Copper Ions Stimulate Polyphosphate Degradation and Phosphate Efflux in Acidithiobacillus ferrooxidans

For some bacteria and algae, it has been proposed that inorganicpolyphosphates and transport of metal-phosphate complexes couldparticipate in heavy metal tolerance. To test for this possibilityin Acidithiobacillus ferrooxidans, a microorganism with a highlevel of resistance to heavy metals, the polyphosphate levelswere determined when the bacterium was grown in or shifted tothe presence of a high copper concentration (100 mM). Underthese conditions, cells showed a rapid decrease in polyphosphatelevels with a concomitant increase in exopolyphosphatase activityand a stimulation of phosphate efflux. Copper in the range of1 to 2 µM greatly stimulated exopolyphosphatase activityin cell extracts from A. ferrooxidans. The same was seen toa lesser extent with cadmium and zinc. Bioinformatic analysisof the available A. ferrooxidans ATCC 23270 genomic sequencedid not show a putative pit gene for phosphate efflux but ratheran open reading frame similar in primary and secondary structureto that of the Saccharomyces cerevisiae phosphate transporterthat is functional at acidic pH (Pho84). Our results supporta model for metal detoxification in which heavy metals stimulatepolyphosphate hydrolysis and the metal-phosphate complexes formedare transported out of the cell as part of a possibly functionalheavy metal tolerance mechanism in A. ferrooxidans.

INTRODUCTION

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Introduction

Acidithiobacillus ferrooxidans (formerly Thiobacillus ferrooxidans)is a chemolithoautotrophic bacterium that obtains its energyfrom the oxidation of ferrous iron, elemental sulfur, or partiallyoxidized sulfur compounds (19, 24). This ability makes it ofgreat industrial importance due to its application in biominingto recover metals such as copper, gold, and uranium (19, 23).These microorganisms are normally subjected to stress in theirenvironment, such as temperature and pH changes and the presenceof toxic heavy metals and nutrient starvation, which affecttheir physiological state (30).

Unlike most heterotrophic bacteria, A. ferrooxidans is capableof resisting high concentrations of heavy metals such as copper,zinc, arsenic, and uranium (9). The genetic basis for mercuryand 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. ferrooxidansis scarce. Although copper-tolerant strains have been obtainedby growth and adaptation to increasingly higher concentrationsof this metal (5, 8, 18), only a few genes were recently identifiedby RNA arbitrarily primed PCR as being induced or repressedin A. ferrooxidans subjected to copper (21). Nevertheless, therole of these genes in the mechanism of copper resistance isstill unclear, and their expression may be related to indirectmetabolic responses to stress (21).

Many heavy metal resistance systems involve either active effluxor detoxification of metal ions by different transformations(27). For copper, these include intracellular complexation,reduced accumulation, extracellular complexation, and sequestrationin the periplasm (13, 25). One of the proposed mechanisms formetal tolerance is the sequestration of metal cations with longpolymers of inorganic polyphosphate (17). Polyphosphate is alinear polymer of hundreds of orthophosphate residues linkedby phosphoanhydride bonds. Several physiological functions havebeen attributed to polyphosphate in addition to being a reservoirof phosphate, such as a substitute for ATP, chelator of metals,and adaptation to stress conditions in the cell (17). The mainenzyme involved in the biosynthesis of polyphosphate is thepolyphosphate kinase, which catalyzes the reversible conversionof the terminal phosphate of ATP into polyphosphate (17). Anexopolyphosphatase (PPX), on the other hand, is known to hydrolyzepolyphosphate, liberating inorganic phosphate (P_i) (17). Theseenzymes have been purified from Escherichia coli, and theirgenes have been identified in several bacteria, including A.ferrooxidans (30). These genes show a relatively high degreeof sequence conservation (7, 28).

