Hyperaccumulation of zinc by zinc-depleted Candida utilis grown in chemostat culture

Abstract

The steady-state levels of zinc in Candida utilis yeast grown in continuous culture under conditions of zinc limitation are 2+/mg dry weight of cells. Unlike carbon-limited cells, zinc-depleted cells from a zinc-limited chemostat possess the capacity to accumulate and store zinc at levels far in excess of the steady-state level of 4 nmol/mg dry biomass observed in carbon-limited chemostat cultures. Zinc uptake is energy-dependent and apparently unidirectional since accumulated ⁶⁵Zn neither exits from preloaded cells nor exchanges with cold Zn²⁺. The transport system exhibits a high affinity for Zn²⁺ (K_m = 0.36 μM) with a V_max of 2.2 nmol per minute per milligram dry weight of cells. Growth during the period of the uptake assay is responsible for the apparent plateau level of 35 nmol Zn²⁺/mg dry weight of cells achieved after 20–30 min in the presence of ⁶⁵Zn at pH 4.5 and 30 °C. Inhibition of growth during the uptake assay by cycloheximide results in a biphasic linear pattern of zinc accumulation where the cellular zinc is about 60 nmol/mg dry weight after 1 h. The enhanced level of accumulated zinc is not inhibitory to growth. Zinc-depleted C. utilis contains elevated amounts of polyphosphate and this anionic polymer must be considered a possible zinc-sequestering and storage agent. Present experimental evidence does not allow discrimination between possible regulation of zinc homeostasis either by inhibition of zinc efflux through control of the membrane carrier or by control of the synthesis of a cytoplasmic zinc-sequestering macromolecule.