Department of Biology, Indiana University, Bloomington, IN, USA.
Département de Microbiologie, Infectiologie et Immunologie, Université de Montréal, Montreal, Quebec, Canada.
Nat Protoc. 2019 Jun;14(6):1803-1819. doi: 10.1038/s41596-019-0162-6. Epub 2019 Apr 26.
Bacteria use surface-exposed, proteinaceous fibers called pili for diverse behaviors, including horizontal gene transfer, surface sensing, motility, and pathogenicity. Visualization of these filamentous nanomachines and their activity in live cells has proven challenging, largely due to their small size. Here, we describe a broadly applicable method for labeling and imaging pili and other surface-exposed nanomachines in live cells. This technique uses a combination of genetics and maleimide-based click chemistry in which a cysteine substitution is made in the major pilin subunit for subsequent labeling with thiol-reactive maleimide dyes. Large maleimide-conjugated molecules can also be used to physically interfere with the dynamic activity of filamentous nanomachines. We describe parameters for selecting cysteine substitution positions, optimized labeling conditions for epifluorescence imaging of pilus fibers, and methods for impeding pilus activity. After cysteine knock-in strains have been generated, this protocol can be completed within 30 min to a few hours, depending on the species and the experiment of choice. Visualization of extracellular nanomachines such as pili using this approach can provide a more comprehensive understanding of the role played by these structures in distinct bacterial behaviors.
细菌利用表面暴露的蛋白质纤维，称为菌毛，进行多种行为，包括水平基因转移、表面感应、运动和致病性。这些丝状纳米机器及其在活细胞中的活性的可视化已被证明具有挑战性，主要是由于它们的体积小。在这里，我们描述了一种广泛适用于标记和成像活细胞中菌毛和其他表面暴露的纳米机器的方法。该技术结合了遗传学和马来酰亚胺基点击化学，其中主要菌毛亚基中的半胱氨酸取代用于随后用巯基反应性马来酰亚胺染料进行标记。大的马来酰亚胺缀合物也可用于物理干扰丝状纳米机器的动态活性。我们描述了选择半胱氨酸取代位置的参数、优化用于菌毛纤维荧光显微镜成像的标记条件，以及阻碍菌毛活性的方法。在生成半胱氨酸敲入菌株后，根据物种和选择的实验，此方案可以在 30 分钟到几个小时内完成。使用这种方法可视化细胞外纳米机器，如菌毛，可以更全面地了解这些结构在不同细菌行为中所起的作用。
