- Email: [email protected]
High level, intrinsic resistance of Natronococcus occultus to potassium tellurite
FEMS Microbiology Letters 174 (1999) 19^23
High level, intrinsic resistance of Natronococcus occultus to potassium tellurite Chad T. Pearion, Peter E. Jablonski * Department of Biological Sciences, Northern Illinois University, DeKalb, IL 60115-2861, USA Received 18 December 1998; received in revised form 23 February 1999; accepted 24 February 1999
Abstract Natronococcus occultus, a haloalkaliphilic archaeon, was examined for its resistance to potassium tellurite. Cells grown in the presence of 1 mM potassium tellurite reduced it to metallic tellurium resulting in the deposition of intracellular crystals in the cytoplasm. The minimal inhibitory concentration for potassium tellurite was 10 mM. N. occultus had an inducible tellurite reductase activity. Cell-free extracts catalyzed the enzymatic reduction of potassium tellurite in a reaction which was dependent on NADH oxidation and a reduced environment. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Tellurite ; Heavy metal resistance; Natronococcus occultus; Archaea
1. Introduction Salts of the rare earth oxyanion tellurite (TeO3 23 ) are toxic to most microorganisms at concentrations as low as 1 Wg ml31 [1,2]. It has been suggested that this toxicity is due to the strong oxidizing ability of tellurite [1]. However, the ability of tellurite to act as an oxidizing agent of any speci¢c components of the cell has not been studied. When grown in the presence of potassium tellurite, bacteria form black colonies due to the deposition of intracellular crystals of what has been shown to be metallic tellurium [3]. In contrast, no member of the domain Archaea has been shown to be naturally resistant to this heavy metal. * Corresponding author. Tel.: +1 (815) 753-3799; Fax: +1 (815) 753-7855; E-mail: [email protected]
Natronococcus occultus is a haloalkaliphilic archaeon which grows under high salt and high pH conditions [4]. Optimum growth occurs at a pH of 9.0^10.0, a NaCl concentration of 3.0^4.0 M and a very low Mg2 concentration. Currently, little is known about the physiology of these organisms with most of the work done thus far concentrating on the lipid composition of these archaeons [4,5]. Here, we present the ¢rst evidence of tellurite resistance in an archaeon.
2. Materials and methods 2.1. Organism N. occultus (ATCC 43101) was grown in de¢ned haloalkaliphile medium [6] containing 1 mM potas-
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 1 1 5 - 9
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C.T. Pearion, P.E. Jablonski / FEMS Microbiology Letters 174 (1999) 19^23
sium tellurite at 40³C with shaking (300 rpm). Cells were harvested by centrifugation at 10 000Ug at 4³C for 15 min, resuspended 1:1 (w/v) in 10 mM Tris (Tris(hydroxymethyl)aminomethane) (pH 7.5, 20³C) containing 3 M KCl and 4 mM 2-mercaptoethanol and stored at 320³C. 2.2. Determination of minimal inhibitory concentrations (MIC) Quantitative susceptibility tests were performed using de¢ned haloalkaliphilic agar medium containing potassium tellurite (1, 2.5, 5, 10, 20 or 40 mM). Each plate was inoculated with approximately 104 ^ 105 cells. The MIC was de¢ned as the lowest concentration of potassium tellurite preventing growth at 40³C after 14 days of incubation. 2.3. Electron microscopy Washed cell pellets of N. occultus, grown in the presence of 1 mM potassium tellurite until the late exponential phase, were ¢xed using glutaraldehyde. Samples were then dehydrated successively in 10, 30, 40, 60, 80 and 90% ethanol for 10 min each, 95 and 100% ethanol, ethanol:acetone (1:1 (v/v)) and ¢nally 100% acetone for 5 min each. Each sample was then embedded in epoxy resin. Sectioned material was left unstained and examined by transmission electron microscopy.
