(IV)-oxalate, as well as by two electron acceptors known to support growth, colloidal sulfur and malate. D. acetoxidans grew in defined anoxic, bicarbonate-buffered medium with acetate as the sole electron donor and poorly crystalline Fe(III) or Mn(IV) as the sole electron acceptor. Magnetite (Fe₃O₄) and siderite (FeCO₃) were the major end products of Fe(III) reduction, whereas rhodochrosite (MnCO₃) was the end product of Mn(IV) reduction. Ethanol, propanol, pyruvate, and butanol also served as electron donors for Fe(III) reduction. In contrast to D. acetoxidans, G. metallireducens could only grow in freshwater medium and it did not conserve energy to support growth from colloidal S⁰ reduction. D. acetoxidans is the first marine microorganism shown to conserve energy to support growth by coupling the complete oxidation of organic compounds to the reduction of Fe(III) or Mn(IV). Thus, D. acetoxidans provides a model enzymatic mechanism for Fe(III) or Mn(IV) oxidation of organic compounds in marine and estuarine sediments. These findings demonstrate that 16S rRNA phylogenetic analyses can suggest previously unrecognized metabolic capabilities of microorganisms.     

Dissimilatory Fe(III) Oxide Reduction by Shewanella alga BrY Requires Adhesion

The Derjaguin-Landau-Verwey-Overbeek (DLVO) theory was used to examine the relationship between adhesion and dissimilatory Fe(III) oxide reduction. Adhesion of Shewanella alga BrY to hydrous ferric oxide (HFO) was correlated with ionic strength and thus was accurately described by the DLVO theory. Reduction of insoluble HFO was also correlated with KCl concentration. In contrast, there was no correlation between soluble Fe(III) reduction and ionic strength. A correlation between HFO reduction rate and adhesion to HFO was observed. These results provide direct evidence that adhesion is requisite for Fe(III) oxide reduction in the absence of soluble electron shuttles. Received: 26 October 1999 / Accepted: 22 November 1999     

Magnetic resonance studies of Mn(II)-, Mn(III)-, and Fe(III)-conalbumin complexes.

Apoconalbumin binds Mn(II) at two sites with association constants of K1 = 7 (+/- 1) X 10(4) and K2 = 0.4 (+/- 0.25) X 10(4) M-1. The binding is tighter in the presence of excess bicarbonate resulting in K1 = 1.8 (+/- 0.2) X 10(5) and K2 = 3 (+/- 2) X 10(4) M-1. The electron paramagnetic resonance spectrum (at both 9 and 35 GHz) of Mn(II) bound at the tight site reveals a rhombic distortion (lambda = E/D approximately equal to 0.25-0.31) in the protein ligand environment of the mental ion. An evaluation of the 1/pT1p, paramagnetic contribution to the longitudinal relaxation rate of solvent protons with Mn(II)-, Mn(III)-, and Fe(III)-derivatives of conalbumin revealed that the mental ion in each site of conalbumin is accessible to one water molecule. For Mn(II)-conalbumin and Mn(III)-conalbumin species, inner coordination sphere protons are rapidly exchanging with the bulk solvent, while slow exchange conditions prevail for Fe(III)-conalbumin.     

Reductive Precipitation of Gold by Dissimilatory Fe(III)-Reducing Bacteria and Archaea

Studies with a diversity of hyperthermophilic and mesophilic dissimilatory Fe(III)-reducing Bacteria and Archaea demonstrated that some of these organisms are capable of precipitating gold by reducing Au(III) to Au(0) with hydrogen as the electron donor. These studies suggest that models for the formation of gold deposits in both hydrothermal and cooler environments should consider the possibility that dissimilatory metal-reducing microorganisms can reductively precipitate gold from solution.     

