Rescue of bacterial motility using two- and three-species FliC chimeras

The bacterial flagellar filament acts as a propeller to drive most bacterial swimming. The filament is made of flagellin, known as FliC in <i>Escherichia coli</i>. FliC consists of four domains, the highly conserved core D0 and D1 domains and the hypervariable outer D2 and D3 domains. The size and structure of the outer domains vary, being completely absent in some bacterial species. Here, we sought to identify outer domains from various species that are compatible with the ability of <i>E. coli</i> K-12 FliC to form filaments capable of supporting motility. We calculated a phylogeny of 210 representative flagellin amino acid sequences and generated a series of FliC variants, including outer domain-deleted forms and 11 chimeric FliC mutants using domains from <i>E. coli K-12</i>, <i>Salmonella</i> Typhimurium, <i>Pseudomonas aeruginosa</i>, <i>Collimonas fungivorans</i>, <i>Helicobacter mustelae</i>, and <i>Mesorhizobium</i> sp. ORS3359 in various combinations. Notably, two of the chimeric <i>fliC</i> mutants rescued motility in a <i>fliC</i>-disrupted <i>E. coli</i> K-12 strain, both of which contained the <i>S</i>. Typhimurium D2 domain. Overall, we demonstrate that, while most FliC chimeras did not support motility, interchangeability of the outer domains can produce filaments that provide motility, providing insights to guide the design of synthetic flagellins.IMPORTANCEFlagellin is a key protein forming the filament of the bacterial flagellar motor which powers most bacterial swimming. Flagellin can have hypervariable domains which can alter motility in different environments and provide immune evasion. Here we engineered two flagellin chimeras that could drive motility. This indicates that the flagellin outer domains can be exchanged, to some degree, allowing us to refine rational design approaches for engineering of bacterial swimming. Our work shows the challenges to overcome when combining flagellins from different species and provides evidence that domain-switched flagellins can form filaments.