INTRODUCTION

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Alcaligenes eutrophus CH34 and related bacteria are adapted to survive in environments with high concentrations of heavy metal ions (8). Strain CH34 contains at least seven determinants encoding resistances to toxic heavy metals; these determinants are located either on the bacterial chromosome or on one of the two indigenous large plasmids, pMOL28 (180 kilobase pairs [kb] [35]) and pMOL30 (238 kb [9, 22]). A. eutrophus has recently been reclassified as Ralstonia eutropha (38). However, strain CH34 and related strains are, though closely related to R. eutropha, different from its type strain. Since classification of all the metal-resistant, CH34-like organisms is under way (18a), strain CH34 should remain classified as A. eutrophus until this work is done.

On plasmid pMOL28, two inducible metal resistance determinants are located adjacent to each other: the cnr determinant encodes resistance to Co²⁺ and Ni²⁺ (17) and physiologically is based on metal cation efflux; the chr determinant gives resistance to chromate (22). Interestingly, in serpentine soils, nickel, chromium, and cobalt are present in high concentrations (1). The adjacent locations of the resistance determinants cnr and chr may be the result of the adaptation of A. eutrophus to such an environment.

The mechanism of chromate resistance is reduced accumulation of chromium (26), but chromate efflux has not been demonstrated. There are three open reading frames in the sequence of the 2.6-kb EcoRI fragment encoding chr: chrB, chrA, and ORF3, which is not essential for chromate resistance and not complete within the 2.6-kb EcoRI fragment (22). ChrA is a membrane-bound protein and probably responsible for the resistance. The function of ChrB, however, is unclear.

In this investigation, regulation of chr and cnr was studied with lacZ fusions. To deepen our understanding of chr induction, a lux-coupled chromate sensor was developed. When the sensor was characterized, interactions among chr-dependent chromate resistance, sulfate metabolism, and chromate reduction were revealed. This shed some light on the unexpected complexity of the chromate metabolism of A. eutrophus.

	MATERIALS AND METHODS

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Bacterial strains, plasmids, and growth conditions. The bacterial strains and plasmids used in this study are listed in Table 1; some plasmid constructs are depicted in Fig. 1. Tris-buffered mineral salts medium (19) was used for growing A. eutrophus. Additionally, a phosphate-buffered mineral salts medium (29) and a HEPES-buffered mineral salts medium were used. The HEPES-buffered medium contained the following (per liter of H₂O): 0.3 mM Na₂KPO₄, 0.2 mM K₂HPO₄, 50 mM HEPES buffer (pH 7.0), 2 g of NH₄Cl, 0.2 g of MgSO₄ · 7H₂O, 10 mg of CaCl₂ · 2H₂O, and 5 mg of FeCl₃ · 6H₂O. Analytical-grade salts of CdCl₂ · H₂O, ZnCl₂, CoCl₂ · 6H₂O, NiCl₂ · 6H₂O, AlCl₃, MnCl₂ · 6H₂O, MnSO₄ · H₂O, CrCl₃, CuCl₂ · 6H₂O, K₂CrO₄, K₂Cr₂O₄, Na₂WO₄, Na₃VO₄, Na₃AsO₄, and Na₂MoO₄ were used to prepare 1 M stock solutions, which were sterilized by filtration. Solid Tris-buffered media contained 2.0% (wt/vol) agar. Nutrient broth (Difco) was used as complex medium for A. eutrophus, and Luria broth (28) was used for Escherichia coli. Metal resistance was tested on solid Tris-buffered mineral salts medium containing 0.2% (wt/vol) sodium gluconate and either 0.2 mM chromate, 1 mM Ni²⁺, or 1 mM Co²⁺.

View larger version (12K): [in this window] [in a new window]   FIG. 1.   Insertion points of Tn5-lacZ transposons in the cnr-chr region of plasmid pMOL28. Plasmid pDNA206 (21), containing the cnr-chr region of plasmid pMOL28, was mutated with ::Tn5-lacZ B20 (33), and derivatives which had lost metal resistance were selected. The points of insertion of the transposons were determined by digestion with EcoRI, BamHI, or HindIII and are indicated by the positions of the circles. The promoterless lacZ gene on the transposon was transcribed either from left to right (circles below the line) or from right to left (circles above the line). Grey circles indicate induction by chromate; the filled triangle indicates the position of the fusion in plasmid pDNA265. Black circles indicate induction by Ni2+; numbers with the circles indicate plasmids pDNA255, pDNA238, pDNA228, and pDNA229. The number 257 and the square give the position of the transposon insertion in plasmid pDNA257, which did not lead to a loss of metal resistance. Physical maps of the cnr determinant (17) with the cnrYXHCBA genes and of the chr determinant (21) with the chrBA genes, ORF3, and the newly proposed ORF13 are drawn to scale above the transposon insertion map.

