Molecular and Cell Biology, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK1
Author for correspondence: Alistair J. P. Brown. Tel: +44 1224 273183. Fax: +44 1224 273144. e-mail: al.brown{at}abdn.ac.uk
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: Yeast physiology, benzoic acid, phosphofructokinase, glycolysis
Abbreviations: Pyk1, pyruvate kinase; PF1K, 6-phosphofructo-1-kinase; PF2K, phosphofructo-2-kinase; Pfk1, PF1K subunit; Pfk2, PF1K ß subunit; Pfk26 and Pfk27, PF2K isozymes; F26BPase, fructose-2,6-bisphosphatase
a Present address: MRC Radiation and Genome Stability Unit, Harwell, Didcot, Oxfordshire OX11 ORD, UK.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Two main mechanisms have been proposed to account for the effects of weak acids upon the growth of yeast. The first model suggests that growth inhibition is due to the intracellular depletion of ATP, rather than the inhibition of glycolytic enzymes such as 6-phosphofructo-1-kinase (PF1K) (Warth, 1988 , 1991
). In response to acidification, the plasma membrane H+-ATPase (Pma1) pumps protons out of the cell in an ATP-dependent manner (Eraso & Gancedo, 1987
; Ramos et al., 1989
; Viegas & Sà-Correia, 1991
). Growth in the presence of a weak acid has been shown to depend upon Pma1 activity (Holyoak et al., 1996
), and the length of the adaptation period, before growth can resume, has been predicted to depend upon the time taken to raise the intracellular pH to neutrality (Lambert & Stratford, 1999
). Recently, it was demonstrated that normal levels of resistance to weak acids depend upon an ABC transporter (Pdr12; Piper et al., 1998
). This transporter pumps benzoate from the yeast cell in an ATP-dependent manner (Holyoak et al., 1999
). Hence, both Pma1 and Pdr12 might contribute to ATP depletion as they combat intracellular accumulation of both protons and benzoate anions, respectively.
A second model suggests that benzoic acid blocks glycolysis, thereby inhibiting growth (Krebs et al., 1983 ). These authors showed that benzoic acid addition leads to a reduction in the rate of glucose fermentation and the accumulation of glycolytic intermediates before PF1K. This was consistent with a block in glycolysis at PF1K. Their basic model was supported and extended by François et al. (1986
, 1988
). They showed that PF1K activity decreased following benzoic acid addition and that there was also a reduction in the levels of fructose 2,6-bisphosphate, a potent activator of PF1K. Benzoic acid was also shown to inhibit purified 6-phosphofructo-2-kinase (PF2K) in vitro (François et al., 1988
). Furthermore, it has been reported that growth in the presence of a weak acid depends upon optimal glycolytic flux (Holyoak et al., 1996
). However, a direct causal relationship between the inhibition of glycolysis and the antimicrobial effects of the weak acid remains to be proven.
In this study we have further examined the relationship between glycolysis and benzoic acid tolerance, and in particular, the influence of PF1K and its activator, fructose-2,6-bisphosphate. We show that (a) wild-type rates of glycolytic flux are not required for growth in the presence of sublethal concentrations of benzoic acid, (b) yeast cells become more sensitive to benzoic acid when PF1K or PF2K levels are lowered artificially, and (c) benzoic acid sensitivity can be suppressed by overexpressing a subunit of PF2K or by inactivating fructose-2,6-bisphosphatase (F26BPase). In this way we have confirmed a causal relationship between the inhibition of glycolysis and the antimicrobial effects of benzoic acid.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Strain construction.
