This study investigates competitive interactions between two bacterial strains using fluorescent markers. The competitor strain outcompetes the reference strain through contact-dependent and contact-independent mechanisms, leading to growth arrest and reduced fluorescence in the reference strain.
Begin with two bacterial strains, a reference and a competitor, each expressing a distinct fluorescent marker for strain identification.
Mix the strains in equal proportions and spot the mixture onto an agar plate.
Incubate the plate to allow the strains to grow and interact.
The competitor outcompetes the reference strain through either contact-dependent or contact-independent mechanisms.
In contact-dependent competition, the competitor uses specialized secretion systems to deliver toxins directly into the reference strain.
In contact-independent competition, the competitor releases toxins that diffuse into the surrounding medium and enter the reference strain.
These toxins disrupt essential processes in the reference strain, including cell wall synthesis, DNA replication, and protein synthesis, leading to growth arrest, cell death, and reduced fluorescence expression.
After incubation, examine the plate under a fluorescence microscope.
Reduced fluorescence in the reference strain, compared to the brighter fluorescence of the competitor, indicates competitive interactions.
To mix the reference strain and competitor strain in a one to one ratio, add 100 microliters of each strain normalized to OD 1.0 to a labeled 1.5 milliliter centrifuge tube and vortex for one to two seconds. As a control, mix the reference strain with a differentially tagged version of itself and vortex as well.
It is critical to achieve the correct starting ratio as it can considerably change the outcome of the experiment. To ensure success, this step may require optimization for a given bacterial species. After repeating this step for each biological replicate, there should be four biological replicates with differentially tagged reference strains as controls and four biological replicates with differentially tagged reference and competitor strains.
To make culture spots that will be used for fluorescence microscopy after the incubation, add 10 microliters of each control and experimental mixture onto a Petri plate containing LBS agar. After the spots have completely dried on the bench, incubate the plates at 24 degrees Celsius for 24 hours.
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