Multiple enzymes can make hydrogen sulfide from cysteine in Treponema denticola

Treponema denticola is a spirochete that is involved in causing periodontal diseases. This bacterium can produce H₂S from thiol compounds found in the gingival crevicular fluid. Determining how H₂S is made by oral bacteria is important since this molecule is present at high levels in periodontally-diseased pockets and the biological effects of H₂S can explain some of the pathologies seen in periodontitis. Thus, it is of interest to identify the enzyme, or enzymes, involved in the synthesis of H₂S by T. denticola. We, and others, have previously identified and characterized a T. denticola cystalysin, called HlyA, which hydrolyzes cysteine into H₂S (and pyruvate and ammonia). However, there have been no studies to show that HlyA is, or is not, the only pathway that T. denticola can use to make H₂S. To address this question, allelic replacement mutagenesis was used to make a deletion mutant (ΔhlyA) in the gene encoding HlyA. The mutant produces the same amount of H₂S from cysteine as do wild type spirochetes, indicating that T. denticola has at least one other enzyme that can generate H₂S from cysteine. To identify candidates for this other enzyme, a BLASTp search of T. denticola strain 33520 was done. There was one gene that encoded an HlyA homolog so we named it HlyB. Recombinant His-tagged HlyB was expressed in E. coli and partially purified. This enzyme was able to make H₂S from cysteine in vitro. To test the role of HlyB in vivo, an HlyB deletion mutant (ΔhlyB) was constructed in T. denticola. This mutant still made normal levels of H₂S from cysteine, but a strain mutated in both hly genes (ΔhlyA ΔhlyB) synthesizes significantly less H₂S from cysteine. We conclude that the HlyA and HlyB enzymes perform redundant functions in vivo and are the major contributors to H₂S production in T. denticola. However, at least one other enzyme can still convert cysteine to H₂S in the ΔhlyA ΔhlyB mutant. An in silico analysis that identifies candidate genes for this other enzyme is presented.