Bacterial Co-Incubation Assay: A Fluorescence Microscopy-Based Technique to Visualize Intraspecific Bacterial Competition at the Single-Cell Level

Intraspecific competition between two bacterial strains causes the inhibitor strain to release toxic effector molecules that kill the target population.

To visualize competitive interactions between two gram-negative bacterial strains, begin with a co-culture comprising an inhibitor and a target strain in equal amounts.

Both strains are engineered to express different-colored fluorescent proteins to visually differentiate them during imaging. Concentrate the culture to maximize cell-to-cell contact between the bacterial strains. Spot the mixed culture onto a coverslip placed at the bottom of a dish.

Place a solidified agarose pad over the spot, allowing bacteria to immobilize, and cover it with another coverslip. Incubate to allow bacterial growth, simulating bacterial crowding conditions.

The cell envelope of the inhibitory strain contains a specialized secretory system - the type six secretion system, or T6SS. It comprises a membrane complex, a baseplate, and an inner tube surrounded by a contractile sheath.

Upon detecting contact, the inhibitor strain activates its T6SS, which causes contraction of the sheath and expulsion of the tube, ultimately leading to the perforation of the target bacterial cell membrane. This facilitates the delivery of the effector molecules into the target bacteria, leading to their death.

Image using a fluorescence microscope to identify differently fluorescing bacterial strains. A round morphology of the target bacteria and a reduction in their number confirm successful killing by the inhibitor bacteria.