Fachbereich Chemie und Zentrum für Synthetische Mikrobiologie, SYNMIKRO, Philipps-Universität Marburg, Hans-Meerwein Strasse 4, 35043 Marburg, Germany.
Mol Biol Cell. 2024 Apr 1;35(4):ar55. doi: 10.1091/mbc.E23-10-0387. Epub 2024 Feb 21.
The bacterial cell wall is a meshwork of crosslinked peptidoglycan strands, with a thickness of up to 50 nm in Firmicutes. Little is known about how proteins move through the cell wall to find sites of enzymatic activity. Cell wall synthesis for cell elongation involves the integration of new peptidoglycan strands by integral membrane proteins, as well as the degradation of existing strands by so-called autolysins, soluble proteins that are secreted through the cell membrane. Autolysins comprise different classes of proteases and glucanases and mostly contain cell-wall binding domains in addition to their catalytic domain. We have studied dynamics of autolysins LytC, a major endopeptidase required for lateral cell wall growth, and LytF, a peptidase acting at the newly formed division site in order to achieve separation of daughter cells. We show that both proteins, fused to moxVenus are present as three distinct populations of different diffusion constants. The fastest population is compatible with free diffusion in a crowded liquid environment, that is similar to that of cytosolic enzymes, likely reflecting autolysins diffusing through the periplasm. The medium mobile fraction can be explained by constrained motion through a polymeric substance, indicating mobility of autolysins through the wall similar to that of DNA-binding proteins within the nucleoid. The slow-mobile fraction are most likely autolysins bound to their specific substrate sites. We show that LytF is more static during exponential phase, while LytC appears to be more active during the transition to stationary phase. Both autolysins became more static in backgrounds lacking redundant other autolysins, suggesting stochastic competition for binding sites. On the other hand, lack of inhibitor IseA or autolysin CwlS lead to an altered preference for polar localization of LytF within the cell wall, revealing that inhibitors and autolysins also affect each other's pattern of localization, in addition to their activity.
细菌细胞壁是交联的肽聚糖链的网格，在厚壁菌门中厚达 50nm。关于蛋白质如何穿过细胞壁找到酶活性位点的知之甚少。细胞壁的延伸合成涉及整合新的肽聚糖链的整合膜蛋白，以及通过所谓的自溶素降解现有链，自溶素是通过细胞膜分泌的可溶性蛋白质。自溶素包含不同类别的蛋白酶和葡聚糖酶，除了其催化结构域外，还主要包含细胞壁结合结构域。我们研究了自溶素 LytC 和 LytF 的动力学，LytC 是横向细胞壁生长所必需的主要内切酶，LytF 是在新形成的分裂部位起作用的肽酶，以实现子细胞的分离。我们表明，两种蛋白质融合到 moxVenus 上存在三种不同扩散常数的不同群体。最快的群体与在拥挤的液体环境中的自由扩散相容，类似于胞质酶，可能反映了自溶素通过周质扩散。中等移动分数可以通过受限的通过聚合物物质的运动来解释，表明自溶素通过细胞壁的迁移类似于核区的 DNA 结合蛋白。缓慢移动的分数很可能是结合其特定底物位点的自溶素。我们表明，在指数生长期，LytF 更为静态，而 LytC 在过渡到静止期时似乎更为活跃。在缺乏冗余其他自溶素的背景下，两种自溶素都变得更加静态，这表明随机竞争结合位点。另一方面，缺乏抑制剂 IseA 或自溶素 CwlS 导致 LytF 在细胞壁内的极性定位偏好发生改变，这表明抑制剂和自溶素除了影响其活性外，还会影响彼此的定位模式。
