Comparative proteomics of a versatile, marine, iron-oxidizing chemolithoautotroph.

This study conducted a comparative proteomic analysis to identify potential genetic markers for the biological function of chemolithoautotrophic iron oxidation in the marine bacterium Ghiorsea bivora. To date, this is the only characterized species in the class Zetaproteobacteria that is not an obligate iron-oxidizer, providing a unique opportunity to investigate differential protein expression to identify key genes involved in iron-oxidation at circumneutral pH. Over 1000 proteins were identified under both iron- and hydrogen-oxidizing conditions, with differentially expressed proteins found in both treatments. Notably, a gene cluster upregulated during iron oxidation was identified. This cluster contains genes encoding for cytochromes that share sequence similarity with the known iron-oxidase, Cyc2. Interestingly, these cytochromes, conserved in both Bacteria and Archaea, do not exhibit the typical ß-barrel structure of Cyc2. This cluster potentially encodes a biological nanowire-like transmembrane complex containing multiple redox proteins spanning the inner membrane, periplasm, outer membrane, and extracellular space. The upregulation of key genes associated with this complex during iron-oxidizing conditions was confirmed by quantitative reverse transcription-PCR. These findings were further supported by electromicrobiological methods, which demonstrated negative current production by G. bivora in a three-electrode system poised at a cathodic potential. This research provides significant insights into the biological function of chemolithoautotrophic iron oxidation.

Widespread energy limitation to life in global subseafloor sediments.

Microbial cells buried in subseafloor sediments comprise a substantial portion of Earth's biosphere and control global biogeochemical cycles; however, the rate at which they use energy (i.e., power) is virtually unknown. Here, we quantify organic matter degradation and calculate the power utilization of microbial cells throughout Earth's Quaternary-age subseafloor sediments. Aerobic respiration, sulfate reduction, and methanogenesis mediate 6.9, 64.5, and 28.6% of global subseafloor organic matter degradation, respectively. The total power utilization of the subseafloor sediment biosphere is 37.3 gigawatts, less than 0.1% of the power produced in the marine photic zone. Aerobic heterotrophs use the largest share of global power (54.5%) with a median power utilization of 2.23 × 10-18 watts per cell, while sulfate reducers and methanogens use 1.08 × 10-19 and 1.50 × 10-20 watts per cell, respectively. Most subseafloor cells subsist at energy fluxes lower than have previously been shown to support life, calling into question the power limit to life.

A Genus Definition for Bacteria and Archaea Based on a Standard Genome Relatedness Index.

Genus assignment is fundamental in the characterization of microbes, yet there is currently no unambiguous way to demarcate genera solely using standard genomic relatedness indices. Here, we propose an approach to demarcate genera that relies on the combined use of the average nucleotide identity, genome alignment fraction, and the distinction between type- and non-type species. More than 3,500 genomes representing type strains of species from >850 genera of either bacterial or archaeal lineages were tested. Over 140 genera were analyzed in detail within the taxonomic context of order/family. Significant genomic differences between members of a genus and type species of other genera in the same order/family were conserved in 94% of the cases. Nearly 90% (92% if polyphyletic genera are excluded) of the type strains were classified in agreement with current taxonomy. The 448 type strains that need reclassification directly impact 33% of the genera analyzed in detail. The results provide a first line of evidence that the combination of genomic indices provides added resolution to effectively demarcate genera within the taxonomic framework that is currently based on the 16S rRNA gene. We also identify the emergence of natural breakpoints at the genome level that can further help in the circumscription of taxa, increasing the proportion of directly impacted genera to at least 43% and pointing at inaccuracies on the use of the 16S rRNA gene as a taxonomic marker, despite its precision. Altogether, these results suggest that genomic coherence is an emergent property of genera in Bacteria and ArchaeaIMPORTANCE In recent decades, the taxonomy of Bacteria and Archaea, and therefore genus designation, has been largely based on the use of a single ribosomal gene, the 16S rRNA gene, as a taxonomic marker. We propose an approach to delineate genera that excludes the direct use of the 16S rRNA gene and focuses on a standard genome relatedness index, the average nucleotide identity. Our findings are of importance to the microbiology community because the emergent properties of Bacteria and Archaea that are identified in this study will help assign genera with higher taxonomic resolution.

Effects of trace element concentrations on culturing thermophiles.

