Global phenotypic analysis and transcriptional profiling defines the weak acid stress response regulon in Saccharomyces cerevisiae

Abstract

Weak organic acids such as sorbate are potent fungistatic agents used in food preservation, but their intracellular targets are poorly understood. We thus searched for potential target genes and signaling components in the yeast genome using contemporary genome-wide functional assays as well as DNA microarray profiling. Phenotypic screening of the EUROSCARF collection revealed the existence of numerous sorbate-sensitive strains. Sorbate hypersensitivity was detected in mutants of the shikimate biosynthesis pathway, strains lacking the PDR12 efflux pump or WAR1, a transcription factor mediating stress induction of PDR12. Using DNA microarrays, we also analyzed the genome-wide response to acute sorbate stress, allowing for the identification of more than 100 genes rapidly induced by weak acid stress. Moreover, a novel War1p- and Msn2p/4p-independent regulon that includes HSP30 was identified. Although induction of the majority of sorbate-induced genes required Msn2p/4p, weak acid tolerance was unaffected by a lack of Msn2p/4p. Ectopic expression of PDR12 from the GAL1-10 promoter fully restored sorbate resistance in a strain lacking War1p, demonstrating that PDR12 is the major target of War1p under sorbic acid stress. Interestingly, comparison of microarray data with results from the phenotypic screening revealed that PDR12 remained as the only gene, which is both stress inducible and required for weak acid resistance. Our results suggest that combining functional assays with transcriptome profiling allows for the identification of key components in large datasets such as those generated by global microarray analysis.

Figure 2.

Clustering of transcript profiles. Hierarchial clustering of induction values representing average inductions over three replicate profiles of each the wild-type (W303-1A) and the isogenic derivatives (war1Δ, msn2Δ msn4Δ, war1Δ msn2Δ msn4Δ) after 20 min of 8 mM potassium sorbate treatment. Color codes refer to different regulatory groups after selection of differently regulated genes as revealed by ratio discrimination.

Figure 1.

(A) FUN34 is induced by sorbic acid in a War1p-dependent manner. Northern analysis of FUN34 under sorbate stress. Sorbic acid, 10 mM, was added to exponentially growing cultures of W303-1A and YAK120 (war1Δ), mRNA was prepared from aliquots taken at the indicated time points. Blots were probed for FUN34 and HSP30 mRNAs, methylene blue-stained rRNA served as a loading control. (B) Sequence logo of a putative consensus War1p DNA-binding motif. An alignment of related sequences detected by AlignACE in the promoter regions of War1p-dependent genes is displayed as a sequence logo. (C) Pdr12p levels are strongly reduced in rpb4Δ mutants. Wild-type and the indicated mutant strains were grown in YPD to the exponential growth phase, stressed with 10 mM sorbic acid and Pdr12p levels were assayed on Western blots. A cross-reacting band served as a loading control.

Figure 6.

Northern analysis of sorbic acid stress. (A) Exponentially growing cells of the indicated background (W303-1A, YAK120 (war1Δ), W303 msn2 msn4 (msn2Δ msn4Δ), and YCS105 (war1Δ msn2Δ msn4Δ) were stressed for 20 min with 10 mM sorbic acid. Strains W303-1A and YAK120 carried a TRP1 gene on the centromeric plasmid YCplac22. mRNA levels of HSP12, HSP30, and PDR12 were determined by simultaneous Northern hybridization. Methylene blue staining of rRNA served as a loading control. (B) HSP30 does not modulate sorbate sensitivity of pdr12Δ cells. Strains lacking the HSP30 and PDR12 genes in all combinations were tested for growth phenotypes on sorbic acid medium pH 4.5. Cells were spotted onto YPD, pH 4.5, with and without 0.3 mM sorbic acid and plates inspected after a 48-h incubation at 30°C.

Figure 3.

War1p, but not Msn2p/4p, is required for weak acid resistance. (A) Lack of MSN2 and MSN4 fails to influence growth properties on sorbic acid. Sensitivity of W303-1A and the mutant strains YBB14 (pdr12Δ), YAK120 (war1Δ), W303 msn2 msn4 (msn2Δ msn4Δ), and YCS105 (war1Δ msn2Δ msn4Δ) was tested on YPD pH4.5 plates. Strains W303-1A, YBB14, and YAK120 carried the TRP1 gene on the centromeric plasmid YCplac22. Growth was monitored after 48 h at 30°C. (B) HSP12, HSP26, and HSP104 are not required for sorbic acid resistance. BY4741 derivatives were spotted onto sorbate-containing plates. Colony growth was recorded after 48 h.

Figure 4.

Pdr12p is necessary and sufficient for sorbic acid resistance. (A) Pdr12p levels as derived from a GAL1-10-PDR12 gene are independent from War1p and sorbate challenge. Cells growing on YPD and YPgalactose were exposed to sorbic acid for 20 min. Immunoblots were decorated using polyclonal anti-Pdr12p antibodies. A cross-reacting band served as a loading control. (B) Cells from logarithmically growing cultures were spotted onto YPD, pH 4.5, and YPGal, pH 4.5, with and without 1 mM sorbic acid. Plates were monitored after a 48-h incubation at 30°C.

Figure 5.

Northern analysis of HSP12 and PDR12 expression during weak acid stress. (A) Cells were grown to the logarithmic growth phase in rich medium and exposed to the indicated weak acids for 20 min. Methylene blue staining of rRNA served as a loading control. (B) Weak acid stress-induced PDR12 promoter activity is dependent on WAR1. Logarithmically growing cells were treated with the indicated concentrations of weak organic acids for 45 min. β-galactosidase activity was determined in crude extracts. Wild-type: YAK3 PDR12-lacZ, war1Δ: YBB27 PDR12-lacZ war1Δ::HisMX6c.