Strain genotypes are presented in Table 1. YKC strains represent a congenic set of PFK1 and PFK2 mutants that were made in S. cerevisiae W303-1B (Pearce et al., 2000
). Congenic
fbp26 mutants were made in W303-1B and YKC14 by targeted gene disruption, as described by Wach et al. (1998)
. Briefly, a
fbp26::kanMX4 cassette was made by PCR amplifying the kanMX4 marker using the primers 5'-TGGGTCGCGCCAATGCGCTCACAAATCTATTCTTATCCCTAACTTACATCAAGCTTGCCTCGTCCCCGCCGGGTC-3' and 5'-TTTGGTGACCTTTGTGCCATAGGCCCTAGGCTCCAGCTTGATCAATGTGTGTCGACACTGGATGGCGGCGTTAG-3' (FBP26 sequences in italics; kanMX4 sequences underlined). The PCR product, which deletes from -191 to +1180 of the 1356 bp FBP26 ORF, was transformed into W303-1B and YKC14 (Gietz & Woods, 1998
), geneticin-resistant colonies were selected, and the fbp26 deletion was confirmed by Southern analysis. The construction of the congenic strains VW-1B and VW.EB-13B was described previously (Müller et al., 1997
). To construct the multicopy plasmid YEpPFK26, YIplacPFK26Asp644 (Müller et al., 1997
) was linearized with HindIII and ligated to the LEU2, 2 µm HindIII fragment from pMA91 (Mellor et al., 1985
).
|
Assays.
Pyruvate kinase (Pyk1) assays were carried out as described previously (Hunsley & Suelter, 1969 ; Yun et al., 1976
). PF1K assays were carried out in duplicate using procedures adapted from Reibstein et al. (1986)
. Reaction mixtures contained 3·6 mM ATP, 2·7 U aldolase, 48 U triosephosphate isomerase, 4·7 U
-glycerophosphate dehydrogenase, 1·4 mM fructose 6-phosphate, 0·1 mM KCl, 8 mM MgCl2, 10 mM NH4Cl, 2·5 mM 2-mercaptoethanol, 5 mM fructose 2,6-bisphosphate and 50 mM PIPES, pH 7·2. Protein extracts were added and after equilibration for 1 min at 23 °C, reactions were initiated by addition of NADH to 0·2 mM. Rates of decrease in A340 from 0·1 to 1·0 min-1 were measured and expressed relative to the amount of total protein used in the assay. Protein determinations were performed using the Bradford assay (Bradford, 1976
). Fructose 2,6-bisphosphate was measured according to Van Schaftingen et al. (1982)
.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Effect of benzoic acid upon PF1K and fructose 2,6-bisphosphate levels
The differing contributions of the PF1K and ß subunits to benzoic acid tolerance could have been due to differential effects of the weak acid upon the expression levels of the individual subunits. However, Western analysis revealed that the levels of these proteins were not significantly affected by exposure to benzoic acid (Fig. 3
). This analysis reconfirmed the reduction in PF1K level in YKC14 compared to its wild-type parent (Fig. 3
; Pearce et al., 2000
).
|
Effect of modulating PF2K levels upon benzoic acid tolerance
If benzoic acid sensitivity is influenced by fructose 2,6-bisphosphate concentration, one would expect changes in PF2K to affect benzoic acid tolerance. In S. cerevisiae, the two PF2K isozymes are encoded by PFK26 and PFK27 (Kretschmer & Fraenkel, 1991 ; Boles et al., 1996
). Therefore, we examined the effects of deleting both genes upon growth in the presence and absence of benzoic acid (Fig. 4
). Even in the absence of benzoic acid, the double
pfk26
pfk27 mutation slowed growth in acidic medium (pH 4·6), and this effect was exacerbated by addition of benzoic acid. Therefore, PF2K is required for normal growth under acidic conditions.
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It has been suggested that growth in the presence of a weak acid depends upon optimal glycolytic flux (Holyoak et al., 1996 ). However, a yeast Pyk1 mutant that displays reduced rates of glycolytic flux (Pearce et al., 2000
) was relatively insensitive to sublethal concentrations of benzoic acid (YKC11, Fig. 2
). Also, a PF1K mutant that displays wild-type rates of glycolytic flux (Pearce et al., 2000
) was relatively sensitive to benzoic acid (YKC14, Fig. 2
). These data indicate that suboptimal glycolytic flux is sufficient for yeast to tolerate at least 1 mM benzoate. They also support previous reports suggesting that PF1K represents a potential target for this weak acid preservative (Krebs et al., 1983
; François et al., 1986
).
