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1 School of Biology, University of Nottingham, University Park, Nottingham NG7 2RD, UK
2 Department of Microbiology, Swedish University of Agricultural Sciences, PO Box 7025, SE-750 07 Uppsala, Sweden
Correspondence
Peter Melin
petter.melin{at}mikrob.slu.se
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) because most yeasts and moulds are unable to grow in the food or packaging environment. Aspergillus niger is a recognized spoilage mould causing post-harvest decay of fruit, and is also among the most common fungi isolated from nuts, especially peanuts, cereals, meat products and cheese (Pitt & Hocking, 1997). A. niger shows resistance to sorbic acid through the degradation of this preservative by germinating spores (Plumridge et al., 2004, 2008). A. niger is also an important industrial organism in citric acid production and has a large number of mutant strains available and a published complete genome sequence (Bos et al., 1988; Pel et al., 2007; Schuster et al., 2002).
To prevent the growth of microbes in food, a very limited range of chemical compounds, with a proven history of safe consumption, has been approved in Europe for use in foods as preservatives (Anon, 1995). Many such preservatives are weak acids, and are collectively known as weak-acid preservatives. These include sorbic and benzoic acids, used in confectionery, dressings and soft drinks, acetic acid which is used as an acidulant in pickles, and propionic acid in bread-based foods. Most commonly, weak organic acids are used to inhibit fungal growth in low-pH foods, although several fungal species, notably in the genus Zygosaccharomyces, show a high level of resistance to preservatives (James & Stratford, 2003). To optimize the dosage and effectiveness of preservatives and to develop improved preservative strategies, it is important to elucidate the mechanisms underlying the antifungal activity of weak-acid preservatives. At present, the exact mechanisms behind the antifungal activity of these acids remain debatable. The classical weak-acid theory suggests that, at low pH, weak acids are uncharged and pass by diffusion through the fungal cell membrane. Once inside the cell, the acids ionize to the charged anionic form, e.g. sorbate, benzoate or acetate, which are unable to diffuse back through the cell membrane and therefore accumulate and acidify the cytoplasm (Neal et al., 1965; Freese et al., 1973; Krebs et al., 1983). This does not explain the widely differing toxicity of acetic and sorbic acids which have identical pKa values, or the far greater toxicity of longer-chain, more lipophilic acids. An effect of weak acids on the cell membrane has been suggested as a contributory toxic mechanism (Stratford & Anslow, 1998).
The A. niger pyrG gene encodes an ornithine decarboxylase enzyme, essential for the biosynthesis of uracil, and subsequent formation of uridine and RNA. pyrG– mutants are therefore cultured in the presence of excess uracil or uridine. In a previous study pyrG was used as a selectable marker for disruption of the padA1 gene in a pyrG– strain of A. niger (Plumridge et al., 2008) in order to confirm that the PadA1 enzyme catalysed the degradation of sorbic acid to 1,3-pentadiene, thereby conferring enhanced resistance to sorbic acid. In this study, we show that pyrG– strains of A. niger are more sensitive to sorbic acid than the wild-type (WT) and we investigate the underlying reason.
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METHODS |
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. All A. niger strains are derived from the same parent strain (N402) referred to here as the WT strain (Bos et al., 1988). The strains of S. cerevisiae used were CEN.PK derivatives (van Dijken et al., 2000). A. niger mutant strains were maintained on Aspergillus minimal medium [AMM: (all l–1) NaNO3, 6 g; KCl, 0.52 g; MgSO4 . 7H2O, 0.52 g; KH2PO4, 1.52 g; FeSO4 . 7H2O, 0.5 mg; ZnSO4 . 7H2O, 0.5 mg; glucose, 20 g; agar, 20 g] plates with the required supplements. The yeast strains were cultured on YPD agar (1 %, w/v, yeast extract, 1 %, w/v, peptone, 2 %, w/v, glucose, 1.5 %, w/v, agar). Conidial suspensions of A. niger were prepared in water containing 0.01 % (v/v) Tween 80 from freshly grown agar plates, whereas yeast inocula were from broth cultures.
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Transformation of A. niger to restore the pyrG+ phenotype.
Transformation of A. niger AB4.1 was performed with an EcoRI-linearized plasmid (van Hartingsveldt et al., 1987) containing the full-length A. niger pyrG gene. A. niger conidia (107 ml–1) were incubated in 400 ml AMM (containing 0.2 %, w/v, glucose, 10 mM uridine) at 28 °C for 16 h. Germlings were collected and washed on a Miracloth filter (Calbiochem). Protoplasting using Glucanex (Sigma) and transformation using PEG and CaCl2 were carried out as described by Melin et al. (2003). Uridine prototrophs from each strain were selected and subcultured at least three times, followed by selection of one colony after plating a diluted spore suspension.
Uridine uptake experiments.