Role of Hydrophobicity in Adhesion of the Dissimilatory Fe(III)-Reducing Bacterium Shewanella alga to Amorphous Fe(III) Oxide

The mechanisms by which the dissimilatory Fe(III)-reducing bacterium Shewanella alga adheres to amorphous Fe(III) oxide were examined through comparative analysis of S. alga BrY and an adhesion-deficient strain of this species, S. alga RAD20. Approximately 100% of S. alga BrY cells typically adhered to amorphous Fe(III) oxide, while less than 50% of S. alga RAD20 cells adhered. Bulk chemical analysis, isoelectric point analysis, and cell surface analysis by time-of-flight secondary-ion mass spectrometry and electron spectroscopy for chemical analysis demonstrated that the surfaces of S. alga BrY cells were predominantly protein but that the surfaces of S. alga RAD20 cells were predominantly exopolysaccharide. Physicochemical analyses and hydrophobic interaction assays demonstrated that S. alga BrY cells were more hydrophobic than S. alga RAD20 cells. This study represents the first quantitative analysis of the adhesion of a dissimilatory Fe(III)-reducing bacterium to amorphous Fe(III) oxide, and the results collectively suggest that hydrophobic interactions are a factor in controlling the adhesion of this bacterium to amorphous Fe(III) oxide. Despite having a reduced ability to adhere, S. alga RAD20 reduced Fe(III) oxide at a rate identical to that of S. alga BrY. This result contrasts with results of previous studies by demonstrating that irreversible cell adhesion is not requisite for microbial reduction of amorphous Fe(III) oxide. These results suggest that the interaction between dissimilatory Fe(III)-reducing bacteria and amorphous Fe(III) oxide is more complex than previously believed.     

Mn(III)-containing acid phosphatase: Properties of Fe(III)-substituted enzyme and function of Mn(III) and Fe(III) in plant and mammalian acid phosphatases

The function of Mn(III) in plant acid phosphatase has been investigated by a metal-substitution study, and some properties of the Fe(III)-substituted enzyme were compared with those of the native Mn(III) enzyme and mammalian Fe(III)-containing acid phosphatases. ¹⁹F nuclear magnetic resonance (NMR) and proton relaxation rate measurements showed that inhibitors such as F⁻ and nitrilotriacetic acid interact with paramagnetic Mn(III) active site. The ³¹P-NMR signal of the enzyme-phosphate complex was also broadened by the paramagnetic effect of Mn(III). In the metal-substitution experiments of the Mn(III)-acid phosphatase with Fe(III), Zn(II) and Cu(II), only the iron gave satisfactory substitution. The Fe(III)-substituted plant acid phosphatase exhibited an absorption maximum at 525 nm (ε = 3000), typical high spin ferric ESR signal at g = 4.39, and lower pH optimum (pH 4.8) than the native Mn(III)-enzyme (pH 5.8). The phosphatase activity of the Fe(III)-substituted enzyme was reduced to about 53% of that of the native enzyme. The substrate specificities of both metallophosphatases were remarkably similar, but different from that of the Fe(III)-containing uteroferrin. The present results indicate that Mn(III) and Fe(IIII) in the acid phosphatase play an important role on effective binding of phosphate and acceleration of hydrolysis of phosphomonoesters at pH 4–6.     

Shewanella putrefaciens mtrB Encodes an Outer Membrane Protein Required for Fe(III) and Mn(IV) Reduction

Iron and manganese oxides or oxyhydroxides are abundant transition metals, and in aquatic environments they serve as terminal electron acceptors for a large number of bacterial species. The molecular mechanisms of anaerobic metal reduction, however, are not understood. Shewanella putrefaciens is a facultative anaerobe that uses Fe(III) and Mn(IV) as terminal electron acceptors during anaerobic respiration. Transposon mutagenesis was used to generate mutants of S. putrefaciens, and one such mutant, SR-21, was analyzed in detail. Growth and enzyme assays indicated that the mutation in SR-21 resulted in loss of Fe(III) and Mn(IV) reduction but did not affect its ability to reduce other electron acceptors used by the wild type. This deficiency was due to Tn5 inactivation of an open reading frame (ORF) designated mtrB. mtrB encodes a protein of 679 amino acids and contains a signal sequence characteristic of secreted proteins. Analysis of membrane fractions of the mutant, SR-21, and wild-type cells indicated that MtrB is located on the outer membrane of S. putrefaciens. A 5.2-kb DNA fragment that contains mtrB was isolated and completely sequenced. A second ORF, designated mtrA, was found directly upstream of mtrB. The two ORFs appear to be arranged in an operon. mtrA encodes a putative 10-heme c-type cytochrome of 333 amino acids. The N-terminal sequence of MtrA contains a potential signal sequence for secretion across the cell membrane. The amino acid sequence of MtrA exhibited 34% identity to NrfB from Escherichia coli, which is involved in formate-dependent nitrite reduction. To our knowledge, this is the first report of genes encoding proteins involved in metal reduction.     

