3.2 | Dynamics of twitching motility in the peripheral cells
We proceeded to measure the magnitude and directionality of twitching motility in R. solanacearum . We observed that these microcolonies grew radially in all directions in successive film-like layers (Fig. 3B). This was evident from the cells exhibiting twitching motility at the edge of the microliter spot. We then studied the dynamics of twitching motility in R. solanacearum under 40X magnification by creating a time-lapse video (Supplementary video 1). Twitching motility in R. solanacearum F1C1 is generally observed in vitropost 12 h of incubation on a nutrient-rich BG media containing 1.5% agar. When bacterial culture is spotted, besides sparse random arrangement of cells within the spotted area, the cells at the border i.e., at the wet-dry interface of the microliter spot area arrange themselves to make a continuous ring-like appearance (Fig. 2A), defining distinctly its perimeter. The cell bodies divide and advance as finger-like projections of individual microcolonies before they ultimately merge into one.
The twitching motility displayed by the bordering cells occurs in a synchronized manner. In the video at the timestamp of 01:03 min and 01:14 min, the twitching motility by the peripheral cells is observed as the emergence of the bacterial cells intermittently from the same region, which gives rise to successive layers. The successive layers of bacterial progression by twitching motility, initiate from the same margin area where the initial layer progression had begun giving the appearance of a multi-layered film (Fig. 2). In a 5.0 μl spot containing 500 cells (~ 105 CFU ml-1) R. solanacearum , the formation of the first layer began at around 12:00 h of incubation. Successive formation of layers was initiated at 19:45 h and 21:45 h respectively over the preceding layers from the same point of initiation of the first layer. So, the duration between the emergence of each successive layer of twitching cells is also minimized progressively. Also, each layer of bacterial cells twitches on top of the other (Supplementary video 1).
The pace with which bacteria twitches on solid surfaces is non-uniform making the edges of its colonies uneven. The rate at which twitching motility progresses follows an exponential curve. R. solanacearumF1C1 displaces 766 μm in just 22 h (Fig. 2C) (Table 1). However, the displacement is not unidirectional. It occurs in all directions of the bacterial microcolony and at a similar pace (Fig. 3). Twitching motility in R. solanacearum F1C1 is not ever continuing. It almost comes to a halt at 7 days post-incubation at room temperature. It has been speculated that at higher cell concentrations when autoinducer levels have reached a threshold, PhcS loses its ability to phosphorylate PhcR, which leads to an upregulated PhcA and a downregulated PehSR By plotting the displacement of the bacteria against time at every hour we found that the progression of twitching motility in R. solanacearumfollows an exponential curve. The bacteria were found to migrate from the origin of inoculation by twitching in multi-directions with a displacement of 766 μm within 22 h of incubation (Table 1). Twitching motility was absent in E. coli .