Weak Acid Resistance A (WarA), a Novel Transcription Factor Required for Regulation of Weak-Acid Resistance and Spore-Spore Heterogeneity in Aspergillus niger

Abstract

Propionic, sorbic, and benzoic acids are organic weak acids that are widely used as food preservatives, where they play a critical role in preventing microbial growth. In this study, we uncovered new mechanisms of weak-acid resistance in molds. By screening a library of 401 transcription factor deletion strains in Aspergillus fumigatus for sorbic acid hypersensitivity, a previously uncharacterized transcription factor was identified and named weak acid resistance A (WarA). The orthologous gene in the spoilage mold Aspergillus niger was identified and deleted. WarA was required for resistance to a range of weak acids, including sorbic, propionic, and benzoic acids. A transcriptomic analysis was performed to characterize genes regulated by WarA during sorbic acid treatment in A. niger Several genes were significantly upregulated in the wild type compared with a ΔwarA mutant, including genes encoding putative weak-acid detoxification enzymes and transporter proteins. Among these was An14g03570, a putative ABC-type transporter which we found to be required for weak-acid resistance in A. niger We also show that An14g03570 is a functional homologue of the Saccharomyces cerevisiae protein Pdr12p and we therefore name it PdrA. Last, resistance to sorbic acid was found to be highly heterogeneous within genetically uniform populations of ungerminated A. niger conidia, and we demonstrate that pdrA is a determinant of this heteroresistance. This study has identified novel mechanisms of weak-acid resistance in A. niger which could help inform and improve future food spoilage prevention strategies.IMPORTANCE Weak acids are widely used as food preservatives, as they are very effective at preventing the growth of most species of bacteria and fungi. However, some species of molds can survive and grow in the concentrations of weak acid employed in food and drink products, thereby causing spoilage with resultant risks for food security and health. Current knowledge of weak-acid resistance mechanisms in these fungi is limited, especially in comparison to that in yeasts. We characterized gene functions in the spoilage mold species Aspergillus niger which are important for survival and growth in the presence of weak-acid preservatives. Such identification of weak-acid resistance mechanisms in spoilage molds will help in the design of new strategies to reduce food spoilage in the future.

Keywords: Aspergillus; food spoilage; fungi; transcription factors; weak-acid resistance.

FIG 1

Screening of A. fumigatus deletant library. (A) Example of A. fumigatus deletant library screen. Conidial suspensions of the different deletants were arrayed in 96-well plates and transferred to growth medium using a 96-pin tool. Examples of putatively sorbic acid sensitive strains are circled in yellow. (B) Sensitivity of A. fumigatus transcription factor deletion strains to sorbic acid. Sixty-two strains were identified from the initial screen in panel A as putatively sorbic acid hypersensitive and subjected to a second round of screening, as outlined in Materials and Methods. Sensitivity to sorbic acid relative to the WT strain is shown (a value of 1 indicates identical sensitivity of the deletion strain to the WT, according to radial growth). ΔwarA refers to the ΔAFUB_000960 mutant.

FIG 2

Growth of A. fumigatus transcription factor deletion strains on medium containing weak acids. (A) Radial growth of A. fumigatus transcription factor deletion strains on agar containing 0.5 mM sorbic acid. Images were captured after 3 days of growth at 37°C. (B) Radial growth of the A. fumigatus ΔwarA mutant and wild type on agar containing weak acids. Plates were inoculated with a 10-fold dilution series of conidial suspensions; approximate numbers of conidia are indicated above the pictures. Images were captured after 2 days of growth at 28°C and are representative of 2 or 3 independent experiments. The concentrations of the acids used are given in Materials and Methods.

FIG 3

Radial growth of the A. niger ΔwarA mutant growing on different weak acids. Plates were inoculated with a 10-fold dilution series of conidial suspensions; approximate numbers of conidia are indicated above the pictures. Images were captured after 2 days of growth at 28°C and are representative of 2 or 3 independent experiments. The concentrations of acids used are given in Materials and Methods.

FIG 4

qRT-PCR of genes differentially regulated in WT and ΔwarA mutant strains of A. niger. Transcript abundances in WT (black bars) and ΔwarA mutant (gray bars) conidia germinated in control medium or in the presence of 1 mM sorbic acid or 1 mM benzoic acid. Error bars are standard deviation of the results from 3 technical replicates. WT and ΔwarA mutant transcript abundances were compared by Student's t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

FIG 5

Radial growth of ΔAn02g09970 and ΔAn14g03570 (ΔpdrA) mutant strains growing on weak acids. Plates were inoculated with a 10-fold dilution series of conidial suspensions.

FIG 6

Growth of S. cerevisiae complemented strains on weak acids. Tenfold dilution series of S. cerevisiae strains (isogenic with the BY4743 wild type) were inoculated onto medium containing weak acids. The Δpdr12 mutant strain was transformed with either empty plasmid (+YEp351), YEp351 plasmid containing PDR12 (+PDR12), or YEp351 plasmid containing the pdrA ORF and PDR12 promoter and terminator (+pdrA). Two independent transformants of the +pdrA strain are shown.

FIG 7

Sorbic acid dose response curves for A. niger conidia. (A) Dose-response curves of germinated (blue lines) and ungerminated (black/gray lines) WT conidia, and comparison of slope values. Dose-response curve slope values were compared with 2-way Welch’s t test (P = 0.0404,) n = 2 or 3. (B) Dose-response curves of WT (black/gray lines) and ΔpdrA mutant (pink/red lines) conidia and comparison of dose-response curve slope values, compared using a 2-way Welch’s t test (P = 0.0468), n = 3. Two representative independent experiments are shown in the dose-response curve. ATAN, arctangent.