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 HS from thiol compounds found in the gingival crevicular fluid. Determining how HS is made by oral bacteria is important since this molecule is present at high levels in periodontally-diseased pockets and the biological effects of HS 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 HS by T. denticola. We, and others, have previously identified and characterized a T. denticola cystalysin, called HlyA, which hydrolyzes cysteine into HS (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 HS. 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 HS from cysteine as do wild type spirochetes, indicating that T. denticola has at least one other enzyme that can generate HS 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 HS 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 HS from cysteine, but a strain mutated in both hly genes (ΔhlyA ΔhlyB) synthesizes significantly less HS from cysteine. We conclude that the HlyA and HlyB enzymes perform redundant functions in vivo and are the major contributors to HS production in T. denticola. However, at least one other enzyme can still convert cysteine to HS in the ΔhlyA ΔhlyB mutant. An in silico analysis that identifies candidate genes for this other enzyme is presented.