Microbiology and Molecular Genetics, McGovern Medical School, Houston, Texas, USA.
J Bacteriol. 2023 Jan 26;205(1):e0037322. doi: 10.1128/jb.00373-22. Epub 2023 Jan 9.
FtsA, a homolog of actin, is essential for cell division of Escherichia coli and is widely conserved among many bacteria. FtsA helps to tether polymers of the bacterial tubulin homolog FtsZ to the cytoplasmic membrane as part of the cytokinetic Z ring. GFP fusions to FtsA have illuminated FtsA's localization in live E. coli, but these fusions have not been fully functional and required the presence of the native FtsA. Here, we characterize "sandwich" fusions of E. coli FtsA to either mCherry or msfGFP that are functional for cell division and exhibit fluorescent rings at midcell that persist throughout constriction until cell separation. FtsA within the Z ring moved circumferentially like FtsZ, and FtsA outside the rings formed highly dynamic patches at the membrane. Notably, both FtsA-mCherry and FtsA-msfGFP acted as mild hypermorphs, as they were not toxic when overproduced, bypassed the essential cell division protein ZipA, and suppressed several thermosensitive alleles, although not as effectively as the prototypical hypermorph FtsA*. Overall, our results indicate that fluorescent FtsA sandwich fusions can be used as the sole FtsA in E. coli and thus should shed new light on FtsA dynamics during the cell division cycle in this model system. FtsA is a key conserved cell division protein, and E. coli is the most well studied model system for bacterial cell division. One obstacle to full understanding of this process is the lack of a fully functional fluorescent reporter for FtsA . Here, we describe a fluorescent fusion to E. coli FtsA that promotes efficient cell division in the absence of the native FtsA and can be used to monitor FtsA dynamics during cell division.
FtsA 是肌动蛋白的同源物，对大肠杆菌的细胞分裂是必不可少的，并且在许多细菌中广泛保守。FtsA 有助于将细菌微管蛋白同源物 FtsZ 的聚合物系链到细胞质膜上，作为细胞分裂的有丝分裂 Z 环的一部分。GFP 与 FtsA 的融合照亮了活大肠杆菌中 FtsA 的定位，但这些融合尚未完全发挥功能，并且需要存在天然的 FtsA。在这里，我们对大肠杆菌 FtsA 与 mCherry 或 msfGFP 的“三明治”融合进行了表征，这些融合对于细胞分裂是功能性的，并且在收缩过程中在中细胞处表现出荧光环，直到细胞分离。Z 环内的 FtsA 像 FtsZ 一样沿圆周运动，而环外的 FtsA 在膜上形成高度动态的斑点。值得注意的是，FtsA-mCherry 和 FtsA-msfGFP 都表现出轻度超量表型，因为它们过量产生时没有毒性，绕过了必需的细胞分裂蛋白 ZipA，并抑制了几个热敏等位基因，尽管不如原型超量表型 FtsA* 那样有效。总体而言，我们的结果表明，荧光 FtsA 三明治融合可以作为大肠杆菌中唯一的 FtsA 使用，因此应该在该模型系统中为细胞分裂周期期间的 FtsA 动力学提供新的见解。FtsA 是一种关键的保守细胞分裂蛋白，大肠杆菌是细菌细胞分裂最深入研究的模型系统。全面理解这一过程的一个障碍是缺乏 FtsA 的完全功能性荧光报告子。在这里，我们描述了一种与大肠杆菌 FtsA 的荧光融合，它在不存在天然 FtsA 的情况下促进有效的细胞分裂，并可用于监测细胞分裂过程中 FtsA 的动力学。
