Genome-wide screening of Saccharomyces cerevisiae to identify genes required for antibiotic insusceptibility of eukaryotes

Abstract

The adverse reactions provoked by many antibiotics in humans are well documented but are generally poorly understood at the molecular level. To elucidate potential genetic defects that could give rise to susceptibility to prokaryote-specific antibiotics in eukaryotes, we undertook genome-wide screens using the yeast Saccharomyces cerevisiae as a model of eukaryotes; our previous work with a small number of yeast mutants revealed some specific gene functions required for oxytetracycline resistance. Here, the complete yeast deletion strain collection was tested for growth in the presence of a range of antibiotics. The sensitivities of mutants revealed by these screens were validated in independent tests. None of the approximately 4,800 defined deletion strains tested were found to be sensitive to amoxicillin, penicillin G, rifampin, or vancomycin. However, two of the yeast mutants were tetracycline sensitive and four were oxytetracycline sensitive; encompassed among the latter were mutants carrying deletions in the same genes that we had characterized previously. Seventeen deletion strains were found to exhibit growth defects in the presence of gentamicin, with MICs for the strains being as low as 32 micro g ml(-1) (the wild type exhibited no growth defects at any gentamicin concentration tested up to 512 micro g ml(-1)). Strikingly, 11 of the strains that were most sensitive to gentamicin carried deletions in genes whose products are all involved in various aspects of vacuolar and Golgi complex (or endoplasmic reticulum) function. Therefore, these and analogous organelles, which are also the principal sites of gentamicin localization in human cells, appear to be essential for normal resistance to gentamicin in eukaryotes. The approach and data described here offer a new route to gaining insight into the potential genetic bases of antibiotic insusceptibilities in eukaryotes.

FIG. 1.

Screening for gentamicin sensitivity using the S. cerevisiae deletion strain collection. Strains were cultured in liquid YEPD medium in a 96-well format and replica inoculated onto YEPD agar supplemented with gentamicin (256 μg ml⁻¹). The results are for 1 strain set (strain set 4_3; Euroscarf) of a total of 76 strain sets examined with each antibiotic after incubation for 3 days at 30°C. Circles highlight strains that exhibited slight (position C12; gcs1Δ) and strong (position G6; luv1Δ) sensitivities to gentamicin relative to their growth on the control plate lacking gentamicin. The gentamicin sensitivities of these strains were subsequently validated (Fig. 2 and Table 1). Empty inocula on the control plate correspond to positions at which essential open reading frames were originally deleted, producing nonviable mutants.

FIG. 2.

Validation and quantification of antibiotic (gentamicin) sensitivity. All 19 putative gentamicin-sensitive strains identified during initial screening of the deletion strain collection were tested quantitatively for antibiotic sensitivity. (A) Grid of putative gentamicin-sensitive mutants identified from screening (WT, wild type). (B) Mutants of interest were cultured in liquid YEPD medium and adjusted to an OD₆₀₀ of ∼0.01 before they were spotted (4 μl) onto unsupplemented and gentamicin-supplemented YEPD agar (the strains in the grid correspond to those shown in panel A). (C) MIC determination. Mutants were cultured as described above for panel B and adjusted to an OD₆₀₀ of ∼0.03 before replica inoculation with a 1- to 2-μl pin tool onto YEPD agar supplemented with a range of gentamicin concentrations (1 to 512 μg ml⁻¹); the results obtained with 0, 64, and 512 μg of gentamicin ml⁻¹ are shown. All plates were incubated for 3 days at 30°C before examination. Typical results from one of several replicates are shown.