19 Bacterial Metal-Responsive Elements and Their Use in Biosensors for Monitoring of Heavy Metals Ibolya Bontidean and Elisabeth Cso ¨ regi Lund University, Lund, Sweden Philippe Corbisier Institute for Reference Materials and Measurements, Geel, Belgium Jonathan R. Lloyd and Nigel L. Brown The University of Birmingham, Edgbaston, Birmingham, United Kingdom 1. INTRODUCTION Society is learning to adapt to pollution by heavy metals in the environment, and is now attempting to remediate, control, and minimize such pollution wherever possible. To do this, there is a need for methods of assessing the amount of heavy metal pollution in the natural and industrial environments. Although it is relatively straightforward to use the techniques of analytical chemistry to detect total amounts of heavy metal in a given location, this rarely tells you how much Copyright © 2002 Marcel Dekker, Inc. of this metal is a biological hazard. To achieve this, biological methods may offer distinct advantages over chemical methods. It is really only since the industrial revolution that large numbers of peo- ple have been exposed to significant levels of toxic metals, although at least since Roman times heavy metals have been used in medicine and cosmetics (1). In contrast, microorganisms have always lived with ‘‘pollution’’ by heavy metals, as they have evolved to occupy ecological niches in which these toxic metals naturally occur and in which they may be released by geochemical processes. Consequently, bacteria in particular, but also yeasts, fungi, and many plants, have developed specific mechanisms to tolerate or detoxify heavy metals. This chapter describes some of the ways in which we and others have begun to exploit these biological mechanisms to determine the amount of ‘‘bio- available’’ heavy metal in natural and industrial environments. These methods are still in their infancy compared with the techniques of analytical chemistry, but may offer some advantages in ease of use as well as in biological relevance. In particular, we are attempting to couple the high specificity of biological sys- tems with the high sensitivity of modern microelectronics in the development of biosensors. Elsewhere in this book, you will find information on the occurrence of heavy metals in the natural environment, and we will not repeat it here. However, knowledge of the occurrence and amount of heavy metal ions is important in many fields, such as environmental monitoring, clinical toxicology, wastewater treatment, and industrial process monitoring. Therefore, many spectroscopic methods, including atomic absorption and emission spectroscopy (2), flame atomic absorption spectrometry (3), and inductively coupled plasma mass spec- troscopy (2,4), have been developed and are commercially available. These meth- ods exhibit good sensitivity, selectivity, reliability, and accuracy, but they often require sophisticated instrumentation and trained personnel. Electrochemical methods like ion selective electrodes, polarography, and other voltammetric methods (5) are much simpler and require less complex instrumentation, but are often unable to monitor at very low concentrations. None of these techniques can define or quantify the amount of heavy metal that is bioavailable and therefore likely to be a risk to living organisms. To achieve that, one needs a measurement that is biologically relevant, and the development of biosensors offers consider- able promise in this respect. A biosensor is a combination of a highly selective biological recognition element, responsible for the selectivity of the device, and a detection system (the transducer) for quantifying the reaction between the biological component and the target substance (analyte) to be monitored. In this chapter, we describe the background of bacterial interactions with heavy metals, and illustrate how that information is being used in the development of biosensors for heavy metals. Copyright © 2002 Marcel Dekker, Inc. 2. BACTERIAL RESISTANCES TO HEAVY METALS Heavy metals interact with living organisms in a variety of ways. A number of metals (e.g., Cu, Fe, Zn, V, Ni) are essential components of metalloenzymes (6). Others (e.g., Hg, Pb, Cd) are highly toxic with no known beneficial function. Metal-binding proteins are synthesized by many cell types in response to the presence of specific metals (7). Both prokaryotic and eukaryotic cells have mech- anisms to transport essential metals to the sites of synthesis of metalloproteins, and many bacterial cells have specific systems for conferring resistance to heavy metals. These transport or resistance systems may be inducible by the metal, and therefore gene regulatory systems may be required that recognize the metal (8). The best understood of these systems at present are those responsible for confer- ring resistance to heavy metals in bacterial systems. A list of some of the determinants of resistance to heavy metals found in bacteria is given in Table 1. These include resistances to cations and to oxyanions of metals in their most common physiological forms, and many of these resistance determinants have been described in recent reviews (9–12). These resistance de- terminants confer specificity to one or a few related metal ions, unlike most eukar- yotic systems, where resistance is due to sequestration by relatively broad-range determinants, such as metallothioneins or phytochelatins. Metallothioneins have rarely been identified in bacterial systems (13,14). For all the resistance determi- nants in Table 1, the genes have been sequenced and the identities of the proteins conferring resistance have been predicted. The mechanisms of metal tolerance and resistance vary, but the majority are due to efflux of the toxic metal from the cell (15). Some of these efflux systems are part of the normal metal homeostasis systems of the bacterial cell and the efflux pumps are encoded on the bacterial chromosome. These can be considered as proteins that confer the normal metal tolerance of the bacterial cells in which they occur. Examples of such proteins are the ZntA zinc transporter (16) and the CopA copper transporter (17) in Escherichia coli, or the CopA and CopB copper transporters in Enterococcus hirae (18). These proteins and some of the metal resistance proteins [e.g., CadA from Staphylococcus aureus, which confers Cd(II) resistance (19), or PbrA from Ralstonia metallidurans, which is part of the lead resistance determinant (20)] have similar structures. They are P-type ATPases, with eight transmembrane helices, one of which contains the amino acid sequence Cys-Pro-(Cys/His/Ser), and are known as CPx-ATPases (21). The N-terminus of about 100 amino acids shows some sequence similarity to the periplasmic mercury-resistance protein, MerP (see below), and contains a Cys-X-X-Cys motif associated with heavy metal binding (where X is any amino acid) (21). This N-terminal MerP-like region may be repeated and was thought to confer metal specificity on the transporter. Copyright © 2002 Marcel Dekker, Inc. T ABLE 1 Some Heavy Metal Resistance Determinants in Bacteria Metal Organism Mechanism Location Ref. Hg Ps. aeruginosa (and large number Uptake of Hg II and reduction to Plasmids and transposons 25 of other gram-negative and Hg 0 by mercuric reductase gram-positive genera) Cd Staph. aureus Efflux CPx-ATPase Chromosome 19 Ralstonia sp. Efflux pumps (czc, Cd, Zn, and Co; Plasmid 70 cnr, Cd and Ni) Ps. aeruginosa CMG103 Efflux pumps (czr, Cd, Zn) Plasmid 71 Zn Ralstonia sp. See Cd, czc system Plasmid 70 E. coli Efflux CPx-ATPase Chromosome 16 Ps. aeruginosa CMG103 See Cd, czr system Plasmid 71 Cu Ps. syringae Surface sequestration (cop sys- Plasmid 72 tem) E. coli ? Surface sequestration/efflux Plasmid 73 (pco system); efflux CPx-ATPase (copA) Chromosome 17 E. hirae Efflux CPx-ATPase (copA/B) Chromosome 74 Ralstonia sp. Efflux CPx-ATPase Plasmid Co Ralstonia sp. Efflux pumps (czc Cd, Zn, and Co) Plasmid 70 Synechocystis Efflux pump (coaT) Chromosome 75 Ni Ralstonia sp. Efflux pump (cnr Cd and Ni) Plasmid 76 Pb Ralstonia sp. Possible efflux and sequestration Plasmid 20 As Staphylococcus aureus Arsenate reductase and arsenite ef- Plasmid 77 flux Cr Ralstonia sp. Efflux Plasmid 78 Ps. aeruginosa Efflux Plasmid 79 Bacillus sp. Efflux ? 