Microbe-Mineral Interaction and Novel Proteins for Iron Oxide Mineral Reduction in the Hyperthermophilic Crenarchaeon Pyrodictium delaneyi.

Dissimilatory iron reduction by hyperthermophilic archaea occurs in many geothermal environments and generally relies on microbe-mineral interactions that transform various iron oxide minerals. In this study, the physiology of dissimilatory iron and nitrate reduction was examined in the hyperthermophilic crenarchaeon type strain Pyrodictium delaneyi Su06. Iron barrier experiments showed that P. delaneyi required direct contact with the Fe(III) oxide mineral ferrihydrite for reduction. The separate addition of an exogenous electron shuttle (anthraquinone-2,6-disulfonate), a metal chelator (nitrilotriacetic acid), and 75% spent cell-free supernatant did not stimulate growth with or without the barrier. Protein electrophoresis showed that the c-type cytochrome and general protein compositions of P. delaneyi changed when grown on ferrihydrite relative to nitrate. Differential proteomic analyses using tandem mass tagged protein fragments and mass spectrometry detected 660 proteins and differential production of 127 proteins. Among these, two putative membrane-bound molybdopterin-dependent oxidoreductase complexes increased in relative abundance 60- to 3,000-fold and 50- to 100-fold in cells grown on iron oxide. A putative 8-heme c-type cytochrome was 60-fold more abundant in iron-grown cells and was unique to the Pyrodictiaceae There was also a >14,700-fold increase in a membrane transport protein in iron-grown cells. For flagellin proteins and a putative nitrate reductase, there were no changes in abundance, but a membrane nitric oxide reductase was more abundant on nitrate. These data help to elucidate the mechanisms by which hyperthermophilic crenarchaea generate energy and transfer electrons across the membrane to iron oxide minerals.IMPORTANCE Understanding iron reduction in the hyperthermophilic crenarchaeon Pyrodictium delaneyi provides insight into the diversity of mechanisms used for this process and its potential impact in geothermal environments. The ability of P. delaneyi to reduce Fe(III) oxide minerals through direct contact potentially using a novel cytochrome respiratory complex and a membrane-bound molybdopterin respiratory complex sets iron reduction in this organism apart from previously described iron reduction processes. A model is presented where obligatory H2 oxidation on the membrane coupled with electron transport and either Fe(III) oxide or nitrate reduction leads to the generation of a proton motive force and energy generation by oxidative phosphorylation. However, P. delaneyi cannot fix CO2 and relies on organic compounds from its environment for biosynthesis.

The complete genome sequence of Staphylothermus marinus reveals differences in sulfur metabolism among heterotrophic Crenarchaeota.

BACKGROUND: Staphylothermus marinus is an anaerobic, sulfur-reducing peptide fermenter of the archaeal phylum Crenarchaeota. It is the third heterotrophic, obligate sulfur reducing crenarchaeote to be sequenced and provides an opportunity for comparative analysis of the three genomes. RESULTS: The 1.57 Mbp genome of the hyperthermophilic crenarchaeote Staphylothermus marinus has been completely sequenced. The main energy generating pathways likely involve 2-oxoacid:ferredoxin oxidoreductases and ADP-forming acetyl-CoA synthases. S. marinus possesses several enzymes not present in other crenarchaeotes including a sodium ion-translocating decarboxylase likely to be involved in amino acid degradation. S. marinus lacks sulfur-reducing enzymes present in the other two sulfur-reducing crenarchaeotes that have been sequenced -- Thermofilum pendens and Hyperthermus butylicus. Instead it has three operons similar to the mbh and mbx operons of Pyrococcus furiosus, which may play a role in sulfur reduction and/or hydrogen production. The two marine organisms, S. marinus and H. butylicus, possess more sodium-dependent transporters than T. pendens and use symporters for potassium uptake while T. pendens uses an ATP-dependent potassium transporter. T. pendens has adapted to a nutrient-rich environment while H. butylicus is adapted to a nutrient-poor environment, and S. marinus lies between these two extremes. CONCLUSION: The three heterotrophic sulfur-reducing crenarchaeotes have adapted to their habitats, terrestrial vs. marine, via their transporter content, and they have also adapted to environments with differing levels of nutrients. Despite the fact that they all use sulfur as an electron acceptor, they are likely to have different pathways for sulfur reduction.

Magnetite formation from ferrihydrite by hyperthermophilic archaea from Endeavour Segment, Juan de Fuca Ridge hydrothermal vent chimneys.

