A simple CO2 equilibration method for measuring blood oxygen isotope compositions.

RATIONALE: Blood water oxygen isotope compositions can provide valuable insights into physiological processes and ecological patterns. While blood samples are commonly drawn for medical or scientific purposes, blood fractions are infrequently measured for oxygen isotopic compositions (δ18 O) because such measurements are time consuming and expensive. METHODS: We sampled blood from sheep, goats, and iguanas raised in field and animal laboratories into serum, EDTA, heparin, and uncoated plastic vials commonly used in medical and scientific research, then separated red blood cell (RBC) and plasma or serum blood fractions. These were injected into helium-flushed Exetainer tubes where they naturally outgassed endogenous CO2 (goat blood), or into He- and CO2 -flushed tubes (iguana blood). The CO2 gas was sampled on a GasBench II system, and δ18 O was measured by an isotope ratio mass spectrometer (IRMS). RESULTS: Repeated δ18 O measurements were stable over multiple days. The addition of desiccated blood solids to water standards had little impact on their δ18 O measurements, suggesting that organic molecular constituents within blood serum and plasma do not interfere with blood water δ18 O values. We observed slight but statistically significant δ18 O offsets between plasma, serum and RBC fractions. Mass-dependent body water turnover times for iguanas were derived from the data. CONCLUSIONS: We demonstrate that a simple blood-CO2 equilibration method using the GasBench can quickly, reliably and accurately characterize water δ18 O in the plasma, RBC, and whole blood fractions of mammalian and reptilian blood samples (precision ≤ 0.1‰). This method will expand the application of blood stable isotope analysis in physiological and medical research.

Determinants of blood water δ 18O variation in a population of experimental sheep: implications for paleoclimate reconstruction.

Mammalian body, blood and hard tissue oxygen isotope compositions (δ 18O values) reflect environmental water and food sources, climate, and physiological processes. For this reason, fossil and archaeological hard tissues, which originally formed in equilibrium with body chemistry, are a valuable record of past climate, landscape paleoecology, and animal physiology and behavior. However, the environmental and physiological determinants of blood oxygen isotope composition have not been determined experimentally from large herbivores. This class of fauna is abundant in Cenozoic terrestrial fossil assemblages, and the isotopic composition of large herbivore teeth has been central to a number of climate and ecological reconstructions. Furthermore, existing models predict blood water, or nearly equivalently body water, δ 18O values based on environmental water sources. These have been evaluated on gross timescales, but have not been employed to track seasonal variation. Here we report how water, food, and physiology determine blood water δ 18O values in experimental sheep (Ovis aries) subjected to controlled water switches. We find that blood water δ 18O values rapidly reach steady state with environmental drinking water and reflect transient events including weaning, seasons, and snowstorms. Behavioral and physiological variation within a single genetically homogenous population of herbivores results in significant inter-animal variation in blood water δ 18O values at single collection times (1 s.d. = 0.1-1.4 ‰, range = 3.5 ‰) and reveals a range of water flux rates (t1/2 = 2.2-2.9 days) within the population. We find that extant models can predict average observed sheep blood δ 18O values with striking fidelity, but predict a pattern of seasonal variation exactly opposite of that observed in our population for which water input variation was controlled and the effect of physiology was more directly observed. We introduce to these models an evaporative loss term that is a function of environmental temperatures. The inclusion of this function produces model predictions that mimic the observed seasonal fluctuations and match observations to within 1.0 ‰. These results increase the applicability of available physiological models for paleoseasonality reconstructions from stable isotope measurements in fossil or archaeological enamel, the composition of which is determined in equilibrium with blood values. However, significant blood δ 18O variation in this experimentally controlled population should promote caution when interpreting isotopic variation in the archaeological and paleontological record.

Metaproteomics reveals functional partitioning and vegetational variation among permafrost-affected Arctic soil bacterial communities.

