Fachbereich Chemie und Zentrum für Synthetische Mikrobiologie, SYNMIKRO, Philipps-Universität Marburg, Marburg, Germany.
J Bacteriol. 2022 Mar 15;204(3):e0057221. doi: 10.1128/jb.00572-21. Epub 2021 Dec 20.
In competent Gram-negative and Gram-positive bacteria, double-stranded DNA is taken up through the outer cell membrane and/or the cell wall and is bound by ComEA, which in Bacillus subtilis is a membrane protein. DNA is converted to single-stranded DNA and transported through the cell membrane via ComEC. We show that in Bacillus subtilis, the C terminus of ComEC, thought to act as a nuclease, is important not only for DNA uptake, as judged from a loss of transformability, but also for the localization of ComEC to the cell pole and its mobility within the cell membrane. Using single-molecule tracking, we show that only 13% of ComEC molecules are statically localized at the pole, while 87% move throughout the cell membrane. These experiments suggest that recruitment of ComEC to the cell pole is mediated by a diffusion/capture mechanism. Mutation of a conserved aspartate residue in the C terminus, likely affecting metal binding, strongly impairs transformation efficiency, suggesting that this periplasmic domain of ComEC could indeed serve a catalytic function as a nuclease. By tracking fluorescently labeled DNA, we show that taken-up DNA has a similar mobility as a protein, in spite of being a large polymer. DNA dynamics are similar within the periplasm as those of ComEA, suggesting that most taken-up molecules are bound to ComEA. We show that DNA can be highly mobile within the periplasm, indicating that this subcellular space can act as reservoir for taken-up DNA, before its entry into the cytosol. Bacteria can take up DNA from the environment and incorporate it into their chromosome, which is termed "natural competence" that can result in the uptake of novel genetic information. We show that fluorescently labeled DNA moves within the periplasm of competent Bacillus subtilis cells, with similar dynamics as DNA receptor ComEA. This finding indicates that DNA can accumulate in the periplasm, likely bound by ComEA, and thus can be stored before uptake at the cell pole, via integral membrane DNA permease ComEC. Assembly of ComEC at the cell pole likely occurs by a diffusion-capture mechanism. DNA uptake into cells thus takes a detour through the entire periplasm and involves a high degree of free diffusion along and within the cell membrane.
在有能力的革兰氏阴性和革兰氏阳性细菌中，双链 DNA 通过外细胞膜和/或细胞壁被摄取，并被 ComEA 结合，在枯草芽孢杆菌中，ComEA 是一种膜蛋白。DNA 被转化为单链 DNA，并通过 ComEC 穿过细胞膜进行运输。我们表明，在枯草芽孢杆菌中，ComEC 的 C 端，被认为起核酸内切酶的作用，不仅对 DNA 的摄取很重要，正如转化能力的丧失所判断的那样，而且对 ComEC 定位于细胞极和在细胞膜内的流动性也很重要。使用单分子追踪，我们表明，只有 13%的 ComEC 分子静态地定位于极，而 87%的分子在整个细胞膜内移动。这些实验表明，ComEC 向细胞极的募集是通过扩散/捕获机制介导的。突变 C 端保守的天冬氨酸残基，可能影响金属结合，强烈损害转化效率，这表明 ComEC 的这个周质域实际上可以作为核酸内切酶发挥催化作用。通过跟踪荧光标记的 DNA，我们表明，摄取的 DNA 与蛋白质具有相似的流动性，尽管它是一种大聚合物。尽管 DNA 动态与 ComEA 相似，但在周质中，这表明大多数摄取的分子与 ComEA 结合。我们表明，DNA 可以在周质内高度移动，这表明这个亚细胞空间可以作为摄取 DNA 的储库，然后再进入细胞质。细菌可以从环境中摄取 DNA 并将其整合到染色体中，这被称为“天然感受态”，可以导致新遗传信息的摄取。我们表明，荧光标记的 DNA 在有能力的枯草芽孢杆菌细胞的周质中移动，与 DNA 受体 ComEA 的动力学相似。这一发现表明，DNA 可以在周质中积累，可能与 ComEA 结合，因此可以在通过整合膜 DNA 透性酶 ComEC 在细胞极摄取之前储存。ComEC 在细胞极的组装可能通过扩散/捕获机制发生。因此，DNA 进入细胞需要通过整个周质绕道而行，并涉及到沿细胞膜和细胞膜内的高度自由扩散。
