Data Sheet 1_The bacterial phosphotransferase system-mediated rifampicin phosphorylation: ancestral links to rifampicin-inactivating enzyme.pdf

IntroductionThe bacterial phosphotransferase system (PTS) transports and phosphorylates sugars. Some PTS proteins share structural motifs with rifampicin phosphotransferases (RPHs), which inactivate rifampicin by phosphorylation. This homology suggests that the PTS may represent an evolutionary ancestor of the multi domain RPHs, though direct biochemical evidence has been lacking.
MethodsBacillus subtilis strains lacking genes encoding PTS proteins were evaluated in growth assays in the absence/presence of rifampicin; liquid chromatography-mass spectrometry was used to monitor the ability of B. subtilis PTS proteins to phosphorylate rifampicin; thermophoresis was employed to characterize protein–rifampicin interactions.
ResultsDeletion of B. subtilis ptsH, ptsI genes (encoding PTS proteins: HPr and EI) or rphT (encoding RphT-B. subtilis RPH) impaired growth in the presence of rifampicin. In vitro, the PTS complex (HPr, EI, MtlF, and PckA) phosphorylated rifampicin, with EI alone sufficient for this activity. However, no rifampicin phosphorylation by EI was detected in vivo. Heterologous expression of rphT then strongly increased rifampicin resistance, while ptsH/ptsI expression did not.
ConclusionThis study shows that part of the PTS, protein EI, can phosphorylate rifampicin, supporting its evolutionary link to RPHs. We also establish that RphT, a putative rifampicin phosphotransferase misannotated as phosphoenolpyruvate synthase (Pps), is a bona fide rifampicin-modifying enzyme in B. subtilis. Finally, we demonstrate that derepressing RphT or its horizontal transfer confers high-level resistance to rifampicin.

引言 细菌磷酸转移酶系统（phosphotransferase system, PTS）负责介导糖类的转运与磷酸化修饰。部分PTS蛋白与利福平磷酸转移酶（rifampicin phosphotransferases, RPHs）存在共同的结构基序，后者可通过磷酸化作用使利福平失活。这种同源性提示PTS可能是多结构域RPHs的进化祖先，但此前尚未有直接的生物化学证据支持这一假说。
方法 针对缺失PTS蛋白编码基因的枯草芽孢杆菌（Bacillus subtilis）菌株，分别在含利福平与不含利福平的培养基中开展生长实验以评估其表型；采用液相色谱-质谱联用法（liquid chromatography-mass spectrometry, LC-MS）检测枯草芽孢杆菌PTS蛋白对利福平的磷酸化能力；利用热泳技术表征蛋白与利福平之间的相互作用。
结果 敲除枯草芽孢杆菌的ptsH、ptsI基因（分别编码PTS蛋白HPr和EI）或rphT基因（编码枯草芽孢杆菌RPH家族的RphT）后，菌株在利福平存在条件下的生长受到显著抑制。体外实验表明，PTS复合物（HPr、EI、MtlF与PckA）可磷酸化利福平，且仅EI蛋白即可独立完成该催化活性。但在体内未检测到EI介导的利福平磷酸化现象。异源表达rphT可显著提升菌株的利福平耐药性，而ptsH/ptsI的表达则无此效果。
结论 本研究证实PTS的组成蛋白EI可磷酸化利福平，为二者的进化关联提供了实验依据。本研究同时明确，此前被误注释为磷酸烯醇式丙酮酸合酶（phosphoenolpyruvate synthase, Pps）的推定利福平磷酸转移酶RphT，实为枯草芽孢杆菌中真正的利福平修饰酶。最后，本研究证实解除RphT的表达调控抑制或通过水平基因转移获取该基因，均可赋予菌株高水平的利福平耐药性。