The two PF1K subunits displayed differential sensitivities to benzoic acid (Fig. 2). Mutants that only contained the
subunit were relatively sensitive, whereas those that only contained the ß subunit were not. This suggests that the PF1K
subunit is inhibited by benzoic acid (Fig. 2
). Heinisch et al. (1996)
have reported that the PF1K
and ß subunits are activated to similar extents by fructose 2,6-bisphosphate. Here we report that the weak acid did not significantly affect the level of either subunit (Fig. 3
). Hence, benzoic acid appears to exert a direct effect upon the activity, rather than the synthesis or degradation, of the PF1K
subunit.
Benzoic acid addition led to a dramatic decline in fructose 2,6-bisphosphate concentration in YKC14 (Fig. 3), confirming a previous report by François et al. (1986)
. This suggested that, in addition to direct inhibition of the PF1K
subunit, benzoic acid might also exert an indirect effect upon glycolysis by reducing the levels of this important allosteric activator of PF1K. Yeast mutants with altered PF1K levels display compensatory changes in the concentration of the allosteric activator, fructose 2,6-bisphosphate (Davies & Brindle, 1992
; Pearce et al., 2000
). Fructose 2,6-bisphosphate levels are abnormally high in YKC14, and this is thought to allow normal rates of glycolytic flux in this strain, despite its low PF1K levels (Pearce et al., 2000
). Therefore, the sensitivity of YKC14 to benzoic acid might be due to the combined effects of this weak acid upon the fructose 2,6-bisphosphate concentration and the PF1K
subunit.
Further examination of the relationship between benzoic acid and fructose-2,6-bisphosphate confirmed this view. Acid sensitivity was increased by inactivating PF2K (pfk26
pfk27; Fig. 4
). In addition, the benzoic acid sensitivity of YKC14 was suppressed by overexpressing PF2K (PFK26Asp644; Fig. 5
), or by inactivating F26BPase (
fbp26; Fig. 6
). These data strongly suggest that benzoic acid mediates its effects upon yeast growth, at least in part, by decreasing fructose 2,6-bisphosphate levels. This decrease is probably mediated by the stimulation of F26BPase combined with the inhibition of PF2K (François et al., 1986
, 1988
).
Müller et al. (1997) showed that yeast cells lacking PF2K (
pfk26
pfk27) are able to grow at normal rates under fermentative conditions, suggesting that under these conditions they are able to sustain glycolytic flux in the absence of fructose 2,6-bisphosphate. Our data indicate that the stimulatory activity of fructose 2,6-bisphosphate is required to maintain adequate glycolytic flux when yeast cells are exposed to weak acid stress.
Our data support the model of Krebs et al. (1983) , which suggests that the antimicrobial effects of benzoic acid are mediated, at least partly, by inhibition of glycolysis at PF1K. However, our data do not invalidate the alternative idea that growth is inhibited as a result of the intracellular depletion of ATP (Warth, 1988
). Pma1 (Eraso & Gancedo, 1987
; Ramos et al., 1989
; Viegas and Sà-Correia, 1991
; Serrano, 1991
) and Pdr12 (Piper et al., 1998
; Holyoak et al., 1999
), probably expend a large proportion of total cellular ATP pumping protons and benzoate anions from the cell.
Hence, the antimicrobial effects of benzoic acid appear to be mediated by a combination of factors. The growth of yeast cells is probably inhibited by their reduced capacity to generate ATP (via the inhibition of glycolytic flux), combined with their need to expend considerable amounts of ATP in their attempt to maintain homeostasis.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bartrons, R., Van Schaftingen, E., Vissers, S. & Hers, H.-G.(1982). The stimulation of yeast phosphofructokinase by fructose-2,6-bisphosphate. FEBS Lett 143, 137-140.[Medline]
Boles, E., Göhlmann, H. W. H. & Zimmermann, F. K.(1996). Cloning of a second gene encoding 6-phosphofructo-2-kinase in yeast, and characterisation of mutant strains without fructose-2,6-bisphosphate. Mol Microbiol 20, 65-76.[Medline]
Booth, I. R. & Kroll, R. G.(1989). The preservation of foods by low pH. In Mechanisms of Action of Food Preservation Procedures , pp. 119-160. Edited by G. W. Gould. London:Elsevier.
