Biosynthetic and catabolic pathways control amino acid δ2H values in aerobic heterotrophs.

The hydrogen isotope ratios (δ2HAA values) of amino acids in all organisms are substantially fractionated relative to growth water. In addition, they exhibit large variations within microbial biomass, animals, and human tissues, hinting at rich biochemical information encoded in such signals. In lipids, such δ2H variations are thought to primarily reflect NADPH metabolism. Analogous biochemical controls for amino acids remain largely unknown, but must be elucidated to inform the interpretation of these measurements. Here, we measured the δ2H values of amino acids from five aerobic, heterotrophic microbes grown on different carbon substrates, as well as five Escherichia coli mutant organisms with perturbed NADPH metabolisms. We observed similar δ2HAA patterns across all organisms and growth conditions, which-consistent with previous hypotheses-suggests a first-order control by biosynthetic pathways. Moreover, δ2HAA values varied systematically with the catabolic pathways activated for substrate degradation, with variations explainable by the isotopic compositions of important cellular metabolites, including pyruvate and NADPH, during growth on each substrate. As such, amino acid δ2H values may be useful for interrogating organismal physiology and metabolism in the environment, provided we can further elucidate the mechanisms underpinning these signals.

Oceanic nutrient rise and the late Miocene inception of Pacific oxygen-deficient zones.

The modern Pacific Ocean hosts the largest oxygen-deficient zones (ODZs), where oxygen concentrations are so low that nitrate is used to respire organic matter. The history of the ODZs may offer key insights into ocean deoxygenation under future global warming. In a 12-My record from the southeastern Pacific, we observe a >10‰ increase in foraminifera-bound nitrogen isotopes (15N/14N) since the late Miocene (8 to 9 Mya), indicating large ODZs expansion. Coinciding with this change, we find a major increase in the nutrient content of the ocean, reconstructed from phosphorus and iron measurements of hydrothermal sediments at the same site. Whereas global warming studies cast seawater oxygen concentrations as mainly dependent on climate and ocean circulation, our findings indicate that modern ODZs are underpinned by historically high concentrations of seawater phosphate.

Novel sulfur isotope analyses constrain sulfurized porewater fluxes as a minor component of marine dissolved organic matter.

Marine dissolved organic matter (DOM) is a major reservoir that links global carbon, nitrogen, and phosphorus. DOM is also important for marine sulfur biogeochemistry as the largest water column reservoir of organic sulfur. Dissolved organic sulfur (DOS) can originate from phytoplankton-derived biomolecules in the surface ocean or from abiotically "sulfurized" organic matter diffusing from sulfidic sediments. These sources differ in 34S/32S isotope ratios (δ34S values), with phytoplankton-produced DOS tracking marine sulfate (21‰) and sulfurized DOS mirroring sedimentary porewater sulfide (∼0 to -10‰). We measured the δ34S values of solid-phase extracted (SPE) DOM from marine water columns and porewater from sulfidic sediments. Marine DOMSPE δ34S values ranged from 14.9‰ to 19.9‰ and C:S ratios from 153 to 303, with lower δ34S values corresponding to higher C:S ratios. Marine DOMSPE samples showed consistent trends with depth: δ34S values decreased, C:S ratios increased, and δ13C values were constant. Porewater DOMSPE was 34S-depleted (∼-0.6‰) and sulfur-rich (C:S ∼37) compared with water column samples. We interpret these trends as reflecting at most 20% (and on average ∼8%) contribution of abiotic sulfurized sources to marine DOSSPE and conclude that sulfurized porewater is not a main component of oceanic DOS and DOM. We hypothesize that heterogeneity in δ34S values and C:S ratios reflects the combination of sulfurized porewater inputs and preferential microbial scavenging of sulfur relative to carbon without isotope fractionation. Our findings strengthen links between oceanic sulfur and carbon cycling, supporting a realization that organic sulfur, not just sulfate, is important to marine biogeochemistry.

Sulfur cycling at natural hydrocarbon and sulfur seeps in Santa Paula Creek, CA.