The majority of microorganisms in natural environments resist laboratory cultivation. Sometimes referred to as 'unculturable', many phylogenetic groups are known only by fragments of recovered DNA. As a result, the ecological significance of whole branches of the 'tree of life' remains a mystery; this is particularly true when regarding genetic material retrieved from extreme environments. Geochemically relevant media have been used to improve the success of culturing Archaea and Bacteria, but these efforts have focused primarily on optimizing pH, alkalinity, major ions, carbon sources, and electron acceptor-donor pairs. Here, we cultured thermophilic microorganisms from 'Sylvan Spring' (Yellowstone National Park, USA) on media employing different trace element solutions, including one that mimicked the source fluid of the inocula. The growth medium that best simulated trace elements found in 'Sylvan Spring' produced a more diverse and faster growing mixed culture than media containing highly elevated trace element concentrations. The elevated trace element medium produced fewer phylotypes and inhibited growth. Trace element concentrations appear to influence growth conditions in extreme environments. Incorporating geochemical data into cultivation attempts may improve culturing success.

Energy sources for chemolithotrophs in an arsenic- and iron-rich shallow-sea hydrothermal system.

The hydrothermally influenced sediments of Tutum Bay, Ambitle Island, Papua New Guinea, are ideal for investigating the chemolithotrophic activities of micro-organisms involved in arsenic cycling because hydrothermal vents there expel fluids with arsenite (As(III)) concentrations as high as 950 µg L(-1) . These hot (99 °C), slightly acidic (pH ~6), chemically reduced, shallow-sea vent fluids mix with colder, oxidized seawater to create steep gradients in temperature, pH, and concentrations of As, N, Fe, and S redox species. Near the vents, iron oxyhydroxides precipitate with up to 6.2 wt% arsenate (As(V)). Here, chemical analyses of sediment porewaters from 10 sites along a 300-m transect were combined with standard Gibbs energies to evaluate the energy yields (-ΔG(r)) from 19 potential chemolithotrophic metabolisms, including As(V) reduction, As(III) oxidation, Fe(III) reduction, and Fe(II) oxidation reactions. The 19 reactions yielded 2-94 kJ mol(-1) e(-) , with aerobic oxidation of sulphide and arsenite the two most exergonic reactions. Although anaerobic As(V) reduction and Fe(III) reduction were among the least exergonic reactions investigated, they are still potential net metabolisms. Gibbs energies of the arsenic redox reactions generally correlate linearly with pH, increasing with increasing pH for As(III) oxidation and decreasing with increasing pH for As(V) reduction. The calculated exergonic energy yields suggest that micro-organisms could exploit diverse energy sources in Tutum Bay, and examples of micro-organisms known to use these chemolithotrophic metabolic strategies are discussed. Energy modeling of redox reactions can help target sampling sites for future microbial collection and cultivation studies.

Energetics of overall metabolic reactions of thermophilic and hyperthermophilic Archaea and bacteria.

Thermophilic and hyperthermophilic Archaea and Bacteria have been isolated from marine hydrothermal systems, heated sediments, continental solfataras, hot springs, water heaters, and industrial waste. They catalyze a tremendous array of widely varying metabolic processes. As determined in the laboratory, electron donors in thermophilic and hyperthermophilic microbial redox reactions include H2, Fe(2+), H2S, S, S2O3(2-), S4O6(2-), sulfide minerals, CH4, various mono-, di-, and hydroxy-carboxylic acids, alcohols, amino acids, and complex organic substrates; electron acceptors include O2, Fe(3+), CO2, CO, NO3(-), NO2(-), NO, N2O, SO4(2-), SO3(2-), S2O3(2-), and S. Although many assimilatory and dissimilatory metabolic reactions have been identified for these groups of microorganisms, little attention has been paid to the energetics of these reactions. In this review, standard molal Gibbs free energies (DeltaGr(0)) as a function of temperature to 200 degrees C are tabulated for 370 organic and inorganic redox, disproportionation, dissociation, hydrolysis, and solubility reactions directly or indirectly involved in microbial metabolism. To calculate values of DeltaGr(0) for these and countless other reactions, the apparent standard molal Gibbs free energies of formation (DeltaG(0)) at temperatures to 200 degrees C are given for 307 solids, liquids, gases, and aqueous solutes. It is shown that values of DeltaGr(0) for many microbially mediated reactions are highly temperature dependent, and that adopting values determined at 25 degrees C for systems at elevated temperatures introduces significant and unnecessary errors. The metabolic processes considered here involve compounds that belong to the following chemical systems: H-O, H-O-N, H-O-S, H-O-N-S, H-O-C(inorganic), H-O-C, H-O-N-C, H-O-S-C, H-O-N-S-C(amino acids), H-O-S-C-metals/minerals, and H-O-P. For four metabolic reactions of particular interest in thermophily and hyperthermophily (knallgas reaction, anaerobic sulfur and nitrate reduction, and autotrophic methanogenesis), values of the overall Gibbs free energy (DeltaGr) as a function of temperature are calculated for a wide range of chemical compositions likely to be present in near-surface and deep hydrothermal and geothermal systems.