Microbial activity in Arctic soils controls the cycling of significant stores of organic carbon and nutrients. We studied in situ processes in Alaskan soils using original metaproteomic methods in order to relate important heterotrophic functions to microbial taxa and to understand the microbial response to Arctic greening. Major bacterial groups show strong metabolic specialization in organic topsoils. α-/ß-/γ-Proteobacteria specialized in the acquisition of small, soluble compounds, whereas Acidobacteria, Actinobacteria, and other detritosphere groups specialized in the degradation of plant-derived polymers. α-/ß-/γ-Proteobacteria dominated the expression of transporters for common root exudates and limiting nitrogenous compounds, supporting an ecological model of dependence upon plants for carbon and competition with plants for nitrogen. Detritosphere groups specialized in distinct substrates, with Acidobacteria producing the most enzymes for hemicellulose depolymerization. Acidobacteria was the most active group across the three plant ecotypes sampled-the largely nonvascular, lower biomass intertussock and the largely vascular, higher biomass tussock and shrub. Functional partitioning among bacterial groups was stable between plant ecotypes, but certain functions associated with α-/ß-/γ-Proteobacteria were more strongly expressed in higher biomass ecotypes. We show that refined metaproteomic approaches can elucidate soil microbial ecology as well as biogeochemical trajectories of major carbon stocks. IMPORTANCE The Arctic is warming twice as fast as the rest of the planet, and Arctic soils currently store twice as much carbon as the entire atmosphere-two facts that make understanding how Arctic soil microbial communities are responding to climate change particularly urgent. Greening of vegetation cover across the Arctic landscape is one of the most prominent climate-driven shifts in Arctic terrestrial ecology, with potentially profound effects on biogeochemical cycling by the soil microbiome. Here we use metaproteomics to document microbial metabolic functions that drive soil carbon and nutrient cycling processes in an Arctic tundra landscape. We identify functional roles among bacterial taxonomic groups that are largely stable across vegetation types, with certain functions strongly expressed by rhizosphere groups reflecting a community metabolic response to greening.

Pressure baseline correction and high-precision CO2 clumped-isotope (∆47) measurements in bellows and micro-volume modes.

RATIONALE: CO(2) 'clumped-isotope' measurements (tracking enrichment of (16)O(13)C(18)O, reported as ∆(47) values, on CO(2) derived from carbonate minerals or the atmosphere) are becoming central to a wide range of geochemical investigations. We present a novel approach to address problems with instrument stability, external precision, and the analysis of small samples that have hampered the advancement of Δ(47) measurements. METHODS: We measured Δ(47) values on CO(2) gases introduced via dual inlet to an isotope ratio mass spectrometer. We developed a method for determining the 'pressure baseline' and integrating a correction to ion beam intensity measurements during analysis. We then tested this approach for both bellows and micro-volume modes of sample introduction. Heated gas and equilibrated gas lines (Δ(47) vs. δ(47)) established the effectiveness of this correction. RESULTS: We have determined that drift in instrument calibration that compromises Δ(47) measurements results from a shift in the baseline signal on sensitive collectors (m/z 47, 48, and 49) that occurs when gas is admitted to the ion source. Applying a 'pressure baseline' (PBL) correction significantly stabilizes ∆(47) measurements and reduces the dependence of ∆(47) values on δ(47) values by up to an order of magnitude. CONCLUSIONS: PBL-corrected heated gas and equilibrated gas calibrations in bellows and micro-volume modes are nearly identical and stable through time. Introduction of the PBL correction, a revision to the absolute reference frame approach to determining Δ(47) values, dramatically improves the external precision of Δ(47) measurements to near instrumental analytical uncertainty (6-8 ppm (1σ) in bellows mode; 10-12 ppm in micro-volume mode).

Phosphorus Release and Regeneration Following Laboratory Lysis of Bacterial Cells.

The availability of phosphorus limits primary production in large regions of the oceans, and marine microbes use a variety of strategies to overcome this limitation. One strategy is the production of alkaline phosphatase (APase), which allows hydrolysis of larger dissolved organic phosphorus (DOP) compounds in the periplasm or at the cell surface for transport of orthophosphate into the cell. Cell lysis, driven by grazing and viral infection, releases phosphorus-containing cell components, along with active enzymes that could persist after lysis. The importance of this continued enzymatic activity for orthophosphate regeneration is unknown. We used three model bacteria - Escherichia coli K-12 MG1655, Synechococcus sp. WH7803, and Prochlorococcus sp. MED4 - to assess the impact of continued APase activity after cell lysis, via lysozyme treatment, on orthophosphate regeneration. Direct release of orthophosphate scaled with cell size and was reduced under phosphate-starved conditions where APase activity continued for days after lysis. All lysate incubations showed post-lysis orthophosphate generation suggesting phosphatases other than APase maintain activity. Rates of DOP hydrolysis and orthophosphate remineralization varied post-lysis among strains. Escherichia coli K-12 MG1655 rates of remineralization were 0.6 and 1.2 amol cell-1hr-1 under deplete and replete conditions; Synechococcus WH7803 lysates ranged from 0.04 up to 0.3 amol cell-1hr-1 during phosphorus deplete and replete conditions, respectively, while in Prochlorococcus MED4 lysates, rates were stable at 0.001 amol cell-1hr-1 in both conditions. The range of rates of hydrolysis and regeneration underscores the taxonomic and biochemical variability in the process of nutrient regeneration and further highlights the complexity of quantitatively resolving the major fluxes within the microbial loop.