2.4. Tellurite reductase assay Cell-free extracts of N. occultus were prepared by French pressure cell disruption at 20 000 psi. The resulting cell extract was centrifuged as described above and the supernatant £uid was used for enzyme assays. The tellurite reduction was assayed spectrophotometrically as described [7]. The reaction mixture (1 ml) contained 10 mM Tris (pH 7.5, 40³C) and 0.1 mM potassium tellurite. Cell-free extract was added to initiate the reaction. The reduction of tellurite was determined by measuring the change in A500 . 3. Results and discussion Cells of N. occultus, when grown in the presence of 1 mM potassium tellurite, accumulated electrondense, black intracellular crystals within the cytoplasm and not speci¢cally membrane-associated (Fig. 1A). Cells grown in the absence of tellurite did not exhibit similar crystals (Fig. 1B). Such crystals have previously been shown to consist of elemental tellurium in several strains of Eubacteria [3,8,9]. The localization of crystals in N. occultus is in contrast to other systems such as Escherichia coli where tellurium crystals have a marked tendency towards local deposition near the cytoplasmic membrane [9]. The addition of bromine water to a pellet
Fig. 1. Transmission electron micrograph through sections of N. occultus cells grown in the presence (A) or absence (B) of 1 mM potassium tellurite. Scale bar represents 1 Wm.
FEMSLE 8712 15-4-99
C.T. Pearion, P.E. Jablonski / FEMS Microbiology Letters 174 (1999) 19^23 Table 1 Tellurite reductase activity in cell-free extracts of N. occultus Assay Conditionsa
Activityb
CFE (uninduced cells) CFEi CFEi +NADH CFEi +2-mercaptoethanol CFEi +NADH+2-mercaptoethanol CFEi (70³C, 15 min) CFEi (100³C, 10 min) CFEi +1% (w/v) SDS (10 min) Membrane fraction CFEi dialyzed against 30% sorbitol
6 0.001 6 0.001 6 0.001 6 0.001 0.237 6 0.001 6 0.001 6 0.001 0.020 0.220
a CFE, cell-free extract (induced cells); 1 mM NADH ; 4 mM 2-mercaptoethanol b vA500 min31
of tellurite grown N. occultus resulted in the rapid oxidation of the black crystals to a colorless compound(s) as previously reported for metallic tellurium [10]. Taken together, these results suggest that N. occultus transported the oxyanion potassium tellurite and subsequently reduced it to black metallic tellurium. To determine the intrinsic level of resistance, the MIC of potassium tellurite was determined by plating 104 ^105 cells on DH medium containing graded concentrations of potassium tellurite. The range used (1^40 mM) covered those concentrations typically used in studies of metal resistance in Eubacteria. After 14 days of incubation at 40³C, the MIC was determined to be 10 mM (2571 Wg ml31 ) for N.occultus. MICs for Natronobacterium magadii and N. gregoryi, two related strains of haloalkaliphilic archaea, were 20 mM (5142 Wg ml31 ). These MICs are the highest reported values to date for the resistance to tellurite. A constitutive high level resistance at concentrations of 800 and 1200 Wg ml31 was described for Rhodobacter capsulatus and Rhodobacter sphaeroides, respectively [11,12]. Recently, three species of Erythromicrobium were shown to have MICs to tellurite of between 2300 and 2700 Wg ml31 [13]. It is important to note that the availability of certain metals can be in£uenced by the microbial growth medium as reported by Ramamoorthy and Kushner [14]. In particular, casamino acids show a high level of binding activity for Hg2 , Pb2 , Cu2 and Cd2 . Measurement of tellurite concentrations in a de¢ned haloalkaliphilic medium not containing cells using
21
the diethyldithiocarbamate (DDTC) assay, indicated that there was insigni¢cant binding to any medium components. The growth of N. occultus was similar in the presence or absence of 10 mM potassium tellurite (data not shown) suggesting an intrinsic resistance in this strain. Cells grown in a de¢ned haloalkaliphilic medium without tellurite to the early stationary phase and then transferred to medium containing 1 mM tellurite exhibited the same initial growth rate. Cells grown in medium containing tellurite to the early stationary phase were inoculated into fresh medium containing tellurite and exhibited nearly the original growth rate. Similarly, when cells previously grown in the presence of 10 mM tellurite were inoculated into medium without tellurite, comparable growth rates were observed. A noticeable uptake of potassium tellurite by N. occultus was not observed using the established DDTC colorimetric assay [15] over a 14-day period (data not shown). Cell-free controls con¢rmed that the medium components did not interfere with the assay. As a positive control, E. coli JM109 harboring the tellurite resistance determinant R478 [16] was tested for its tellurite uptake. A decrease in tellurite in the medium was observed using the DDTC assay. This suggests that a di¡erent resistance mechanism in N. occultus may exist. Several possibilities are likely. N. occultus may exclude a large portion of the tellurite thus accounting for resistance. While reducing tellurite to tellurium intracellularly, the cells may also transport unoxidized tellurite back into the medium. It is also possible that additional unidenti¢ed water soluble forms of tellurite may be produced, transported out of the cell and react in the DDTC assay. While neither a reduced uptake nor an increased e¥ux is responsible for the tellurite resistance in Escherichia coli [17], further investigation will be necessary to de¢ne the system in N. occultus. In addition, the methylation of tellurium is theoretically a mechanism of resistance. Microorganisms including Thermus thermophilus and Pseudomonas aeruginosa produce a garlic odor (probably dimethyl telluride) [1,18] while E. coli does not [1]. N. occultus does indeed produce a garlic odor when grown in the presence of tellurite suggesting the production of dimethyl telluride. Further experiments are needed to determine if methylation by N. occultus occurs. The
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C.T. Pearion, P.E. Jablonski / FEMS Microbiology Letters 174 (1999) 19^23
tellurite uptake in E. coli appears to occur at last partly via the cellular phosphate transport systems [19]. In uptake experiments with N. occultus using radiolabelled phosphate, tellurite did not compete with the phosphate transport (data not shown). Clearly, a number of resistance mechanisms are possible and remain to be elucidated in the haloalkaliphilic archaea. N. occultus showed an inducible tellurite reductase activity (Table 1). Cells that were not previously exposed to tellurite did not contain this activity. Cellfree extracts of tellurite grown cells catalyzed the reduction of tellurite to metallic tellurium only upon the addition of NADH and under reducing conditions. The enzyme activity was destroyed by heat treatment at 70³C for 15 min. Detergent treatment with sodium dodecyl sulfate (SDS) also eliminated the activity. These results suggest that the reduction of tellurite was catalyzed by an enzyme(s). Maintenance of the tellurite reductase activity in cell extracts was dependent on the presence of KCl in the breakage bu¡er although the salt was not required in enzyme assay bu¡ers. In the case of the tellurite reductase from N. occultus, KCl can be replaced in the breakage bu¡er by 30% sorbitol and the basically full enzyme activity is maintained (Table 1). Small soluble thiols may account for the reduction of tellurite in whole cells [20]. In dialyzed cell-free extract, soluble thiols should be removed, however, we cannot discount the possibility that bound thiols may account for reduction. Sucrose gradient centrifugation of cell extract con¢rmed that the enzyme activity was probably not associated with a membrane fraction but rather was either loosely membrane bound or cytoplasmic (Table 1). Only 8% of the original activity of cell-free extracts was seen in the membrane fraction. The enzyme from N. occultus shares a number of features with characterized tellurite reductases. Thermus thermophilus HB8 cell extracts contain three tellurite-reducing activities, one of which has been puri¢ed to homogeneity [7]. This enzyme is NADHdependent and inhibited by SDS. More recently, a number of tellurite-reducing activities have been characterized from Bacillus stearothermophilus V [21]. Crude extracts of the bacterium catalyze the NADH-dependent, protease-sensitive reduction of potassium tellurite. Given the presence of a number
of di¡erent tellurite-reducing systems in these bacteria, puri¢cation of the enzyme(s) of N. occultus is needed for further insight into the method(s) of resistance to tellurite of this organism. Only one study has examined heavy metal resistances in extreme halophiles [22]. Nieto et al. examined a number of halophilic archaeons for their tolerance to common heavy metal contaminants but not tellurite. The reduction of soluble tellurite to elemental tellurium could be an important mechanism for the removal of this element from polluted hypersaline environments. The haloalkaliphilic archaea, being able to transform this toxic anion to non-toxic tellurium at very high concentrations, may be promising candidates for heavy metal-contaminated waste brines.
Acknowledgments We thank Simon Silver for useful comments and encouragement, Dian Molsen for the electron micrography support and David Kelly for providing tellurite-resistant E. coli strains.