Calculation of the standard molal thermodynamic properties of aqueous biomolecules at elevated temperatures and pressures II. Unfolded proteins.

Equations of state for completely unfolded proteins have been generated from group additivity algorithms and the revised Helgeson-Kirkham-Flowers (HKF) equations of state to compute the standard molal thermodynamic properties of these molecules at elevated temperatures and pressures. The requisite equations of state parameters were computed from those of groups retrieved by regression of experimental calorimetric and densimetric data reported in the literature. This approach permits calculation of the standard molal thermodynamic properties as a function of temperature and pressure for any completely unfolded protein for which the amino acid sequence is known. Calculations of this kind have been carried out for 11 thermophilic proteins. The thermodynamic properties reported below can be combined with those for protein unfolding to compute the corresponding properties of completely folded (i.e. native) proteins.

Energetics of amino acid synthesis in hydrothermal ecosystems.

Thermodynamic calculations showed that the autotrophic synthesis of all 20 protein-forming amino acids was energetically favored in hot (100 degrees C), moderately reduced, submarine hydrothermal solutions relative to the synthesis in cold (18 degrees C), oxidized, surface seawater. The net synthesis reactions of 11 amino acids were exergonic in the hydrothermal solution, but all were endergonic in surface seawater. The synthesis of the requisite amino acids of nine thermophilic and hyperthermophilic proteins in a 100 degreesC hydrothermal solution yielded between 600 and 8000 kilojoules per mole of protein, which is energy that is available to drive the intracellular synthesis of enzymes and other biopolymers in hyperthermophiles thriving in these ecosystems.

Systemic responses to SFHS-infusion in hemorrhaged dogs.

Anesthetized mongrel (weight range: 16-27 Kg) dogs were prepared for monitoring hemodynamics, blood flow distribution, plasma colloid osmotic pressure and renal functional parameters at various intervals. Removal of 35 ml/Kg blood resulted in marked drop and only partial spontaneous recovery in systemic and pulmonary arterial pressures, cardiac output and organ blood flows (> 50% flow-decrements occurred in kidney, spleen, heart, gut and pancreas); plasma colloid osmotic pressure as well as urine output and creatinine clearance also fell. Group I (n = 6) of dogs was transfused after 45 minutes of hypovolemia with their own anticoagulated blood, while Group II (n = 6) received an equal volume of unmodified 6% stromafree hemoglobin solution (SFHS). Comparison of the two groups' responses to resuscitation yielded some differences. There was a significant overshoot (30 mmHg) in systemic arterial blood pressure accompanied by bradycardia in Group II only. Cardiac output recovered in both groups but was less well sustained in Group II. Cerebral blood flow rose higher and hepatic arterial flow-increment was less in Group II than in Group I; the responses to resuscitation in the other organs were comparable. Colloid osmotic pressure decreased in Group I whereas it rose immediately after resuscitation in Group II, declining thereafter with a converging trend and 30 minutes thereafter the differences were not significant between the groups. Urine excretion and creatinine clearance recovered to comparable extents in both groups, but N-acetyl-beta-D-glucosaminidase (N.A.G.) excretion rose over 10-fold higher in Group II than in Group I. These experiments have defined the response of bled animals to resuscitation with unmodified, unpurified SFHS, when compared to resuscitation with whole blood, showing a less well sustained but adequate hemodynamic and renal functional recovery while revealing indications of early renal tubular cellular injury, providing baseline comparison for testing highly purified and modified hemoglobin solutions.

Use of the internal mammary artery as a graft in emergency coronary artery bypass grafting after failed PTCA.

