γ-proteobacteria swimming slows at later growth stages due to loss of flagella.

(A) Swimming speeds of P. shigelloides, V. fischeri, and S. enterica sv. Typhimurium at increasing cell density. Speed relative to initial speed at OD600 0.2 are represented. Error bars indicate standard error. (B) Representative negative-stain EM images of cells grown to three different cell densities of P. shigelloides and V. fischeri, revealing loss of polar Na+-driven flagella yet no loss of peritrichous Salmonella flagella. Scale bars are 1 μm. (C) Mean number of flagella, counted from 150 cells (50 per biological replicate) at increasing cell densities suggests loss of polar flagella. The error bars indicate a 95% t-based confidence interval. (D) The absolute number of attached flagella in the population calculated from the mean number of flagella and CFU suggests flagellar ejection by P. shigelloides and V. fischeri. The error bars indicate a 95% t-based confidence interval. (E) Negative-stain EM images of flagella recovered from the supernatant of stationary phase P. shigelloides confirms consistent flagellar loss from the base of the hook. Inset shows close-up of a hook. Scale bar is 100 nm. (F) Negative-stain EM images of flagella recovered from the supernatant of stationary phase Salmonella. Inset shows close-up of a broken filament; no hooks were observed in isolated Salmonella flagella. Scale bar is 100 nm. (G) SDS-PAGE gel of P. shigelloides and Salmonella cells grown to stationary phase confirms ejection of polar Na+-driven flagella at the base of their hook. Polar FliC (filament) and FlgE (hook) proteins are seen in the S of the P. shigelloides culture. Underlying data in S1 Data. CFU, colony-forming unit; EM, electron microscopy; OD, optical density; P, pellet; S, supernatant.