References [1] Summers, A.O. and Jacoby, G.A. (1977) Plasmid-determined resistance to tellurium compounds. J. Bacteriol. 129, 276^281. [2] Summers, A.O. and Silver, S. (1978) Microbial transformation of metals. Annu. Rev. Microbiol. 32, 637^672. [3] Taylor, D.E., Walter, E.G., Sherburne, R. and Bazett-Jones, D.P. (1988) Structure and location of tellurium deposited in Escherichia coli cells harboring tellurite resistance plasmids. J. Ultrastruct. Mol. Struct. Res. 99, 18^26. [4] Tindall, B.J., Ross, H.N.M. and Grant, W.D. (1984) Natronobacterium gen. nov. and Natronococcus gen. nov., two new genera of haloalkaliphilic archaebacteria. Syst. Appl. Microbiol. 5, 41^57. [5] DeRosa, M., Gambacorta, A., Grant, W.D., Lanzotti, V. and Nicolaus, B. (1988) Polar lipids and glycine betaine from haloalkaliphilic archaebacteria. J. Gen. Microbiol. 134, 205^211. [6] Desmarais, D., Jablonski, P.E., Fedarko, N.S. and Roberts, M.F. (1997) 2-Sulfotrehalose, a novel osmolyte in haloalkaliphilic archaea. J. Bacteriol. 179, 3146^3153. [7] Chiong, M., Gonzaèlez, E., Barra, R. and Vaèasquez, C. (1988) Puri¢cation and biochemical characterization of tellurite-reducing activities from Thermus thermophilus HB8. J. Bacteriol. 170, 3269^3273. [8] Bradley, D., Grewal, K., Taylor, D. and Whelan, J. (1988)
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[9]
[10]
[11]
[12]
[13]
[14]
[15]
Characterization of RP4 tellurite-resistance transposon Tn521. J. Gen. Microbiol. 134, 2009^2018. Lloyd-Jones, G., Osborn, A., Ritchie, D., Strike, P., Hobman, J., Brown, N. and Rouch, D. (1994) Accumulation and intracellular fate of tellurite in tellurite-resistant Escherichia coli: a model for the mechanism of resistance. FEMS Microbiol. Lett. 118, 113^120. Morton, H. and Anderson, T. (1941) Electron microscopic studies of biological reactions. I. Reduction of potassium tellurite by Corynebacterium diptheriae. Proc. Soc. Exp. Biol. Med. 46, 272^276. Moore, M.D. and Kaplan, S. (1992) Identi¢cation of intrinsic high-level resistance to rare-earth oxides and oxyanions in members of the class Proteobacteria: Characterization of tellurite, selenite, and rhodium sesquioxide reduction in Rhodobacter sphaeroides. J. Bacteriol. 174, 1505^1514. Moore, M. and Kaplan, S. (1994) Members of the family Rhodospirillaceae reduce heavy-metal oxyanions to maintain redox poise during photosynthetic growth. ASM News 60, 17^24. Yurkov, V., Jappeè, J. and Vermeèglio, A. (1996) Tellurite resistance and reduction by obligately aerobic photosynthetic bacteria. Appl. Environ. Microbiol. 62, 4195^4198. Ramamoorthy, S. and Kushner, D. (1975) Binding of mercury and other heavy metal ions by microbial growth media. Microb. Ecol. 2, 162^176. Turner, R., Weiner, J. and Taylor, D. (1992) Use of diethyl-
[16]
[17]
[18]
[19]
[20]
[21]
[22]
23
dithiocarbamate for quantitative determination of tellurite uptake by bacteria. Anal. Biochem. 204, 292^295. Whelan, K.F., Colleran, E. and Taylor, D.K. (1995) Phage inhibition, colicin resistance, and tellurite resistance are encoded by a single cluser of genes on the IncH12 plasmid R478. J. Bacteriol. 177, 5016^5027. Turner, R., Weiner, J. and Taylor, D. (1995) Neither reduced uptake nor increased e¥ux is encoded by tellurite resistance determinants expressed in Escherichia coli. Can. J. Microbiol. 41, 92^98. Chiong, M., Barra, R., Gonzaèlez, E. and Vaèsquez, C. (1988) Resistance of Thermus spp. to potassium tellurite. Appl. Environ. Microbiol. 54, 610^612. Tomas, J.M. and Kay, W.W. (1986) Tellurite susceptibility and non-plasmid-mediated resistance in Escherichia coli. Antimicrob. Agents Chemother. 30, 127^131. Turner, R.J., Weiner, J.H. and Taylor, D.E. (1995) The tellurite-resistance determinants tehAtehB and klaAklaBtelB have di¡erent biochemical requirements. Microbiol. 141, 3133^3140. Moscoso, H., Saavedra, C., Loyola, C., Pichuantes, S. and Vaèsquez, C. (1998) Biochemical characterization of telluritereducing activities of Bacillus stearothermophilus V. Res. Microbiol. 149, 389^397. Nieto, J.J., Ventosa, A. and Ruiz-Berraquero, F. (1987) Susceptibility of halobacteria to heavy metals. Appl. Environ. Microbiol. 53, 1199^1202.
