Fig. 4: Base cell contribution to microcolony motility.

a A clay model elaborates the potential base cell mechanism of movement based on a proposed transition in orientation and mode of movement from a single bacterium gliding on substrate to a base cell engaging in pinwheel movement at the base of a microcolony. The angle between the rotation axis of the base cell (the black dash-dot line) and the normal of the substrate (the green dash-dot line) could convert the pinwheel movement to a repetitive tapping contact movement, defined as stroking. It’s worth noting the simulation is not limited to this specific mechanism. b A microscopic image (z direction) shows a typical microcolony from the hyper-motile mutant fjoh_0352 (the black circle). The focal plane was set on cells at the base of the microcolony (the yellow circle). (Callout) Threshold-processed, amplified image of substrate-proximal poles (i.e., the bright dots) of the base cells. Scale bar, 100 μm. The experiments were performed three times for the representative result. c Number of base cells (N, the bright dots in the callout of b and later shown as red dots in d) selected by ImageJ (Supplementary Fig. 5a) as a function of microcolony diameter (D) (12 h after initiating culture growth). Microcolonies with D varying from ~10 to ~100 μm were counted. The solid line shows the power fitting, revealing a nonlinear relationship. d Net force percentage (NFP) of base cells obtained by computational simulations. Two algorithms were established to evaluate and compare the net propulsion force on a microcolony (F_{Prop,microcolony}) and its theoretical maximum (Max. F_{Prop,microcolony}) with NFP defined as F_{Prop,microcolony}/Max. F_{Prop,microcolony} × 100%. First, the stroking force of a base cell (F_Stroking) was determined from the gliding force (F_Gliding) of F. johnsoniae based on the area in contact with the surface, i.e., S_L = L × W for a cell lying flat shown in white, S_W = π (W/2)² for a cell perpendicular to the surface shown in red, where L and W are the cell length and width, respectively. Gliding bacteria were arranged parallel to each other with an experimental density in a cropped microdrop (r = 18 μm in radius), moving in the same direction (from left to right) at a velocity of v = 2 μm/s. F_Stroking was evaluated for the bacterium at the center of a microdrop. Max. F_{Prop,microcolony} was then determined as F_Stroking × N, corresponding to 100% NFP, i.e., all base cells generate force in the same direction. Second, F_{Prop,microcolony} was estimated from the measured microcolony velocity. microcolonies (50 μm in diameter, N = 41 obtained in c were arranged randomly with an experimental density across a microdrop, moving in random directions at an average velocity of v = 0.031 μm/s. F_{Prop,microcolony} was estimated for the microcolony at the center of the microdrop. Here, the average velocity was used to reflect the average level of base cell participation in force generation. Given the low Reynolds number (Re ≈ 1e−6 in this study), F_Gliding or F_{Prop,microcolony} is balanced with the drag force (F_Drag) exerted by the fluidic environment⁵², and hence can be evaluated from F_Drag. The color contour (unit: Pa) denotes the distribution of total stress in the fluid around the objects solved numerically by generalized moving least squares (GMLS) with an adaptive resolution in the discretization (Methods). Error bars, mean (the yellow bar) ± s.d. (the red bar). The simulated results were obtained based on the averaged input data from three biological replicates. Source data are provided as a Source Data file.