Flagellar Motility is Mutagenic

Abstract

Flagella are highly complex rotary molecular machines that enable bacteria to not only migrate to optimal environments but to also promote range expansion, competitiveness, virulence, and antibiotic survival. Flagellar motility is an energy-demanding process, where the sum of its production (biosynthesis) and operation (rotation) costs has been estimated to total ~10% of the entire energy budget of an E. coli cell. The acquisition of such a costly adaptation process is expected to secure short-term benefits by increasing competitiveness and survival, as well as long-term evolutionary fitness gains. While the role of flagellar motility in bacterial survival has been widely reported, its direct influence on the rate of evolution remains unclear. We show here that both production and operation costs contribute to elevated mutation frequencies. Our findings suggest that flagellar movement may be an important player in tuning the rate of bacterial evolution.

Keywords: E. coli; Flagella; Motility; Mutation.

Fig. 1.. Flagellar motility and mutation frequency are positively correlated.

(A) Illustration of the flagellar apparatus and chemotaxis system derived from structures of experimentally resolved components from E. coli and related bacteria (drawn to scale). PDB IDs are: 7CGO (S. enterica hook, L/ P/MS-rings, rod and export apparatus); 8UOX (S. enterica C ring composed of FliG-FliM-FliN); 1UCU (S. enterica FliC filament); 6YKM (C. jejuni MotAB), 8C5V (E. coli chemoreceptor apparatus consisting of MCP, CheY, and CheW); 1F4V (E. coli CheY); 1KMI (E. coli CheZ). See SI Methods for details. The genes mutated in this study are labeled in red. (B) Operational and production costs of deletion (∆) and overexpression (↑) strains, calculated as per (5) and (7). See SI Methods for calculation details. (C) Swimming motility (6h) of various strains assayed on soft agar (n=6); v, empty vector control for the overexpression strains. (D) Mutation frequencies (MF) of indicated strains measured as Rif⁵⁰-resistant CFUs per unit CFU (n=9). The ±fold changes in MF (in parenthesis) compared to WT are: ∆motA (−3.5), ∆fliC (−3), ∆motA∆fliC (−10), ∆flhDC (−10), fliT↑ (−2), flhDC↑ (+4.5). All p values were calculated from Mann-Whitney tests by comparing each strain with respective WT controls; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 2.. Mutagenicity of flagellar motility is ROS derived.

(A) ROS levels in different strains using CellROX redox sensor (n=6 for each strain). p values were calculated from Mann-Whitney tests. (B) Brightfield (BF) and ROS green fluorescence images of different strains. Mid-log cells were used for staining with Cell-ROX dye and imaged. (C) Energy metabolism was estimated by staining the cells with CTC dye (n=6 for each strain). The p values were calculated from Wilcoxon rank-sum tests. (D) MF was measured as in Fig. 1D; ±GSH, 50 mM glutathione. The right panel shows restoration of MF in ∆motA and ∆fliC strains after complementation with motA and fliC expressed from plasmids. p values were calculated from Wilcoxon rank sum tests (left) or Mann-Whitney tests (right); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (E) Correlation of MF and flagellar energy cost of all strains (except WT/ fliT↑) using data shown in Fig. 1D and 1B respectively; R²=0.9341.