The motility defect caused by absence of the transcriptional regulator LdtR in Sinorhizobium meliloti is restored by mutations in the motility genes motA and motS

The flagellar motor is a powerful macromolecular machine used to propel bacteria through various environments. Flagellar motility of the alpha-proteobacterium Sinorhizobium meliloti is nearly abolished in the absence of the transcriptional regulator LdtR, which is involved in peptidoglycan remodeling. We report that LdtR does not regulate motility gene transcription. Remarkably, the motility defects of the DldtR mutant can be restored by secondary mutations in the motility gene motA or a previously uncharacterized gene in the flagellar regulon, which we named motS. MotS is not essential for S. meliloti motility and may serve an accessory role in flagellar motor function. Structural modeling predicts that MotS is comprised of an N-terminal transmembrane segment, a long-disordered region, and a conserved ß-sandwich domain. The C-terminus of MotS is localized in the periplasm. Genetics-based substitution of MotA with a MotAG12S variant protein also restored the ?ldtR motility defect. The MotAG12S variant causes a local polarity shift at the periphery of the MotAB stator units. We propose that MotS may be required for optimal alignment of stators in wild-type flagellar motors but becomes detrimental in cells with altered peptidoglycan. Similarly, the polarity shift in the stator units composed of MotB/MotAG12S might stabilize its interaction with altered peptidoglycan. Overall design: To investigate the differential gene expression profiles of Sinorhizobium meliloti ?ldtR cells compared to wild-type under motility-promoting conditions, we grew three replicates of each strain (six samples total) in Bromfield overlay plates to early log phase and harvested RNA from equivalent amounts of bacteria. RNA-seq analysis was performed for both strains (three replicates) and the data used to perform comparative gene expression analysis between the two strains.

鞭毛马达（flagellar motor）是一类强大的大分子机器，用于驱动细菌在各类环境中运动。苜蓿中华根瘤菌（Sinorhizobium meliloti，一种α-变形菌门（alpha-proteobacterium）细菌）的鞭毛运动能力，在缺失参与肽聚糖重塑的转录调控因子LdtR后几乎完全丧失。本研究发现，LdtR并不会调控运动基因的转录。值得注意的是，ΔldtR缺失突变体的运动缺陷可通过运动基因motA的次级突变，或是鞭毛调控子中此前未被鉴定的基因motS（我们将其命名为motS）的突变得以恢复。MotS并非苜蓿中华根瘤菌运动所必需的基因，可能在鞭毛马达功能中发挥辅助作用。结构建模预测，MotS包含N端跨膜区段、一段长无序区域以及一个保守的β-折叠桶结构域（β-sandwich domain）。MotS的C端定位于周质中。通过遗传学手段将MotA替换为MotAG12S变体蛋白，同样可恢复ΔldtR菌株的运动缺陷。该MotAG12S变体可在MotAB定子单元的外围引发局部极性偏移。我们提出假说：野生型鞭毛马达中，定子的最优排布需要MotS的参与，但在肽聚糖发生改变的细胞中，MotS会产生有害影响。类似地，由MotB/MotAG12S构成的定子单元所产生的极性偏移，或许可稳定其与改变后的肽聚糖的相互作用。实验设计：为探究在运动促进条件下，苜蓿中华根瘤菌ΔldtR突变体与野生型菌株的基因表达谱差异，我们将两种菌株各设置3个生物学重复（共计6个样本），在Bromfield覆盖平板中培养至早期对数生长期，随后从等量的细菌中提取RNA。对两种菌株各3个重复样本进行RNA测序（RNA-seq）分析，并利用所得数据开展两菌株间的比较基因表达分析。