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.

Evolving challenges and strategies for fungal control in the food supply chain

“…, 2013 ). Furthermore, rare cells or spores can be hyper-resistant to the thresholds of food preservatives allowed in different products, such as soft drinks ( Geoghegan et al. , 2020 ; Stratford et al.…”

Transcription factors and ABC transporters: from pleiotropic drug resistance to cellular signaling in yeast

“…Like S. cerevisiae , the distally related spoilage mold Aspergillus niger also displays resistance to lipophilic weak acids. The zinc cluster TF WarA and the ABC transporter PdrA were identified in A. niger that function in an analogous manner to War1 and Pdr12 in S. cerevisiae despite low sequence identity [128]. Complementation of a S. cerevisiae Δpdr12 strain with pdrA recovered resistance to weak acids in a similar manner to PDR12 [128].…”

“…The zinc cluster TF WarA and the ABC transporter PdrA were identified in A. niger that function in an analogous manner to War1 and Pdr12 in S. cerevisiae despite low sequence identity [128]. Complementation of a S. cerevisiae Δpdr12 strain with pdrA recovered resistance to weak acids in a similar manner to PDR12 [128]. While Pdr12 mediated weak acid export is necessary for survival during lipophilic weak acid stress, complete adaptation requires plasma membrane alterations to limit diffusional uptake of the weak acids.…”

Natural Variation and the Role of Zn2Cys6 Transcription Factors SdrA, WarA and WarB in Sorbic Acid Resistance of Aspergillus niger

“…The transcription factor 'weak-acid resistance A' (WarA) has been recently described in A. niger, and is required for resistance against a range of weak-acid preservatives [33]. The knock-out strain (∆warA) showed sensitivity to propionic, butanoic, pentanoic, hexanoic, sorbic and benzoic acids.…”

“…In S. cerevisiae Pdr12p is regulated by War1p, a Zn 2 Cys 6 -finger transcription factor, which binds to the weakacid response element, WARE, in the Pdr12p promotor [30]. It should be noted that the S. cerevisiae War1p and A. niger WarA proteins are both Zn 2 Cys 6 transcription factors, but do not show significant sequence similarity apart from the DNA binding domain [33].…”