Using Coculture to Detect Chemically Mediated Interspecies Interactions

Summary

Bacteria produce secreted compounds that have the potential to affect the physiology of their microbial neighbors. Here we describe a coculture screen that allows detection of such chemically mediated interspecies interactions by mixing soil microbes with fluorescent transcriptional reporter strains of Bacillus subtilis on solid media.

Abstract

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by secreting metabolites. These metabolites have the potential to modulate the physiology and differentiation of their microbial neighbors and are likely important factors in the establishment and maintenance of complex microbial communities. We have developed a fluorescence-based coculture screen to identify such chemically mediated microbial interactions. The screen involves combining a fluorescent transcriptional reporter strain with environmental microbes on solid media and allowing the colonies to grow in coculture. The fluorescent transcriptional reporter is designed so that the chosen bacterial strain fluoresces when it is expressing a particular phenotype of interest (i.e. biofilm formation, sporulation, virulence factor production, etc.) Screening is performed under growth conditions where this phenotype is not expressed (and therefore the reporter strain is typically nonfluorescent). When an environmental microbe secretes a metabolite that activates this phenotype, it diffuses through the agar and activates the fluorescent reporter construct. This allows the inducing-metabolite-producing microbe to be detected: they are the nonfluorescent colonies most proximal to the fluorescent colonies. Thus, this screen allows the identification of environmental microbes that produce diffusible metabolites that activate a particular physiological response in a reporter strain. This publication discusses how to: a) select appropriate coculture screening conditions, b) prepare the reporter and environmental microbes for screening, c) perform the coculture screen, d) isolate putative inducing organisms, and e) confirm their activity in a secondary screen. We developed this method to screen for soil organisms that activate biofilm matrix-production in Bacillus subtilis; however, we also discuss considerations for applying this approach to other genetically tractable bacteria.

Introduction

We are interested in understanding how the metabolites that bacteria secrete affect the physiology and development of neighboring microbes. Many metabolites have been characterized for their bioactive effects on other microbes. Two well-described examples include antibiotics, which inhibit the growth of other microbes, and quorum sensing molecules, which alter the global gene expression of other microbes. However, bacteria produce many other small molecule natural products that have no known bioactivities¹. We hypothesize that bacteria have evolved and preserved the ability to produce some of these metabolites because they allow them to modulate the cellula....

Protocol

1. Select a Reporter Gene and Construct a Fluorescent Transcriptional Reporter

For B. subtilis:

1. See the JoVE article in reference ¹⁹ for a protocol describing the construction of fluorescent transcriptional reporters in Bacillus subtilis.

For other bacterial species:

1. Identify a gene that is upregulated during the physiological response of interest. This can be based on existing literature or transcriptional analysis of the microbe under particular conditions.
2. Construct a fluorescent transcriptional reporter for this gene to act as a ....

Results

This screen was used to identify soil organisms secreting compounds that alter the physiology of B. subtilis. The results described here focus on the matrix-producing cell type of B. subtilis, which produces the protein and exopolysaccharide that are required for robust biofilm formation in this bacterium. We selected the promoter of the tapA-sipW-tasA operon for our fluorescent reporter construct (P_tapA-yfp). This operon encodes the protein structural component of the matri.......

Discussion

One of the inherent limitations of this protocol is that it relies on the cultivability of microbial organisms. As has been well documented²⁴, most microbial life on the planet cannot (yet) be grown under the culturing conditions explored to date. Thus, a huge number of interactions between microbial species that are occurring in natural settings will go undetected using this approach. However, since our desire is to not only identify the existence of such interactions, but then also study the mechanisms and m.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Spectrophotometer			Any spectrophotomer capable of measuing OD600 absorbance values.
Luria broth, Lennox	VWR	80017-484	Alternative media sources may be necessary.
Glass beads, 3 mm	VWR	26396-508
Gel loading tips, round	VWR	29442-666
Glass rods	VWR	59060-069
Fluorescence dissecting stereoscope	Zeiss	N/A	The author used a Zeiss Stemi SV6 dissection stereoscope with an EXFO X-cite 120 fluorescent light source, a long-pass YFP filter cube, an achromat 0.63X objective, 10X eyepieces, and an Axio HRC HR digital camera. Most screening was done with the focusing mount at 2.0-3.2X. Any dissecting stereoscope with fluorescence capabilities is fine, provided you have the correct filters for the FP you are using. It is best if there is a shutter that allows you to easily switch between brightfield and fluorescense, as well as a stage that allows illumination from above and below. If you want to capture images, an attached camera is also necessary.