It has been proposed that polyphosphate sequesters the heavymetals, thereby reducing their intracellular concentration and,on the other hand, that the hydrolysis of polyphosphate detoxifiesthe metals (1, 14). Van Veen (29) has shown that the inorganicphosphate transport system (Pit) in E. coli and Acinetobacterjohnsonii can reversibly transport metal phosphates. Later,Keasling and Hupf (15), using genetically engineered strainsof E. coli, obtained results indicating that not only a largequantity of intracellular polyphosphate but also the abilityto synthesize and degrade polyphosphate is important for toleranceto heavy metals. Based on these results and those mentionedabove, Keasling (14) proposed a model in which the intracellularcation concentration in bacteria would regulate the activityof PPX, which would in turn degrade polyphosphate and the P_igenerated accompanied by cation transport out of the cell throughthe Pit system.

In the present work, it was found that A. ferrooxidans normallyaccumulates high amounts of polyphosphate granules and thatthe levels of intracellular polyphosphate are greatly reducedwhen the bacterium is grown in or shifted to 100 mM Cu²⁺ ions.In the presence of this metal, PPX activity and P_i efflux increasedgreatly. Our results support a model for metal tolerance mediatedthrough polyphosphate in A. ferrooxidans.

MATERIALS AND METHODS

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Materials and Methods

Bacterial strains and growth conditions.
A. ferrooxidans ATCC 19859 was grown in spherical prills, 0.15to 0.20 cm in diameter, of elementary sulfur (2) in the presenceof 1.75 mM P_i (phosphate-sufficient conditions), unless otherwiseindicated. Growth was monitored by measuring cell numbers orthe optical density at 600 nm (OD₆₀₀). Experiments were conductedwhen cells reached the late exponential phase (OD₆₀₀ = 0.2).Cultures were adapted to grow in the presence of CuSO₄ by continuoussubculturing. During the first culture in the presence of Cu²⁺(10 mM), a 4- to 5-day lag period was observed. Bacteria werecompletely adapted to grow in the presence of Cu²⁺ (10 to 100mM) in the second subculture. It is a known phenomenon thatA. ferrooxidans cells adapted to grow under these conditionslose this tolerance when they are cultured again in medium withoutcupric ions (18).

Electron microscopy and X-ray microanalyses.
Unstained and unfixed cells were examined for the presence ofelectron-dense granules by transmission electron microscopy(12). A suspension (10 µl) of A. ferrooxidans cells (OD₆₀₀= 0.25) was placed on a Formvar-coated grid and left for 2 minto allow the cells to sediment. Excess liquid was removed witha piece of filter paper, and the grids were air dried. For analysis,a transmission electron microscope (Philips Tecnai 12) operatingat 80 kV was used. Energy-dispersive X-ray microanalysis (EDAX)was performed with an EDAX-PV 9800 energy-dispersive microanalyzerat an accelerating voltage of 120 kV (11).

Polyphosphate quantification.
Polyphosphate was quantified with a two-step conversion of polyphosphateinto ATP by polyphosphate kinase and quantification of the ATPformed by using luciferase to generate light (3). First, polyphosphatewas extracted from cell extracts with Glassmilk and then assayedby the reverse reaction of E. coli polyphosphate kinase in ADPexcess. Finally, the ATP content was assayed with the fireflyluciferase ATP assay, and luminescence was measured with a luminometer(BioScan Lumi/96). The concentration of polyphosphate is givenin terms of P_i residues.

Preparation of crude cell extracts from A. ferrooxidans.
Cultures (200 ml) grown to the late exponential phase were harvestedby centrifugation (10,000 x g for 20 min), and the cell pelletswere resuspended in 500 µl of a buffer containing 50 mMTris-HCl (pH 7) and 10% sucrose and lysed by four cycles offreezing (–80°C) and thawing by sonication. The lysatewas centrifuged (5,000 x g for 5 min) to eliminate cellulardebris, and the supernatant (crude cell extract) was used tomeasure PPX activity. These cell extracts were also obtainedfrom nonadapted cells shifted to copper.

