B22D-01 INVITED
The Diverse Microbiology of Anaerobic Fe(II) Oxidation
Although anaerobic microbial oxidation of Fe(II) has been know for over a decade there is still a paucity of information available on this important metabolic process or the organisms involved. Recent studies have indicated that the metabolism is ubiquitous and a broad diversity of organisms are capable of oxidizing Fe(II) in the absence of oxygen. Our previous studies demonstrated the existence of geochemical conditions conducive to supporting the activity of nitrate-dependent Fe(II) oxidizing bacteria (NFoB) in sedimentary environments. As part of these studies we isolated and characterized several novel NFoBs. Three of these organisms, Diaphorobacter sp. strain TPSY, Ferrutens nitratireducens strain 2002 and Azospira suillum strain PS are currently undergoing whole genome shotgun sequencing in an effort to gain insight into the biochemistry and molecular biology of this geochemically important metabolism. These organisms represent diverse genera capable of anaerobically oxidizing Fe(II) using nitrate as the electron acceptor. Two of these organisms, strain 2002 and strain TPSY, are also capable of the anaerobic nitrate-dependent oxidation of U(IV) to U(VI). Diaphorobacter sp. strain TPSY was isolated from uranium and nitrate contaminated groundwater and is a member of the Comamonadaceae family in the beta subclass of the Proteobacteria, closely related to Diaphorobacter nitroreducens. It represents the first example of an anaerobic Fe(II)-oxidizer from the Comamonadaceae family and grows mixotrophically requiring an organic carbon source when growing with Fe(II) and nitrate as the electron donor and acceptor respectively. F. nitratireducens strain 2002 was isolated from aquatic sediment and is the type strain of a new genus, Ferrutens, in the beta class of the Proteobacteria. Its closest relative is Chromobacterium violaceum, a common soil bacterium. In contrast to C. violaceum, F. nitratireducens is non-fermentative and does not produce free cyanide (CN-) or the purple/violet pigments indicative of violacein production, a characteristic of Chromobacterium species. F. nitratireducens strain 2002 is currently the only described mesophilic autotrophic nitrate-dependent Fe(II)-oxidizing organism and biogenically produces green rust as a primary end-product of this metabolism. Azospira suillum strain PS is a member of the beta subclass of the Proteobacteria and was isolated based on its ability to grow anaerobically by dissimilatory perchlorate reduction from animal waste lagoons. It represents one of the dominant perchlorate-reducing genera in the natural environment. Although this organism does not grow by Fe(II) oxidation, phenotypic studies revealed its ability to couple Fe(II) oxidation to nitrate reduction and it was the first organism shown to be capable of the oxidation of the Fe(II) content of silicaceous Fe(II) minerals including almandine and staurolite. It was also the first organism shown to be capable of biogenic magnetite production through anaerobic Fe(II) oxidative processes. These varied organisms demonstrate both the phylogenetic and metabolic diversity of organisms involved in the anaerobic bio-oxidation of Fe(II) and raise many questions regarding the underlying mechanisms involved in this important metabolism in the natural environment.
B22D-02
Anaerobic Nitrate-Dependent Metal Bio-Oxidation
Direct biological oxidation of reduced metals (Fe(II) and U(IV)) coupled to nitrate reduction at circumneutral pH under anaerobic conditions has been recognized in several environments as well as pure culture. Several phylogentically diverse mesophilic bacteria have been described as capable of anaerobic, nitrate-dependent Fe(II) oxidation (NFOx). Our recent identification of a freshwater mesophilic, lithoautotroph, Ferrutens nitratireducens strain 2002, capable of growth through NFOx presents an opportunity to further study metal bio- oxidation. Continuing physiological studies revealed that in addition to Fe(II) oxidation, strain 2002 is capable of oxidizing U(IV) (4 μM) in washed cell suspensions with nitrate serving as the electron acceptor. Pasteurized cultures exhibited abiotic oxidation of 2 μM U(IV). Under growth conditions, strain 2002 catalyzed the oxidation of 12 μM U(IV) within a two week period. Cultures amended with sodium azide, an electron transport inhibitor, demonstrated limited oxidation (7 μM) similar to pasteurized cultures, supporting the direct role of electron transport in U(IV) bio-oxidation. The oxidation of U(IV) coupled denitrification at circumneutral pH would yield enough energy to support anaerobic microbial growth (ΔG°'= -460.36 kJ/mole). It is currently