Biogeochemical cycling of sulfur is relatively understudied in terrestrial environments compared to marine environments. However, the comparative ease of access, observation, and sampling of terrestrial settings can expand our understanding of organisms and processes important in the modern sulfur cycle. Furthermore, these sites may allow for the discovery of useful process analogs for ancient sulfur-metabolizing microbial communities at times in Earth's past when atmospheric O2 concentrations were lower and sulfide was more prevalent in Earth surface environments. We identified a new site at Santa Paula Creek (SPC) in Ventura County, CA-a remarkable freshwater, gravel-bedded mountain stream charged with a range of oxidized and reduced sulfur species and heavy hydrocarbons from the emergence of subsurface fluids within the underlying sulfur- and organic-rich Miocene-age Monterey Formation. SPC hosts a suite of morphologically distinct microbial biofacies that form in association with the naturally occurring hydrocarbon seeps and sulfur springs. We characterized the geology, stream geochemistry, and microbial facies and diversity of the Santa Paula Creek ecosystem. Using geochemical analyses and 16S rRNA gene sequencing, we found that SPC supports a dynamic sulfur cycle that is largely driven by sulfide-oxidizing microbial taxa, with contributions from smaller populations of sulfate-reducing and sulfur-disproportionating taxa. This preliminary characterization of SPC revealed an intriguing site in which to study geological and geochemical controls on microbial community composition and to expand our understanding of sulfur cycling in terrestrial environments.

Position-specific carbon isotope analysis of serine by gas chromatography/Orbitrap mass spectrometry, and an application to plant metabolism.

RATIONALE: Position-specific 13 C/12 C ratios within amino acids remain largely unexplored in environmental samples due to methodological limitations. We hypothesized that natural-abundance isotope patterns in serine may serve as a proxy for plant metabolic fluxes including photorespiration. Here we describe an Orbitrap method optimized for the position-specific carbon isotope analysis of serine to test our hypothesis and discuss the generalizability of this method to other amino acids. METHODS: Position-specific carbon isotope ratios of serine were measured using a Thermo Scientific™ Q Exactive™ GC Orbitrap™. Amino acids were hydrolyzed from Arabidopsis biomass, purified from potential matrix interferences, and derivatized alongside standards. Derivatized serine (N,O-bis(trifluoroacetyl)methyl ester) was isolated using gas chromatography, trapped in a reservoir, and purged into the electron ionization source over tens of minutes, producing fragment ions containing different combinations of atoms from the serine-derivative molecule. The 13 C/12 C ratios of fragments with monoisotopic masses of 110.0217, 138.0166, and 165.0037 Da were monitored in the mass analyzer and used to calculate position-specific δ13 C values relative to a working standard. RESULTS: This methodology constrains position-specific δ13 C values for nanomole amounts of serine isolated from chemically complex mixtures. The δ13 C values of fragment ions of serine were characterized with ≤1‰ precisions, leading to propagated standard errors of 0.7-5‰ for each carbon position. Position-specific δ13 C values differed by up to ca 28 ± 5‰ between serine molecules hydrolyzed from plants grown under contrasting pCO2 , selected to promote different fluxes through photosynthesis and photorespiration. The method was validated using pure serine standards characterized offline. CONCLUSIONS: This study presents the first Orbitrap-based measurements of natural-abundance, position-specific carbon isotope variation in an amino acid isolated from a biological matrix. We present a method for the precise characterization of isotope ratios in serine and propose applications probing metabolism in plants. We discuss the potential for extending these approaches to other amino acids, paving the way for novel applications.

Simultaneous, High-Precision Measurements of δ2H and δ13C in Nanomole Quantities of Acetate Using Electrospray Ionization-Quadrupole-Orbitrap Mass Spectrometry.