Assay for PPX activity.
PPX activity was determined as previously reported (16). The20-µl reaction mixture contained 50 mM Tris-HCl (pH 6.5),1 mM MgCl₂, 100 mM KCl, 250 µM [³³P]polyP₇₅₀ (polyphosphatewith an average of 750 P_i residues). After incubation of themixture at 30°C for 60 min, the reaction was stopped byloading the mixture in polyethyleneimine-cellulose plates forthin-layer chromatography (Aldrich) and development in 0.75M KH₂PO₄, pH 3.5. Radioactive spots corresponding to the P_iliberated by the hydrolysis of polyphosphate were visualizedand quantified with a phosphor imager (Molecular Imager FX;Bio-Rad). [³³P]polyP₇₅₀ was synthesized in vitro by the methodof Ault-Riché et al. (3) as described in Cardona et al.(7). One unit of enzyme was defined as the amount releasing1 pmol of P_i from polyphosphate per min.

In vivo labeling of A. ferrooxidans with ³²Pi.
Cells were grown in sulfur medium to the late exponential phasein P_i-sufficient conditions (1.75 mM). Cells were collectedby centrifugation and resuspended at a higher cell density (10¹⁰cells/ml) in medium with reduced P_i (0.18 mM P_i). To label thecells, H₃³²PO₄ (100 µCi/ml) was added, and the microorganismswere further incubated for 17 h, after which the radioactivelylabeled cells were harvested by centrifugation.

P_i efflux measurements.
The ³²P_i-labeled cells were exhaustively washed by resuspensionand centrifugation with fresh medium containing sufficient P_i(1.75 mM) to eliminate the nonincorporated radioactive labeland finally resuspended in the same medium to an OD₆₀₀ of 0.26(10⁹ cells/ml) in the presence or absence of CuSO₄. To determinethe amount of ³²P_i released into the medium, samples (1.5 ml)were taken periodically, and the radioactivity in the supernatantsobtained by centrifugation at 12,000 x g for 10 min (1.0 ml)was determined by scintillation counting.

Genome sequence analysis.
Preliminary sequence data for A. ferrooxidans strain 23270 wasobtained from the Institute for Genomic Research website athttp://www.tigr.org. Identity and similarity searches in thedatabases were done with the tBlastn program from NCBI (http://www.ncbi.nlm.nih.gov).The finished available A. ferrooxidans ATCC 23270 genomic sequence(http://www.tigr.org), which is not yet annotated, was used.The amino acid sequences of the PitA and PitB transporters fromE. coli and the Pho84 transporter from Saccharomyces cerevisiaewere used as probes. The possible presence of transmembranedomains in the open reading frames analyzed was studied withthe Top-Pred program (http://bioweb.pasteur.fr/seqanal/interfaces/toppred.html).

RESULTS AND DISCUSSION

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Results and Discussion

Accumulation of polyphosphate in A. ferrooxidans.
Previously, it was demonstrated that A. ferrooxidans possessesa polyphosphate kinase activity and the corresponding gene,suggesting the existence of polyphosphate in this bacterium(30). Typically, accumulation of polyphosphates in the formof electron-dense granules has been reported in many bacteria(12). To detect the presence of such granules in A. ferrooxidans,cells grown in sulfur medium in a P_i-sufficient condition wereanalyzed by transmission electron microscopy. As Fig. 1 shows,abundant spherical electron-dense granules were observed. Allcells grown under these conditions presented at least two granules,and over 90% of the cells presented three to four granules.These granules disappeared when the cells were subjected tophosphate starvation (results not shown).

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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.

To confirm the chemical nature of the electron-dense granules,an elemental analysis with EDAX in the transmission electronmicroscopy mode was carried out. As can be seen in Fig. 1, theEDAX spectra showed that the electron-dense bodies present inA. ferrooxidans were mainly composed of phosphorus and oxygen(Fig. 1, left spectrum), in contrast to the elemental compositionof a cytoplasmic area (Fig. 1, right spectrum), indicating thatA. ferrooxidans synthesizes and accumulates electron-dense granulescontaining phosphate, which is most likely polyphosphate. Cellsgrown under control conditions (P_i sufficient) showed a veryhigh level of polyphosphate, determined enzymatically, of approximately400 nmol of P_i residues/mg of protein (Fig. 2). This high levelof the polymer was in agreement with the presence of abundantelectron-dense granules in the cells (Fig. 1). A. ferrooxidanscan therefore be considered a polyphosphate-accumulating microorganism,like Acinetobacter johnsonnii (29).

