Department of Biological Sciences, University of Dundee, Dundee DD1 4HN, UK1
Author for correspondence: Geoffrey M. Gadd. Tel:+44 1382 344266. Fax:+44 1382 344275. e-mail: g.m.gadd{at}dundee.ac.uk
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ABSTRACT |
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Keywords: sulphate-reducing bacteria, copper binding, zinc binding, metal binding, Desulfococcus multivorans
Abbreviations: SRB, sulphate-reducing bacteria
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INTRODUCTION |
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Micro-organisms release a diverse range of specific and non-specific metal-binding compounds into the environment, which can act to ameliorate the presence of toxic metals and/or mediate uptake. Non-specific metal-binding microbial metabolites, e.g. organic acids, can complex metals and affect mobility and thus toxicity (Burgstaller & Schinner, 1993 ; Sayer & Gadd, 1997
; Gadd, 1999
). Undefined, microbially produced macromolecules can also bind significant amounts of potentially toxic metals. These include humic and fulvic acids arising from lignocellulose degradation and extracellular polymeric substances (EPS), a mixture of polysaccharides, mucopolysaccharides and proteins produced by bacteria, algae and fungi (Schreiber et al., 1990
; Beech & Cheung, 1995
; Spark et al., 1997
). The composition of EPS produced by SRB has been shown to be modified by the presence of carbon steel, which may increase its metal-binding capacity (Zinkevich et al., 1996
). In contrast, specific metal-binding compounds are produced directly in response to high levels of toxic metals. Intracellular, low-molecular-mass, metal-binding proteins produced by mammals (metallothioneins) and also plants and algae (phytochelatins) have been well studied (Birch & Bachofen, 1990
; Rauser, 1995
; Howe et al., 1997
). However, some micro-organisms are known to produce extracellular, specific metal-binding compounds. Examples include Sarcina urea, which has been shown to produce metal-binding proteins in response to Pd2+, Sr2+, In2+ and Ce4+ (Beveridge, 1986
), a Pseudomonas sp. which has been shown to produce cadmium-binding proteins in response to cadmium (Rayner & Sadler, 1989
) and Vibrio alginolyticus, which produces copper-binding proteins in response to high levels of copper (Harwood-Sears & Gordon, 1990
). Specific extracellular metal-binding compounds can also be produced by micro-organisms in response to low levels of metals, in order to facilitate the uptake of essential metals. The most studied system is the production of siderophores in response to low environmental iron concentrations. Siderophores are low-molecular-mass Fe(III) coordination compounds (5001000 Da) produced by many micro-organisms and act by complexing and solubilizing insoluble Fe(III) in a form which can be transported into the cell using specific transport mechanisms (Neilands, 1981
). Although siderophores are iron(III)-binding compounds, they are also able to bind other metals such as magnesium, manganese, chromium(III), gallium(III) and radionuclides such as plutonium(IV) (Bulman, 1978
; Birch & Bachofen, 1990
). However, siderophores are not produced under the anaerobic conditions in which SRB grow and where iron is predominately in the Fe(II) form (Guerinot, 1994
).
It can be theorized that, since metals are effectively immobilized by the sulphide produced by SRB, these organisms must require a specific metal-binding and/or uptake mechanism in order to obtain essential metals such as copper and zinc. Such a metal acquisition system should also have an affinity greater than or comparable to that of sulphide in order to compete successfully for any available metal cations with the sulphide present. This study was therefore undertaken to determine whether SRB produce extracellular, soluble metal-binding compounds in order to provide further understanding of metal metabolism in these organisms.
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METHODS |
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Polarographic measurement of metal binding by SRB spent medium.