unknown whether or not strain 2002 can couple this metabolism to growth. The growth of F. nitratireducens strain 2002 utilizing Fe(II) as the sole electron donor was previously demonstrated. The amount of U(IV) (~12 μM) that strain 2002 oxidized under similar autotrophic growth conditions yields 0.0019 kJ, enough energy for the generation of ATP (5.3 x 10-20 kJ ATP-1), but not enough energy for cell replication as calculated for nitrate-dependent Fe(II) oxidizing conditions (0.096 kJ) assuming a similar metabolism. In addition to F. nitratireducens strain 2002, a nitrate-dependent Fe(II) oxidizing bacterium isolated from U contaminated groundwater, Diaphorobacter sp. strain TPSY, was also capable of nitrate- dependent U(IV) oxidation (8 μM over 24 hours, pseudo first order rate constant of 0.12 ± 0.02 hr-1) in washed cell suspensions. Further biochemical investigation of nitrate-dependent U(IV) oxidation in strain TPSY revealed the expression of several putative high molecular weight proteins specific to this metabolism. Together with the previously described metabolic ability of Geobacter metallireducens (Finneran et al. 2002) and Thiobacillus denitrificans (Beller 2005), these data indicate that anaerobic, metal oxidation may be a ubiquitous microbial metabolism.
B22D-03 INVITED
Constraining the role of anoxygenic phototrophic Fe(II)-oxidizing bacteria in deposition of BIFs
Banded Iron Formations (BIFs) are Precambrian sedimentary deposits of alternating iron oxide and silica mineral layers. Their presence in the rock record ca.3.8-2.2 Ga makes them particularly intriguing formations for the debate over when oxygen became dominant on Earth. The mechanism(s) of BIF deposition is still unclear; suggestions including both abiotic and biotic processes. We are interested in constraining one of these proposed mechanisms; the direct biological oxidation of Fe(II) via anoxygenic Fe(II)-oxidizing autophototrophs. In order to find the limitations of photoferrotrophic BIF deposition, we take a holistic approach, investigating the oxidation of Fe(II) by modern Fe(II)-oxidizing phototrophs, the precipitation of Fe(III) (hydr)oxides, and the fate of the cell-mineral aggregates in the water column and at the basin floor. Specifically, physiology experiments with Fe(II)-oxidizing phototrophs under various conditions of light intensity, pH, Fe(II) concentration and temperature allow us to determine the environmental limits of such organisms. We carry out precipitation experiments to characterize the sedimentation rates, aggregate size and composition in order to resolve the effect of reactions in the water column. Finally, we simulate the diagenetic fate of these aggregates on the basin floor by placing them in gold capsules under T and P conditions relevant for the Transvaal Supergroup BIFs of South Africa. Recently, we have developed a tank simulating the Archean ocean in which the strains grow in continuous culture and collect the aggregates formed under various geochemical conditions. We aim to model the extent of and limitations to photoferrotrophs in BIF deposition. This information will help constrain whether biotic processes were dominant in the Archean ocean and will offer insight to the evolution of the early biogeosphere.
B22D-04
Physiology and Mechanism of Phototrophic Fe(II) Oxidation by Rhodopseudomonas palustris TIE-1
Phototrophic Fe(II)-oxidizing bacteria use electrons from ferrous iron [Fe(II)] and energy from light to drive reductive CO2 fixation. This metabolism is thought to be ancient in origin, and plays an important role in environmental iron cycling. It has been implicated in the deposition of Banded Iron Formations, a class of ancient sedimentary iron deposits. Consistent with this hypothesis, we discovered that hydrogen gas, a thermodynamically favorable electron donor to Fe(II), in an Archean atmosphere would not have inhibited phototrophic Fe(II) oxidation. To understand this physiology and the connection to BIF formation at the molecular level, the mechanisms of phototrophic Fe(II) oxidation were examined in a model organism Rhodopseudomonas palustris TIE-1. Increased expression of a putative decaheme c-type cytochrome, encoded by pioA, was observed when cells were grown under Fe(II)-oxidizing conditions. Two genes located immediately downstream of pioA in the same operon, pioB and pioC, encode a putative outer membrane beta-barrel protein and a putative high potential iron-sulfur protein, respectively. Deletion studies demonstrated that all three genes are involved in phototrophic Fe(II) oxidation. This study provides our first insight into the molecular mechanisms of this metabolism, which will be further characterized by in vitro biochemical studies.