Hyperthermophilic iron reducers are common in hydrothermal chimneys found along the Endeavour Segment in the northeastern Pacific Ocean based on culture-dependent estimates. However, information on the availability of Fe(III) (oxyhydr) oxides within these chimneys, the types of Fe(III) (oxyhydr) oxides utilized by the organisms, rates and environmental constraints of hyperthermophilic iron reduction, and mineral end products is needed to determine their biogeochemical significance and are addressed in this study. Thin-section petrography on the interior of a hydrothermal chimney from the Dante edifice at Endeavour showed a thin coat of Fe(III) (oxyhydr) oxide associated with amorphous silica on the exposed outer surfaces of pyrrhotite, sphalerite, and chalcopyrite in pore spaces, along with anhydrite precipitation in the pores that is indicative of seawater ingress. The iron sulfide minerals were likely oxidized to Fe(III) (oxyhydr) oxide with increasing pH and Eh due to cooling and seawater exposure, providing reactants for bioreduction. Culture-dependent estimates of hyperthermophilic iron reducer abundances in this sample were 1740 and 10 cells per gram (dry weight) of material from the outer surface and the marcasite-sphalerite-rich interior, respectively. Two hyperthermophilic iron reducers, Hyperthermus sp. Ro04 and Pyrodictium sp. Su06, were isolated from other active hydrothermal chimneys on the Endeavour Segment. Strain Ro04 is a neutrophilic (pH opt 7-8) heterotroph, while strain Su06 is a mildly acidophilic (pH opt 5), hydrogenotrophic autotroph, both with optimal growth temperatures of 90-92 °C. Mössbauer spectroscopy of the iron oxides before and after growth demonstrated that both organisms form nanophase (<12 nm) magnetite [Fe3 O4 ] from laboratory-synthesized ferrihydrite [Fe10 O14 (OH)2 ] with no detectable mineral intermediates. They produced up to 40 mm Fe(2+) in a growth-dependent manner, while all abiotic and biotic controls produced <3 mm Fe(2+) . Hyperthermophilic iron reducers may have a growth advantage over other hyperthermophiles in hydrothermal systems that are mildly acidic where mineral weathering at increased temperatures occurs.

Di-myo-inositol phosphate and novel UDP-sugars accumulate in the extreme hyperthermophile Pyrolobus fumarii.

The archaeon Pyrolobus fumarii, one of the most extreme members of hyperthermophiles known thus far, is able to grow at temperatures up to 113 degrees C. Over a decade after the description of this organism our knowledge about the structures and strategies underlying its remarkable thermal resistance remains incipient. The accumulation of a restricted number of charged organic solutes is a common response to heat stress in hyperthermophilic organisms and accordingly their role in thermoprotection has been often postulated. In this work, the organic solute pool of P. fumarii was characterized using 1H, 13C, and 31P NMR. Di-myo-inositol phosphate was the major solute (0.21 micromol/mg protein), reinforcing the correlation between the occurrence of this solute and hyperthermophily; in addition, UDP-sugars (total concentration 0.11 micromol/mg protein) were present. The structures of the two major UDP-sugars were identified as UDP-alpha-GlcNAc3NAc and UDP-alpha-GlcNAc3NAc-(4<--1)-beta-GlcpNAc3NAc. Interestingly, the latter compound appears to be derived from the first one by addition of a 2,3-N-acetylglucoronic acid unit, suggesting that these UDP-sugars are intermediates of an N-linked glycosylation pathway. To our knowledge the UDP-disaccharide has not been reported elsewhere. The physiological roles of these organic solutes are discussed.

The genome of Hyperthermus butylicus: a sulfur-reducing, peptide fermenting, neutrophilic Crenarchaeote growing up to 108 degrees C.

Hyperthermus butylicus, a hyperthermophilic neutrophile and anaerobe, is a member of the archaeal kingdom Crenarchaeota. Its genome consists of a single circular chromosome of 1,667,163 bp with a 53.7% G+C content. A total of 1672 genes were annotated, of which 1602 are protein-coding, and up to a third are specific to H. butylicus. In contrast to some other crenarchaeal genomes, a high level of GUG and UUG start codons are predicted. Two cdc6 genes are present, but neither could be linked unambiguously to an origin of replication. Many of the predicted metabolic gene products are associated with the fermentation of peptide mixtures including several peptidases with diverse specificities, and there are many encoded transporters. Most of the sulfur-reducing enzymes, hydrogenases and electron-transfer proteins were identified which are associated with energy production by reducing sulfur to H(2)S. Two large clusters of regularly interspaced repeats (CRISPRs) are present, one of which is associated with a crenarchaeal-type cas gene superoperon; none of the spacer sequences yielded good sequence matches with known archaeal chromosomal elements. The genome carries no detectable transposable or integrated elements, no inteins, and introns are exclusive to tRNA genes. This suggests that the genome structure is quite stable, possibly reflecting a constant, and relatively uncompetitive, natural environment.