Stable hydrogen isotope compositions (2H/1H ratios) have been an invaluable tool for studying biogeochemical processes in nature, but the diversity of molecular targets amenable to such analysis is limited. Here, we demonstrate a new technique for measuring δ2H of biomolecules via Orbitrap mass spectrometry (MS) using acetate as a model analyte. Acetate was chosen as a target molecule because its production and consumption are central to microbial carbon cycling, yet the mechanisms behind acetate turnover remain poorly understood. δ2H of acetate could provide a useful constraint on these processes; however, it remains uncharacterized in nature due to analytical challenges. Electrospray ionization (ESI)-Orbitrap MS circumvents these challenges and delivers methyl-specific H-isotope compositions of acetate with nanomole sensitivity, enough to enable analyses of environmental samples. This approach quantifies the methyl-specific δ2H and molecular-average δ13C of acetate simultaneously while achieving <3 and <0.5‰ uncertainty, respectively. Using optimized ionization and Orbitrap parameters, this level of precision is obtained within 15 min using only 15 nmol of acetate. As a demonstration of our analytical approach, we cultured three acetogenic bacteria and found a large 2H-fractionation between acetate and water (>310‰ depletion) associated with the Wood-Ljungdahl pathway, while fermentation expressed a muted (∼80‰) fractionation. With its high precision and sensitivity, Orbitrap MS is a promising tool for investigating these signals in nature after offline purification. Furthermore, the ESI-Orbitrap method presented here could be applied to other molecules amenable to ESI, including central metabolites and sugars, greatly expanding the molecular targets used in hydrogen isotope biogeochemistry.

Microbial succession and dynamics in meromictic Mono Lake, California.

Mono Lake is a closed-basin, hypersaline, alkaline lake located in Eastern Sierra Nevada, California, that is dominated by microbial life. This unique ecosystem offers a natural laboratory for probing microbial community responses to environmental change. In 2017, a heavy snowpack and subsequent runoff led Mono Lake to transition from annually mixed (monomictic) to indefinitely stratified (meromictic). We followed microbial succession during this limnological shift, establishing a two-year (2017-2018) water-column time series of geochemical and microbiological data. Following meromictic conditions, anoxia persisted below the chemocline and reduced compounds such as sulfide and ammonium increased in concentration from near 0 to ~400 and ~150 µM, respectively, throughout 2018. We observed significant microbial succession, with trends varying by water depth. In the epilimnion (above the chemocline), aerobic heterotrophs were displaced by phototrophic genera when a large bloom of cyanobacteria appeared in fall 2018. Bacteria in the hypolimnion (below the chemocline) had a delayed, but systematic, response reflecting colonization by sediment "seed bank" communities. Phototrophic sulfide-oxidizing bacteria appeared first in summer 2017, followed by microbes associated with anaerobic fermentation in spring 2018, and eventually sulfate-reducing taxa by fall 2018. This slow shift indicated that multi-year meromixis was required to establish a sulfate-reducing community in Mono Lake, although sulfide oxidizers thrive throughout mixing regimes. The abundant green alga Picocystis remained the dominant primary producer during the meromixis event, abundant throughout the water column including in the hypolimnion despite the absence of light and prevalence of sulfide. Our study adds to the growing literature describing microbial resistance and resilience during lake mixing events related to climatic events and environmental change.

Sulfur isotope analysis of cysteine and methionine via preparatory liquid chromatography and elemental analyzer isotope ratio mass spectrometry.

RATIONALE: Sulfur isotope analysis of organic sulfur-containing molecules has previously been hindered by challenging preparatory chemistry and analytical requirements for large sample sizes. The natural-abundance sulfur isotopic compositions of the sulfur-containing amino acids, cysteine and methionine, have therefore not yet been investigated despite potential utility in biomedicine, ecology, oceanography, biogeochemistry, and other fields. METHODS: Cysteine and methionine were subjected to hot acid hydrolysis followed by quantitative oxidation in performic acid to yield cysteic acid and methionine sulfone. These stable, oxidized products were then separated by reversed-phase high-performance liquid chromatography (HPLC) and verified via offline liquid chromatography/mass spectrometry (LC/MS). The sulfur isotope ratios (δ34 S values) of purified analytes were then measured via combustion elemental analyzer coupled to isotope ratio mass spectrometry (EA/IRMS). The EA was equipped with a temperature-ramped chromatographic column and programmable helium carrier flow rates. RESULTS: On-column focusing of SO2 in the EA/IRMS system, combined with reduced He carrier flow during elution, greatly improved sensitivity, allowing precise (0.1-0.3‰ 1 s.d.) δ34 S measurements of 1 to 10 µg sulfur. We validated that our method for purification of cysteine and methionine was negligibly fractionating using amino acid and protein standards. Proof-of-concept measurements of fish muscle tissue and bacteria demonstrated differences up to 4‰ between the δ34 S values of cysteine and methionine that can be connected to biosynthetic pathways. CONCLUSIONS: We have developed a sensitive, precise method for measuring the natural-abundance sulfur isotopic compositions of cysteine and methionine isolated from biological samples. This capability opens up diverse applications of sulfur isotopes in amino acids and proteins, from use as a tracer in organisms and the environment, to fundamental aspects of metabolism and biosynthesis.

