A metabolic puzzle: Consumption of C₁ compounds and thiosulfate in Hyphomicrobium denitrificans X^T

Many obligately heterotrophic methylotrophs oxidize thiosulfate as an additional electron source during growth on C₁ compounds. Although two different pathways of thiosulfate oxidation are implemented in Hyphomicrobium denitrificans X^T, a pronounced negative effect on growth rate is observed when it is cultured in the simultaneous presence of methanol and thiosulfate. In this model organism, periplasmic thiosulfate dehydrogenase TsdA catalyzes formation of the dead-end product tetrathionate. By reverse genetics we verified the second pathway that also starts in the periplasm where SoxXA catalyzes the oxidative fusion of thiosulfate to SoxYZ, from which sulfate is released by SoxB. Sulfane sulfur is then further oxidized in the cytoplasm by the sulfur-oxidizing heterodisulfide reductase-like system (sHdr) which is produced constitutively in a strain lacking the transcriptional repressor sHdrR. When exposed to thiosulfate, the ΔshdrR strain exhibited a strongly reduced growth rate even without thiosulfate in the pre-cultures. When grown on methanol, cells exhibit significantly increased NAD⁺/NADH ratios in the presence of thiosulfate. In contrast, thiosulfate did not exert any negative effect on growth rate or increase NAD⁺ levels during growth on formate. On both C₁ substrates, excretion of up to 0.5 mM sulfite as an intermediate of thiosulfate (2 mM) oxidation was recorded. Sulfite is known to form adducts with pyrroloquinoline quinone, the cofactor of periplasmic methanol dehydrogenase. We rationalize that this causes specific inhibition of methanol degradation in the presence of thiosulfate while formate metabolism in the cytoplasm remains unaffected.