The use of the internal mammary artery (IMA) is recommended in elective aorto-coronary bypass grafting (CABG) because of favourable long-term patency results. In emergency CABG many surgeons prefer revascularization only with venous grafts due to the shorter operation time and higher initial flow rates of this type of graft. We investigated whether use of the IMA influences operative and mid-term results of emergency CABG after failed percutaneous transluminal coronary angioplasty (PTCA). From January 1990, to March, 1993, 56 emergency CABGs were performed in patients from 7 different cardiological centres where PTCA had failed. In 23 patients (Group A), the IMA was used as a bypass graft. In most of these patients the left IMA was anastomosed with the left anterior descending artery (n = 19). In one case both IMAs were used as bypass grafts. Venous grafts only were applied in 33 patients (Group B). Due to preparation of the IMA, aortic cross-clamp and bypass times were approximately 15 mins longer in Group A patients, although there was no significant difference in the number of grafts (1.7 +/- 0.8 in Group A vs. 1.5 +/- 0.7 in Group B). All patients of Groups A and B underwent echocardiographical investigations 14.6 +/- 8.2 months postoperatively. The overall mortality in Group A was 13% (n = 3) compared to 9% (n = 3) in Group B (p = 0.58). Significant predictors for death were age 65 years and over, diabetes mellitus, dilatation of the RCX and stenoses unfavourable for PTCA.(ABSTRACT TRUNCATED AT 250 WORDS)

Coronary artery bypass grafting after failed coronary angioplasty: risk factors and long-term results.

In this case-control study we investigated the preoperative risk factors and long-term results in patients undergoing emergency coronary artery bypass grafting (CABG) after failed percutaneous transluminal coronary angioplasty (PTCA). From January 1990 to March 1993, 56 emergency CABGs (Group A) were performed in patients from 7 different cardiology centres--six of these located outside our hospital within a radius of 19 km-1 h to 114 h (phi 7.3 h) after failed PTCA. No special stand-by was given and patients were operated in the next available theatre. Criteria of anatomically unfavourable stenoses for PTCA were defined as follows: Long stenoses, occlusions of other major vessels, tandem stenoses, stenoses at or near a bend or branch and, finally, eccentric stenoses. Thirty-two percent met one criterion, 35.8% two, 6.9% three and 1.8% four criteria. All patients were examined echocardiographically 14.6 +/- 8.2 months postoperatively. Patients who had been operated on electively within the same period served as a control (Group B; n = 56; case-control study). The patients were matched for age, sex, EF, LVEDP, body surface area, reoperation status, and concomitant diseases. The mortality rate for Group A patients was 10.7% (n = 6); for Group B, 1.8% (n = 1). Five of the Group A patients who died came from our own hospital and only one from a hospital 1 km away (mortality: 14.3% vs. 4.8%). There were no deaths among patients with an anatomically favourable stenosis for a PTCA; in patients with more than one criterion for an unfavourable stenosis mortality increased to 19.0% (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship between domoic acid levels in the blue mussel (Mytilus edulis) and toxicity in mice.

Monitoring of eastern blue mussels (Mytilus edulis), contaminated with domoic acid, involved mouse bioassays and quantitative analysis using HPLC. Mice undergo a typical scratching syndrome at sublethal as well as lethal doses of domoic acid. The onset of scratching behaviour and time of death in mice were inversely related to the dosage of domoic acid. An LD50 (i.p.) of 3.6 mg domoic acid/kg mouse was calculated. Toxic mussels held in tanks and flushed with uncontaminated sea water showed a decline in domoic acid concentration in mussel tissue with time. In addition, domoic acid concentrations in mussels from two infected rivers declined to negligible levels in 40-50 days under normal environmental conditions. The bulk of domoic acid and toxicity was located in the hepatopancreas which also contained large amounts of chlorophyll-A, an algae biomass indicator, relative to control mussels. These results support the conclusion that domoic acid was the primary causative factor in the shellfish poisonings from Prince Edward Island mussels in late 1987.

Changes in the caecal flora associated with the onset of laminitis.

Caecal fluid samples collected 8 and 24 hours after carbohydrate overload were quantitatively compared to control samples in terms of aerobic and anaerobic bacteria. Concomitant increases in lactic acid-producing bacteria and decreases in Gram negative bacteria were substantiated during the onset of acute laminitis. Progressive decreases in caecal fluid pH were also quantitated. Although endotoxin assays of caecal fluid and blood were not done, the caecal flora changes suggest its presence during the onset of acute laminitis.