Studies on weak organic acid resistance in yeast

Certain yeasts can grow at low pH in the presence of the highest levels of weak organic acid preservatives permitted in foods, leading to spoilage of many manufactured products. This study sought to gain further understanding into the weak acid mechanisms of the yeast Saccharomyces cerevisiae, to help identify novel routes to inhibit the growth of this yeast. Weak acids are thought to act by reducing intracellular pH (pHi), thus disrupting cellular homeostasis. To definitively link pHi to weak acid inhibition, a new fluorescent in-vivo pHi method was developed (Chapter 3) to study the effects of this stress on pHi and growth. The inhibitory action of sorbic acid was found to cause disruption of pHi, necessitating induction of an energy-consuming response to rebalance homeostasis. The latter in turn reduced the available free energy for growth and division. To determine other key factors involved in yeast weak acid resistance, the proteome of cells under sorbate stress was investigated using high-resolution two-dimensional gels (Chapter 4). This identified eight cytosolic proteins that were up-regulated. However, contrary to hypothesis, phenotypic evaluation of the knock out deletion mutants of the identified proteins revealed that none were hypersensitive to sorbate. To gain further insight into these results, the two genetically almost identical strains used in the above proteomic and phenotypic studies were compared. Results showed that the strain used for the proteomic study (Chapter 4) was far more sorbate sensitive than the other used for the physiological measurements (Chapter 3). Since the only major difference was that the former was a tryptophan auxotroph and the latter a tryptophan phototroph, it was investigated in Chapter 5 whether this increased weak acid sensitivity was due to the auxotrophic requirement for tryptophan (i.e. the strain was trp-). High levels of exogenous tryptophan suppressed this enhanced sensitivity of trp- cells (Chapter 5). It is apparent therefore that increased weak acid sensitivity arises because the acids strongly inhibit the uptake of the aromatic acids from the medium, rendering trp- cells extremely sensitive to their requirement to catalyse this uptake. This identification of this trp- effect has implications for the choice of strains used for future weak acid studies. It has allowed us to reassess the findings of previous studies, where gene functions have been assigned based upon studies using trp- strains