Bradford, M. M.(1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Anal Biochem 72, 248-254.[Medline]
Church, G. M. & Gilbert, W.(1984). Genomic sequencing. Proc Natl Acad Sci USA 81, 1991-1995.[Abstract]
Davies, S. E. C. & Brindle, K. M.(1992). Effects of overexpression of phosphofructokinase on glycolysis in the yeast Saccharomyces cerevisiae. Biochemistry 31, 4729-4735.[Medline]
Eraso, P. & Gancedo, J. M.(1987). Activation of yeast plasma membrane ATPase by acid pH during growth. FEBS Lett 224, 187-192.[Medline]
Feinberg, A. P. & Vogelstein, B.(1983). A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132, 6-13.[Medline]
François, J., Van Schaftingen, E. & Hers, H.-G.(1986). Effect of benzoate on the metabolism of fructose-2,6-bisphosphate in yeast. Eur J Biochem 154, 141-145.[Abstract]
François, J., Van Schaftingen, E. & Hers, H.-G.(1988). Characterisation of phosphofructokinase 2 and of enzymes involved in the degradation of fructose-2,6-bisphosphate in yeast. Eur J Biochem 171, 599-608.[Abstract]
Gietz, R. D. & Woods, R. A.(1998). Transformation of yeast by the lithium acetate/single-stranded carrier DNA/PEG method. In Yeast Gene Analysis: Methods in Microbiology , pp. 53-66. Edited by A. J. P. Brown & M. F. Tuite. London:Academic Press.
Heinisch, J. J., Boles, E. & Timpel, C.(1996). A yeast phosphofructokinase insensitive to the allosteric activator fructose-2,6-bisphosphate. J Biol Chem 271, 15928-15933.
Hoffman, C. S. & Winston, F.(1987). A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57, 267-272.[Medline]
Holyoak, C. D., Stratford, M., McMullin, Z., Cole, M. B., Crimmins, K., Brown, A. J. P. & Coote, P. J.(1996). Activity of the plasma membrane H+-ATPase and optimal glycolytic flux are required for rapid adaptation and growth of Saccharomyces cerevisiae in the presence of the weak acid preservative, sorbic acid. Appl Environ Microbiol 62, 3158-3164.[Abstract]
Holyoak, C., Bracey, D., Piper, P. W., Kuchler, K. & Coote, P.(1999). The Saccharomyces cerevisiae weak-acid-inducible ABC transporter Pdr12 transports fluorescein and preservative anions from the cytosol by an energy-dependent mechanism. J Bacteriol 181, 4644-4652.
Hunsley, J. R. & Suelter, C. H.(1969). Yeast pyruvate kinase: kinetic properties. J Biol Chem 244, 4819-4822.
Krebs, H. A., Wiggins, D., Stubbs, M., Sols, A. & Bedoya, A.(1983). Studies on the mechanism of the antifungal action of benzoate. Biochem J 214, 657-663.[Medline]
Kretschmer, M. & Fraenkel, D. G.(1991). Yeast 6-phosphofructo-2-kinase: sequence and mutant. Biochemistry 30, 10663-10672.[Medline]
Lambert, R. J. & Stratford, M.(1999). Weak-acid preservatives: modelling microbial inhibition and response. J Appl Microbiol 86, 157-164.[Medline]
Mellor, J. E., Dobson, M. J., Roberts, N. A., Kingsman, A. J. & Kingsman, S. M.(1985). Factors affecting heterologous gene expression in Saccharomyces cerevisiae. Gene 33, 215-226.[Medline]
Müller, S., Zimmermann, F. K. & Boles, E.(1997). Mutant studies of phosphofructo-2-kinases do not reveal an essential role of fructose-2,6-bisphosphate in the regulation of carbon fluxes in yeast cells. Microbiology 143, 3055-3061.[Abstract]
Paravicini, G. & Kretschmer, M.(1992). The yeast FBP26 gene codes for a fructose-2,6-bisphosphatase. Biochemistry 31, 7126-7133.[Medline]
Pearce, A. K., Crimmins, K., Groussac, E., Hewlins, M. J. E., Dickinson, J. R., Francois, J., Booth, I. R. & Brown, A. J. P.(2000). Pyruvate kinase (Pyk1) levels influence both the rate and direction of carbon flux in yeast under fermentative conditions. Microbiology 147, 391-401.