Fresh suspensions of WT A. niger conidia (107 ml–1 in 200 ml ACM, pH 4.0, supplemented with 0.1 mM uridine) were incubated for 5 h at 28 °C to allow the spores to initiate growth and nutrient uptake. At this stage the conidia were swollen but no emerging germ tubes could be observed. The culture was then divided equally into four 50 ml tubes and the conidia were collected by centrifugation. Each pellet was resuspended and washed three times in succinate buffer (30 mM succinic acid, pH 4.0, supplemented with 0.2 %, w/v, glucose). Finally, all conidia were collected and the volume was adjusted to 4 ml and divided into four 2.0 ml Eppendorf tubes that were kept on ice for up to 40 min. One millilitre of the conidial suspension (about 5x107 ml–1) was added to 9.0 ml pre-warmed (28 °C) succinate buffer supplemented with 0.01 mM uridine and [2-14C]uridine (1.7x104 Bq; 0.10 µM; Sigma). Temperature was maintained at 28 °C using a water bath and the spore suspension was kept homogeneous with a magnetic stirrer. Samples of 2.0 ml were taken after 1, 10, 20 and 30 min. Sorbic acid was added to a final concentration of 5.0 mM after 15 min. Samples (2.0 ml) were filtered through 2.5 cm glass fibre filters (Whatman GF/B; pore size 1.0 µm). Filters were pre-treated before sampling with 1 ml ice-cold succinate buffer containing 1.0 mM uridine. After each sample had been filtered, the filter was washed five times with 1 ml ice-cold succinate buffer containing 1.0 mM uridine. For analysis, filters were placed into scintillation vials with 10 ml Emulsifier-Safe (Perkin-Elmer) scintillation fluid. Activities were estimated using a liquid scintillation analyser (Tri-Carb 2100TR; Packard).
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RESULTS |
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). After 72 h, both the mutant and the WT formed large colonies on the control plates. However, on plates supplemented with 1.0 mM sorbic acid, a pyrG mutant colony was only observed at the highest inoculum level, whereas the WT colonies were only slightly smaller than on the control plate (Fig. 1). At 2.0 mM sorbic acid, no growth was observed for the AB4.1 mutant, while the WT grew at all inoculation levels (data not shown).
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Since MIC values alone do not provide full information on whether the growth (rate or yield) of an organism is reduced at subinhibitory concentrations, we complemented this study by estimating growth rates over 96 h, and biomass yields after 16 days. This study was also carried out using a sorbic acid analogue, hexanoic acid, which is not a substrate for PadA1 and therefore eliminates any potential experimental complications due to weak-acid degradation (Plumridge et al., 2008). The growth rates of A. niger WT and AB4.1 strains were similar in control media (i.e. without weak-acid supplementation). The AB4.1 strain had a much slower growth rate than WT when cultured in the presence of either 1.0 mM sorbic or hexanoic acids (Fig. 2a and b). However, if given sufficient time, the slow-growing AB4.1 strain in the presence of 1.0 mM sorbic or hexanoic acids eventually reached the same biomass yield as WT strains (Fig. 2c). Therefore, while the amount of uridine may not limit the yield of biomass, uptake of this base appears to limit the growth rate in strain AB4.1.
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), showing only a marginal increase in resistance with increased uridine. Replacing uridine with uracil in the medium, however, resulted in increased resistance to sorbic and hexanoic acids in the uridine auxotroph AB4.1 compared to the WT strain (Fig. 3b).
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). At the end of the control experiment, less than 10 % of the added radiolabelled uridine had been incorporated into the swollen spores. There was no evidence of leakage of radiolabel from cells after sorbic acid inhibition of uptake. The concentration of sorbic acid used in this experiment (5.0 mM) was the minimal concentration required to prevent germination of AB4.1 in an overnight experiment using a conidial concentration of 5x107 ml–1 (data not shown).
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). To learn whether the increased sensitivity (compared to WT) was dependent on the hydrophobicity or chain length of the fatty acid, we exposed cells to the aliphatic series of acids from C2 (acetic acid) up to C10 (decanoic acid). For both the WT and strain AB4.1, the MIC values were reduced with increased length, and hydrophobicity, of the acid. Strain AB4.1 was sensitive to all acids tested (Table 2). The MIC value of the WT strain for decanoic acid was above the solubility limit at this pH, preventing a numerical comparison.
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). In contrast, the nicotinic-acid- and all amino-acid-auxotrophic strains tested (see Table 1) showed an overall similar level of resistance as the WT (data not shown). In addition we could not observe any significant difference in biomass yield compared to the WT at subinhibitory concentrations (data not shown).
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ura3) of S. cerevisiae. Using a similar experimental procedure as used for A. niger, the MIC values of acetic, sorbic and decanoic acids were found to be similar in the S. cerevisiae WT strains and uridine auxotrophs (data not shown), suggesting that uridine uptake is not inhibited by weak acids in S. cerevisiae.