FEMSLE 8712 15-4-99
High level, intrinsic resistance of Natronococcus occultus to potassium tellurite Chad T. Pearion, Peter E. Jablonski * Department of Biological Sciences, Northern Illinois University, DeKalb, IL 60115-2861, USA Received 18 December 1998; received in revised form 23 February 1999; accepted 24 February 1999
Abstract Natronococcus occultus, a haloalkaliphilic archaeon, was examined for its resistance to potassium tellurite. Cells grown in the presence of 1 mM potassium tellurite reduced it to metallic tellurium resulting in the deposition of intracellular crystals in the cytoplasm. The minimal inhibitory concentration for potassium tellurite was 10 mM. N. occultus had an inducible tellurite reductase activity. Cell-free extracts catalyzed the enzymatic reduction of potassium tellurite in a reaction which was dependent on NADH oxidation and a reduced environment. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Tellurite ; Heavy metal resistance; Natronococcus occultus; Archaea
1. Introduction Salts of the rare earth oxyanion tellurite (TeO3 23 ) are toxic to most microorganisms at concentrations as low as 1 Wg ml31 [1,2]. It has been suggested that this toxicity is due to the strong oxidizing ability of tellurite [1]. However, the ability of tellurite to act as an oxidizing agent of any speci¢c components of the cell has not been studied. When grown in the presence of potassium tellurite, bacteria form black colonies due to the deposition of intracellular crystals of what has been shown to be metallic tellurium [3]. In contrast, no member of the domain Archaea has been shown to be naturally resistant to this heavy metal. * Corresponding author. Tel.: +1 (815) 753-3799; Fax: +1 (815) 753-7855; E-mail: [email protected]
Natronococcus occultus is a haloalkaliphilic archaeon which grows under high salt and high pH conditions [4]. Optimum growth occurs at a pH of 9.0^10.0, a NaCl concentration of 3.0^4.0 M and a very low Mg2 concentration. Currently, little is known about the physiology of these organisms with most of the work done thus far concentrating on the lipid composition of these archaeons [4,5]. Here, we present the ¢rst evidence of tellurite resistance in an archaeon.
2. Materials and methods 2.1. Organism N. occultus (ATCC 43101) was grown in de¢ned haloalkaliphile medium [6] containing 1 mM potas-
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 1 1 5 - 9
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sium tellurite at 40³C with shaking (300 rpm). Cells were harvested by centrifugation at 10 000Ug at 4³C for 15 min, resuspended 1:1 (w/v) in 10 mM Tris (Tris(hydroxymethyl)aminomethane) (pH 7.5, 20³C) containing 3 M KCl and 4 mM 2-mercaptoethanol and stored at 320³C. 2.2. Determination of minimal inhibitory concentrations (MIC) Quantitative susceptibility tests were performed using de¢ned haloalkaliphilic agar medium containing potassium tellurite (1, 2.5, 5, 10, 20 or 40 mM). Each plate was inoculated with approximately 104 ^ 105 cells. The MIC was de¢ned as the lowest concentration of potassium tellurite preventing growth at 40³C after 14 days of incubation. 2.3. Electron microscopy Washed cell pellets of N. occultus, grown in the presence of 1 mM potassium tellurite until the late exponential phase, were ¢xed using glutaraldehyde. Samples were then dehydrated successively in 10, 30, 40, 60, 80 and 90% ethanol for 10 min each, 95 and 100% ethanol, ethanol:acetone (1:1 (v/v)) and ¢nally 100% acetone for 5 min each. Each sample was then embedded in epoxy resin. Sectioned material was left unstained and examined by transmission electron microscopy.
