Formate-driven isoprene reduction enables energetic independence in Pelotomaculum schinkii: Evidence from metatranscriptomic and functional analyses
Isoprene, a volatile hydrocarbon emitted in vast quantities by terrestrial vegetation, can serve as a terminal electron acceptor in anoxic environments. Here, we investigated the molecular and physiological basis of isoprene reduction in an anaerobic enrichment culture highly enriched in Pelotomaculum. Substrate-exclusion assays revealed that formate is indispensable for isoprene reduction: cultures lacking formate showed no isoprene reduction, whereas formate alone sustained both reduction activity and the highest enrichment of Pelotomaculum. Metatranscriptomic analyses confirmed that Pelotomaculum schinkii was the most transcriptionally active species under isoprene-amended conditions, whereas Sedimentibacter saalensis was most enriched in unamended controls. Isoprene-treated cultures exhibited strong upregulation of [NiFe]-hydrogenase maturation factors (hypA/hybF, hypB), energy-conserving complexes (ATP synthase, H /Na - translocating pyrophosphatases), and transport systems (FeoB, ModBC), consistent with a formate-driven respiratory pathway. In contrast, genes for oxidative stress and metal detoxification were enriched in controls, suggesting physiological stress in the absence of isoprene. Thermodynamic calculations further supported this mechanism, showing that formate oxidation coupled to isoprene reduction is highly favorable ({Delta}G{degrees}' {approx} -135 kJ/mol), with an energy yield far exceeding that of formate-driven hydrogen production. Together, these findings support two mechanistic models: (i) direct isoprene reduction by group 4b [NiFe]-hydrogenase or (ii) indirect reduction via hydrogen transfer to an isrA-like oxidoreductase, analogous to Acetobacterium wieringae ISORED-2. Our results demonstrate that P. schinkii, a metabolically versatile bacterium, couples formate oxidation to isoprene respiration, indicating that isoprene can serve as an alternative electron sink. This allows P. schinkii to escape obligate syntropy with methanogens and alters microbial activity and energy conservation in anoxic ecosystems.