This study investigates the competitive interactions between two bacterial strains, focusing on the mechanisms of cell killing via the type six secretion system (T6SS). The use of fluorescent proteins allows for visual differentiation of the strains during imaging.
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.
Measure and record the optical density at 600 nanometers for all samples. Normalize each sample to an optical density of 1 by diluting the culture with LBS medium.
Mix the two competing strains at an equal ratio by adding 30 microliters of each normalized strain to a labeled 1.5-milliliter tube. Vortex the mixed strain culture for 1 to 2 seconds. Mix the competing strains for each biological replicate and treatment to generate a total of 4 mixed-strain tubes.
Concentrate each mixed culture three-fold by centrifuging, to ensure sufficiently dense competing cells for contact-dependent killing during co-incubation. Discard the supernatant. Resuspend each pellet and 20 microliters of LBS medium, and perform concentration procedures for each sample.
If imaging on an inverted microscope, begin by spotting two microliters of a mixed culture onto the #1.5 coverslip bottom of a 35-millimeter Petri dish and place the agarose pad over the co-incubation spot. Place a 12-millimeter circular glass coverslip over the agarose pad. Repeat spotting and placing of coverslip for the remaining 3 mixed cultures, which will result in 4 dishes to be imaged.
Before proceeding ahead, let the slides sit on the benchtop for about 5 minutes to allow the cells to settle on the agar pad, to avoid movement during the imaging process.
Begin by focusing on cells using DIC to minimize photobleaching effects. Based on the average size of a single bacterial cell, use a 100x oil objective. Adjust the exposure time and acquisition settings for each channel with minimal background detection. Select at least 5 fields of view, and acquire images in each appropriate channel.
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