Sewage water. Artificial sewage water was composed of the following components (per liter): 10 mg of aniline, 5 mg of nitrobenzol, 10 mg of phenol, 2 mg of toluene, 50 mg of acetone, 50 mg of ethanol, 100 mg of isopropanol, 300 mg of methanol, 29 mg of urea, 6.5 g of chloride anions, 138 mg of nitrate anions, 1.7 mg of phosphate anions, and 3.5 g of sulfate anions. This artificial sewage water resembles in its composition real sewage water from an industrial plant.

Enzymatic activities. -Galactosidase activity in permeabilized cells was determined (24), with 1 U defined as the activity forming 1 nmol of o-nitrophenol per min at 30°C. The bioluminescence of slow-growing cells was measured with a Lumac/3M M2010A biocounter. Cells were grown for 48 h at 28°C in HEPES medium plus tetracycline (7.5 µg ml¹), harvested, and adjusted to 10⁹ CFU/ml. A volume of 0.5 ml of the cell suspension was used for each determination and incubated in polystyrene tubes. The light output was integrated for 10 s. The bioluminescence of fast-growing cells was measured with a Lumistox luminometer (Dr. Lange, Berlin, Germany) in Tris-buffered mineral salts medium containing 9 mM sodium gluconate as the carbon source.

Chromate reduction and uptake. The cells were cultivated for 18 h at 30°C in Tris-buffered mineral salts medium containing 30 µM or 3 mM disodium sulfate and 46 mM sodium gluconate as the carbon source. The cells were harvested by centrifugation, washed, and suspended in 10 mM Tris HCl, pH 7.0, containing 46 mM sodium gluconate, and 50 µM [⁵¹Cr]chromate (specific activity, 17.6 GBq/g; Du Pont de Nemours, Bad Homburg, Germany) was added. Samples (1 ml) were removed and centrifuged. The cells were discarded. The total chromium concentration in the supernatant was determined with a Beckman LS6500 scintillation counter with 100-µl samples. The total chromate in the supernatant was measured with diphenylcarbazide as described previously (10). The amount of reduced chromium in the supernatant was the difference between the total chromium in the supernatant and the total chromate in the supernatant. Since the cells previously removed by centrifugation were responsible for chromate reduction, this value was divided by the dry weight of the cells at the time the sample was taken. The uptake of chromium was determined by filtration as previously described (26).

Genetic techniques. Standard molecular genetic (28) and previously published (23) techniques were used. For conjugal gene transfer, overnight cultures of donor strain E. coli S17/1 (32) and of the A. eutrophus recipient strains grown at 30°C in complex medium were mixed (1:1) and plated onto nutrient broth agar. After overnight growth, the bacteria were suspended in saline and plated onto selective media as previously described (23). For transposon mutagenesis, E. coli S17/1(pDNA206) was infected with ::Tn5-lacZ B20 (33) and plated onto Luria broth agar containing 50 µg of kanamycin per ml. The resulting transposon mutants were conjugated by replica plating with A. eutrophus AE104 on Tris-gluconate-tetracycline agar (2 g of sodium gluconate per liter, 12.5 µg of tetracycline per ml). AE104 transconjugants were replica plated onto nutrient broth agar containing 3 mg of kanamycin and 0.1 mg of X-Gal (5-bromo-4-chloro-3-indolyl--D-galactopyranoside) per ml. Kanamycin-resistant strains which were sensitive to 1 mM Ni²⁺ or to 0.2 mM chromate were purified and analyzed.

Construction of the broad-host-range chromate sensor plasmid. By using the XhoI site, which is a single site in the kanamycin resistance genes of both plasmids, plasmid pRME1 (11) was cloned into the broad-host-range plasmid pVK102 (15). A hybrid plasmid encoding kanamycin resistance was isolated, and the cos site was deleted with PstI and BglII. The resulting plasmid, pEBZ112, consists of plasmid pVK102 with the multiple-cloning site of pRME1 replacing the cos site. By using a BamHI site, the Vibrio fischeri luxCDABE operon of Tn4431 from plasmid pUCD623 (30) was cloned into plasmid pEBZ112, resulting in plasmid pEBZ116. Finally, the 1.6-kb chrBA' EcoRI-XbaI fragment of plasmid pECD352 was cloned as an EcoRI blunt-end fragment into pEBZ116 upstream of lux, resulting in plasmid pEBZ141 (Fig. 2). Plasmid pECD352 carries the 5' end of chr from the EcoRI site at position 1 up to bp 1697 in the middle of chrA (22).

View larger version (25K): [in this window] [in a new window]   FIG. 2.   Map of the chromate sensor plasmid pEBZ141. The genes encoding resistances to tetracycline (tet) and kanamycin (kan) and the chrBA'::lux gene fusion are indicated. Restriction endonucleases were SalI (S), XbaI (A), BamHI (B), PstI (P), KpnI (K), SacI (Sc), EcoRI (E), and XhoI (X).