80 Ag Salmonella Sequestration and efflux Plasmid 81 Copyright © 2002 Marcel Dekker, Inc. Expression of the metal homeostasis proteins is usually regulated as part of the mechanisms whereby the bacterial cell adjusts the intracellular concentra- tion of the individual metal. ZntA and CopA are regulated by activator proteins (ZntR and CueR), which respond to the specific metal (22; 22a), and the E. hirae system involves a complex interaction of the regulatory proteins CopY and CopZ (23). Resistance determinants per se are frequently plasmid-borne and may inter- act with the chromosomally encoded systems for metal homeostasis if the metal is also an essential nutrient. For example, the copper resistance determinant of E. coli appears to require proteins that are part of the normal homeostasis system of E. coli (24) and are therefore encoded on the chromosome. Determinants of mercuric ion resistance, on the other hand, appear to require only those genes carried on the mercury-resistance plasmid (25). The proteins required for metal homeostasis or metal resistance are often expressed by the bacteria in response to metal ion concentration (26). For ex- ample, many resistance determinants are expressed only in the presence of the specific metal ions at high subtoxic concentration (8). This involves specific regulatory proteins, either repressors or activators, that bind the metal ion and alter transcription of the structural genes responsible for metal sequestration, transport, or modification. Metal-resistance determinants and the chromosomal determinants of metal homeostasis contain metal-responsive genetic elements responsible for expression of structural gene products that bind and/or transport the metal ions. These regulatory elements, the regulatory proteins, or the products of the structural genes could be used in the construction of metal-specific biosen- sors. Probably the best understood of all metal resistances is the widespread group of mercury-resistance (mer) determinants (25,27). Mercury is not an es- sential nutrient and the resistance determinants are often found in plasmids or transposons. Among the simplest of these mer determinants is that of transposon Tn501 from Pseudomonas aeruginosa. This is shown in Figure 1. Three structural genes, encoding (a) a small periplasmic protein, MerP, (b) an inner membrane transport protein, MerT, and (c) the enzyme mercuric reductase, are expressed under the regulation of the activator protein, MerR, which binds Hg(II) and activates gene expression (25). Possibly because of our detailed knowledge of this system, several different components of the mer system have been used in the design of biosensors. These include the NADPH-dependent mercuric reductase in an enzyme-linked biosensor (28), the mer regulatory region in a whole cell biosensor (29), and the MerR protein in a capacitance biosensor (30). We believe that, as a general principle, we can use many of the bacterial resistance determinants for other metals in the development of biosensors. Some examples of the creation of such biosensors are given below. Copyright © 2002 Marcel Dekker, Inc. F IGURE 1 Diagram of the mercuric ion resistance (mer) operon of transposon Tn501 showing the genes, gene products, and regulatory sequences. The regulatory region, the mercuric reductase enzyme, and the MerR protein have all been used in the development of biosensors for Hg(II). 3. OTHER METAL-REGULATED SYSTEMS A number of heavy-metal-regulated bacterial systems not directly related to heavy-metal-resistance mechanisms have been described. Those systems are mainly involved in the intracellular regulation of essential transition metals ions such as iron, nickel, molybdenum, and magnesium. The transport and regulation of iron concentration in bacteria has been stud- ied in detail. This essential metal for cellular metabolism is needed as a cofactor for a large number of enzymes, but is not easily available to microorganisms in aerobic environments. Therefore, most aerobic bacteria produce and secrete low- molecular-weight compounds termed siderophores to capture Fe 3ϩ from the extra- cellular medium. The iron uptake has to be very well regulated to maintain the intracellular concentration of the metal between desirable limits, since