Dersch Simon, Mehl Johanna, Stuckenschneider Lisa, Mayer Benjamin, Roth Julian, Rohrbach Alexander, Graumann Peter L
SYNMIKRO, LOEWE-Zentrum für Synthetische Mikrobiologie, Philipps-Univetsität Marburg, Marburg, Germany.
Fachbereich Chemie, Philipps-Univetsität Marburg, Marburg, Germany.
Front Microbiol. 2020 Aug 20;11:1946. doi: 10.3389/fmicb.2020.01946. eCollection 2020.
The movement of filamentous, actin-like MreB and of enzymes synthesizing the bacterial cell wall has been proposed to be highly coordinated. We have investigated the motion of MreB and of RodA and PbpH cell wall synthesis enzymes at 500 ms and at 20 ms time scales, allowing us to compare the motion of entire MreB filaments as well as of single molecules with that of the two synthesis proteins. While all three proteins formed assemblies that move with very similar trajectory orientation and with similar velocities, their trajectory lengths differed considerably, with PbpH showing shortest and MreB longest trajectories. These experiments suggest different on/off rates for RodA and PbpH at the putative peptidoglycan-extending machinery (PGEM), and during interaction with MreB filaments. Single molecule tracking revealed distinct slow-moving and freely diffusing populations of PbpH and RodA, indicating that they change between free diffusion and slow motion, indicating a dynamic interaction with the PGEM complex. Dynamics of MreB molecules and the orientation and speed of filaments changed markedly after induction of salt stress, while there was little change for RodA and PbpH single molecule dynamics. During the stress adaptation phase, cells continued to grow and extended the cell wall, while MreB formed fewer and more static filaments. Our results show that cell wall synthesis during stress adaptation occurs in a mode involving adaptation of MreB dynamics, and indicate that cell wall extension involves an interplay of enzymes with distinct binding kinetics to sites of active synthesis.
有人提出，丝状的、肌动蛋白样的MreB以及合成细菌细胞壁的酶的运动是高度协调的。我们研究了MreB、RodA和PbpH细胞壁合成酶在500毫秒和20毫秒时间尺度下的运动，这使我们能够将整个MreB丝以及单个分子的运动与两种合成蛋白的运动进行比较。虽然这三种蛋白质都形成了组装体，它们以非常相似的轨迹方向和相似的速度移动，但它们的轨迹长度差异很大，PbpH的轨迹最短，MreB的轨迹最长。这些实验表明，在假定的肽聚糖延伸机制（PGEM）处以及与MreB丝相互作用期间，RodA和PbpH的开启/关闭速率不同。单分子追踪揭示了PbpH和RodA明显的缓慢移动和自由扩散群体，表明它们在自由扩散和缓慢运动之间变化，这表明与PGEM复合物存在动态相互作用。在盐胁迫诱导后，MreB分子的动力学以及丝的方向和速度发生了显著变化，而RodA和PbpH单分子动力学变化很小。在应激适应阶段，细胞继续生长并扩展细胞壁，而MreB形成的丝更少且更静态。我们的结果表明，应激适应期间的细胞壁合成以涉及MreB动力学适应的模式发生，并表明细胞壁延伸涉及具有不同结合动力学的酶与活性合成位点之间的相互作用。