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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.

Effect of CuSO₄ on the growth of A. ferrooxidans and its polyphosphate levels.
To evaluate the effect of Cu²⁺ on polyphosphate levels, cellswere grown in the presence of different concentrations of Cu²⁺and the cellular polyphosphate content was determined. The bacterialcells used in these experiments were previously adapted to growat different concentrations of copper. There was a small decreasein the bacterial growth rates, and the curves reached the plateausat slightly lower cell densities when cells were grown in thepresence of increasing Cu²⁺ concentrations compared with thecontrol culture in the absence of the metal (Fig. 2A). On theother hand, polyphosphate levels showed a clear drop (more than50%) only when the Cu²⁺ concentration was raised to 100 mM (Fig.2B). These results indicate a possible relationship betweenthe polyphosphate level and the adaptation of A. ferrooxidansto growth in the presence of Cu²⁺.

Polyphosphate levels in cells shifted to medium containing copper ions.
To determine the effect of Cu²⁺ in unadapted A. ferrooxidans,cells grown under P_i-sufficient conditions to accumulate a largeamount of polyphosphate (Fig. 2B) were shifted to the presenceof 20 mM Cu²⁺, and the polyphosphate levels were determinedat different times postshift (Fig. 3A). A great decrease inpolyphosphate levels was seen after 1 h, reaching the lowestlevel by 4 h, with polyphosphate rapidly dropping to about 20%of the level at time zero, and this level was maintained forthe next 24 h. These results indicate that the presence of Cu²⁺ions affects the polyphosphate content in A. ferrooxidans, probablyby stimulating the degradation of this polymer, as has beensuggested in other systems (1, 14).

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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 ( ${circ}$ ), and an equal volume of fresh medium was added to the control sample ( ${blacksquare}$ ). 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 ( ${blacksquare}$ ) and cells exposed to 20 mM copper ( ${circ}$ ), and the PPX activity was determined. The error bars represent the standard deviations.

Effect of copper ions on PPX activity.
A possible mechanism to explain the observed decrease in polyphosphatelevels when cells were exposed to copper ions is an increasein PPX activity. Previously, a putative ppx gene was found inA. ferrooxidans which appears to be part of a Pho regulon inthis microorganism (30). To investigate the effect of copperions on PPX activity, nonadapted cells were shifted to the presenceof 20 mM CuSO₄. Cells were collected at different time intervals,and PPX activity was measured in the cell extracts. A rapidincrease in PPX activity was seen when cells were exposed tocopper (Fig. 3B). This increase corresponded in time to thedecrease in polyphosphate levels seen in Fig. 3A, strongly suggestingthat the decrease in polyphosphate levels observed when cellswere shifted to the presence of copper could be due to an increasein this exopolyphosphatase activity.

To further analyze the previous phenomenon, the in vitro effectof copper and other metals on the PPX activity present in cellextracts of nonadapted A. ferrooxidans was determined, as shownin Fig. 4. It is clear that copper greatly stimulated PPX activityat very low concentrations. The same maximal PPX activity wasreached with both copper and magnesium. However, the metal concentrationrequired for this maximal activity was 1 to 2 µM for copper(Fig. 4) and 1,000 µM for magnesium (results not shown).At concentrations of copper higher than 5 µM, there wasan inhibition of the activity, whereas magnesium continued tostimulate PPX. Cadmium and zinc also stimulated PPX activityat concentrations of 1 to 2 µM. However, this effect wasonly half that seen with copper (not shown). These results suggestan effect of the heavy metals on PPX activity and polyphosphatehydrolysis.

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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%.