too high an intracellular concentration of iron can catalyze Fenton reactions and generate toxic species of oxygen. An understanding of how bacteria regulate iron transport (31) through the Fur protein (for ferric uptake regulation) was gained by mapping, cloning, and eventually sequencing the fur gene (32). The Fur protein has been purified (33), and recently the abundance of the Fur protein, the form of interac- tion with target DNA sequences, and the involvement of Fur in many cell func- Copyright © 2002 Marcel Dekker, Inc. tions indicate that the Fur protein performs more like a general regulator than a specific repressor (34). The cooperative binding of the Fur protein in extended promoter regions would explain how a relatively simple protein controls a com- plex regulon in a gradual fashion. The type of regulation described for Fur appears to be very similar to that of other metal-dependent repressors. Zinc is also an essential element that, de- pending on the concentration, becomes a potent toxin. In addition to the regula- tion of zinc efflux by ZntR (22), the regulation of zinc uptake by the Zur protein has been described in E. coli (35). The genes involved were named znuACB (for zinc uptake) and localized at 42 min on the genetic map of E. coli.AznuA-lacZ operon fusion was repressed by 5 µM zinc and showed a more than 20-fold increase in β-galactosidase activity when zinc was bound to a zinc chelator; this was under the control of the zur (zinc uptake regulator) gene. High-affinity 65 Zn transport of the constitutive zur mutant was 10-fold higher than that of the unin- duced parental strain. An in vivo titration assay suggested that Zur binds to the bidirectional promoter region of znuA and znuCB. The Zur protein showed 27% sequence identity with the iron regulator Fur and is very similar to the Bacillus subtilis (36) and Listeria monocytogenes (37) homologs. The Zur and Fur proteins have significant sequence identities (24% in B. subtilis and 27% in E. coli), and Zur-binding sequences have been described for promoters of genes related to zinc uptake that are similar to the Fur box. More- over, the fact that Fur has recently been defined as a zinc metalloprotein con- taining one structural ion of zinc per polypeptide (38) makes the relation between these proteins even more complex. Another example, in addition to Fur and Zur, is SirR—a novel iron-depen- dent repressor in Staphylococcus epidermidis with homology to the DtxR family of metal-dependent repressor proteins (39). SirR functions as a divalent metal- cation-dependent transcriptional repressor and is widespread among the staphylo- cocci. In B. subtilis the PerR regulon (40) has been shown to respond to iron as well as the genes involved in the response to oxidative stress such as katA (encod- ing catalase A) and aphC (alkyl hydroperoxide reductase). However, the Per boxes are associated with oxidative stress genes in several gram-positive bacteria rather than with iron transport. The Fur, Zur, SirR, and PerR are all proteins that can be used as potential biological components of biosensor devices. Other nonspecific metal-regulated genes related to global stress responses have also been described and used as biological components of biosensor devices (41). Heat shock gene expression is induced by a variety of environmental stresses, including the presence of metal ions. Escherichia coli heat shock pro- moters for dnaK and grpE were fused to the lux genes of Vibrio fischeri, and it has been suggested that biosensors constructed in this manner have potential for environmental monitoring (42). Copyright © 2002 Marcel Dekker, Inc. 4. BIOSENSORS FOR HEAVY METALS Biosensors are often cheap analytical devices in which a simple biological event is transduced into an electronic signal in a quantitative fashion, and ideally these should show high sensitivity and high specificity, and should work robustly in complex matrices, such as soil, water, and biological material. The selectivity of a biosensor depends on the biological component, and its sensitivity depends on the response of that component and the ease with which this can be transduced into a measurable signal. A large