School of Biotechnology and Biomolecular Sciences, UNSW, Kensington, Australia.
J Bacteriol. 2024 Oct 24;206(10):e0014024. doi: 10.1128/jb.00140-24. Epub 2024 Sep 16.
Powered by ion transport across the cell membrane, conserved ion-powered rotary motors (IRMs) drive bacterial motility by generating torque on the rotor of the bacterial flagellar motor. Homologous heteroheptameric IRMs have been structurally characterized in ion channels such as Tol/Ton/Exb/Gld, and most recently in phage defense systems such as Zor. Functional stator complexes synthesized from chimeras of PomB/MotB (PotB) have been used to study flagellar rotation at low ion-motive force achieved via reduced external sodium concentration. The function of such chimeras is highly sensitive to the location of the fusion site, and these hybrid proteins have thus far been arbitrarily designed. To date, no chimeras have been constructed using interchange of components from Tol/Ton/Exb/Gld and other ion-powered motors with more distant homology. Here, we synthesized chimeras of MotAB, PomAPotB, and ExbBD to assess their capacity for cross-compatibility. We generated motile strains powered by stator complexes with B-subunit chimeras. This motility was further optimized by directed evolution. Whole-genome sequencing of these strains revealed that motility-enhancing residue changes occurred in the A-subunit and at the peptidoglycan binding domain of the B-unit, which could improve motility. Overall, our work highlights the complexity of stator architecture and identifies the challenges associated with the rational design of chimeric IRMs.
Ion-powered rotary motors (IRMs) underpin the rotation of one of nature's oldest wheels, the flagellar motor. Recent structures show that this complex appears to be a fundamental molecular module with diverse biological utility where electrical energy is coupled to torque. Here, we attempted to rationally design chimeric IRMs to explore the cross-compatibility of these ancient motors. We succeeded in making one working chimera of a flagellar motor and a non-flagellar transport system protein. This had only a short hybrid stretch in the ion-conducting channel, and function was subsequently improved through additional substitutions at sites distant from this hybrid pore region. Our goal was to test the cross-compatibility of these homologous systems and highlight challenges arising when engineering new rotary motors.
离子跨细胞膜运输提供动力，保守的离子驱动旋转马达（IRM）通过在细菌鞭毛马达的转子上产生扭矩来驱动细菌的运动性。同源异七聚体 IRM 已在托利/顿/外泌体/古尔德等离子通道中进行了结构表征，最近在噬菌体防御系统中也有研究，如 zor。从 PomB/MotB（PotB）嵌合体合成的功能性定子复合物已被用于研究在通过降低外部钠离子浓度获得的低离子动力下的鞭毛旋转。这种嵌合体的功能对融合部位的位置高度敏感，到目前为止，这些杂合蛋白都是任意设计的。迄今为止，尚未使用托利/顿/外泌体/古尔德和其他具有更远同源性的离子驱动马达的组件互换构建嵌合体。在这里，我们合成了 MotAB、PomAPotB 和 ExbBD 的嵌合体，以评估它们的交叉兼容性。我们生成了由定子复合物驱动的运动菌株，该复合物的 B 亚基嵌合体。通过定向进化进一步优化了这种运动性。对这些菌株的全基因组测序显示，在 A 亚基和 B 亚基的肽聚糖结合域中发生了增强运动性的残基变化，这可能会提高运动性。总的来说，我们的工作强调了定子结构的复杂性，并确定了与理性设计嵌合 IRM 相关的挑战。
离子驱动旋转马达（IRM）为自然界最古老的轮子之一——鞭毛马达的旋转提供动力。最近的结构表明，这种复杂的结构似乎是一种具有多种生物用途的基本分子模块，其中电能与扭矩相耦合。在这里，我们试图理性设计嵌合 IRM 来探索这些古老马达的交叉兼容性。我们成功地制造了一个工作的鞭毛马达和一个非鞭毛运输系统蛋白的嵌合体。这个嵌合体在离子传导通道中只有一个很短的杂合片段，功能随后通过在远离这个杂合孔区域的其他位置进行额外的取代得到改善。我们的目标是测试这些同源系统的交叉兼容性，并突出在设计新型旋转马达时出现的挑战。
