Studying bacterial swarming motility using atomic force microscopy

Understanding the molecular mechanisms underlying bacterial swarming motility requires studying the factors that induce and control flagella expression in swarming cells. Moreover, bacterial flagella are currently recognized to mediate a number of functions besides their important role in motility, including acting as attachment organelles, secretory systems, and as stimuli of the innate immune response. Therefore, the analysis of flagella and other cell surface components has become an important challenge, especially in Gram-positive bacteria. Different methodologies are available for visualizing bacterial flagella using either optical or electronic microscopy, but none of these techniques combine versatility and easiness, with quantitative and high-resolution information. Here, we report an atomic force microscopy (AFM)-based approach for the fast imaging of bacterial phenotypes (cell shape, flagella expression) in swarming motility studies, focusing on the Gram-positive, rod-shaped, spore-forming bacterium Bacillus thuringiensis. This bacterium is an entomopathogen used worldwide as bioinsecticide which shares important genetic background with other two human pathogens, i.e. Bacillus anthracis, the etiological agent of the lethal disease anthrax, and Bacillus cereus, the food contaminant and opportunistic human pathogen. Because B. thuringiensis is safe for humans, it represents an ideal model to study swarming in this group of microorganisms with biomedical significance. B. thuringiensis sv. israelensis cells were inoculated on energy-rich media containing increasing agar concentrations. Following swarming assays (2 days), the cell morphology and the amount of flagella were directly observed by AFM imaging in air. Consistent with the macroscopic swarming behavior, cells harvested from the rim of colonies spreading on soft agar (0.35%) were hyperflagellated, elongated and arranged in chains. Increasing the agar concentration (up to 1.5%) led to much lower amounts of flagella and to shorter rod-shaped cells, a finding consistent with the slower swarming motility of the cells. Cells taken from colony centers on soft and hard agar surfaces were generally non-flagellated, rod-shaped, rarely arranged in chains, and exhibited sporulation and lysis. We next addressed the question as to whether cell-aging and nutrient limitation modulate the morphology of swarming cells, by observing B. thuringiensis sv. israelensis cells harvested from the rim and center of colonies developed on soft agar after 10 days of inoculation. AFM-imaging showed that aging of swarming-cells leads to a loss of flagella and to endospore formation. In addition, we found that aged cells developed at higher agar concentrations displayed complex colony architectures reminiscent of biofilms. Our results sustain previous findings for Bacillus subtilis indicating that flagellum formation is a prerequisite for biofilm formation in early stages. This study shows that AFM imaging can readily discriminate between swarming and non-swarming cells, and quantify their morphological details, thus offering an important tool to study the dynamics of bacterial populations