Andrianova Ekaterina P, Dobbins Amanda L, Erhardt Marc, Hendrixson David R, Zhulin Igor B
Department of Microbiology, The Ohio State University, Columbus, Ohio, USA.
Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
bioRxiv. 2025 May 6:2025.05.06.652439. doi: 10.1101/2025.05.06.652439.
The bacterial flagellum is a complex nanomachine essential for motility, environmental sensing, and host colonization. While many of its core components have been well characterized, the relevance of proteins such as FliO, which are inconsistently annotated and poorly conserved at the sequence level, has remained ambiguous in their evolutionary and functional status. Here, we present a comprehensive phylogenomic and structural analysis of FliO across >30,000 representative genomes spanning >100 bacterial phyla. Additionally, during this analysis, we found that approximately 40% of bacterial genomes contain flagellar genes - significantly fewer than previously reported. Using a custom pipeline combining low-threshold HMM searches, operon context analysis, and structural information, we demonstrate that FliO is present in ~95% of genomes encoding the core flagellar components FliP, FliQ, and FliR. This suggests that FliO is a nearly ubiquitous and ancestral core component of the flagellar type III secretion system (fT3SS). FliO exhibits considerable structural diversity, including lineage-specific acquisitions of LysM and AMIN domains. We identify FliO homologs not only in canonical flagellar systems but also in some virulence-associated T3SS and even some non-flagellated organisms, suggesting functional repurposing and highlighting its functional plasticity. Functional studies in reveal that FliO and its AMIN domain are critical for efficient bipolar flagellation, membrane stability of the export gate component FlhB, and colonization of the host. These findings establish FliO as a core, yet evolutionarily dynamic, component of flagella and provide new insights into the evolution and diversification of bacterial secretion systems.
细菌鞭毛是一种复杂的纳米机器，对于运动性、环境感知和宿主定殖至关重要。虽然其许多核心组件已得到充分表征，但诸如FliO等蛋白质，在序列水平上注释不一致且保守性较差，其进化和功能状态仍不明确。在此，我们对跨越100多个细菌门的30000多个代表性基因组进行了全面的系统发育基因组学和结构分析。此外，在该分析过程中，我们发现约40%的细菌基因组含有鞭毛基因——显著少于先前报道。通过结合低阈值隐马尔可夫模型搜索、操纵子上下文分析和结构信息的定制流程，我们证明FliO存在于约95%编码核心鞭毛组件FliP、FliQ和FliR的基因组中。这表明FliO是鞭毛III型分泌系统（fT3SS）几乎普遍存在的祖先核心组件。FliO表现出相当大的结构多样性，包括LysM和AMIN结构域的谱系特异性获得。我们不仅在典型的鞭毛系统中鉴定出FliO同源物，还在一些与毒力相关的III型分泌系统甚至一些无鞭毛生物中鉴定出，这表明其功能发生了重新利用，并突出了其功能可塑性。 中的功能研究表明，FliO及其AMIN结构域对于高效的双极鞭毛形成、输出门组件FlhB的膜稳定性以及宿主定殖至关重要。这些发现确立了FliO作为鞭毛的核心但在进化上具有动态性的组件，并为细菌分泌系统的进化和多样化提供了新见解。