	RESULTS

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Induction of chr and cnr. To develop metal-sensing bacterial strains and to study the regulation of chr and cnr, plasmid pDNA206 (21), which contains both determinants on a 30-kb fragment of pMOL28, was mutated with ::Tn5-lacZ B20 (33). Thirty-eight Co²⁺-, Ni²⁺-, and/or chromate-sensitive derivatives of pDNA206 were identified in plasmid-free A. eutrophus AE104. The mutant strains were either chromate sensitive (10 strains) or sensitive to Co²⁺ and Ni²⁺ (28 strains). No strains were sensitive to all three metal ions.

A chromate sensor strain. Of all the fusions in the chr-cnr region tested, fusions in chrA were the most specific and gave the strongest responses (data not shown). To measure chr induction in whole cells, plasmid pEBZ141 (Fig. 2), with a chrBA'::luxCDABE transcriptional fusion, was constructed. The plasmid was transferred into A. eutrophus CH34(pMOL28, pMOL30) and into its plasmid-free derivative AE104. To compare the influences of various carbon sources on the light emissions of the resulting transconjugants, CH34(pMOL30, pMOL28, pEBZ141) and AE104(pEBZ141) were cultivated at 28°C in HEPES medium containing 10 µM chromate, 7.5 µg of tetracycline per ml, and 4 g of a carbon source per liter. Poor or no growth and low light emission were observed for both strains in acetate, citrate, formate, pyruvate, and succinate (data not shown). In gluconate, light emission increased during the exponential phase of growth to about 300 relative light units and decreased again during the stationary phase. In glycerol, growth of both strains was slow. Light emission by CH34(pMOL30, pMOL28, pEBZ141) was poor (about 20 relative light units); however, AE104(pEBZ141) achieved light emissions of about 6,000 relative light units (data not shown). Therefore, glycerol as the carbon source and strain AE104(pEBZ141) were considered optimal for the study of chr induction in whole cells.

View larger version (13K): [in this window] [in a new window]   FIG. 3.   AE104(pEBZ141) as a chromate sensor. Strain AE104(pEBZ141) was precultivated in glycerol-HEPES medium containing various sulfate concentrations (A) or 1 mM sulfate (B). When the early stationary phase was reached, the cell density was adjusted to 109/ml. A volume of 0.5 ml of this cell suspension was tested with various concentrations of chromate and other metal ions, and light emission (in relative light units [RLU]) by the products of the chr::lux operon fusion was determined at 28°C. (A) The cells were precultivated in the presence of 1,000 (), 500 (), 250 (), or 125 () µM sulfate, and light emission was measured after induction with 10 µM chromate. (B) Inductions by 1 () and 10 () µM chromate were compared with inductions by () and 10 () µM dichromate or by 1 () and 10 () µM Cr3+ and with that in cells incubated without chromium ().

Interaction between chr induction and the sulfate concentration. Since sulfate starvation derepresses the transport systems responsible for sulfate and chromate uptake (25), sulfate starvation should have increased the sensitivity of the chromate sensor, but the opposite was observed (Fig. 3A). Growth of A. eutrophus CH34 in glycerol was very slow (0.02 h¹) compared to growth in gluconate (0.2 h¹ [data not shown]). To characterize the influence of sulfate on induction of chr in Tris-buffered mineral salts medium with gluconate as the carbon source (conditions normally used to cultivate the strain) plasmid pEBZ141 was transferred into A. eutrophus AE126(pMOL28). The control plasmid pEBZ116, which contains a promoterless lux operon but not chrBA' upstream of it, was transferred into strains AE104 and AE126(pMOL28). Cells of all four strains, AE104(pEBZ141), AE104(pEBZ116), AE126(pMOL28, pEBZ141), and AE126(pMOL28, pEBZ116), were cultivated in Tris-buffered mineral salts medium, and induction of chr::lux was determined in exponentially growing cells (shown in Fig. 4 for pEBZ141; data not shown for pEBZ116).

View larger version (16K): [in this window] [in a new window]   FIG. 4.   Induction of chr::lux in growing cells. Cells of strain AE104(pEBZ141) ( and ) and of strain AE126(pMOL28, pEBZ141) ( and ), both containing a chr::lux fusion on plasmid pEBZ141, were cultivated in gluconate-Tris medium containing either 3 mM or 30 µM sulfate in the presence ( and ) or absence ( and ) of 10 µM chromate, and the light intensities (in relative light units [RLU]) were determined.