Probing the Ecology and Climate of the Eocene Southern Ocean With Sand Tiger Sharks Striatolamia macrota.

Many explanations for Eocene climate change focus on the Southern Ocean-where tectonics influenced oceanic gateways, ocean circulation reduced heat transport, and greenhouse gas declines prompted glaciation. To date, few studies focus on marine vertebrates at high latitudes to discern paleoecological and paleoenvironmental impacts of this climate transition. The Tertiary Eocene La Meseta (TELM) Formation has a rich fossil assemblage to characterize these impacts; Striatolamia macrota, an extinct (†) sand tiger shark, is abundant throughout the La Meseta Formation. Body size is often tracked to characterize and integrate across multiple ecological dimensions. †S. macrota body size distributions indicate limited changes during TELMs 2-5 based on anterior tooth crown height (n = 450, mean = 19.6 ± 6.4 mm). Similarly, environmental conditions remained stable through this period based on δ18OPO4 values from tooth enameloid (n = 42; 21.5 ± 1.6‰), which corresponds to a mean temperature of 22.0 ± 4.0°C. Our preliminary ε Nd (n = 4) results indicate an early Drake Passage opening with Pacific inputs during TELM 2-3 (45-43 Ma) based on single unit variation with an overall radiogenic trend. Two possible hypotheses to explain these observations are (1) †S. macrota modified its migration behavior to ameliorate environmental changes related to the Drake Passage opening, or (2) the local climate change was small and gateway opening had little impact. While we cannot rule out an ecological explanation, a comparison with climate model results suggests that increased CO2 produces warm conditions that also parsimoniously explain the observations.

'That which does not kill us only makes us stronger': the role of carbon monoxide in thermophilic microbial consortia.

Carbon monoxide (CO), while a potent toxin, is also a key intermediate in major autotrophic pathways such as methanogenesis and acetogenesis. The ability of purple sulfur bacteria to use CO as an energy source was first described by Uffen in 1976. The prototype extremely thermophilic carboxydotroph Carboxydothermus hydrogenoformans was described in 1991. Eight bacteria and one archaeon that utilize CO have since been isolated and described from diverse geothermal environments. They derive energy from the oxidation of CO with water to form CO(2) and H(2). Most of these isolates thrive with headspace CO partial pressures around 1 atm, which is grossly elevated relative to CO concentrations in geothermal effluents. To account for this, we suggest that under consortial growth conditions the carboxydotrophs occupy microniches in which biogenic CO accumulates locally to high concentrations. CO oxidizers dissipate these potentially toxic CO hot spots with the production of H(2), CO(2) and acetate whose subsequent oxidation fuels other thermophiles. The identification of genes related to anaerobic CO oxidation in many metagenomic databases attests to widespread distribution of carboxydotrophs. Current evidence suggests that CO-oxidizing bacteria and archaea hold a vital niche in thermophilic ecosystems.

Diversity and expression of nitrogen fixation genes in bacterial symbionts of marine sponges.

Marine sponges contain complex assemblages of bacterial symbionts, the roles of which remain largely unknown. We identified diverse bacterial nifH genes within sponges and found that nifH genes are expressed in sponges. This is the first demonstration of the expression of any protein-coding bacterial gene within a sponge. Two sponges Ircinia strobilina and Mycale laxissima were collected from Key Largo, Florida and had delta(15)N values of c. 0-1 per thousand and 3-4 per thousand respectively. The potential for nitrogen fixation by symbionts was assessed by amplification of nifH genes. Diverse nifH genes affiliated with Proteobacteria and Cyanobacteria were detected, and expression of nifH genes affiliated with those from cyanobacteria was detected. The nifH genes from surrounding seawater were similar to those of Trichodesmium and clearly different from the cyanobacterial nifH genes detected in the two sponges. This study advances understanding of the role of bacterial symbionts in sponges and suggests that provision of fixed nitrogen is a means whereby symbionts benefit sponges in nutrient-limited reef environments. Nitrogen fixation by sponge symbionts is possibly an important source of new nitrogen to the reef environment that heretofore has been neglected and warrants further investigation.

Corrigendum: Regulation of multiple carbon monoxide consumption pathways in anaerobic bacteria.

[This corrects the article on p. 147 in vol. 2, PMID: 21808633.].

