Lozano Anali Migueles, Asp Merrill, Rocha Sofia T, Li Jiaqi, Fanouraki Georgia, Sun Aden D, Zhang Lichun, Waldbauer Jacob R, Hong Jiarong, Shrivastava Abhishek, Yan Jing, Mukherjee Sampriti
Department of Molecular Genetics & Cell Biology, University of Chicago, Chicago, IL, USA.
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, USA.
bioRxiv. 2025 Jul 29:2025.07.29.667523. doi: 10.1101/2025.07.29.667523.
Evolutionary constraints governing flagellar number in bacterial pathogens remain poorly understood. While related species are hyperflagellated, maintains strict monoflagellation through the FleQ-FleN regulatory circuit. Here, we demonstrate that FleN dosage is essential for maintaining monoflagellation and bacterial fitness. Wild-type consistently displayed unipolar monoflagellation, while Δ mutants developed over two-to-five flagella per cell in uni- or bipolar arrangements. Hyperflagellated Δ cells exhibited severe fitness defects including reduced growth rates, attenuated virulence in nematode infection models, and competitive disadvantages in co-culture experiments. Remarkably, Δ cells rapidly evolved suppressor mutations in that partially restored growth and motility without always restoring monoflagellation. Five independent suppressor alleles mapped to critical FleQ functional domains (four in the AAA+ ATPase domain, one in the DNA-binding domain), suggesting reduced protein activity that rebalances the disrupted regulatory circuit. Single-cell motility analysis revealed that suppressor strains exhibit heterogeneous swimming dynamics, with subpopulations achieving wild-type speeds despite carrying multiple flagella. Proteomic analysis demonstrated that hyperflagellation triggers extensive cellular reprogramming beyond flagellar components, affecting metabolic pathways, stress responses, and signaling networks. While hyperflagellated cells suffered complete loss of pathogenicity in animal infection models, environmental selection under viscous conditions could drive wild-type cells to evolve enhanced motility through specific mutations. These findings suggest that bacterial flagellar regulatory circuits function as evolutionary capacitors, normally constraining phenotypic variation but enabling rapid adaptation to alternative motility configurations when environmental pressures exceed the performance limits of standard monotrichous flagellation.
目前人们对控制细菌病原体鞭毛数量的进化限制仍知之甚少。虽然相关物种有多个鞭毛，但通过FleQ-FleN调节回路保持严格的单鞭毛状态。在此，我们证明FleN的剂量对于维持单鞭毛状态和细菌适应性至关重要。野生型始终表现为单极单鞭毛，而Δ突变体在单极或双极排列中每个细胞长出两到五根鞭毛。多鞭毛的Δ细胞表现出严重的适应性缺陷，包括生长速率降低、线虫感染模型中毒力减弱以及共培养实验中的竞争劣势。值得注意的是，Δ细胞在中迅速进化出抑制突变，部分恢复了生长和运动能力，但并不总是恢复单鞭毛状态。五个独立的抑制等位基因定位于关键的FleQ功能域（四个在AAA+ATP酶结构域，一个在DNA结合结构域），表明蛋白质活性降低，从而重新平衡了被破坏的调节回路。单细胞运动分析表明，抑制菌株表现出异质的游动动力学，尽管携带多根鞭毛，但亚群达到了野生型速度。蛋白质组学分析表明，多鞭毛状态引发了鞭毛成分之外的广泛细胞重编程，影响了代谢途径、应激反应和信号网络。虽然多鞭毛细胞在动物感染模型中完全丧失了致病性，但在粘性条件下的环境选择可以驱使野生型细胞通过特定突变进化出增强的运动能力。这些发现表明，细菌鞭毛调节回路起着进化电容器的作用，通常限制表型变异，但当环境压力超过标准单鞭毛状态的性能极限时，能够使细菌快速适应替代的运动配置。