2.4. Tellurite reductase assay Cell-free extracts of N. occultus were prepared by French pressure cell disruption at 20 000 psi. The resulting cell extract was centrifuged as described above and the supernatant £uid was used for enzyme assays. The tellurite reduction was assayed spectrophotometrically as described [7]. The reaction mixture (1 ml) contained 10 mM Tris (pH 7.5, 40³C) and 0.1 mM potassium tellurite. Cell-free extract was added to initiate the reaction. The reduction of tellurite was determined by measuring the change in A500 . 3. Results and discussion Cells of N. occultus, when grown in the presence of 1 mM potassium tellurite, accumulated electrondense, black intracellular crystals within the cytoplasm and not speci¢cally membrane-associated (Fig. 1A). Cells grown in the absence of tellurite did not exhibit similar crystals (Fig. 1B). Such crystals have previously been shown to consist of elemental tellurium in several strains of Eubacteria [3,8,9]. The localization of crystals in N. occultus is in contrast to other systems such as Escherichia coli where tellurium crystals have a marked tendency towards local deposition near the cytoplasmic membrane [9]. The addition of bromine water to a pellet
Fig. 1. Transmission electron micrograph through sections of N. occultus cells grown in the presence (A) or absence (B) of 1 mM potassium tellurite. Scale bar represents 1 Wm.
FEMSLE 8712 15-4-99
C.T. Pearion, P.E. Jablonski / FEMS Microbiology Letters 174 (1999) 19^23 Table 1 Tellurite reductase activity in cell-free extracts of N. occultus Assay Conditionsa
Activityb
CFE (uninduced cells) CFEi CFEi +NADH CFEi +2-mercaptoethanol CFEi +NADH+2-mercaptoethanol CFEi (70³C, 15 min) CFEi (100³C, 10 min) CFEi +1% (w/v) SDS (10 min) Membrane fraction CFEi dialyzed against 30% sorbitol
6 0.001 6 0.001 6 0.001 6 0.001 0.237 6 0.001 6 0.001 6 0.001 0.020 0.220
a CFE, cell-free extract (induced cells); 1 mM NADH ; 4 mM 2-mercaptoethanol b vA500 min31
of tellurite grown N. occultus resulted in the rapid oxidation of the black crystals to a colorless compound(s) as previously reported for metallic tellurium [10]. Taken together, these results suggest that N. occultus transported the oxyanion potassium tellurite and subsequently reduced it to black metallic tellurium. To determine the intrinsic level of resistance, the MIC of potassium tellurite was determined by plating 104 ^105 cells on DH medium containing graded concentrations of potassium tellurite. The range used (1^40 mM) covered those concentrations typically used in studies of metal resistance in Eubacteria. After 14 days of incubation at 40³C, the MIC was determined to be 10 mM (2571 Wg ml31 ) for N.occultus. MICs for Natronobacterium magadii and N. gregoryi, two related strains of haloalkaliphilic archaea, were 20 mM (5142 Wg ml31 ). These MICs are the highest reported values to date for the resistance to tellurite. A constitutive high level resistance at concentrations of 800 and 1200 Wg ml31 was described for Rhodobacter capsulatus and Rhodobacter sphaeroides, respectively [11,12]. Recently, three species of Erythromicrobium were shown to have MICs to tellurite of between 2300 and 2700 Wg ml31 [13]. It is important to note that the availability of certain metals can be in£uenced by the microbial growth medium as reported by Ramamoorthy and Kushner [14]. In particular, casamino acids show a high level of binding activity for Hg2 , Pb2 , Cu2 and Cd2 . Measurement of tellurite concentrations in a de¢ned haloalkaliphilic medium not containing cells using
21
the diethyldithiocarbamate (DDTC) assay, indicated that there was insigni¢cant binding to any medium components. The growth of N. occultus was similar in the presence or absence of 10 mM potassium tellurite (data not shown) suggesting an intrinsic resistance in this strain. Cells grown in a de¢ned haloalkaliphilic medium without tellurite to the early stationary phase and then transferred to medium containing 1 mM tellurite exhibited the same initial growth rate. Cells grown in medium containing tellurite to the early stationary phase were inoculated into fresh medium containing tellurite and exhibited nearly the original growth rate. Similarly, when cells previously grown in the presence of 10 mM tellurite were inoculated into medium without tellurite, comparable growth rates were observed. A noticeable uptake of potassium tellurite by N. occultus was not observed using the established DDTC colorimetric assay [15] over a 14-day period (data not shown). Cell-free controls con¢rmed that the medium components did not interfere with the assay. As a positive control, E. coli JM109 harboring the tellurite resistance determinant