Zuke Jason D, Erickson Rachel, Hummels Katherine R, Burton Briana M
Department of Bacteriology, University of Wisconsin - Madison.
Microbiology Doctoral Training Program, University of Wisconsin - Madison.
bioRxiv. 2023 May 27:2023.05.26.542325. doi: 10.1101/2023.05.26.542325.
The first step in the process of bacterial natural transformation is DNA capture. Although long-hypothesized based on genetics and functional experiments, the pilus structure responsible for initial DNA-binding had not yet been visualized for Bacillus subtilis. Here, we visualize functional competence pili in Bacillus subtilis using fluorophore-conjugated maleimide labeling in conjunction with epifluorescence microscopy. In strains that produce pilin monomers within ten-fold of wild type levels, the median length of detectable pili is 300nm. These pili are retractile and associate with DNA. Analysis of pilus distribution at the cell surface reveals that they are predominantly located along the long axis of the cell. The distribution is consistent with localization of proteins associated with subsequent transformation steps, DNA-binding and DNA translocation in the cytosol. These data suggest a distributed model for B. subtilis transformation machinery, in which initial steps of DNA capture occur throughout the long axis of the cell and subsequent steps may also occur away from the cell poles.
细菌自然转化过程的第一步是DNA捕获。尽管基于遗传学和功能实验早就提出了这一假说，但负责初始DNA结合的菌毛结构尚未在枯草芽孢杆菌中观察到。在这里，我们使用荧光团共轭马来酰亚胺标记结合落射荧光显微镜，观察到了枯草芽孢杆菌中的功能性感受态菌毛。在产生菌毛单体水平为野生型水平十倍以内的菌株中，可检测到的菌毛的中位长度为300纳米。这些菌毛是可收缩的，并与DNA相关联。对细胞表面菌毛分布的分析表明，它们主要沿细胞的长轴分布。这种分布与后续转化步骤、DNA结合以及胞质溶胶中DNA易位相关蛋白的定位一致。这些数据表明了枯草芽孢杆菌转化机制的一种分布式模型，其中DNA捕获的初始步骤发生在细胞的整个长轴上，后续步骤也可能发生在远离细胞两极的地方。