Stable Isotope Analysis of Intact Oxyanions Using Electrospray Quadrupole-Orbitrap Mass Spectrometry.

The stable isotopes of sulfate, nitrate, and phosphate are frequently used to study geobiological processes of the atmosphere, ocean, as well as land. Conventionally, the isotopes of these and other oxyanions are measured by isotope-ratio sector mass spectrometers after conversion into gases. Such methods are prone to various limitations on sensitivity, sample throughput, or precision. In addition, there is no general tool that can analyze several oxyanions or all the chemical elements they contain. Here, we describe a new approach that can potentially overcome some of these limitations based on electrospray hyphenated with Quadrupole Orbitrap mass spectrometry. This technique yields an average accuracy of 1-2‰ for sulfate δ34S and δ18O and nitrate δ15N and δ18O, based on in-house and international standards. Less abundant variants such as δ17O, δ33S, and δ36S, and the 34S-18O "clumped" sulfate can be quantified simultaneously. The observed precision of isotope ratios is limited by the number of ions counted. The counting of rare ions can be accelerated by removing abundant ions with the quadrupole mass filter. Electrospray mass spectrometry (ESMS) exhibits high-throughput and sufficient sensitivity. For example, less than 1 nmol sulfate is required to determine 18O/34S ratios with 0.2‰ precision within minutes. A purification step is recommended for environmental samples as our proposed technique is susceptible to matrix effects. Building upon these initial provisions, new features of the isotopic anatomy of mineral ions can now be explored with ESMS instruments that are increasingly available to bioanalytical laboratories.

2H/1H variation in microbial lipids is controlled by NADPH metabolism.

The hydrogen-isotopic compositions (2H/1H ratios) of lipids in microbial heterotrophs are known to vary enormously, by at least 40% (400‰) relative. This is particularly surprising, given that most C-bound H in their lipids appear to derive from the growth medium water, rather than from organic substrates, implying that the isotopic fractionation between lipids and water is itself highly variable. Changes in the lipid/water fractionation are also strongly correlated with the type of energy metabolism operating in the host. Because lipids are well preserved in the geologic record, there is thus significant potential for using lipid 2H/1H ratios to decipher the metabolism of uncultured microorganisms in both modern and ancient ecosystems. But despite over a decade of research, the precise mechanisms underlying this isotopic variability remain unclear. Differences in the kinetic isotope effects (KIEs) accompanying NADP+ reduction by dehydrogenases and transhydrogenases have been hypothesized as a plausible mechanism. However, this relationship has been difficult to prove because multiple oxidoreductases affect the NADPH pool simultaneously. Here, we cultured five diverse aerobic heterotrophs, plus five Escherichia coli mutants, and used metabolic flux analysis to show that 2H/1H fractionations are highly correlated with fluxes through NADP+-reducing and NADPH-balancing reactions. Mass-balance calculations indicate that the full range of 2H/1H variability in the investigated organisms can be quantitatively explained by varying fluxes, i.e., with constant KIEs for each involved oxidoreductase across all species. This proves that lipid 2H/1H ratios of heterotrophic microbes are quantitatively related to central metabolism and provides a foundation for interpreting 2H/1H ratios of environmental lipids and sedimentary hydrocarbons.

Role of APS reductase in biogeochemical sulfur isotope fractionation.