Interaction between chr and chromate reduction. To find a reason for the unexpected effect of sulfate on chr induction, cells of the plasmid-free strain AE104 were cultivated without chromate in the presence of 3 mM or 30 µM sulfate, and the levels of chromate accumulation and chromate reduction were determined (Fig. 5). Strain AE104 was able to reduce chromate (Fig. 5A). Sulfate starvation led to increased chromate uptake but also to increased chromate reduction. Other sulfur sources (cysteine, methionine, taurine, sulfite, and thiosulfate) were also tested, but due to interference with the chromate determination assay, these experiments yielded no results (data not shown). Thus, A. eutrophus reduced chromate, and the sulfate concentration in the growth medium influences chromate uptake and reduction.

View larger version (14K): [in this window] [in a new window]   FIG. 5.   Chromate uptake and chromate reduction in strain AE104. Cells of the plasmid-free A. eutrophus strain AE104 were cultivated 18 h with shaking at 30°C in gluconate-Tris medium containing 1% sodium gluconate and 3 mM () or 30 µM () sulfate. The cells were washed once and suspended in 10 mM Tris HCl buffer, pH 7.0, containing 1% sodium gluconate. Potassium [51Cr]chromate (50 µM) was added, and incubation was continued with shaking at 30°C. Samples were removed and used to determine the amount of chromate reduced by the cells (A) and the amount of cell-bound chromium (B). Each point is the mean for three experiments; the bars give the standard deviations. d.w., dry weight.

	DISCUSSION

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A. eutrophus AE104(pEBZ141) may be readily used as a biosensor for chromate, even in unnatural environments, such as industrial sewage water. The chromate-sensing process is highly specific. The main inducers are chromate and dichromate; Cr³⁺ is 10-fold less active as an inducer. Other oxyanions show activities which are less than 1% of the activity obtained with chromate. The data gathered during development and characterization of the chromate sensor also shed some light on regulation of the chr and the cnr resistance determinants and on the chromium metabolism of strain CH34, which is the wild-type counterpart of the plasmid-free strain AE104 (19).

Cells of A. eutrophus starved for sulfur are known to derepress the sulfate and chromate uptake systems (25). Therefore, it was expected that incubation of the sensor strain AE104(pEBZ141) with sulfate starvation would lead to enhanced sensitivity of the sensor bacteria. Surprisingly, the opposite was the case: sulfate starvation repressed induction of the chr::lux reporter by chromate. To explain this result, the interaction between chr and chromate reduction was investigated. Many bacteria reduce chromate (3, 5, 6, 12, 13, 18, 31, 34, 36), and now it has been shown that A. eutrophus does so also. The product of the reduction should be Cr(III) or Cr(II), since metallic chromium, Cr(IV), and Cr(V) should not be stable in aqueous environments at neutral pH values (37).

In strain AE104, sulfate starvation induced uptake and reduction of chromate (Fig. 5). Thus, chromate reduction by strain AE104 might be catalyzed by the sulfate reduction pathway. Since chromate is rapidly reduced by sulfate-starved AE104, the intracellular concentration of chromate, probably the inducer of chr, might be lower in sulfate-starved cells than in sulfate-saturated cells. This may explain the repression of chr induction in sulfate-starved cells.

This result could indicate a connection between a chromosomally encoded sulfate reduction pathway and the plasmid-borne chr chromate resistance system. Two known products of chr are essential for chromate resistance, which is based on reduced accumulation of chromium (26). The first is ChrA, a membrane-bound protein with various transmembrane-spanning alpha-helices (16), which is encoded at the 3' end of chr. Deletion of chrA leads to chromate sensitivity and loss of the mechanism leading to reduced accumulation of the metal ion (22). Thus, ChrA is probably a chromate efflux protein. Genes with products homologous to ChrA have been found in Synechococcus (20), Synechocystis (14), Methanococcus jannaschii (2), Pseudomonas (4), and Vibrio cholerae (27). Expression of the ChrA-homologous SrpC protein of Synechococcus is induced by sulfate starvation (20); therefore, regulation is different from ChrA regulation in A. eutrophus. Gene products homologous to ChrB, the second product of chr, have not been found in other organisms until now. ChrB is also essential for chromate resistance: expression of a chr derivative with a deletion in the 5' end of chrB led to hyperaccumulation of chromium (22).

Chromate might be rapidly transported into sulfate-starved cells of A. eutrophus but also reduced very quickly to a less toxic form inside the cells. This makes sense, because effluxed chromate comes back into the cell while reduced chromium is detoxified permanently. The chr resistance system is not induced under these conditions and may not be required. In sulfate-saturated cells, however, chromate is not reduced because the sulfate reduction pathway is repressed; chromate accumulates and becomes toxic, the chr system is induced, and ChrA pumps out the chromate. In this case, ChrA would be a safety valve for chromate, which would explain the high specificity of chr induction by chromate. The data currently available do not justify further speculations, but chromate reduction seems to be part of the chromate detoxification system in A. eutrophus.