Effect of copper ions on efflux of P_i from A. ferrooxidans cells.
The decrease in polyphosphate levels due to increased PPX activityin A. ferrooxidans cells subjected to Cu²⁺ should generate freeP_i. To evaluate if this P_i was transported out of the cells,A. ferrooxidans was grown to the late exponential phase andlabeled in vivo with ³²Pi as indicated in Materials and Methods.After exhaustive washing of the radioactively labeled cellsto eliminate nonincorporated label, they were resuspended insulfur medium with or without Cu²⁺ ions, and P_i efflux was determined,as shown in Fig. 5. There was a continuous basal release oflabel from the control cells. However, an increase in the P_iefflux over this basal level was observed when cells were exposedto Cu²⁺ ions. The amount of ³²Pi released into the medium washigher in cells exposed to 50 mM Cu²⁺. By analyzing the radioactivityreleased into the medium by thin-layer chromatography on polyethyleneimine-cellulose,it was found that it consisted mainly of P_i (not shown). Noother radioactively labeled cellular metabolites appeared onthe thin-layer chromatograph, indicating that the label releasedinto the medium was not the product of cellular lysis duringtreatment with copper. These results strongly suggest that atleast part of the P_i generated from polyphosphate hydrolysisin the presence of Cu²⁺ ions was transported out of the cells.

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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.

In silico search for P_i transporters in A. ferrooxidans.
The proposed model for metal ion detoxification based on thehydrolysis of polyphosphate involves the transport of metal-phosphatecomplexes out of the cell. It has been proposed that the inorganicphosphate transport (Pit) system is a candidate for this purposebecause it can reversibly transport metal-phosphate complexes(14, 29). P_i transporters have not been described in A. ferrooxidans,although the bacterium possesses a putative Pho regulon (30).A Pit-like phosphate transport system was searched for in theavailable genome of A. ferrooxidans ATCC 23270. A Pit-like transporterwas not found in this bacterium, but instead we found an openreading frame coding for a protein similar to the Pho84 P_i transporterfrom S. cerevisiae. An alignment of the predicted amino acidsequences of the putative Pho84-like protein of A. ferrooxidanswith Pho84 from S. cerevisiae showed 26% identity and 43% similarity,with the same number of highly conserved transmembrane segments(results not shown). On the other hand, experimental evidenceindicates that yeast Pho84, like Pit, transports metal-phosphatecomplexes (22).

Pho84 and Pho89 are the major P_i transporters in S. cerevisiae.Pho84, like Pit, belongs to the family of P_i:H⁺ symporters andis a member of the major facilitator superfamily (20). The Pho84transporter is functional only in acidic environmental conditions(22). Although currently there is no experimental evidence fora Pho84-like transporter in A. ferrooxidans, it is remarkablethat this microorganism, an acidophilic bacterium, possessesa putative P_i transporter of this kind. In this regard, genesencoding proteins similar to Pho84 but not to Pit (or Pho89)were also found in the genomes of other acidophilic microorganisms,such as Sulfolobus tokodaii, Sulfolobus solfataricus, Thermoplasmaacidophilum, Thermoplasma volcanicum, and Ferroplasma acidarmanus(results not shown).

Finally, the A. ferrooxidans ATCC 23270 genome sequence alsoshows the presence of a putative CopA uptake Cu⁺ P-type ATPaseand the CopB efflux Cu⁺ P-type ATPase present in other bacteria(4). This suggests that A. ferrooxidans might have a copperhomeostasis mechanism similar to that of other microorganisms,but no experimental evidence supporting this proposal is availablefor this bacterium. Irrespective of the existence of such metalcation uptake and efflux mechanisms, it is plausible that apolyphosphate-mediated metal tolerance mechanism such as theone described here is also of great functional survival valuefor this extremophilic microorganism.

ACKNOWLEDGMENTS

This work was supported in part by grants from ICGEB (projectCRP/CHI00-04, contract 01/001, and ICM-P99-031-F).

We acknowledge Arthur Kornberg for supplying E. coli strainNR 100.

FOOTNOTES

* Corresponding author. Mailing address: Laboratorio de Microbiología Molecular y Biotecnología, Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago, Chile. Phone: (56 2) 678 73 76. Fax: (56 2) 271 29 83. E-mail: cjerez{at}uchile.cl.

REFERENCES

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