Abstract
Bifunctional dihaem cytochrome c thiosulfate dehydrogenases/tetrathionate reductases (TsdA) exhibit different catalytic properties depending on the source organism. In the human food-borne intestinal pathogen Campylobacter jejuni, TsdA functions as a tetrathionate reductase enabling respiration with tetrathionate as an alternative electron acceptor. In the present study, evidence is provided that Cys138 and Met255 serve as the sixth ligands of Haem 1 and Haem 2 respectively, in the oxidized CjTsdA wt protein. Replacement of Cys138 resulted in a virtually inactive enzyme, confirming Haem 1 as the active site haem. Significantly, TsdA variants carrying amino acid exchanges in the vicinity of the electron-transferring Haem 2 (Met255, Asn254 and Lys252) exhibited markedly altered catalytic properties of the enzyme, showing these residues play a key role in the physiological function of TsdA. The growth phenotypes and tetrathionate reductase activities of a series of ΔtsdA/*tsdA complementation strains constructed in the original host C. jejuni 81116, showed that invivo, the TsdA variants exhibited the same catalytic properties as the pure, recombinantly produced enzymes. However, variants that catalysed tetrathionate reduction more effectively than the wild-type enzyme did not allow better growth.
Highlights
Tetrathionate ( − O3S–S–S–SO3 − ) has long been known to be used by some bacteria as an electron acceptor under anaerobic conditions, recent evidence suggests that the biochemical and environmental significance of tetrathionate respiration has been hugely underestimated [1,2]
Anaerobic growth of C. jejuni 81116 wt, tsdA mutant and complementation strains C. jejuni cells of 81116 wt (81116), tsdA mutant and the different tsdA/*tsdA complementation strains (*tsdA WT, *tsdA C138G, *tsdA M255G, *tsdA N254G, *tsdA K252G) were grown under oxygen-limited conditions at 42 ◦C in almost completely filled 500 ml shake flasks containing brain heart infusion medium supplemented with serine (BHI-S) medium plus 20 mM sodium formate and 15 mM tetrathionate
For enzyme activity assays with crude cell extract (B) C. jejuni cells of 81116 wt (81116), tsdA mutant and the different complementation strains (*tsdA WT, *tsdA C138G, *tsdA M255G, *tsdA N254G, *tsdA K252G) were disrupted by bead beating
Summary
Tetrathionate ( − O3S–S–S–SO3 − ) has long been known to be used by some bacteria as an electron acceptor under anaerobic conditions, recent evidence suggests that the biochemical and environmental significance of tetrathionate respiration has been hugely underestimated [1,2]. The high relevance of tetrathionate as an in vivo electron acceptor for bacterial pathogenesis is emphasized by the finding that the human intestinal pathogen Salmonella typhimurium induces host-driven formation of tetrathionate from thiosulfate by reactive oxygen species produced during inflammation [4]. It has long been known that Citrobacter and Proteus species are able to perform tetrathionate respiration [5], the mechanism and in vivo significance of this have not been studied
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