Global Phenotypic Analysis and Transcriptional Profiling Defines the Weak Acid Stress Response Regulon inSaccharomyces 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 EURO-SCARF 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.

Novel stress responses facilitate Saccharomyces cerevisiae growth in the presence of the monocarboxylate preservatives

“…Resistances are induced to Acetic, but not propionic, sorbic or benzoic acids [19] Propionic, sorbic, benzoic acids, but not acetic acid [1,7] Key induced signalling pathway and activity HOG pathway signalling, leading to Hog1p MAP kinase activation [19] No evidence for a signalling pathway involvement; acid anion may act directly on the War1p transcription factor [13] Key action/target of the induced activity needed for the acquisition of resistance Phosphorylation of Fps1p, leading to Fps1p endocytosis [20] PDR12 gene induction, leading to an elevated plasma membrane level and activity of the Pdr12p ABC transporter [23,28] Probable related stress response Hog1p-directed downregulation of metalloid entry through the Fps1p channel [40] War1p activation in media containing leucine, methionine or phenylalanine as sole nitrogen source [9] Normal physiological function of the major target of the response Fps1p is the major glycerol channel of the plasma membrane, important for osmoadaptation [10,22,38] Pdr12p is also involved in export of the potentially toxic aromatic and branched-chain organic acids produced during amino acid catabolism [9] (2), a MAP kinase constitutively bound to, and that directly phosphorylates, Fps1p (3). This phosphorylation provides the signal for Fps1p endocytosis and degradation, eliminating this source of the acetic acid entry to the cell.…”

“…Acquisition of resistance to these larger acids appears to involve, not the loss of a membrane pore, but the induction of an activity catalysing active efflux of the acid anion from the cell (Figure 2b). Inhibitory amounts of propionic, sorbic and benzoic acid induce the activity of a zinc-finger transcription factor, War1p [7,13,28] (Table 1). Only a small number of genes are subject to War1p regulation and, of these, it seems that just a single gene -that encoding the Pdr12p plasma membrane ATP binding cassette (ABC) transporter -is of any major importance for weak organic acid resistance.…”

“…Only a small number of genes are subject to War1p regulation and, of these, it seems that just a single gene -that encoding the Pdr12p plasma membrane ATP binding cassette (ABC) transporter -is of any major importance for weak organic acid resistance. [28] No evidence has emerged for any involvement of a signal transduction process in War1p activation, so the direct binding of the preservative anion to this transcription factor may be the activation mechanism ( Table 1). The Zn 2 Cys 6 family yeast transcription factors, of which War1p is a member, are often found to be regulated by the binding of low molecular weight effector molecules.…”

“…This is an ABC transporter that provides resistance to those carboxylate compounds that can, to a reasonable extent, partition into both lipid and aqueous phases. [7,11,13,23,28] It may therefore initially bind acid anions that have become incorporated within the inner leaflet of the plasma membrane, anions orientated with their polar carboxylate groups exposed on the cytosolic surface of this membrane, then act by transporting these to the opposite (periplasmic) side of the membrane (i.e. act as a 'flippase'; Figure 2b).…”

Yeast ABC transporters – A tale of sex, stress, drugs and aging

“…War1p controls a rather small regulon [73], forms homodimers, is activated by stress-triggered hyper-phosphorylation (Frohner et al, in preparation), and decorates a cis-acting weak acid response element in the PDR12 promotor [72,73].…”