Piper, P. W., Mahe, Y., Thompson, S., Pandjaitan, R., Holyoak, C., Egner, R., Muhlbauer, M., Coote, P. & Kuchler, K.(1998). The Pdr12 ABC transporter is required for the development of weak organic acid resistance in yeast. EMBO J 17, 4257-4265.
Ramos, S. M., Balbin, M., Raposo, E. & Pardo, L. A.(1989). The mechanism of intracellular acidification induced by glucose in Saccharomyces cerevisiae. J Gen Microbiol 135, 2413-2422.[Medline]
Reibstein, D., den Hollander, J. A., Pilkis, S. J. & Shulman, R. G.(1986). Studies on the regulation of yeast phosphofructo-1-kinase: its role in aerobic and anaerobic glycolysis. Biochemistry 25, 219-227.[Medline]
Roe, A. J., McLaggan, D., Davidson, I., OByrne, C. & Booth, I. R.(1998). Perturbation of anion balance during inhibition of growth of Escherichia coli by weak acids. J Bacteriol 180, 767-772.
Serrano, R.(1991). Transport across yeast vacuolar and plasma membranes. In The Molecular Biology of the Yeast Saccharomyces: Genome Dynamics, Protein Synthesis and Energetics , pp. 523-585. Edited by J. N. Strathern, E. W. Jones & J. R. Broach. Cold Spring Harbor, NY:Cold Spring Harbor Laboratory.
Thomas, B. J. & Rothstein, R.(1989). The genetic control of direct-repeat recombination in Saccharomyces: the effect of rad52 and rad1 on mitotic recombination at GAL10, a transcriptional regulation gene. Genetics 123, 725-738.
Towbin, H., Staehelin, T. & Gordon, J.(1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76, 4350-4354.[Abstract]
Van Schaftingen, E., Lederer, B., Bartrons, R. & Hers, H.-G.(1982). A kinetic study of pyrophosphate: fructose-6-phosphate phosphotransferase from potato tubers. Eur J Biochem 129, 191-195.[Abstract]
Viegas, C. A. & Sà-Correia, I.(1991). Activation of plasma membrane ATPase of Saccharomyces cerevisiae by octanoic acid. J Gen Microbiol 137, 645-651.[Medline]
Wach, A., Brachat, A., Rebischung, C., Steiner, S., Pokorni, K, te Heesen, S. & Philippsen, P.(1998). PCR-based gene targeting in Saccharomyces cerevisiae. In Yeast Gene Analysis: Methods in Microbiology , pp. 67-81. Edited by A. J. P. Brown & M. F. Tuite. London:Academic Press.
Warth, A. D.(1988). Effect of benzoic acid on growth yield of yeasts differing in their resistance to preservatives. Appl Environ Microbiol 54, 2091-2095.
Warth, A. D.(1991). Effect of benzoic acid on glycolytic metabolite levels and intracellular pH in Saccharomyces cerevisiae. Appl Environ Microbiol 57, 3415-3417.[Medline]
Wicksteed, B. L., Collins, I., Dershowitz, A., Stateva, L. I., Green, R. P., Oliver, S. G., Brown, A. J. P. & Newlon, C. S.(1994). A physical comparison of chromosome III in six strains of Saccharomyces cerevisiae. Yeast 10, 39-57.[Medline]
Yun, S. L., Aust, A. E. & Suelter, C. H.(1976). A revised preparation of yeast (Saccharomyces cerevisiae) pyruvate kinase. J Biol Chem 251, 124-128.[Abstract]
Received 31 August 2000;
revised 3 November 2000;
accepted 22 November 2000.