MIC of acids, aldehydes, alcohols and antibiotics
The adenine-auxotrophic A. niger strain A890 and uridine-auxotrophic strains were more sensitive to fatty acids than the WT. Further tests examined their sensitivities towards aromatic weak acids, alcohols, aldehydes and two antibiotics. The inhibitory effect of all tested acids, including the structurally unrelated benzoic acid, was stronger in both the adenine and uridine auxotrophs than in the WT (Table 3), and reduced growth at subinhibitory concentrations was also observed (data not shown). In contrast, when we treated strains with sorbic aldehyde and decanol, two compounds that both showed strong antifungal activity and are structurally related to sorbic and decanoic acid, respectively, the observed MICs were similar in the mutants and the WT (Table 3). Furthermore, no additional sensitivity to the two antibiotics hygromycin B and amphotericin B could be observed in the auxotrophs (Table 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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In contrast to A. niger, uridine-auxotrophic strains of S. cerevisiae did not show abnormal sensitivity to weak acids, in accordance with a previous report where the authors screened the S. cerevisiae gene-deletion strain collection and did not observe any hypersensitivity to sorbic acid in strains deleted in any gene involved in nucleotide synthesis (Mollapour et al., 2004). It therefore appears that the uridine transporter in A. niger is sensitive to weak-acid inhibition, whereas the S. cerevisiae homologue is not. It is probable that the adenine transporter is also weak-acid-sensitive in A. niger. Conversely, there may be other transporters in S. cerevisiae that are specifically weak-acid-sensitive. It has been reported that a tryptophan auxotroph in S. cerevisiae was abnormally sensitive to sorbic acid (Bauer et al., 2003) although, in this instance, supplying the medium with tryptophan in excess could circumvent the effect. Since the trp marker is commonly used for selection in yeast, this led to several erroneous reports of gene interactions in weak-acid resistance. Here, no such general sensitivity to a variety of weak acids was seen in the trpA auxotroph of A. niger. It has also been observed that in nitrogen-starved Penicillium chrysogenum mycelia, weak acids inhibit leucine transport (Hunter & Segel, 1973), but this study is hard to interpret and compare with ours since no acid concentrations were given. Weak acids have also been shown to reduce the uptake of several amino acids in the bacterium Bacillus subtilis (Sheu et al., 1972), but this appears to be a general effect on active transport (Freese et al., 1973).
The mechanisms behind the observed sensitivity to weak acids of A. niger strains auxotrophic for nucleotide bases are likely to be due to inhibited uptake of the nucleotide, since we could not observe any uridine uptake into sorbic-acid-treated swollen spores. In S. cerevisiae, there exist separate but homologous transporters for uridine and uracil, and an unrelated, non-homologous adenine transporter (Nelissen et al., 1997) and homologous proteins in A. niger can be found in the recently annotated genome sequence (Pel et al., 2007). The data presented here indicate a strong effect of sorbic acid on the A. niger uridine transporter and an as yet unproven effect on the adenine and uracil transporters.
The mechanism by which weak acids inhibit the transporters remains speculative at present. Weak acids or their hydrophilic anions could interact directly with the aqueous portions of transport proteins protruding from either face of the plasma membrane. Alternatively, the observed effect could be caused by the weak acids integrating into the lipid bilayer of the membrane, thereby interacting with the hydrophobic portions of the transporters. The partition coefficients of lipophilic weak acids suggest that they may accumulate to significant levels in membranes, depending on the acid (Leo et al., 1971). Since it is known that transporters may be affected by their lipid environment (Keenan et al., 1982), it is tempting to speculate that the altered environment of a weak-acid-containing membrane may inhibit the activity of the A. niger uridine transporter. In that case, some of the transporters (e.g. uridine) would be more sensitive to the lipid environment than others (e.g. amino acids). Other mechanisms whereby weak acids may impair the uptake of uridine may also be involved, but data are not available to inform a choice between possibilities. At present, the most likely explanation is that the lipophilic weak acids affect the environment of transmembrane transporters within the membrane and that some transporters (e.g. for uridine) are more sensitive than others.
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ACKNOWLEDGEMENTS |
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Edited by: H. A. B. Wösten
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REFERENCES |
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Plumridge, A., Stratford, M., Lowe, K. C. & Archer, D. B. (2008). The weak-acid preservative sorbic acid, is decarboxylated and detoxified by a phenylacrylic acid decarboxylase, PadA1, in the spoilage mold Aspergillus niger. Appl Environ Microbiol 74, 550–552.
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Received 25 October 2007;
revised 18 January 2008;
accepted 24 January 2008.
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, N402 (control); 
, N402 (sorbic acid); 
, AB4.1 (control); 
, AB4.1 (sorbic acid). (b) Yield after growth in 1.0 mM hexanoic acid. 