Fe(III) and S0 reduction by Pelobacter carbinolicus.

There is a close phylogenetic relationship between Pelobacter species and members of the genera Desulfuromonas and Geobacter, and yet there has been a perplexing lack of physiological similarities. Pelobacter species have been considered to have a fermentative metabolism. In contrast, Desulfuromonas and Geobacter species have a respiratory metabolism with Fe(III) serving as the common terminal electron acceptor in all species. However, the ability of Pelobacter species to reduce Fe(III) had not been previously evaluated. When a culture of Pelobacter carbinolicus that had grown by fermentation of 2,3-butanediol was inoculated into the same medium supplemented with Fe(III), the Fe(III) was reduced. There was less accumulation of ethanol and more production of acetate in the presence of Fe(III). P. carbinolicus grew with ethanol as the sole electron donor and Fe(III) as the sole electron acceptor. Ethanol was metabolized to acetate. Growth was also possible on Fe(III) with the oxidation of propanol to propionate or butanol to butyrate if acetate was provided as a carbon source. P. carbinolicus appears capable of conserving energy to support growth from Fe(III) respiration as it also grew with H2 or formate as the electron donor and Fe(III) as the electron acceptor. Once adapted to Fe(III) reduction, P. carbinolicus could also grow on ethanol or H2 with S0 as the electron acceptor. P. carbinolicus did not contain detectable concentrations of the c-type cytochromes that previous studies have suggested are involved in electron transport to Fe(III) in other organisms that conserve energy to support growth from Fe(III) reduction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biomineralization of Poorly Crystalline Fe(III) Oxides by Dissimilatory Metal Reducing Bacteria (DMRB)

Dissimilatory metal reducing bacteria (DMRB) catalyze the reduction of Fe(III) to Fe(II) in anoxic soils, sediments, and groundwater. Two-line ferrihydrite is a bioavailable Fe(III) oxide form that is exploited by DMRB as a terminal electron acceptor. A wide variety of biomineralization products result from the interaction of DMRB with 2-line ferrihydrite. Here we describe the state of knowledge on the biotransformation of synthetic 2-line ferrihydrite by laboratory cultures of DMRB using select published data and new experimental results. A facultative DMRB is emphasized ( Shewanella putrefaciens ) upon which most of this work has been performed. Key factors controlling the identity of the secondary mineral suite are evaluated including medium composition, electron donor and acceptor concentrations, ferrihydrite aging/recrystallization status, sorbed ions, and co-associated crystalline Fe(III) oxides. It is shown that crystalline ferric (goethite, hematite, lepidocrocite), ferrous (siderite, vivianite), and mixed valence (magnetite, green rust) iron solids are formed in anoxic, circumneutral DMRB incubations. Some products are well rationalized based on thermodynamic considerations, but others appear to result from kinetic pathways driven by ions that inhibit interfacial electron transfer or the precipitation of select phases. The primary factor controlling the nature of the secondary mineral suite appears to be the Fe(II) supply rate and magnitude, and its surface reaction with the residual oxide and other sorbed ions. The common observation of end-product mineral mixtures that are not at global equilibrium indicates that microenvironments surrounding respiring DMRB cells or the reaction-path trajectory (over Eh-pH space) may influence the identity of the final biomineralization suite.