The study of the bacterial periplasm requires techniques with sufficient spatial resolution and sensitivity to resolve the components and processes within this subcellular compartment. Peroxidase-mediated biotinylation has enabled targeted labeling of proteins within subcellular compartments of mammalian cells. We investigated whether this methodology could be applied to the bacterial periplasm. In this study, we demonstrated that peroxidase-mediated biotinylation can be performed in mycobacteria and Escherichia coli. To eliminate detection artifacts from natively biotinylated mycobacterial proteins, we validated two alternative labeling substrates, tyramide azide and tyramide alkyne, which enable biotin-independent detection of labeled proteins. We also targeted peroxidase expression to the periplasm, resulting in compartment-specific labeling of periplasmic versus cytoplasmic proteins in mycobacteria. Finally, we showed that this method can be used to validate protein relocalization to the cytoplasm upon removal of a secretion signal. This novel application of peroxidase-mediated protein labeling will advance efforts to characterize the role of the periplasm in bacterial physiology and pathogenesis.

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通过过氧化物酶介导的生物素化作用对细菌周质蛋白进行区室特异性标记

细菌周质的研究需要具有足够空间分辨率和灵敏度的技术，以解决该亚细胞区室中的成分和过程。过氧化物酶介导的生物素化已使哺乳动物细胞亚细胞区隔内的蛋白质有针对性地标记。我们调查了这种方法是否可以应用于细菌周质。在这项研究中，我们证明了过氧化物酶介导的生物素化可以在分枝杆菌和大肠杆菌中进行。为了消除天然生物素化分枝杆菌蛋白的检测伪影，我们验证了两种替代性标记底物，叠氮化酪胺和炔烃炔烃，可实现不依赖生物素的标记蛋白检测。我们还将过氧化物酶表达靶向周质，导致分枝杆菌中周质与细胞质蛋白的区室特异性标记。最后，我们表明该方法可用于去除分泌信号后验证蛋白质重新定位到细胞质中。过氧化物酶介导的蛋白质标记的这一新应用将促进表征周质在细菌生理学和发病机理中的作用。