Sulfur isotope fractionation resulting from microbial sulfate reduction (MSR) provides some of the earliest evidence of life, and secular variations in fractionation values reflect changes in biogeochemical cycles. Here we determine the sulfur isotope effect of the enzyme adenosine phosphosulfate reductase (Apr), which is present in all known organisms conducting MSR and catalyzes the first reductive step in the pathway and reinterpret the sedimentary sulfur isotope record over geological time. Small fractionations may be attributed to low sulfate concentrations and/or high respiration rates, whereas fractionations greater than that of Apr require a low chemical potential at that metabolic step. Since Archean sediments lack fractionation exceeding the Apr value of 20‰, they are indicative of sulfate reducers having had access to ample electron donors to drive their metabolisms. Large fractionations in post-Archean sediments are congruent with a decline of favorable electron donors as aerobic and other high potential metabolic competitors evolved.

Towards measuring growth rates of pathogens during infections by D2 O-labeling lipidomics.

RATIONALE: Microbial growth rate is an important physiological parameter that is challenging to measure in situ, partly because microbes grow slowly in many environments. Recently, it has been demonstrated that generation times of S. aureus in cystic fibrosis (CF) infections can be determined by D2 O-labeling of actively synthesized fatty acids. To improve species specificity and allow growth rate monitoring for a greater range of pathogens during the treatment of infections, it is desirable to accurately quantify trace incorporation of deuterium into phospholipids. METHODS: Lipid extracts of D2 O-treated E. coli cultures were measured on liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) instruments equipped with time-of-flight (TOF) and orbitrap mass analyzers, and used for comparison with the analysis of fatty acids by isotope-ratio gas chromatography (GC)/MS. We then developed an approach to enable tracking of lipid labeling, by following the transition from stationary into exponential growth in pure cultures. Lastly, we applied D2 O-labeling lipidomics to clinical samples from CF patients with chronic lung infections. RESULTS: Lipidomics facilitates deuterium quantification in lipids at levels that are useful for many labeling applications (>0.03 at% D). In the E. coli cultures, labeling dynamics of phospholipids depend largely on their acyl chains and between phospholipids we notice differences that are not obvious from absolute concentrations alone. For example, cyclopropyl-containing lipids reflect the regulation of cyclopropane fatty acid synthase, which is predominantly expressed at the beginning of stationary phase. The deuterium incorporation into a lipid that is specific for S. aureus in CF sputum indicates an average generation time of the pathogen on the order of one cell doubling per day. CONCLUSIONS: This study demonstrates how trace level measurement of stable isotopes in intact lipids can be used to quantify lipid metabolism in pure cultures and provides guidelines that enable growth rate measurements in microbiome samples after incubation with a low percentage of D2 O.

Refining the Application of Microbial Lipids as Tracers of Staphylococcus aureus Growth Rates in Cystic Fibrosis Sputum.

Chronic lung infections in cystic fibrosis (CF) could be treated more effectively if the effects of antimicrobials on pathogens in situ were known. Here, we compared changes in the microbial community composition and pathogen growth rates in longitudinal studies of seven pediatric CF patients undergoing intravenous antibiotic administration during pulmonary exacerbations. The microbial community composition was determined by counting rRNA with NanoString DNA analysis, and growth rates were obtained by incubating CF sputum with heavy water and tracing incorporation of deuterium into two branched-chain ("anteiso") fatty acids (a-C15:0 and a-C17:0) using gas chromatography-mass spectrometry (GC/MS). Prior to this study, both lipids were thought to be specific for Staphylococcaceae; hence, their isotopic enrichment was interpreted as a growth proxy for Staphylococcus aureus Our experiments revealed, however, that Prevotella is also a relevant microbial producer of a-C17:0 fatty acid in some CF patients; thus, deuterium incorporation into these lipids is better interpreted as a more general pathogen growth rate proxy. Even accounting for a small nonmicrobial background source detected in some patient samples, a-C15:0 fatty acid still appears to be a relatively robust proxy for CF pathogens, revealing a median generation time of ∼1.5 days, similar to prior observations. Contrary to our expectation, pathogen growth rates remained relatively stable throughout exacerbation treatment. We suggest two straightforward "best practices" for application of stable-isotope probing to CF sputum metabolites: (i) parallel determination of microbial community composition in CF sputum using culture-independent tools and (ii) assessing background levels of the diagnostic metabolite.IMPORTANCE In chronic lung infections, populations of microbial pathogens change and mature in ways that are often unknown, which makes it challenging to identify appropriate treatment options. A promising tool to better understand the physiology of microorganisms in a patient is stable-isotope probing, which we previously developed to estimate the growth rates of S. aureus in cystic fibrosis (CF) sputum. Here, we tracked microbial communities in a cohort of CF patients and found that anteiso fatty acids can also originate from other sources in CF sputum. This awareness led us to develop a new workflow for the application of stable-isotope probing in this context, improving our ability to estimate pathogen generation times in clinical samples.