Synchrotron imaging and Markov Chain Monte Carlo reveal tooth mineralization patterns.

The progressive character of tooth formation records aspects of mammalian life history, diet, seasonal behavior and climate. Tooth mineralization occurs in two stages: secretion and maturation, which overlap to some degree. Despite decades of study, the spatial and temporal pattern of elemental incorporation during enamel mineralization remains poorly characterized. Here we use synchrotron X-ray microtomography and Markov Chain Monte Carlo sampling to estimate mineralization patterns from an ontogenetic series of sheep molars (n = 45 M1s, 18 M2s). We adopt a Bayesian approach that posits a general pattern of maturation estimated from individual- and population-level mineral density variation over time. This approach converts static images of mineral density into a dynamic model of mineralization, and demonstrates that enamel secretion and maturation waves advance at nonlinear rates with distinct geometries. While enamel secretion is ordered, maturation geometry varies within a population and appears to be driven by diffusive processes. Our model yields concrete expectations for the integration of physiological and environmental signals, which is of particular significance for paleoseasonality research. This study also provides an avenue for characterizing mineralization patterns in other taxa. Our synchrotron imaging data and model are available for application to multiple disciplines, including health, material science, and paleontological research.

Evidence for horizontal gene transfer of anaerobic carbon monoxide dehydrogenases.

Carbon monoxide (CO) is commonly known as a toxic gas, yet both cultivation studies and emerging genome sequences of bacteria and archaea establish that CO is a widely utilized microbial growth substrate. In this study, we determined the prevalence of anaerobic carbon monoxide dehydrogenases ([Ni,Fe]-CODHs) in currently available genomic sequence databases. Currently, 185 out of 2887, or 6% of sequenced bacterial and archaeal genomes possess at least one gene encoding [Ni,Fe]-CODH, the key enzyme for anaerobic CO utilization. Many genomes encode multiple copies of [Ni,Fe]-CODH genes whose functions and regulation are correlated with their associated gene clusters. The phylogenetic analysis of this extended protein family revealed six distinct clades; many clades consisted of [Ni,Fe]-CODHs that were encoded by microbes from disparate phylogenetic lineages, based on 16S rRNA sequences, and widely ranging physiology. To more clearly define if the branching patterns observed in the [Ni,Fe]-CODH trees are due to functional conservation vs. evolutionary lineage, the genomic context of the [Ni,Fe]-CODH gene clusters was examined, and superimposed on the phylogenetic trees. On the whole, there was a correlation between genomic contexts and the tree topology, but several functionally similar [Ni,Fe]-CODHs were found in different clades. In addition, some distantly related organisms have similar [Ni,Fe]-CODH genes. Thermosinus carboxydivorans was used to observe horizontal gene transfer (HGT) of [Ni,Fe]-CODH gene clusters by applying Kullback-Leibler divergence analysis methods. Divergent tetranucleotide frequency and codon usage showed that the gene cluster of T. carboxydivorans that encodes a [Ni,Fe]-CODH and an energy-converting hydrogenase is dissimilar to its whole genome but is similar to the genome of the phylogenetically distant Firmicute, Carboxydothermus hydrogenoformans. These results imply that T carboxydivorans acquired this gene cluster via HGT from a relative of C. hydrogenoformans.

Regulation of multiple carbon monoxide consumption pathways in anaerobic bacteria.

Carbon monoxide (CO), well known as a toxic gas, is increasingly recognized as a key metabolite and signaling molecule. Microbial utilization of CO is quite common, evidenced by the rapid escalation in description of new species of CO-utilizing bacteria and archaea. Carbon monoxide dehydrogenase (CODH), the protein complex that enables anaerobic CO-utilization, has been well-characterized from an increasing number of microorganisms, however the regulation of multiple CO-related gene clusters in single isolates remains unexplored. Many species are extraordinarily resistant to high CO concentrations, thriving under pure CO at more than one atmosphere. We hypothesized that, in strains that can grow exclusively on CO, both carbon acquisition via the CODH/acetyl CoA synthase complex and energy conservation via a CODH-linked hydrogenase must be differentially regulated in response to the availability of CO. The CO-sensing transcriptional activator, CooA is present in most CO-oxidizing bacteria. Here we present a genomic and phylogenetic survey of CODH operons and cooA genes found in CooA-containing bacteria. Two distinct groups of CooA homologs were found: one clade (CooA-1) is found in the majority of CooA-containing bacteria, whereas the other clade (CooA-2) is found only in genomes that encode multiple CODH clusters, suggesting that the CooA-2 might be important for cross-regulation of competing CODH operons. Recombinant CooA-1 and CooA-2 regulators from the prototypical CO-utilizing bacterium Carboxydothermus hydrogenoformans were purified, and promoter binding analyses revealed that CooA-1 specifically regulates the hydrogenase-linked CODH, whereas CooA-2 is able to regulate both the hydrogenase-linked CODH and the CODH/ACS operons. These studies point to the ability of dual CooA homologs to partition CO into divergent CO-utilizing pathways resulting in efficient consumption of a single limiting growth substrate available across a wide range of concentrations.

