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

Area of Science:

• Microbiology
• Structural Biology
• Protein Engineering

Background:

• Bacterial flagellar filaments, composed of flagellin (FliC), are essential for motility.
• FliC proteins feature conserved core domains (D0, D1) and variable outer domains (D2, D3) that influence filament structure and function.
• Variations in outer domain size and presence exist across bacterial species.

Purpose of the Study:

• To investigate the compatibility of outer domains from various bacterial flagellins with the Escherichia coli K-12 FliC structure.
• To determine if chimeric FliC proteins, combining domains from different species, can form functional flagellar filaments that support motility.

Main Methods:

• Phylogenetic analysis of 210 flagellin sequences.
• Construction and testing of FliC variants, including outer domain-deleted mutants and 11 chimeric FliC proteins.
• Assessing motility in a fliC-disrupted E. coli K-12 strain using engineered FliC variants.

Main Results:

• Most chimeric FliC proteins did not restore motility in the E. coli K-12 strain.
• Two chimeric FliC mutants, each containing the D2 domain from Salmonella Typhimurium, successfully rescued motility.
• Demonstrated interchangeability of FliC outer domains, albeit with limitations, for filament formation and motility.

Conclusions:

• The outer domains of flagellin can be exchanged between species to some extent, enabling the formation of motile flagellar filaments.
• This study provides insights into the structural requirements for functional flagellar assembly and guides rational design of synthetic flagellins.
• Highlights challenges in interspecies flagellin domain recombination while confirming the potential for engineering bacterial motility.