Minimal Influence of [NiFe] Hydrogenase on Hydrogen Isotope Fractionation in H2-Oxidizing Cupriavidus necator.

Fatty acids produced by H2-metabolizing bacteria are sometimes observed to be more D-depleted than those of photoautotrophic organisms, a trait that has been suggested as diagnostic for chemoautotrophic bacteria. The biochemical reasons for such a depletion are not known, but are often assumed to involve the strong D-depletion of H2. Here, we cultivated the bacterium Cupriavidus necator H16 (formerly Ralstonia eutropha H16) under aerobic, H2-consuming, chemoautotrophic conditions and measured the isotopic compositions of its fatty acids. In parallel with the wild type, two mutants of this strain, each lacking one of two key hydrogenase enzymes, were also grown and measured. In all three strains, fractionations between fatty acids and water ranged from -173‰ to -235‰, and averaged -217‰, -196‰, and -226‰, respectively, for the wild type, SH- mutant, and MBH- mutant. There was a modest increase in δD as a result of loss of the soluble hydrogenase enzyme. Fractionation curves for all three strains were constructed by growing parallel cultures in waters with δDwater values of approximately -25‰, 520‰, and 1100‰. These curves indicate that at least 90% of the hydrogen in fatty acids is derived from water, not H2. Published details of the biochemistry of the soluble and membrane-bound hydrogenases confirm that these enzymes transfer electrons rather than intact hydride (H-) ions, providing no direct mechanism to connect the isotopic composition of H2 to that of lipids. Multiple lines of evidence thus agree that in this organism, and presumably others like it, environmental H2 plays little or no direct role in controlling lipid δD values. The observed fractionations must instead result from isotope effects in the reduction of NAD(P)H by reductases with flavin prosthetic groups, which transfer two electrons and acquire H+ (or D+) from solution. Parallels to NADPH reduction in photosynthesis may explain why D/H fractionations in C. necator are nearly identical to those in many photoautotrophic algae and bacteria. We conclude that strong D-depletion is not a diagnostic feature of chemoautotrophy.

Comparison of three methods for the methylation of aliphatic and aromatic compounds.

RATIONALE: Methylation protocols commonly call for acidic, hot conditions that are known to promote organic 1 H/2 H exchange in aromatic and aliphatic C-H bonds. Here we tested two such commonly used methods and compared a third that avoids these acidic conditions, to quantify isotope effects with each method and to directly determine acidic-exchange rates relevant to experimental conditions. METHODS: We compared acidic and non-acidic methylation approaches catalyzed by hydrochloric acid, acetyl chloride and EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide)/DMAP (4-dimethylaminopyridine), respectively. These were applied to two analytes: phthalic acid (an aromatic) and octacosanoic acid (an aliphatic). We analyzed yield by gas chromatography/flame ionization (GC/FID) and hydrogen and carbon isotopic compositions by isotope ratio mass spectrometry (GC/IRMS). We quantified the 1 H/2 H exchange rate on dimethyl phthalate under acidic conditions with proton nuclear magnetic resonance (1 H-NMR) measurements. RESULTS: The δ2 H and δ13 C values and yield were equivalent among the three methods for methyl octacosanoate. The two acidic methods resulted in comparable yield and isotopic composition of dimethyl phthalate; however, the non-acidic method resulted in lower δ2 H and δ13 C values perhaps due to low yields. Concerns over acid-catalyzed 1 H/2 H exchange are unwarranted as the effect was trivial over a 12-h reaction time. CONCLUSIONS: We find product isolation yield and evaporation to be the main concerns in the accurate determination of isotopic composition. 1 H/2 H exchange reactions are too slow to cause measurable isotope fractionation over the typical duration and reaction conditions used in methylation. Thus, we are able to recommend continued use of acidic catalysts in such methylation reactions for both aliphatic and aromatic compounds.