Rapid environmental change over the past decade revealed by isotopic analysis of the California mussel in the northeast Pacific.

The anthropogenic input of fossil fuel carbon into the atmosphere results in increased carbon dioxide (CO(2)) into the oceans, a process that lowers seawater pH, decreases alkalinity and can inhibit the production of shell material. Corrosive water has recently been documented in the northeast Pacific, along with a rapid decline in seawater pH over the past decade. A lack of instrumentation prior to the 1990s means that we have no indication whether these carbon cycle changes have precedence or are a response to recent anthropogenic CO(2) inputs. We analyzed stable carbon and oxygen isotopes (δ(13)C, δ(18)O) of decade-old California mussel shells (Mytilus californianus) in the context of an instrumental seawater record of the same length. We further compared modern shells to shells from 1000 to 1340 years BP and from the 1960s to the present and show declines in the δ(13)C of modern shells that have no historical precedent. Our finding of decline in another shelled mollusk (limpet) and our extensive environmental data show that these δ(13)C declines are unexplained by changes to the coastal food web, upwelling regime, or local circulation. Our observed decline in shell δ(13)C parallels other signs of rapid changes to the nearshore carbon cycle in the Pacific, including a decline in pH that is an order of magnitude greater than predicted by an equilibrium response to rising atmospheric CO(2), the presence of low pH water throughout the region, and a record of a similarly steep decline in δ(13)C in algae in the Gulf of Alaska. These unprecedented changes and the lack of a clear causal variable underscores the need for better quantifying carbon dynamics in nearshore environments.

Phosphate oxygen isotope analysis on microsamples of bioapatite: removal of organic contamination and minimization of sample size.

Modern and fossil teeth record seasonal information on climate, diet, and migration through stable isotope compositions in enamel and dentine. Climatic signals such as seasonal variation in meteoric water isotopic composition can be recovered through a microscale histology-based sampling and isotopic analysis of enamel phosphate oxygen. The phosphate moiety in bioapatite is particularly resistant to post mortem diagenesis. In order to determine the phosphate oxygen isotope composition of enamel, phosphate must be chemically purified from other oxygen sources in the enamel lattice and matrix, mainly hydroxyl and carbonate ions, and trace quantities of organics. We present a wet chemical technique for purifying phosphate from microsampled enamel and dentine. This technique uses a sodium hypochlorite oxidation step to remove interferences from residual organic constituents of the enamel and/or dentine scaffold, isolates phosphate as relatively large and easily manipulated Ag(3)PO(4) crystals by using a strongly buffered, moderate-temperature microprecipitation, and preserves the oxygen isotope composition of the initial tooth phosphate. The reproducibility of phosphate oxygen isotope compositions thus determined (measured as delta(18)O, V-SMOW scale) is typically 0.2-0.3 per thousand (1 s.d.) on samples as small as 300 microg of enamel or dentine, a considerable improvement over available techniques for analyses of bioapatite phosphate oxygen.

Marine phosphate oxygen isotopes and organic matter remineralization in the oceans.

We show that the isotopic composition of oxygen (delta18O) in dissolved inorganic phosphate (Pi) reveals the balance between Pi transport and biological turnover rates in marine ecosystems. Our delta18Op of Pi (delta18Op) measurements herein indicate the importance of cell lysis in the regeneration of Pi in the euphotic zone. Depth profiles of the delta18Op in the Atlantic and Pacific Oceans are near a temperature-dependent isotopic equilibrium with water. Small deviations from equilibrium below the thermocline suggest that P remineralization in the deep ocean is a byproduct of microbial carbon and energy requirements. However, isotope effects associated with phosphohydrolase enzymes involved in P remineralization are quite large and could potentially lead to significant disequilibration of Pi oxygen. The observed near equilibration of deep water Pi likely calls for continued slow rates of microbial uptake and release of Pi and/or extracellular pyrophosphatase-mediated oxygen exchange between water and Pi along the deep water flow path.