B22D-05
Biochemistry and Ecology of Novel Cytochromes Catalyzing Fe(II) Oxidation by an Acidophilic Microbial Community
An acidophilic microbial community found in the Richmond Mine at Iron Mountain, CA forms abundant biofilms in extremely acidic (pH<1) and toxic metal conditions. In this ecosystem, biological Fe(II) oxidation is critical to the metabolic functioning of the community, and in turn this process generates acid mine drainage, causing an environmental catastrophe. Two conspicuous novel proteins isolated from these biofilms were identified as gene products of Leptospirillum group II and were characterized as cytochromes with unique properties. Sulfuric acid extraction of biofilm samples liberated one of these proteins, a 16 kDa cytochrome with an unusual alpha-band absorption at 579 (Cyt579). Genomic sequencing of multiple biofilms indicated that several variants of Cyt579 were present in Leptospirillum strains. Intact protein MS analysis identified the dominant variants in each biofilm and documented multiple N-terminal cleavage sites for Cyt579. By combining biochemical, geochemical and microbiological data, we established that the sequence variation and N-terminal processing of Cyt579 are selected by ecological conditions. In addition to the soluble Cyt579, the second cytochrome appears as a much larger protein complex of ~210 kDa predominant in the biofilm membrane fraction, and has an alpha-band absorption at 572 nm. The 60 kDa cytochrome subunit, Cyt572, resides in the outer membrane of LeptoII, and readily oxidizes Fe(II) at low pH (0.95 - 3.0). Several genes encoding Cyt572 were localized within a recombination hotspot between two strains of LeptoII, causing a large range of variation in the sequences. Genomic sequencing and MS proteomic studies established that the variants were also selected by ecological conditions. A general mechanistic model for Fe(II) oxidation has been developed from these studies. Initial Fe(II) oxidation by Cyt572 occurs at the outer membrane. Cyt572 then transfers electrons to Cyt579, perhaps representing an initial step in energy flow to the biofilm community. Amino acid variations and post-translational modifications of these unique cytochromes may represent fine-tuning of function in response to local environmental conditions.
B22D-06
Expression of Genes Involved in Iron and Sulfur Respiration in a Novel Thermophilic Crenarchaeon Isolated from Acid-Sulfate-Chloride Geothermal Systems
Acidic geothermal springs within Yellowstone National Park (YNP) provide an excellent opportunity to study microbial populations and their relationship with geochemical processes such as redox cycling and biomineralization of iron. Fourteen acid-sulfate-chloride (ASC) and acid-sulfate (AS) geothermal springs located in (YNP) have been extensively characterized for aqueous chemistry, solid phase mineral deposition and microbial diversity and distribution. The oxidation of Fe(II) with oxygen as an electron acceptor is exergonic under these conditions, consequently, Fe(II) may be an important electron donor driving primary production in ASC and AS habitats, and products of biomineralization (e.g. Fe[III]-oxides of varying crystallinity and structure, as well as jarosite in some cases) are common in the outflow channels of these environments. Recently, we isolated a novel Metallosphaera-like microorganism (Metallosphaera strain MK1) from an ASC spring in Norris Geyser Basin, YNP. Clone libraries (16S rRNA gene) from multiple sites suggest that microorganisms closely related to strain MK1 (between 98-100 percent similarity) dominate many spring locations between 55-80 C. The in situ abiotic oxidation rate of Fe(II) has been shown to be very slow in these systems and Metallosphaera strain MK1 has been directly implicated in biotic Fe(II) oxidation. Metallosphaera strain MK1 has been submitted for full genome sequencing and is yielding gene sequences related to the terminal oxidases SOXABC and SOXM super-complex. In addition, sequences from a recently characterized terminal oxidase FOX complex involved in Fe(II) and pyrite oxidation from Sulfolobus metallicus have been found in Metallosphaera strain MK1. A protein complex analogous to Metallosphaera sedula has been identified in strain MK1 and this complex has also been expressed in cells grown on pyrite and Fe(II). Other sequences identified in Metallosphaera strain MK1 that are involved in respiration are the TQO complex (thiosulfate:quinone oxidoreductase) related to the Acidianus ambivalens DOXAD complex and a sulfur reductase (SRE) complex related to one found in Sulfolobus solfataricus and Acidianus ambivalens. Here we report on the RNA expression of seven gene sequences from each of the above mentioned complexes for Metallosphaera strain MK1 grown aerobically on pyrite, sulfur, Fe(II)-ferrihydrite, and anaerobically with yeast extract and sulfur. In addition, expression studies are also compared to in situ samples collected from the geothermal Fe-mats.
