Transhydrogenase and Growth Substrate Influence Lipid Hydrogen Isotope Ratios in Desulfovibrio alaskensis G20

Abstract

Microbial fatty acids preserve metabolic and environmental information in their hydrogen isotope ratios (2H/1H). This ratio is influenced by parameters that include the 2H/1H of water in the microbial growth environment, and biosynthetic fractionations between water and lipid. In some microbes, this biosynthetic fractionation has been shown to vary systematically with central energy metabolism, and controls on fatty acid 2H/1H may be linked to the intracellular production of NADPH. We examined the apparent fractionation between media water and the fatty acids produced by Desulfovibrio alaskensis G20. Growth was in batch culture with malate as an electron donor for sulfate respiration, and with pyruvate and fumarate as substrates for fermentation and for sulfate respiration. A larger fractionation was observed as a consequence of respiratory or fermentative growth on pyruvate than growth on fumarate or malate. This difference correlates with opposite apparent flows of electrons through the electron bifurcating/confurcating transhydrogenase NfnAB. When grown on malate or fumarate, mutant strains of D. alaskensis G20 containing transposon disruptions in a copy of nfnAB show different fractionations than the wild type strain. This phenotype is muted during fermentative growth on pyruvate, and it is absent when pyruvate is a substrate for sulfate reduction. All strains and conditions produced similar fatty acid profiles, and the 2H/1H of individual lipids changed in concert with the mass-weighted average. Unsaturated fatty acids were generally depleted in 2H relative to their saturated homologs, and anteiso-branched fatty acids were generally depleted in 2H relative to straight-chain fatty acids. Fractionation correlated with growth rate, a pattern that has also been observed in the fractionation of sulfur isotopes during dissimilatory sulfate reduction by sulfate-reducing bacteria.

Hydrogen isotope composition ofThermoanaerobacterium saccharolyticumlipids: comparing wild type to anfn-transhydrogenase mutant

“…We compare the wild-type strain to a transhydrogenase-deficient mutant to determine phenotypic effects on growth rate, lipid profile, and magnitude of hydrogen isotopic fractionation between medium water and lipid. Our findings show patterns similar to those observed for D. alaskensis G20 (Leavitt et al, 2016).…”

“…Cells were harvested at early stationary phase by way of centrifugation and were lyophilized. Lipid extraction, derivatization and analysis protocols were identical to those reported by Leavitt et al (2016). Lipid retention times and peak areas were determined by gas chromatography-mass spectrometry (GC-MS), the • 2 H values of lipids measured by GC isotope ratio mass spectrometry (GC-IRMS) and the • 2 H of water samples by duel-inlet IRMS and cavity ring-down spectroscopy (CRDS), following Leavitt et al (2016).…”

“…Lipid extraction, derivatization and analysis protocols were identical to those reported by Leavitt et al (2016). Lipid retention times and peak areas were determined by gas chromatography-mass spectrometry (GC-MS), the • 2 H values of lipids measured by GC isotope ratio mass spectrometry (GC-IRMS) and the • 2 H of water samples by duel-inlet IRMS and cavity ring-down spectroscopy (CRDS), following Leavitt et al (2016). The • 2 H values are reported relative to V-SMOW (Vienna Standard Mean Ocean Water) and fractionation is reported as apparent fractionation between medium water and lipid from the equation: , where .…”

“…The correlation has been inferred to relate to the mechanisms controlling the production of intracellular electron carriers such as NADPH and NADH. In some anaerobic bacteria the pattern of fractionation is more complicated, and does not strongly correlate with central carbon metabolism (Dawson et al, 2015; Leavitt et al, 2016; Osburn et al, 2016). One potential explanation for this complexity relates to the importance of flavin-based electron bifurcation by transhydrogenase in anaerobes (Demmer et al, 2015).…”

“…These enzymes may impose a large isotope effect, which could overprint signals that relate primarily to carbon metabolism. Examination of transhydrogenase mutants in Desulfovibrio alaskensis G20 showed that on substrates such as malate and fumarate, perturbed transhydrogenase significantly affected the • 2 H values of lipids (Leavitt et al, 2016).…”