Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates

Bacteria encode diverse mechanisms for engaging in interbacterial competition. Here, we present a culture-based protocol for characterizing competitive interactions between bacterial isolates and how they impact the spatial structure of a mixed population.

This manuscript describes a culture-based, coincubation assay for detecting and characterizing competitive interactions between two bacterial populations. This method employs stable plasmids that allow each population to be differentially tagged with distinct antibiotic resistance capabilities and fluorescent proteins for selection and visual discrimination of each population, respectively. Here, we describe the preparation and coincubation of competing Vibrio fischeri strains, fluorescence microscopy imaging, and quantitative data analysis. This approach is simple, yields quick results, and can be used to determine whether one population kills or inhibits the growth of another population, and whether competition is mediated through a diffusible molecule or requires direct cell-cell contact. Because each bacterial population expresses a different fluorescent protein, the assay permits the spatial discrimination of competing populations within a mixed colony. Although the described methods are performed with the symbiotic bacterium V. fischeri using conditions optimized for this species, the protocol can be adapted for most culturable bacterial isolates.

This manuscript outlines a culture-based method to determine whether two bacterial isolates are capable of competitive interactions. When studying mixed populations, it is important to assess the extent to which the bacterial isolates interact, particularly whether isolates are directly competing through interference mechanisms. Interference competition refers to interactions where one population directly inhibits the growth or kills a competitor population¹. These interactions are important to identify because they can have profound effects on a microbial community’s structure and function^2,

1. Prepare Strains for Coincubation

1. Choose an appropriate reference strain that will serve as the target for bacterial competition during the coincubation assay. See Discussion for best practices when selecting a reference strain and how the reference strain will impact results. In this protocol, V. fischeri strain ES114 will serve as the reference strain.
2. Determining which selection and screening methods will be used to distinguish between the isolates in coincubat.......

In order to assess competitive interactions between bacterial populations, a coincubation assay protocol was developed and optimized for V. fischeri. This method utilizes stable plasmids that encode antibiotic resistance genes and fluorescent proteins, allowing for differential selection and visual discrimination of each strain. By analyzing the data collected from the coincubation assay, the competitive outcome of an interaction and the mechanism of the interaction can be identi.......

The coincubation assay described above provides a powerful method to discover interbacterial competition. This approach allowed for the identification of intraspecific competition among V. fischeri isolates and characterization of the competitive mechanism¹⁹. Although the method described was optimized for the marine bacterium V. fischeri, it can be easily modified to accommodate other bacterial species including clinical and environmental isolates. It is important to note that c.......

We would like to thank reviewers for their helpful feedback. A.N.S. was supported by the Gordon and Betty Moore Foundation through Grant GBMF 255.03 to the Life Sciences Research Foundation.