Monitoring microbial reactions in highly opaque or autofluorescent environments like soils, seawater, and wastewater remains challenging. To develop a simple approach for observing post-translational reactions within microbes situated in environmental matrices, we designed a methyl halide transferase (MHT) fragment complementation assay that reports by synthesizing an indicator gas. We show that backbone fission within regions of high sequence variability in the Rossmann domain yields split MHT (sMHT) AND gates whose fragments cooperatively associate to synthesize CH₃Br. Additionally, we identify a sMHT whose fragments require fusion to pairs of interacting partner proteins for maximal activity. We also show that sMHT fragments fused to FKBP12 and the FKBP-rapamycin binding domain of mTOR display significantly enhanced CH₃Br production in the presence of rapamycin. This gas production is reversed in the presence of the competitive inhibitor of FKBP12/FKPB dimerization, indicating that sMHT is a reversible reporter of post-translational reactions. This sMHT represents the first genetic AND gate that reports on protein–protein interactions via an indicator gas. Because indicator gases can be measured in the headspaces of complex environmental samples, this assay should be useful for monitoring the dynamics of diverse molecular interactions within microbes situated in hard-to-image marine and terrestrial matrices.

中文翻译：

---

通过合成指示气体报告的分裂甲基卤化物转移酶和门

在土壤、海水和废水等高度不透明或自发荧光环境中监测微生物反应仍然具有挑战性。为了开发一种简单的方法来观察位于环境基质中的微生物内的翻译后反应，我们设计了一种卤代甲烷转移酶 (MHT) 片段互补分析，该分析通过合成指示剂气体进行报告。我们表明，罗斯曼域中高序列变异性区域内的骨干裂变产生分裂的 MHT (sMHT) 和门，其片段协同关联以合成 CH ₃兄弟 此外，我们确定了一个 sMHT，其片段需要与成对的相互作用伙伴蛋白融合才能获得最大活性。我们还表明，与 FKBP12 融合的 sMHT 片段和 mTOR 的 FKBP-雷帕​​霉素结合域显示显着增强了 CH ₃在雷帕霉素存在下产生溴。在 FKBP12/FKPB 二聚化的竞争性抑制剂存在的情况下，这种气体产生被逆转，表明 sMHT 是翻译后反应的可逆报告分子。这个 sMHT 代表了第一个通过指示气体报告蛋白质-蛋白质相互作用的遗传与门。由于可以在复杂环境样品的顶空测量指示剂气体，因此该测定应有助于监测位于难以成像的海洋和陆地基质中微生物内不同分子相互作用的动态。