Copper ions stimulate polyphosphate degradation and phosphate efflux in Acidithiobacillus ferrooxidans

Abstract

For some bacteria and algae, it has been proposed that inorganic polyphosphates and transport of metal-phosphate complexes could participate in heavy metal tolerance. To test for this possibility in Acidithiobacillus ferrooxidans, a microorganism with a high level of resistance to heavy metals, the polyphosphate levels were determined when the bacterium was grown in or shifted to the presence of a high copper concentration (100 mM). Under these conditions, cells showed a rapid decrease in polyphosphate levels with a concomitant increase in exopolyphosphatase activity and a stimulation of phosphate efflux. Copper in the range of 1 to 2 microM greatly stimulated exopolyphosphatase activity in cell extracts from A. ferrooxidans. The same was seen to a lesser extent with cadmium and zinc. Bioinformatic analysis of the available A. ferrooxidans ATCC 23270 genomic sequence did not show a putative pit gene for phosphate efflux but rather an open reading frame similar in primary and secondary structure to that of the Saccharomyces cerevisiae phosphate transporter that is functional at acidic pH (Pho84). Our results support a model for metal detoxification in which heavy metals stimulate polyphosphate hydrolysis and the metal-phosphate complexes formed are transported out of the cell as part of a possibly functional heavy metal tolerance mechanism in A. ferrooxidans.

FIG. 1.

Transmission electron microscopy and energy dispersive X-ray analysis of A. ferrooxidans. Unstained and unfixed cells taken from sulfur-containing medium were examined directly for the presence of electron-dense granules. The elemental composition of a granule (left spectrum) and a cytoplasmic area (right spectrum) was analyzed by energy dispersive X-ray analysis. Arrows indicate the signals corresponding to oxygen and phosphorus.

FIG. 2.

Growth and polyphosphate levels of A. ferrooxidans in the presence of copper ions. A. ferrooxidans cultures were inoculated in sulfur medium with 1.75 mM P_i in the presence of the indicated concentration of CuSO₄, and cells were counted daily (A). To determine polyphosphate levels (B), the cells in A were harvested in the early stationary phase, and polyphosphate was extracted and quantified by the nonradioactive enzymatic method. Two independent determinations were performed. The error bars represent the standard deviations.

FIG. 3.

(A) Reduction in polyphosphate content during exposure to copper ions. A. ferrooxidans cells grown in sulfur medium in the absence of copper to the early stationary phase were divided into two portions. CuSO₄ (20 mM final concentration) was added to one sample (○), and an equal volume of fresh medium was added to the control sample (▪). Both were then incubated at 30°C. Aliquots were taken at the indicated times, and polyphosphate was quantified. (B) PPX activity in cells of A. ferrooxidans shifted to copper. A. ferrooxidans was cultured and transferred to medium with copper as in A. Cell extracts were then prepared at each of the indicated postshift times from control cells (▪) and cells exposed to 20 mM copper (○), and the PPX activity was determined. The error bars represent the standard deviations.

FIG. 4.

PPX response to divalent cations in vitro. PPX activity was determined in the standard assay with cell extracts from A. ferrooxidans grown in the absence of copper. The indicated amounts of MgSO₄ or CuSO₄ were added. The enzyme activity in the absence of added metal was set at 100%.

FIG. 5.

Effect of copper ions on the efflux of P_i from A. ferrooxidans cells. A. ferrooxidans was grown in sulfur medium with 1.75 mM P_i to the exponential phase. These cells were then labeled in vivo with H₃³²PO₄ (100 μCi/ml) for 17 h in the presence of 0.18 mM P_i, as indicated in Materials and Methods. After the cells were exhaustively washed with unlabeled standard medium, they were shifted to the same fresh medium containing the indicated concentrations of CuSO₄. At the times indicated, the cells were removed by centrifugation, and the radioactive P_i released into the supernatants was determined.