Natural abundance 13C-nuclear magnetic resonance spectroscopic analysis of acyclic polyol and trehalose accumulation by several yeast species in response to salt stress.

Analysis of seventeen yeast strains by 13C-NMR spectroscopy has confirmed the significance of glycerol as the sole osmoregulatory solute under salt-stressed conditions, and has shown arabitol to be present in most of the osmotolerant species. Ribitol was detected in some species, including Debaryomyces hansenii, although ribitol accumulation did not correlate with the osmotic pressure of the medium. Relative amounts of arabitol and ribitol decreased in relation to glycerol when the external osmotic pressure was increased. Trehalose was present during exponential growth of some species.

Silver tolerance and accumulation in yeasts.

Debaryomyces hansenii (NCYC 459 and strain 75-21), Candida albicans (3153A), Saccharomyces cerevisiae (X2180-1B), Rhodotorula rubra (NCYC 797) and Aureobasidium pullulans (IMI 45533 and ATCC 42371) were grown on solid medium supplemented with varying concentrations of AgNO3. Although Ag+ is highly toxic towards yeasts, growth on solid media was still possible at Ag concentrations of 1-2 mM. Further subculture on higher Ag concentrations (up to 5 mM) resulted in elevated tolerance. The extent of Ag tolerance depended on whether Ag-containing plates were exposed to light prior to inoculation since light-mediated reduction of Ag+ to Ag0 resulted in the production of a less toxic silver species. Experimental organisms exhibited blackening of colonies and the surrounding agar during growth on AgNO3-containing medium especially at the highest Ag concentrations tested. All organisms accumulated Ag from the medium; electron microscopy revealed that silver was deposited as electron-dense granules in and around cell walls and in the external medium. X-ray microprobe analysis indicated that these granules were metallic Ag0 although AgCl was also present in some organisms. Volatile and non-volatile reducing compounds were produced by several test organisms which presumably effected Ag+ reduction to Ag0.

Toxicity of organotins towards the marine yeastDebaryomyces hansenii.

Of nine organotin compounds tested towards the marine yeastDebaryomyces hansenii, only triphenyltin chloride (Ph3SnCl) and mono-, di-, and tributyltin chloride induced significant K(+) release from cells which was symptomatic of viability loss. The general order of toxicity of the butylated compounds was tributyltin chloride (Bu3SnCl) > monobutyltin chloride (BuSnCl3) ≫ dibutyltin chloride (Bu2SnCl2). The overall toxicity of Ph3SnCl was similar to BuSnCl3. Release of K(+) induced by butylated tin compounds or by Ph3SnCl was strongly dependent on the external pH. Maximal toxicity occurred at pH 6.5 for Bu3SnCl, BuSnCl3, and Ph3SnCl, whereas maximal toxicity of Bu2SnCl2 occurred at pH 5.0. Toxicity was decreased above or below these values. The toxicity of BuSnCl3, Bu3SnCl, and Ph3SnCl was reduced at salinity levels approximating to sea water conditions. Prior growth ofD. hansenii in 3% (w/v) NaCl also resulted in reduced sensitivity to Bu3SnCl as evidenced by a decreased rate and extent of K(+) efflux. Bu3SnCl-induced Na(+) release from cells grown in the absence or presence of 3% (w/v) NaCl was low and similar in both cases. It appeared that the monovalent cation was important in the reduction of Bu3SnCl toxicity since Na2SO4 had a similar protective effect as NaCl while CsCl completely prevented K(+) efflux. Thus, the effects of external NaCl were related both to Na(+) and to Cl(-). These results emphasize that cellular and environmental factors influence the toxic effects of organotins and suggests that these compounds may be more effective antimicrobial agents in some environmental niches than in others.

Osmotic significance of glycerol accumulation in exponentially growing yeasts.

Natural-abundance 13C-nuclear magnetic resonance spectroscopy has shown glycerol to be the major osmotically significant low-molecular-weight solute in exponentially growing, salt-stressed cells of the yeasts Saccharomyces cerevisiae, Zygosaccharomyces rouxii, and Debaromyces hansenii. Measurement of the intracellular nonosmotic volume (i.e., the fraction of the cell that is osmotically unresponsive) by using the Boyle-van't Hoff relationship (for nonturgid cells, the osmotic volume is directly proportional to the reciprocal of the external osmotic pressure) showed that the nonosmotic volume represented up to 53% of the total cell volume; the highest values were recorded in media with maximum added NaCl. Determinations of intracellular glycerol levels with respect to cell osmotic volumes showed that increases in intracellular glycerol may counterbalance up to 95% of the external osmotic pressure due to added NaCl. The lack of other organic osmotica in 13C-nuclear magnetic resonance spectra indicates that inorganic ions may constitute the remaining component of intracellular osmotic pressure.