William D. Leavitt | Search bioRxiv

bioRxiv2016

Transhydrogenase and growth substrate influence lipid hydrogen isotope ratios in Desulfovibrio alaskensis G20

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 homologues, 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.

Microbiology↗

bioRxiv2015

Sulfur Isotope Effects of Dissimilatory Sulfite Reductase

The precise interpretation of environmental sulfur isotope records requires a quantitative understanding of the biochemical controls on sulfur isotope fractionation by the principle isotope-fractionating process within the S cycle, microbial sulfate reduction (MSR). Here we provide the only direct observation of the major (34S/32S) and minor (33S/32S, 36S/32S) sulfur isotope fractionations imparted by a central enzyme in the energy metabolism of sulfate reducers, dissimilatory sulfite reductase (DsrAB). Results from in vitro sulfite reduction experiments allow us to calculate the in vitro DsrAB isotope effect in 34S/32S (hereafter, 34{varepsilon}DsrAB) to be 15.3{+/-}2{per thousand}, 2{sigma}.The accompanying minor isotope effect in 33S, described as 33{lambda}DsrAB, is calculated to be 0.5150{+/-}0.0012, 2{sigma}. These observations facilitate a rigorous evaluation of the isotopic fractionation associated with the dissimilatory MSR pathway, as well as of the environmental variables that govern the overall magnitude of fractionation by natural communities of sulfate reducers. The isotope effect induced by DsrAB upon sulfite reduction is a factor of 0.3 to 0.6 times prior indirect estimates, which have ranged from 25 to 53{per thousand} in 34{varepsilon}DsrAB. The minor isotope fractionation observed from DsrAB is consistent with a kinetic or equilibrium effect. Our in vitro constraints on the magnitude of 34{varepsilon}DsrAB is similar to the median value of experimental observations compiled from all known published work, where 34{varepsilon}r-p = 16.1{per thousand} (r - p indicates reactant versus product, n = 648). This value closely matches those of MSR operating at high sulfate reduction rates in both laboratory chemostat experiments (34{varepsilon}SO4-H2S = 17.3{+/-}1.5{per thousand}) and in modern marine sediments (34{varepsilon}SO4-H2S = 17.3{+/-}3.8{per thousand}). Targeting the direct isotopic consequences of a specific enzymatic processes is a fundamental step toward a biochemical foundation for reinterpreting the biogeochemical and geobiological sulfur isotope records in modern and ancient environments.

Microbiology↗