Differential pulse polarography was carried out using a Metrohm 663 VA stand incorporating a multi-wavelength electrode set to the static mercury drop electrode facility. The analysis was carried out using Autolab General Purpose Electrochemical System (GPES) for Windows, version 4.3 (Ecochemie, The Netherlands). Measurements of free metal ion concentrations were carried out in 25 ml electrolyte containing 1 M KCl, buffered with either 10 mM PIPES (pH 6·0) or 50 mM MES (pH 5·5) buffer, adjusted to the appropriate pH value using tetramethylammonium hydroxide. These buffers were chosen for their negligible metal-binding activities (Good et al., 1986 ). In most experiments, free metal cation concentrations, from added metal chloride solutions, were measured immediately after the reaction mixture was purged with N2. The electrolyte was purged for 180 s and the current measured over three scans between potentials of 0 and -0·4 V for copper or -0·8 and -1·2 V for zinc with a step potential of 0·0036 V. Each metal has a characteristic half wave potential, -0·18 V for copper and -0·97 V for zinc, with the measured current being proportional to the amount of metal ion present. Free metal concentrations were calculated with respect to a standard curve over the range 10100 µM. Two approaches were used to measure the metal-binding activity present in SRB culture filtrates. The first involved measuring the decrease in free metal concentration on the addition of a known volume of sample and calculating the concentration of metal-binding compound as the amount of metal bound (ml culture filtrate)-1 (Shuman, 1994
). This method was used to measure the increase in metal-binding activity over time in Dc. multivorans batch cultures and to identify which HPLC fractions contained metal-binding activity. The second approach (the reverse of the method described above) involved the addition of aliquots of metal ion solution to buffered culture filtrate. These titration data could then be transformed using the method of Ruzic (1982)
and van den Berg (1982)
to obtain stability constants for the metal-binding ligands.
Purification of a metal-binding compound from SRB spent media.
The metal-binding compound was extracted and purified using a method suggested by Professor R. O. Hallberg, University of Stockholm (personal communication). Each sample of spent medium was extracted by shaking for 1 h with approximately 10% (v/v) dichloromethane (CH2Cl2), the organic phase being collected using a separating funnel, and the extraction repeated. Both extracts were pooled and evaporated under nitrogen to a volume of less than 1 ml; acetonitrile (CH3CN) was added and the residual CH2Cl2 was removed under nitrogen. All the apparatus used for the purification was washed with dichloromethane and rinsed with Milli-Q water.
HPLC was carried out using a 5 µM adsorbosphere, C18 reverse-phase column (Alltech) linked to a Waters 600 system controller, a Waters 486 programmable multiwavelength detector, a Waters 600 pump and a Waters 717plus autosampler controlled by Millipore (Waters) Millennium chromatography software. The mobile phase consisted of an initial gradient from 10 to 100% (v/v) CH3CN over 10 min followed by isocratic 100% CH3CN for 15 min. The detector monitored the A318 of the eluate (R. O. Hallberg, personal communication). Timed fractions of eluate were collected at 30 s intervals using a Pharmacia FRAC-100 fraction collector. Fractions from three runs were collected in the same tubes and most of the acetonitrile was removed by evaporation under nitrogen (final volume 0·1 ml) in order to prevent interference with the detection of the copper-binding compound. Each fraction was tested for copper-binding activity as the decrease in free ion concentration.
Analytical techniques.
The Bradford method was used to estimate protein (Bradford, 1976 ). The reagent consisted of a filtered solution of 100 mg Coomassie BBG-250 l-1 dissolved in 5% (v/v) ethanol acidified with 10% (v/v) phosphoric acid. One hundred microlitres of an appropriate dilution of the unknown sample was mixed with 1 ml reagent and incubated in the dark for 2 min before measuring the A595 in a Unicam SP600 series 2 spectrophotometer. Bovine serum albumin (BSA) was used to construct a standard curve over the range 110 µg (100 µl)-1. Sulphide was measured using polarography. Twenty millilitres of 0·1 M NaOH electrolyte was purged with N2 for 300 s, the unknown sample was added and the current was measured over three scans between potentials of -0·6 and -1 V, using a step potential of 0·0036 V. The electrolyte was purged for 10 s after the sample had been added to mix the solution but was not purged between scans in order to minimize sulphide loss during the analysis. Sodium sulphide (2·08 mM in 0·1 M NaOH) was used to construct a standard curve over the range 050 µM.