R478 [16] was tested for its tellurite uptake. A decrease in tellurite in the medium was observed using the DDTC assay. This suggests that a di¡erent resistance mechanism in N. occultus may exist. Several possibilities are likely. N. occultus may exclude a large portion of the tellurite thus accounting for resistance. While reducing tellurite to tellurium intracellularly, the cells may also transport unoxidized tellurite back into the medium. It is also possible that additional unidenti¢ed water soluble forms of tellurite may be produced, transported out of the cell and react in the DDTC assay. While neither a reduced uptake nor an increased e¥ux is responsible for the tellurite resistance in Escherichia coli [17], further investigation will be necessary to de¢ne the system in N. occultus. In addition, the methylation of tellurium is theoretically a mechanism of resistance. Microorganisms including Thermus thermophilus and Pseudomonas aeruginosa produce a garlic odor (probably dimethyl telluride) [1,18] while E. coli does not [1]. N. occultus does indeed produce a garlic odor when grown in the presence of tellurite suggesting the production of dimethyl telluride. Further experiments are needed to determine if methylation by N. occultus occurs. The
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tellurite uptake in E. coli appears to occur at last partly via the cellular phosphate transport systems [19]. In uptake experiments with N. occultus using radiolabelled phosphate, tellurite did not compete with the phosphate transport (data not shown). Clearly, a number of resistance mechanisms are possible and remain to be elucidated in the haloalkaliphilic archaea. N. occultus showed an inducible tellurite reductase activity (Table 1). Cells that were not previously exposed to tellurite did not contain this activity. Cellfree extracts of tellurite grown cells catalyzed the reduction of tellurite to metallic tellurium only upon the addition of NADH and under reducing conditions. The enzyme activity was destroyed by heat treatment at 70³C for 15 min. Detergent treatment with sodium dodecyl sulfate (SDS) also eliminated the activity. These results suggest that the reduction of tellurite was catalyzed by an enzyme(s). Maintenance of the tellurite reductase activity in cell extracts was dependent on the presence of KCl in the breakage bu¡er although the salt was not required in enzyme assay bu¡ers. In the case of the tellurite reductase from N. occultus, KCl can be replaced in the breakage bu¡er by 30% sorbitol and the basically full enzyme activity is maintained (Table 1). Small soluble thiols may account for the reduction of tellurite in whole cells [20]. In dialyzed cell-free extract, soluble thiols should be removed, however, we cannot discount the possibility that bound thiols may account for reduction. Sucrose gradient centrifugation of cell extract con¢rmed that the enzyme activity was probably not associated with a membrane fraction but rather was either loosely membrane bound or cytoplasmic (Table 1). Only 8% of the original activity of cell-free extracts was seen in the membrane fraction. The enzyme from N. occultus shares a number of features with characterized tellurite reductases. Thermus thermophilus HB8 cell extracts contain three tellurite-reducing activities, one of which has been puri¢ed to homogeneity [7]. This enzyme is NADHdependent and inhibited by SDS. More recently, a number of tellurite-reducing activities have been characterized from Bacillus stearothermophilus V [21]. Crude extracts of the bacterium catalyze the NADH-dependent, protease-sensitive reduction of potassium tellurite. Given the presence of a number
of di¡erent tellurite-reducing systems in these bacteria, puri¢cation of the enzyme(s) of N. occultus is needed for further insight into the method(s) of resistance to tellurite of this organism. Only one study has examined heavy metal resistances in extreme halophiles [22]. Nieto et al. examined a number of halophilic archaeons for their tolerance to common heavy metal contaminants but not tellurite. The reduction of soluble tellurite to elemental tellurium could be an important mechanism for the removal of this element from polluted hypersaline environments. The haloalkaliphilic archaea, being able to transform this toxic anion to non-toxic tellurium at very high concentrations, may be promising candidates for heavy metal-contaminated waste brines.
Acknowledgments We thank Simon Silver for useful comments and encouragement, Dian Molsen for the electron micrography support and David Kelly for providing tellurite-resistant E. coli strains.
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