Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates

Summary

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.

Abstract

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.

Introduction

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,

Protocol

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.......

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
1.5 mL Microcentrifuge Tubes	Fisher	05-408-129
10 μL multichannel pipette
100 μL multichannel pipette
300 μL multichannel pipette
10 μL single channel pipette
20 μL single channel pipette
200 μL single channel pipette
1000 μL single channel pipette
24-well plates	Fisher	07-200-84	sterile with lid
96-well plates	VWR	10062-900	sterile with lid
Calculator
Chloramphenicol	Sigma	C0378	stock (20 mg/mL in Ethanol); final concentration in media (2 μg /mL LBS)
Fluorescence dissecting microscope with camera and imaging software
forceps	Fisher	08-880
Kanamycin Sulfate	Fisher	BP906-5	stock (100 mg/mL in water, filter sterilize); final concentration in media (1 μg/mL LBS)
Nitrocellulose membrane (FS MCE, 25MM, NS)	Fisher	SA1J788H5	0.22 μm nitrocellulose membrane (pk of 100)
petri plates	Fisher	FB0875713	sterile with lid
Spectrophotometer
Semi-micro cuvettes	VWR	97000-586
TipOne 0.1-10 μL starter system	USA Scientific	1111-3500	10 racks
TipOne 200 μL starter system	USA Scientific	1111-500	10 racks
TipOne 1000 μL starter system	USA Scientific	1111-2520	10 racks
Vortex
Name	Company	Catalog Number	Comments
LBS media
1M Tris Buffer (pH ~7.5)			50 mL 1 M stock buffer (62 mL HCl, 938 mL DI water, 121 g Trizma Base)
Agar Technical	Fisher	DF0812-17-9	15 g (Add only for plates)
DI water			950 mL
Sodium Chloride	Fisher	S640-3	20 g
Tryptone	Fisher	BP97265	10 g
Yeast Extract	Fisher	BP9727-2	5 g