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RESULTS AND DISCUSSION |
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The titration curve produced on the addition of trace metal (M) can be described using the equation
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This can be rearranged to give
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and if MT equals CM+M, then this last equation can be rearranged to give
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Plotting free metal ion concentration/bound metal ion concentration, [M]/(MT-[M]), against free metal ion concentration, [M], produces a straight line with a gradient of 1/CL and a y intercept of 1/KCL. The titration data plotted in Fig. 3(a) were thereby transformed and the graph of free copper/bound copper against free copper (Fig. 3b
) was used to calculate ligand concentrations and binding constants for Dc. multivorans culture filtrates, assuming 1:1 metalligand binding. A copper-binding ligand concentration of 3·64±0·33 µmol ml-1 was obtained which compared to that of 2·90±0·62 µmol ml-1 calculated by titration (n=4). Stability constants (K=[ML]/[M][L]) are traditionally compared as their log values and the log10K for the copper ligand complex was calculated as 5·68±0·64 (n=4). This value is similar to that obtained for the binding of copper by oxalic acid (log10K=6·23; Smith & Martell, 1976
) but several orders of magnitude lower than that of copper or iron(III) complexed by organic ligands in sea water [copper log10K
912; iron(III) log10K
21; van den Berg & Donat, 1992
; van den Berg, 1995
] and either that of copper bound by sulphide (log10K=36·1; Smith & Martell, 1976
) or typical siderophores. For example, catecholate-type siderophores isolated from the marine bacterium Alteromonas luteoviolacea and marine cyanobacterium Synechococcus were found to bind iron(III) with a log10K of 37·643·6 (Lewis et al., 1995
) and hydroxamate siderophores have been shown to form iron(III) complexes with stability constants in the range 2932·5 (Crumbliss, 1991
). The relatively low stability constant obtained for the copper-binding compound from Dc. multivorans culture filtrates as compared to metal-sulphide binding suggests that unless the stability constant has been underestimated due to other ligands in the culture filtrate, the copper-binding compound identified is rather inefficient and therefore unlikely to have a role in the acquisition of essential metals for metabolism. It can be postulated that there is an alternative, more competitive specific metal-binding and uptake system, possibly similar to one of the range of cell surface (as opposed to extracellular) metal transport systems that have been characterized in microbes for both the import and export of ions. Metal ions can be transported as either free ions (e.g. the transport of Cu+ by Saccharomyces cerevisiae) or metalligand complexes [e.g. siderophore-mediated Fe(III) transport]. S. cerevisiae has surface metal reductase enzymes that convert insoluble extracellular Fe3+ to the more soluble Fe2+ and Cu2+ into Cu+, for uptake into the cell. Each metal is taken up using either a high-affinity system which is expressed at low metal levels or a low-affinity system which is expressed under high levels of metal (Eide, 1998
). Cell membrane metal-ion transport systems tend to involve one or more transmembrane proteins and often other substrate or ATP-binding proteins. Transport requires energy, either in the form of ATP (e.g. the cadmium and copper ATPases found in Gram-positive bacteria) or by the chemiosmotic co-transport of ions such as H+ (e.g. the arsenite efflux system in Gram-negative bacteria; Silver & Phung, 1996
).
Both the ligand concentrations and stability constants obtained for Dc. multivorans culture filtrates were for complexes formed over a few minutes, i.e. the culture filtrate was added to metal ion solution (or vice versa) and the free metal ion concentration was measured almost immediately. However, work carried out by van den Berg (1995) on complexation of copper in sea water allowed the reaction mixtures to equilibrate over 5 h before polarographic analysis. In order to find out if long-term incubation influenced the copperligand complexes, copper-binding capacities of Dc. multivorans culture filtrate were measured after 0, 2, 5 and 72 h (data not shown). It was found that the ligand concentrations and stability constants obtained were similar to those described above and there was no apparent change with time.
Purification of the metal-binding compound from Dc. multivorans culture filtrate
The metal-binding compound from Dc. multivorans culture filtrate was partly purified by organic solvent extraction. The copper-binding capacity and sulphide concentration of the original culture filtrate and the dichloromethane extract transferred into acetonitrile are given in Table 2. Data presented in Table 2
show that extraction of the Dc. multivorans culture filtrate using dichloromethane resulted in the separation of non-dichloromethane-soluble medium constituents such as sulphide from the copper-binding activity. The extraction process resulted in an approximate 1/33 concentration in volume with approximately 6% of the total activity being extracted. The absence of sulphide in the extract confirms that the metal-binding capacity measured in Dc. multivorans culture filtrates was not due to the presence of sulphide.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Beech, I. B. & Cheung, C. W. S. (1995). Interactions of exopolymers produced by sulphate-reducing bacteria with metal ions. Int Biodeterior Biodegrad 35, 59-72.
van den Berg, C. M. G. (1982). Determination of copper complexation with natural organic ligands in seawater by equilibration with MnO2. 1. Theory. Mar Chem 11, 307-322.
van den Berg, C. M. G. (1995). Evidence for organic complexation of iron in seawater. Mar Chem 50, 139-157.
van den Berg, C. M. G. & Donat, J. R. (1992). Determination and data evaluation of copper complexation by organic ligands in sea water using cathodic stripping voltammetry at varying detection windows. Anal Chim Acta 257, 281-291.