B22D-07 INVITED
Biogeochemistry of Iron Oxidation in a Circumneutral Freshwater Habitat
Iron(II) oxidation in natural waters at circumneutral pH, often regarded as an abiotic process, may be biologically- mediated when it occurs in iron-rich redox gradients. West Berry Creek, a small circumneutral tributary flowing through a mixed coniferous forest in Big Basin State Park, California, contains localized iron (hydr)oxide precipitates at points along its course where anoxic groundwater meets oxygenated creek water. These mixing zones establish redox gradients and iron-rich microbial mats that may create microenvironments that promote active biogeochemical cycling of Fe. Water sampling revealed strong correlations between the concentrations of aqueous inorganic species, suggesting a rock-weathering source for most of these solutes. Liquid chromatography-mass spectrometry detected significant concentrations of organic exudates, including low molecular mass organic acids and siderophores, indicating active biogeochemical cycling of iron is occurring in the creek. X-ray diffraction and elemental analysis showed the iron precipitates to be amorphous minerals, such as ferrihydrite. Microbial biofilm communities are associated with the iron (hydr)oxide deposits. Clone libraries developed from 16s rDNA sequences revealed the presence of microorganisms related to the neutrophilic iron- oxidizing bacteria Gallionella and Siderooxidans; in addition, micrographs suggest the widespread presence of sheath-forming bacteria (e.g., Leptothrix). Sequences from these libraries also indicated the presence of significant populations of organisms related to bacteria in the genera Pseudomonas, Sphingomonas, and Nitrospira. These geosymbiotic systems appear to be significant not only for the biogeochemical cycling of iron in the creek, but also for the cycling of organic species, inorganic nutrients, and trace metals.
B22D-08
Energy Filtering Transmission Electron Tomography (EFTET) of Bacteria-Mineral Associations within the Deep sea Hydrothermal Vent Shrimp Rimicaris exoculata.
The chemical and temperature conditions around deep sea hydrothermal vents are both dynamic and extreme, yet the shrimp Rimicaris exoculata flourishes around these environments on the Mid--Atlantic Ridge (MAR). Epibiotic bacteria and minerals found within the branchial chamber (BC) of the shrimp are of great interest in the search for a chemical model for the Rainbow MAR hydrothermal vent site. Here we examine the close, three-- dimensional (3D) relationship between bacteria (on the inner surface of the BC wall) and the minerals that surround them. The morphology and chemistry of the minerals were analysed by Energy filtering Transmission Electron Microscopy (EFTEM, on a LEO--912 microscope) and X-ray Nano-analysis (EDXN, on a JEOL--2010 FEG microscope) respectively, and the 3D organization was determined by Transmission Electron Tomography (TET) and EFTET. Consecutive thin and semi--thin sections of 50--80nm (for EFTEM and EDXN) and 200-–250nm (for TEM and EFTET) were cut through the BC cuticle and mounted on standard microscope grids. Sections were observed initially for morphology, to find broad relationships between bacteria and minerals. EFTET series acquisition was performed under cryo-conditions (-175°C) using a LEO-912 microscope. At each position of interest four tilt series were taken at two degree increments between -55° and +55° at various energy--losses: 1) zero--loss (ref); 2) 720 eV, 3) 690 eV and 4) 670 eV, to reconstruct the 3D location of iron. Tilted series were obtained using the ESIvision program (Soft--Imaging Software, Münster, Germany) with additional in--house scripts for automated acquisition. The 3D EFTET reconstruction volume was produced from the four tilted series using recently developed EFTET--J software (http://www.snv.jussieu.fr/~wboudier/softs.html). In many cases the observed minerals exhibit a sharp boundary against the bacteria, often with a substantial void between bacterial membrane/cell wall and mineral boundary. Mineral layering and zoning are also present. Our findings highlight the potential importance of iron as an energy source for Rimicaris exoculata epibionts at Rainbow, from their close association. The results from this study are contributing to the formulation of a chemical model for the Rainbow hydrothermal vent site (MAR).