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 metalphosphate 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 M 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. Acidithiobacillus ferrooxidans (formerly Thiobacillus ferrooxidans)is a chemolithoautotrophic bacterium that obtains its energy from the oxidation of ferrous iron, elemental sulfur, or partially oxidized sulfur compounds (19,24). This ability makes it of great industrial importance due to its application in biomining to recover metals such as copper, gold, and uranium (19,23). These microorganisms are normally subjected to stress in their environment, such as temperature and pH changes and the presence of toxic heavy metals and nutrient starvation, which affect their physiological state (30).Unlike most heterotrophic bacteria, A. ferrooxidans is capable of resisting high concentrations of heavy metals such as copper, zinc, arsenic, and uranium (9). The genetic basis for mercury and arsenic resistance has been studied in detail in this acidophile (6,26). Copper is an essential trace element for all cells. However, it can cause serious cell damage through radical formation (10). Information regarding copper resistance in A. ferrooxidans is scarce. Although copper-tolerant strains have been obtained by growth and adaptation to increasingly higher concentrations of this metal (5,8,18), only a few genes were recently identified by RNA arbitrarily primed PCR as being induced or repressed in A. ferrooxidans subjected to copper (21). Nevertheless, the role of these genes in the mechanism of copper resistance is still unclear, and their expression may be related to indirect metabolic responses to stress (21).Many heavy metal resistance systems involve either active efflux or detoxification of metal ions by different transformations (27). For copper, these include intracell...

The influence of single and combined effects of Zn, Cu and temperature on microbial growth

“…Similar stimulatory effects of Cu have also been observed for exopolyphosphatase (PPX) activity of A. ferrooxidans up to 1-2 μM Cu, where inhibition in the PXX activity occurred for Cu concentrations greater than 5 μM (Alvarez and Jerez, 2004), and up to 10 μM Cu for the archaeon Sulfolobus metallicus (Remonsellez et al, 2006). Zn was also observed to stimulate the PXX activity of A. ferrooxidans at 1-2 μM concentrations; however, this stimulation effect was only half compared to that of exposure to Cu (Alvarez and Jerez, 2004). Stimulation effect of Zn on the PXX activity was also seen for S. metallicus in the micromolar range, similarly lower than of the Cu effect (Remonsellez et al, 2006), both consistent with our observations with Arthrobacter sp.…”

Copper tolerance of the thermoacidophilic archaeon Sulfolobus metallicus: possible role of polyphosphate metabolism

“…We have recently found that Acidithiobacillus ferrooxidans, a micro-organism that can tolerate very high concentrations of heavy metals in its normal environment, normally accumulates high amounts of polyP granules and that the levels of intracellular polyP are greatly decreased when the bacterium is grown in or shifted to 100 mM Cu 2+ . In the presence of this metal, PPX activity and P i efflux greatly increased, supporting a model for metal tolerance mediated through polyP (Alvarez & Jerez, 2004).…”

“…Radioactively labelled polyP with a chain length of 750 residues was prepared as described by Ault-Riché et al (1998), with the modifications of Cardona et al (2001) and Alvarez & Jerez (2004). The identity and purity of [ 33 P]polyP 750 were determined by its susceptibility to hydrolysis by ScPPX1 as described previously (Ault-Riché et al, 1998;Cardona et al, 2001).…”

“…An exopolyphosphatase (PPX), on the other hand, is known to hydrolyse polyP, liberating inorganic phosphate (P i ) (Akiyama et al, 1993). These enzymes have been purified from Escherichia coli and their genes have been identified in several bacteria (Kornberg et al, 1999;Alvarez & Jerez, 2004).…”

Differential gene expression in response to copper in Acidithiobacillus ferrooxidans strains possessing dissimilar copper resistance

“…As such, much research has been focused on acquiring excellent bioleaching strains that have good traits and a higher copper resistance. Some copper-resistant strains have been obtained (Alvarez and Jerez, 2004); however, little is known about the mechanisms involved in copper resistance. The results of research conducted by Chisholm et al (1998) and Ahmad et al (2002) suggest that the copper-resistance genes of At.ferrooxidans are chromosomal and not plasmid based, but the role of these genes is still not clear.…”