Beveridge, T. J. (1986). The immobilisation of soluble metals by bacterial walls. Biotechnol Bioeng Symp 16, 127-139.
Beveridge, T. J., Hughes, M. N., Lee, H., Leung, K. T., Poole, R. K., Savvaidis, I., Silver, S. & Trevors, J. T. (1997). Metalmicrobe interactions: contemporary approaches. Adv Microb Physiol 38, 177-243.[Medline]
Birch, L. & Bachofen, R. (1990). Complexing agents from microorganisms. Experientia 46, 827-834.
Bradford, M. B. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal Biochem 72, 248-254.[Medline]
Bulman, R. A. (1978). Chemistry of plutonium and the transuranics in the biosphere. Struct Bonding 34, 39-77.
Burgstaller, W. & Schinner, F. (1993). Leaching of metals with fungi. J Biotechnol 27, 91-116.
Crumbliss, A. L. (1991). Aqueous solution equilibrium and kinetic studies of iron siderophore and model siderophore complexes. In CRC Handbook of Microbial Iron Chelates, pp. 177-233. Edited by G. Winkelmann. Boca Raton, FL: CRC Press.
Eide, D. J. (1998). The molecular biology of metal ion transport in Saccharomyces cerevisiae. Annu Rev Nutr 18, 441-469.[Medline]
Ensley, B. D. & Suflita, J. M. (1995). Metabolism of environmental contaminants by mixed and pure cultures of sulphate-reducing bacteria. In Sulphate-Reducing Bacteria, pp. 293-332. Edited by L. L. Barton. New York: Plenum.
Gadd, G. M. (1999). Fungal production of citric and oxalic acid: importance in metal speciation, physiology and biogeochemical processes. Adv Microb Physiol 41, 47-92.[Medline]
Gadd, G. M. & White, C. (1993). Microbial treatment of metal pollution a working biotechnology? Trends Biotechnol 11, 353-359.[Medline]
Good, N. E., Winget, G. D., Winter, W., Connolly, T. N., Izawa, S. & Singh, R. M. M. (1986). Hydrogen ion buffers for biological research. Biochemistry 5, 467-474.
Guerinot, M. L. (1994). Microbial iron transport. Annu Rev Microbiol 48, 743-772.[Medline]
Hansen, T. A. (1993). Carbon metabolism in sulphate-reducing bacteria. In The Sulphate-Reducing Bacteria: Contemporary Perspectives, pp. 21-40. Edited by J. M. Odom & R. Singleton. New York: Springer.
Hao, O. J., Chen, J. M., Huang, L. & Buglass, R. L. (1996). Sulfate-reducing bacteria. Crit Rev Environ Sci Technol 26, 155-187.
Harwood-Sears, V. & Gordon, A. S. (1990). Copper-induced production of copper-binding supernatant proteins by the marine bacterium Vibrio alginolyticus. Appl Environ Microbiol 56, 1327-1332.[Medline]
Howe, R., Evans, R. L. & Ketteridge, S. W. (1997). Copper-binding proteins in ectomycorrhizal fungi. New Phytol 135, 123-131.
Hughes, M. N. & Poole, R. K. (1989). Metals and Micro-organisms. London: Chapman & Hall.
Lewis, B. L., Holt, P. D., Taylor, S. W., Wilhelm, S. W., Trick, C. G., Butler, A. & Luther, G. W. (1995). Voltammetric estimation of iron(III) thermodynamic stability constants for catecholate siderophores isolated from marine bacteria and cyanobacteria. Mar Chem 50, 179-188.
Lide, D. R. (1995). CRC Handbook of Chemistry and Physics, 76th edn. Boca Raton, FL: CRC Press.
