Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins

Summary

Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.  Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides.

Abstract

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms. We have shown that bacterially produced antimicrobial peptides (bacteriocins) dictate the outcome of these competitive interactions. All fully-sequenced pneumococcal strains harbor a bacteriocin-like peptide (blp) locus. The blp locus encodes for a range of diverse bacteriocins and all of the highly conserved components needed for their regulation, processing, and secretion. The diversity of the bacteriocins found in the bacteriocin immunity region (BIR) of the locus is a major contributor of pneumococcal competition. Along with the bacteriocins, immunity genes are found in the BIR and are needed to protect the producer cell from the effects of its own bacteriocin. The overlay assay is a quick method for examining a large number of strains for competitive interactions mediated by bacteriocins. The overlay assay also allows for the characterization of bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. The assay is performed by pre-inoculating an agar plate with a strain to be tested for bacteriocin production followed by application of a soft agar overlay containing a strain to be tested for bacteriocin sensitivity. A zone of clearance surrounding the stab indicates that the overlay strain is sensitive to the bacteriocins produced by the pre-inoculated strain. If no zone of clearance is observed, either the overlay strain is immune to the bacteriocins being produced or the pre-inoculated strain does not produce bacteriocins. To determine if the blp locus is functional in a given strain, the overlay assay can be adapted to evaluate for peptide pheromone secretion by the pre-inoculated strain. In this case, a series of four lacZ-reporter strains with different pheromone specificity are used in the overlay.

Introduction

Streptococcus pneumoniae, a common colonizer of the polymicrobial community of the nasopharynx, is able to compete with other pneumococci and closely related species through the production of bacterially produced antimicrobial peptides (bacteriocins). Every fully-sequenced pneumococcal strain examined to date contains a version of the bacteriocin-like peptide locus, blp. Bacteriocin production by pneumococcus has been demonstrated to be important in the outcome of both in vitro and in vivo competition^1-4. Competitive dynamics are influenced by the expression of d....

Protocol

1. Preparation of Producer Strain

1. Streak a pneumococcal strain to be tested for bacteriocin production on a 5% sheep blood tryptic soy agar plate from -80 °C freezer stocks.
   NOTE: The use of blood plates for initial growth of the strain to be tested for inhibition greatly increases the reproducibility of the assay. Additionally, other non-pneumococcal strains that produce bacteriocins can be used although growth conditions may need to be modified for a specific organism. We have used Streptococcus mitis and Lactococcus lactis in the overlay assay successfully.
2. Incubate the plate overnight (O/N) at 37 °C with 5% ....

Results

There are two possible outcomes in the overlay assay. It should be possible to see growth of the stabbed strain after overnight incubation. In Figure 1A, the overlay strain is sensitive to the bacteriocins being produced. A zone of clearance can be visualized surrounding the stabbed strain. Additionally, swirling of the stabbed strain into the overlay mixture is possible and can be seen in the Figure 1A. In Figure 1B, the overlay strain is immune to the bacteriocins bein.......

Discussion

This overlay assay is a rapid way to determine the range of activity for bacteriocin producers and to demonstrate competitive interactions that may occur among pneumococcal strains. The overlay assay can be adapted to use for screening multiple strains for bacteriocin production on a single plate. We have successfully screened large strain collections with this assay by using a 48-pin replicator to inoculate SBA plates from one half of a 96-well plate. After overnight incubation, growth is transferred to the TSA plate us.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Trypticase Soy Agar with 5% Sheep Blood	Fisher	B21261X	For growth from freezer cultures
Catalase	Sigma	C100-500mg	4,741 Units per plate
Todd Hewitt Broth + Yeast Extract (THY)	Fisher	DF0492-17-6	30 g of THB, 5 g yeast extract, 1 L ddH2O autoclave
Trypticase Soy Broth + Agar plates (TSA)	Fisher	B11768	30 g of TS, 15 g agar, 1 L ddH2O autoclave
5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal)	Sigma	B4252-50MG	Dissolve in dimethylformamide
Petri dishes	Fisher	FB0875712
Spectrophometer			Measures the OD620 of cultures
37 °C water bath
37 °C incubator with 5% CO2
15 ml conical tube