Department of Biochemistry, University of Wisconsin, Madison, Wisconsin, USA.
Cryo-Electron Microscopy Research Center, Department of Biochemistry, University of Wisconsin, Madison, Wisconsin, USA.
J Bacteriol. 2021 Feb 8;203(5). doi: 10.1128/JB.00399-20.
is a Gram-negative alphaproteobacterium that commonly lives in oligotrophic fresh- and saltwater environments. is a host to many bacteriophages, including ϕCbK and ϕCbK-like bacteriophages, which require interaction with the bacterial flagellum and pilus complexes during adsorption. It is commonly thought that the six paralogs of the flagellin gene present in are important for bacteriophage evasion. Here, we show that deletion of specific flagellins in can indeed attenuate ϕCbK adsorption efficiency, although no single deletion completely ablates ϕCbK adsorption. Thus, the bacteriophage ϕCbK likely recognizes a common motif among the six known flagellins in with various degrees of efficiency. Interestingly, we observe that most deletion strains still generate flagellar filaments, with the exception of a strain that contains only the most divergent flagellin, FljJ, or a strain that contains only FljN and FljO. To visualize the surface residues that are likely recognized by ϕCbK, we determined two high-resolution structures of the FljK filament, with and without an amino acid substitution that induces straightening of the filament. We observe posttranslational modifications on conserved surface threonine residues of FljK that are likely O-linked glycans. The possibility of interplay between these modifications and ϕCbK adsorption is discussed. We also determined the structure of a filament composed of a heterogeneous mixture of FljK and FljL, the final resolution of which was limited to approximately 4.6 Å. Altogether, this work builds a platform for future investigations of how phage ϕCbK infects at the molecular level. Bacterial flagellar filaments serve as an initial attachment point for many bacteriophages to bacteria. Some bacteria harbor numerous flagellin genes and are therefore able to generate flagellar filaments with complex compositions, which is thought to be important for evasion from bacteriophages. This study characterizes the importance of the six flagellin genes in for infection by bacteriophage ϕCbK. We find that filaments containing the FljK flagellin are the preferred substrate for bacteriophage ϕCbK. We also present a high-resolution structure of a flagellar filament containing only the FljK flagellin, which provides a platform for future studies on determining how bacteriophage ϕCbK attaches to flagellar filaments at the molecular level.
是一种革兰氏阴性的α变形菌,通常生活在贫营养的淡水和咸水环境中。它是许多噬菌体的宿主,包括ϕCbK 和ϕCbK 样噬菌体,它们在吸附过程中需要与细菌鞭毛和菌毛复合物相互作用。通常认为,中存在的六个鞭毛基因的 paralogs 对于噬菌体逃逸很重要。在这里,我们表明,中特定鞭毛基因的缺失确实可以减弱ϕCbK 的吸附效率,尽管没有单个缺失完全消除ϕCbK 的吸附。因此,噬菌体ϕCbK 可能识别中六种已知鞭毛蛋白之间的共同基序,其识别效率各不相同。有趣的是,我们观察到大多数缺失菌株仍然产生鞭毛丝,除了一种只包含最具差异的鞭毛蛋白 FljJ 的菌株或一种只包含 FljN 和 FljO 的菌株。为了可视化可能被ϕCbK 识别的表面残基,我们确定了 FljK 细丝的两个高分辨率结构,其中一个结构带有诱导细丝变直的氨基酸取代,另一个结构没有。我们观察到 FljK 上保守的表面苏氨酸残基发生翻译后修饰,这些残基可能是 O-连接的聚糖。讨论了这些修饰与ϕCbK 吸附之间相互作用的可能性。我们还确定了由 FljK 和 FljL 组成的异质混合物细丝的结构,其最终分辨率限制在约 4.6Å。总的来说,这项工作为进一步研究噬菌体ϕCbK 如何在分子水平上感染提供了一个平台。细菌鞭毛丝是许多噬菌体最初附着到细菌的一个点。一些细菌拥有许多鞭毛基因,因此能够生成具有复杂组成的鞭毛丝,这被认为对于逃避噬菌体很重要。本研究描述了中六个鞭毛基因对噬菌体ϕCbK 感染的重要性。我们发现含有 FljK 鞭毛蛋白的细丝是噬菌体ϕCbK 的首选底物。我们还呈现了一个仅含有 FljK 鞭毛蛋白的鞭毛细丝的高分辨率结构,这为未来在分子水平上确定噬菌体ϕCbK 如何附着到鞭毛细丝的研究提供了一个平台。