Lund, W. (1986). Electrochemical methods and their limitations for the determination of metal species in natural waters. In The Importance of Chemical Speciation in Environmental Processes, pp. 533-561. Edited by M. Bernhard, P. B. Brinckman & P. J. Sadler. Berlin: Springer.
Neilands, J. B. (1981). Microbial iron compounds. Annu Rev Biochem 50, 715-731.[Medline]
Nurnberg, H. W. (1984). Potentialities of voltammetry for the study of physiochemical aspects of heavy metal complexation in natural waters. In Complexation of Trace Metals in Natural Waters, pp. 1-16. Edited by C. J. M. Kramer & J. C. Duinker. The Hague: Junk Publishers.
Postgate, J. R. (1984). The Sulphate-Reducing Bacteria. Cambridge: Cambridge University Press.
Rauser, W. E. (1995). Phytochelatins and related peptides. Plant Physiol 109, 1141-1149.
Rayner, M. H. & Sadler, P. J. (1989). Cadmium accumulation and resistance mechanisms in bacteria. In MetalMicrobe Interactions, pp. 39-49. Edited by R. K. Poole & G. M. Gadd. Oxford: IRL Press.
Ruzic, I. (1982). Theoretical aspects of the direct titration of natural waters and its information yield for trace metal speciation. Anal Chim Acta 140, 99-113.
Sayer, J. A. & Gadd, G. M. (1997). Solubilization and transformation of insoluble inorganic metal compounds to insoluble metal oxalates by Aspergillus niger. Mycol Res 101, 653-661.
Schreiber, D. R., Millero, F. J. & Gordon, A. S. (1990). Production of an extracellular copper-binding compound by the heterotrophic marine bacterium Vibrio alginolyticus. Mar Chem 28, 275-284.
Shuman, L. M. (1994). Chelate and pH effects on aluminum determined by differential pulse polarography and plant root bioassay. J Environ Sci Health 29, 1423-1438.
Silver, S. & Phung, L. T. (1996). Bacterial heavy metal resistance: new surprises. Annu Rev Microbiol 50, 753-789.[Medline]
Smith, R. M. & Martell, A. E. (1976). Critical Stability Constants, vol. 4, Inorganic Complexes. New York: Plenum.
Spark, K. M., Wells, J. D. & Johnson, B. B. (1997). The interaction of a humic acid with heavy metals. Aust J Soil Res 35, 89-101.
Svehla, G. (1996). Vogels Qualitative Inorganic Analysis, 7th edn. Harlow: Longman Group.
White, C. & Gadd, G. M. (1996). Mixed sulphate-reducing bacterial cultures for bioprecipitation of toxic metals: factorial and response-surface analysis of the effects of dilution rate, sulphate and substrate concentration. Microbiology 142, 2197-2205.
White, C. & Gadd, G. M. (1998). Reduction of metal cations and oxyanions by anaerobic and metal-resistant microorganisms: chemistry, physiology, and potential for the control and bioremediation of toxic metal pollution. In Extremophiles, Microbial Life in Extreme Environments, pp. 233-254. Edited by K. Horikoshi & W. D. Grant. New York: Wiley-Liss.
White, C., Sharman, A. K. & Gadd, G. M. (1998). An integrated microbial process for the bioremediation of soil contaminated with toxic metals. Nat Biotechnol 16, 572-575.[Medline]
Widdel, F. & Hansen, T. A. (1991). The dissimilatory sulphate- and sulphur-reducing bacteria. In The Prokaryotes, 2nd edn, pp. 583624. Edited by A. Balows, H. G. Trüper, M. Dworkin, W. Harder & K. H. Schleifer. New York: Springer.
Widdel, F. & Pfennig, N. (1981). Studies on dissimilatory sulfate-reducing bacteria that decompose fatty-acids. 1. Isolation of new sulfate-reducing bacteria enriched with acetate from saline environments description of Desulfobacter postgatei gen-nov, sp-nov. Arch Microbiol 129, 395-400.[Medline]
Zinkevich, V., Bogdarina, I., Kang, H., Hill, M. A. W., Tapper, R. & Beech, I. B. (1996). Characterization of exopolymers produced by different isolates of marine sulphate-reducing bacteria. Int Biodeterior Biodegrad 37, 163-172.
Received 14 January 1999;
revised 13 May 1999;
accepted 7 June 1999.
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