Concomitant osmotic and chaotropicity-induced stresses in Aspergillus wentii: compatible solutes determine the biotic window.

Whereas osmotic stress response induced by solutes has been well-characterized in fungi, less is known about the other activities of environmentally ubiquitous substances. The latest methodologies to define, identify and quantify chaotropicity, i.e. substance-induced destabilization of macromolecular systems, now enable new insights into microbial stress biology (Cray et al. in Curr Opin Biotechnol 33:228-259, 2015a, doi: 10.1016/j.copbio.2015.02.010 ; Ball and Hallsworth in Phys Chem Chem Phys 17:8297-8305, 2015, doi: 10.1039/C4CP04564E ; Cray et al. in Environ Microbiol 15:287-296, 2013a, doi: 10.1111/1462-2920.12018 ). We used Aspergillus wentii, a paradigm for extreme solute-tolerant fungal xerophiles, alongside yeast cell and enzyme models (Saccharomyces cerevisiae and glucose-6-phosphate dehydrogenase) and an agar-gelation assay, to determine growth-rate inhibition, intracellular compatible solutes, cell turgor, inhibition of enzyme activity, substrate water activity, and stressor chaotropicity for 12 chemically diverse solutes. These stressors were found to be: (i) osmotically active (and typically macromolecule-stabilizing kosmotropes), including NaCl and sorbitol; (ii) weakly to moderately chaotropic and non-osmotic, these were ethanol, urea, ethylene glycol; (iii) highly chaotropic and osmotically active, i.e. NH4NO3, MgCl2, guanidine hydrochloride, and CaCl2; or (iv) inhibitory due primarily to low water activity, i.e. glycerol. At ≤0.974 water activity, Aspergillus cultured on osmotically active stressors accumulated low-M r polyols to ≥100 mg g dry weight(-1). Lower-M r polyols (i.e. glycerol, erythritol and arabitol) were shown to be more effective for osmotic adjustment; for higher-M r polyols such as mannitol, and the disaccharide trehalose, water-activity values for saturated solutions are too high to be effective; i.e. 0.978 and 0.970 (25 ºC). The highly chaotropic, osmotically active substances exhibited a stressful level of chaotropicity at physiologically relevant concentrations (20.0-85.7 kJ kg(-1)). We hypothesized that the kosmotropicity of compatible solutes can neutralize chaotropicity, and tested this via in-vitro agar-gelation assays for the model chaotropes urea, NH4NO3, phenol and MgCl2. Of the kosmotropic compatible solutes, the most-effective protectants were trimethylamine oxide and betaine; but proline, dimethyl sulfoxide, sorbitol, and trehalose were also effective, depending on the chaotrope. Glycerol, by contrast (a chaotropic compatible solute used as a negative control) was relatively ineffective. The kosmotropic activity of compatible solutes is discussed as one mechanism by which these substances can mitigate the activities of chaotropic stressors in vivo. Collectively, these data demonstrate that some substances concomitantly induce chaotropicity-mediated and osmotic stresses, and that compatible solutes ultimately define the biotic window for fungal growth and metabolism. The findings have implications for the validity of ecophysiological classifications such as 'halophile' and 'polyextremophile'; potential contamination of life-support systems used for space exploration; and control of mycotoxigenic fungi in the food-supply chain.

Physiological relationship between food preservatives, environmental factors, ochratoxin and otapksPV gene expression by Penicillium verrucosum.

There is significant interest in trying to understand the relationship between environmental factors, preservative concentration and expression of genes involved in mycotoxin production. However, little information is available on the link between physiological stress factors and expression of genes responsible for mycotoxin production. This study has examined the effect of interactions between ionic and non-ionic water availability modified with glycerol or NaCl (a(w), 0.98, 0.95 and 0.93) and sub-optimal concentrations of calcium propionate and potassium sorbate (150, 300 ppm) at 25 degrees C on growth, ochratoxin A (OTA) and otapksPV gene expression by the mycotoxigenic species Penicillium verrucosum. Growth was inhibited between 25-35% by the preservatives at each a(w) level. However, OTA production was stimulated by 150 and 300 ppm of both preservatives, especially at 0.95 and 0.93 a(w). If water activity as a single stress factor was changed, a typical OTA production and otapksPV expression profile occurred, indicating that OTA biosynthesis is activated under two conditions, i.e., under optimal growth conditions and under weak stress conditions. Temporal analysis of otapksPV expression showed that there was an optimum after 8-9 days incubation. Statistical analyses indicated good correlation between sub-optimal concentrations of preservatives, intermediate a(w) levels and genotypic and phenotypic gene and toxin production. This is the first time that genotypic information has been linked to phenotypic mycotoxin production in relation to such interacting stress factors.