Benzoic acid, a weak organic acid food preservative, exerts specific effects on intracellular membrane trafficking pathways in Saccharomyces cerevisiae

Abstract

Microbial spoilage of food causes losses of up to 40% of all food grown for human consumption worldwide. Yeast growth is a major factor in the spoilage of foods and beverages that are characterized by a high sugar content, low pH, and low water activity, and it is a significant economic problem. While growth of spoilage yeasts such as Zygosaccharomyces bailii and Saccharomyces cerevisiae can usually be retarded by weak organic acid preservatives, the inhibition often requires levels of preservative that are near or greater than the legal limits. We identified a novel synergistic effect of the chemical preservative benzoic acid and nitrogen starvation: while exposure of S. cerevisiae to either benzoic acid or nitrogen starvation is cytostatic under our conditions, the combination of the two treatments is cytocidal and can therefore be used beneficially in food preservation. In yeast, as in all eukaryotic organisms, survival under nitrogen starvation conditions requires a cellular response called macroautophagy. During macroautophagy, cytosolic material is sequestered by intracellular membranes. This material is then targeted for lysosomal degradation and recycled into molecular building blocks, such as amino acids and nucleotides. Macroautophagy is thought to allow cellular physiology to continue in the absence of external resources. Our analyses of the effects of benzoic acid on intracellular membrane trafficking revealed that there was specific inhibition of macroautophagy. The data suggest that the synergism between nitrogen starvation and benzoic acid is the result of inhibition of macroautophagy by benzoic acid and that a mechanistic understanding of this inhibition should be beneficial in the development of novel food preservation technologies.

FIG. 1.

Effects of benzoic acid on macroautophagy. (A) Benzoic acid inhibits autophagy-dependent maturation of of prApe1 in a vac8Δ mutant. Exponentially growing cells (HAY394) were harvested and resuspended in medium with (+) or without (−) nitrogen and various concentrations of benzoic acid. After 2 h of incubation, 0.5 OD₆₀₀ unit of cells was harvested, and protein extracts were blotted and probed with anti-Ape1 antibody. The data are representative of the data from duplicate independent experiments in which identical results were obtained. (B) Benzoic acid inhibits autophagy-dependent maturation of Pho8Δ60. Cells expressing Pho8Δ60 (HAY369) were treated as described above for panel A, and alkaline phosphatase activity was determined in the extracts. One unit was defined as 1 nmol of nitrophenol evolved per mg of protein per min (4). The error bars indicate standard errors (n = 3).

FIG. 2.

Effects of benzoic acid on the trafficking of CPY and Ape1. (A) Benzoic acid at a concentration of 2 mM does not inhibit the Cvt pathway. Exponentially growing wild-type cells (HAY75) were harvested and resuspended in synthetic dextrose medium containing different concentrations of benzoic acid. Following 4 h of incubation, total protein was extracted, and 0.5 OD₆₀₀ equivalent was separated by SDS-PAGE and probed with anti-Ape1 antibody. As a control to identify the relative mobility of prApe1, extracts from an atg1Δ strain, SLY2, were probed in parallel. (B) Benzoic acid at a concentration of 2 mM delays but does not block CPY maturation. Two OD₆₀₀ units of wild-type cells (HAY75) per time point was pulsed for 5 min with 10 μCi of a radioactive cysteine-methionine mixture and chased with unlabeled amino acids plus 2 mM benzoic acid. Samples were taken at different times and separated into intracellular fractions and extracellular fractions. Total protein was extracted and immunoprecipitated with anti-CPY antibody and analyzed by SDS-PAGE and autoradiography.

FIG. 3.

Mutants with ATG1 deleted are defective in adaptation to 2 mM benzoic acid. Wild-type (SLY3) and atg1Δ (SLY2) cells were grown to the log phase, and identical numbers of cells (1 × 10⁶ cells) were serially diluted in a microtiter plate, plated on SD medium plates with 2 mM benzoic acid (pH 4.5), and grown at 26 and 30°C for 5 days. WT, wild type.

FIG. 4.

Nitrogen starvation enhances the toxicity of benzoic acid. Exponentially growing wild-type cells (HAY75 in panels A and B; SLY3 in panel C) were resuspended at a density of 1 OD₆₀₀ in SD medium (pH 4.5) supplemented with 1 mM benzoic acid (A) or 2 mM benzoic acid (B and C) in the presence or absence of a nitrogen source, as well as with nitrogen starvation alone (A). The cell survival at different times is expressed as a percentage of the initial count. Values greater than 100% reflect continued cell growth. The error bars indicate standard deviations (n = 3). BA, benzoic acid; BA-N, benzoic acid with nitrogen starvation.

FIG. 5.

Differential effects of sorbic, acetic, and benzoic acids. (A) Exponentially growing vac8Δ cells (HAY394) were resuspended at a density of 1 OD₆₀₀ unit in SD medium (control) (pH 4.5), nitrogen starvation medium (SD-N) (pH 4.5), or nitrogen starvation medium supplemented with different concentrations of acetic acid (2 to 20 mM), benzoic acid (2 mM), and sorbic acid (2 and 15 mM) at pH 4.5. Following 2 h of incubation, cells were collected, and lysates were prepared and probed for prApe1 maturation by Western blotting as described in Materials and Methods. (B) Exponentially growing wild-type cells (HAY75) were resuspended at a density of 1 OD₆₀₀ unit in citrate-buffered SD medium (pH 3) supplemented with 0, 2, 5, 10, or 20 mM acetic acid in the presence or absence of a nitrogen source. The cell survival at different times is expressed as a percentage of the initial count. Values greater than 100% reflect continued cell growth. The data are representative of the results of two independent experiments. acetic acid-N, acetic acid treatment with nitrogen starvation.