Rapid quantification and isotopic analysis of dissolved sulfur species.

RATIONALE: Dissolved sulfur species are of significant interest, both as important substrates for microbial activities and as key intermediaries in biogeochemical cycles. Species of intermediate oxidation state such as sulfite, thiosulfate, and thiols are of particular interest but are notoriously difficult to analyze, because of low concentrations and rapid oxidation during storage and analysis. METHODS: Dissolved sulfur species are reacted with monobromobimane which yields a fluorescent bimane derivative that is stable to oxidation. Separation by Ultra-Performance Liquid Chromatography (UPLC) on a C18 column yields baseline resolution of analytes in under 5 min. Fluorescence detection (380 nm excitation, 480 nm emission) provides highly selective and sensitive quantitation, and Time-of-Flight Mass Spectrometry (TOF-MS) is used to quantify isotopic abundance, providing the ability to detect stable isotope tracers (either 33 S or 34 S). RESULTS: Sulfite, thiosulfate, methanethiol, and bisulfide were quantified with on-column detection limits of picomoles (µM concentrations). Other sulfur species with unshared electrons are also amenable to analysis. TOF-MS detection of 34 S enrichment was accurate and precise to within 0.6% (relative) when sample and standard had similar isotope ratios, and was able to detect enrichments as small as 0.01 atom%. Accuracy was validated by comparison to isotope-ratio mass spectrometry. Four example applications are provided to demonstrate the utility of this method. CONCLUSIONS: Derivatization of aqueous sulfur species with bromobimane is easily accomplished in the field, and protects analytes from oxidation during storage. UPLC separation with fluorescence detection provides low-µM detection limits. Using high-resolution TOF-MS, accurate detection of as little as 0.01% 34 S label incorporation into multiple species is feasible. This provides a useful new analytical window into microbial sulfur cycling. Copyright © 2017 John Wiley & Sons, Ltd.

Fractionation of Hydrogen Isotopes by Sulfate- and Nitrate-Reducing Bacteria.

Hydrogen atoms from water and food are incorporated into biomass during cellular metabolism and biosynthesis, fractionating the isotopes of hydrogen-protium and deuterium-that are recorded in biomolecules. While these fractionations are often relatively constant in plants, large variations in the magnitude of fractionation are observed for many heterotrophic microbes utilizing different central metabolic pathways. The correlation between metabolism and lipid δ(2)H provides a potential basis for reconstructing environmental and ecological parameters, but the calibration dataset has thus far been limited mainly to aerobes. Here we report on the hydrogen isotopic fractionations of lipids produced by nitrate-respiring and sulfate-reducing bacteria. We observe only small differences in fractionation between oxygen- and nitrate-respiring growth conditions, with a typical pattern of variation between substrates that is broadly consistent with previously described trends. In contrast, fractionation by sulfate-reducing bacteria does not vary significantly between different substrates, even when autotrophic and heterotrophic growth conditions are compared. This result is in marked contrast to previously published observations and has significant implications for the interpretation of environmental hydrogen isotope data. We evaluate these trends in light of metabolic gene content of each strain, growth rate, and potential flux and reservoir-size effects of cellular hydrogen, but find no single variable that can account for the differences between nitrate- and sulfate-respiring bacteria. The emerging picture of bacterial hydrogen isotope fractionation is therefore more complex than the simple correspondence between δ(2)H and metabolic pathway previously understood from aerobes. Despite the complexity, the large signals and rich variability of observed lipid δ(2)H suggest much potential as an environmental recorder of metabolism.

Organic Reference Materials for Hydrogen, Carbon, and Nitrogen Stable Isotope-Ratio Measurements: Caffeines, n-Alkanes, Fatty Acid Methyl Esters, Glycines, L-Valines, Polyethylenes, and Oils.

