Halo Assay for Toxic Peptides and Other Compounds in Microorganisms

We describe an assay for determination of toxicity in S. cerevisiae involving spotting of a toxic peptide on a lawn of yeast cells. This assay may be generalized to determine toxicity of a variety of compounds by substituting a putative toxic compound in place of the peptide. The general protocol may also be used to determine toxicity of any small compound toward another microorganism by replacing S. cerevisiae with the target microbe and modifying growth conditions accordingly.

Di-/tripeptides are one of the major sources of nitrogen, carbon, and amino acids for all organisms. Synthetic peptides containing a toxic amino acid residue provide an experimental approach to measure peptide transport and/or utilization in Saccharomyces cerevisiae. Hydrolysis of internalized peptides by intracellular peptidases or proteases releases the toxic residue leading to an easily detectable zone (halo) of growth arrest on a lawn of cells plated in a Petri plate. For example, upon intracellular hydrolysis the toxic peptide Ala-Eth releases ethionine (Eth), a methionine antagonist which interferes with the incorporation of amino acids into proteins and with the normal methylation of DNA and other methylation pathways, thereby leading to cell death. When spotted onto a lawn of yeast cells, the transported dipeptide Ala-Eth will inhibit growth, and a clear 'halo' will form in the lawn of cells around the region where the Eth-containing toxic peptide is spotted (Figure 1A). The assay described here for determination of peptide toxicity in S. cerevisiae may be generalized as follows: (1) it may be modified to determine toxicity of any substrate by simply using a putative toxic compound in place of a peptide containing a toxic amino acid, or (2) it may be modified to determine toxicity of a substrate toward any microorganism by replacing S. cerevisiae in the assay with the target organism. It is a simple, inexpensive and relatively rapid method for determining substrate toxicities as modified for the specific organism and toxic moiety assayed.