Listeria monocytogenes rotates around its long axis during actin-based motility

Actin-based motility of the Grampositive bacterium Listeria monocytogenes is an analog of eukaryotic cell motility and has long been the subject of biochemical and biophysical investigations (reviewed in [1]). Only one bacterial factor—ActA—is required for the movement of L. monocytogenes [2–4]. Biophysical mechanisms have been proposed to explain the conversion of actin polymerization into propulsive force [5–11], but none predicts a torsional force. Surprisingly, we find that L. monocytogenes rotates around its long axis as it is propelled by actin polymerization. In contrast, Gramnegative bacteria do not rotate if actin-based motility is directed by IcsA (VirG), a Shigella flexneri protein unrelated to ActA [3,12,13]. Through the non-specific coupling of fluorescent microspheres to the bacterial surface we observed the longitudinal rotation of L. monocytogenes moving in extract (Figure 1A; supplementary data ). While beads remained at a fixed distance from the bacterial poles, indicating that they are not mobile on the cell surface, they changed their position with respect to the bacterial long axis. Bacterial speed averaged 0.09 μm/s (S.D. = 0.03 μm/s), and neither speed nor path curvature was affected by bead attachment (by Student’s Ttest, p > 0.05, n > 15). After plotting the length of the bead’s orthogonal projection onto the longitudinal axis (Figure 1B,C), we found that all traces indicated unidirectional rotation. The length of the orthogonal projection reached a maximum, crossed the bacterial longitudinal axis, reached a minimum and returned to a maximum as the bead crossed to the opposite side (n = 40, Figure 1C). Immotile bacteria symmetrically surrounded by actin jittered randomly. The periodicity of rotation was calculated by computing the autocorrelation functions of the orthogonal projections (Figure 1D). Two such calculations were performed on every trajectory for which at least 500 s of data were available (n = 27), one with respect to time and the other with respect to forward distance traveled by the bacterium (translational distance). A Fourier transformation of the autocorrelation (Figure 1E) showed a single peak in distance and time for all but one bacterium, indicating that each bacterium had a nearly constant rotation speed. Rotation was slow compared to forward motion. The average distance a bacterium travelled per rotation was 29.4 ± 11.8 μm (n = 20) and the average time per rotation was 507 ± 160 s (n = 19). Neither the temporal nor the spatial period for rotation was well-correlated with bacterial speed or bacterial length, and the temporal and spatial periods did not correlate with each other (supplemental data). Electron microscopy has shown that the filaments in actin tails are strikingly twisted [14], and several experiments indicate that at least a subset of these filaments is attached to the bacterium [8,15].