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.

齿垢密螺旋体是一种螺旋体，与引起牙周病有关。这种细菌可以利用龈沟液中的硫醇化合物产生 H _{2 S。}确定口腔细菌如何产生 H ₂ S 非常重要，因为这种分子在患有牙周病的牙周袋中含量很高，并且 H ₂ S 的生物效应可以解释牙周炎中所见的一些病理。因此，鉴定参与T合成 H ₂ S的酶是有意义的。齿垢。我们和其他人之前已经识别并表征了T。Denticola Cystalysin，称为 HlyA，可将半胱氨酸水解成 H ₂ S（以及丙酮酸和氨）。然而，尚无研究表明 HlyA 是或不是T的唯一途径。denticola可以用来制造 H ₂ S。为了解决这个问题，使用等位基因替换诱变在编码 HlyA 的基因中制造缺失突变体 (Δ hlyA )。_{突变体从半胱氨酸中产生的 H 2} S量与野生型螺旋体相同，表明T。denticola至少有一种其他酶可以从半胱氨酸产生 H _{2 S。}为了确定这种其他酶的候选者，对T进行 BLASTp 搜索。完成了denticola菌株 33520。有一个基因编码 HlyA 同源物，因此我们将其命名为 HlyB。重组组氨酸标签的 HlyB 在大肠杆菌中表达。大肠杆菌并部分纯化。该酶能够在体外从半胱氨酸产生H ₂ S。为了测试HlyB在体内的作用，在T. denticola中构建了HlyB缺失突变体(Δ hlyB ) 。该突变体仍能从半胱氨酸合成正常水平的 H _{2 S，但两个}hly基因 (Δ hlyA Δ hlyB )突变的菌株从半胱氨酸合成的 H ₂ S显着减少。我们得出结论，HlyA 和 HlyB 酶在体内执行冗余功能，并且是T中 H ₂ S 产生的主要贡献者。齿垢。然而，在Δ hlyA Δ hlyB突变体中，至少一种其他酶仍然可以将半胱氨酸转化为H ₂ S。计算机模拟提出了鉴定该另一种酶的候选基因的分析。