An international project developed, quality-tested, and determined isotope-δ values of 19 new organic reference materials (RMs) for hydrogen, carbon, and nitrogen stable isotope-ratio measurements, in addition to analyzing pre-existing RMs NBS 22 (oil), IAEA-CH-7 (polyethylene foil), and IAEA-600 (caffeine). These new RMs enable users to normalize measurements of samples to isotope-δ scales. The RMs span a range of δ(2)H(VSMOW-SLAP) values from -210.8 to +397.0 mUr or ‰, for δ(13)C(VPDB-LSVEC) from -40.81 to +0.49 mUr and for δ(15)N(Air) from -5.21 to +61.53 mUr. Many of the new RMs are amenable to gas and liquid chromatography. The RMs include triads of isotopically contrasting caffeines, C16 n-alkanes, n-C20-fatty acid methyl esters (FAMEs), glycines, and l-valines, together with polyethylene powder and string, one n-C17-FAME, a vacuum oil (NBS 22a) to replace NBS 22 oil, and a (2)H-enriched vacuum oil. A total of 11 laboratories from 7 countries used multiple analytical approaches and instrumentation for 2-point isotopic normalization against international primary measurement standards. The use of reference waters in silver tubes allowed direct normalization of δ(2)H values of organic materials against isotopic reference waters following the principle of identical treatment. Bayesian statistical analysis yielded the mean values reported here. New RMs are numbered from USGS61 through USGS78, in addition to NBS 22a. Because of exchangeable hydrogen, amino acid RMs currently are recommended only for carbon- and nitrogen-isotope measurements. Some amino acids contain (13)C and carbon-bound organic (2)H-enrichments at different molecular sites to provide RMs for potential site-specific isotopic analysis in future studies.

Trace incorporation of heavy water reveals slow and heterogeneous pathogen growth rates in cystic fibrosis sputum.

Effective treatment for chronic infections is undermined by a significant gap in understanding of the physiological state of pathogens at the site of infection. Chronic pulmonary infections are responsible for the morbidity and mortality of millions of immunocompromised individuals worldwide, yet drugs that are successful in laboratory culture are far less effective against pathogen populations persisting in vivo. Laboratory models, upon which preclinical development of new drugs is based, can only replicate host conditions when we understand the metabolic state of the pathogens and the degree of heterogeneity within the population. In this study, we measured the anabolic activity of the pathogen Staphylococcus aureus directly in the sputum of pediatric patients with cystic fibrosis (CF), by combining the high sensitivity of isotope ratio mass spectrometry with a heavy water labeling approach to capture the full range of in situ growth rates. Our results reveal S. aureus generation times with a median of 2.1 d, with extensive growth rate heterogeneity at the single-cell level. These growth rates are far below the detection limit of previous estimates of CF pathogen growth rates, and the rates are slowest in acutely sick patients undergoing pulmonary exacerbations; nevertheless, they are accessible to experimental replication within laboratory models. Treatment regimens that include specific antibiotics (vancomycin, piperacillin/tazobactam, tobramycin) further appear to correlate with slow growth of S. aureus on average, but follow-up longitudinal studies must be performed to determine whether this effect holds for individual patients.

Sulfate was a trace constituent of Archean seawater.

In the low-oxygen Archean world (>2400 million years ago), seawater sulfate concentrations were much lower than today, yet open questions frustrate the translation of modern measurements of sulfur isotope fractionations into estimates of Archean seawater sulfate concentrations. In the water column of Lake Matano, Indonesia, a low-sulfate analog for the Archean ocean, we find large (>20 per mil) sulfur isotope fractionations between sulfate and sulfide, but the underlying sediment sulfides preserve a muted range of δ(34)S values. Using models informed by sulfur cycling in Lake Matano, we infer Archean seawater sulfate concentrations of less than 2.5 micromolar. At these low concentrations, marine sulfate residence times were likely 10(3) to 10(4) years, and sulfate scarcity would have shaped early global biogeochemical cycles, possibly restricting biological productivity in Archean oceans.