A Specific Ancestral Sequence Reconstruction of Flagellin Can Drive Motility in Salmonella enterica but not Escherichia coli
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The bacterial flagellar filament, built from thousands of flagellin subunits, is essential for flagellar motility. Here, we aimed to use ancestral sequence reconstruction (ASR) to generate conservative, ancestral flagellin sequences that surround the divergency point between Escherichia and Salmonella species and tested their ability to restore motility in flagellin deleted Escherichia coli and Salmonella Typhimurium. Only one (N131) out of three candidates restored motility in S. Typhimurium but not E. coli , despite high structural similarity to native FliC/FljB. S. Typhimurium expressing N131 showed reduced motility relative to wild-type Salmonella FliC, alongside increased tumbling frequency. Serial passage across swim plates produced a derivative, N131.10, containing a single flagellin mutation (M144I), with a larger swim ring diameter but also increased tumbling frequency. Comparison of structural interaction networks among the residues of S. Typhimurium LT2 FliC with those of other filament proteins showed that non-motile candidates (N132 and N134) carried substitutions that disrupted key core-domain atomic interactions. These results demonstrate that flagellar filament assembly tolerates only limited sequence variation and that this capacity for tolerance differs between E. coli and S. Typhimurium despite their close phylogenetic relationship.
Importance
Bacteria swim using long, rotating filaments assembled from thousands of copies of a protein called flagellin. We reconstructed likely ancestral versions of this protein to investigate how evolutionary changes affect swimming. Of three versions tested, only one restored swimming in Salmonella unable to make its own flagellin. The same protein did not work in the closely related bacterium E. coli . After repeated selection for better movement, a single additional change helped the Salmonella spread farther and also altered its swimming behaviour. These findings show that closely related bacteria differ in the changes that their flagellar assembly machinery can tolerate. Understanding these limits helps explain how flagella evolve while retaining their function and provides a foundation for studying the role of flagellar variation in immune recognition and evasion.