variety of biological components and transduc- ers that can be used for heavy metal sensing are summarized in Table 2, showing their main analytical characteristics, such as limit of detection (LOD), dynamic range (DR), and selectivity. As can be seen, these characteristics are highly de- pendent on the type of biological molecule and the transducer used for biosensor design and construction. The various biosensors also display different stability, and those based on immobilized enzymes are characterized by a low operating period. Recently we have been involved in the development of two different types of biosensor (43). In one, bacterial cells are genetically modified to respond to the presence of a heavy metal by the emission of light (44). These whole-cell biosensors (or in vivo biosensors) are now commercially available. The other biosensor uses immobilized bacterial proteins that bind heavy metals and alter the surface properties of an electrode in response to metal binding (30). Such capacitance electrodes show high sensitivity and some selectivity, but are at an early stage of development. Some publications use ‘‘biosensor’’ in the context of detection of toxic compounds by viability assays, of varying types, on whole cells. We eschew such a definition, and use ‘‘biosensor’’ in the context of detection of a specific analyte or a small range of chemically related compounds. 4.1 Whole-Cell Biosensors A significant area of research in bacterial molecular genetics has been the study of the control of gene expression (8,26). As part of these studies, many ‘‘reporter systems’’ have been developed that allow a transcriptional regulatory element to be placed such that it regulates expression of a gene that has a quantifiable prod- uct. Two of the most commonly used systems are the lacZ gene of E. coli and the lux genes of V. fischeri (for example, see ref. 45). The former encodes β- galactosidase, the production of which can be determined in a simple enzyme assay using the chromogenic substrate o-nitrophenol-β-d-galactose (46). This is of some use in the laboratory, but of little use in making a biosensor. The lux genes of V. fischeri are much more useful in the construction of biosensors, as they produce and oxidize long-chain aldehydes and generate photons as part of the reaction (47). The light that is emitted can be measured. Copyright © 2002 Marcel Dekker, Inc. T ABLE 2 Heavy Metal Biosensors and Their Properties Transducer Operating Biological molecule type conditions M 2ϩ LOD DR Ref Whole cell Mosses Sphagnum sp. Stripping differ- Acetate pH 6.0, Pb 2ϩ 2 ng/ml 5–125 ng/ml 82 ential pulse IS 0.7, 10% voltammetry moss, carbon paste elec- trodes E. coli ϩ mer pro- Optical detection Bioluminescence Hg 2ϩ 0.1 µM20nM–4µM83 moter ϩ lux is measured at genes from V. fi- 28°C, 30 min scheri response time under aeration E. coli ϩ lux genes Optical detection Hg 2ϩ 0.1 µM84 from V. fischeri Cu 2ϩ 0.1µM R. silverii ϩ lux op- Optical detection 23°C, 0.2% ace- Cu 2ϩ 2 µM2–40µM 43,50, eron from V. fi- tate, 20 mM Zn 2ϩ 5 µM 5–250 µM 55,85 scheri MOPS, pH 7.0, Cd 2ϩ 5 µM 5–200 µM 20 µg/ml tet- Cr 6ϩ 1 µM1µM–40 µM racycline Pb 2ϩ 1 µM1µM–40 µM Tl ϩ Ni 2ϩ Bacteria R. silverii ϩ lux op- Optical detection Microorganisms Cu 2ϩ 1 µM1 86 eron from V. fi- immobilized in scheri polymer matri- ces, 25°C E. coli ϩ lux operon Optical detection 30°C, M9 Hg 2ϩ 10 nM 52 medium Cu 2ϩ 1 µM E. coli ϩ firefly lucif- Optical detection Luminescence is Hg 2ϩ 0.1 fM 0.1 fM–0.1 µM87 erase gene measured in microtiter plate after 60 min at 30°C Copyright © 2002 Marcel Dekker, Inc. T ABLE 2 Continued Transducer Operating Biological molecule type conditions M 2ϩ LOD DR Ref Staph. aureus ϩ Optical detection Luminescence is AsO 43 1 µM1–5µM49 firefly luciferase measured in Cd 2ϩ 1 µM1–20µM gene scintillation counter after 60 min Staphy. aureus ϩ Optical detection Luminescence is Cd 2ϩ 10 nM–1 µM88 firefly luciferase measured in Pb 2ϩ 33 nM–330 µM gene microtiter Hg 2ϩ 33–100 nM plates, 30°C B. subtilis ϩ firefly Optical detection Luminescence is Cd 2ϩ 3.3 nM–1 µm88 luciferase measured in Zn 2ϩ 1–33 µM microtiter plates, 30°C Enzyme Urease ISFET Inhibition of ure- Cu 2ϩ 1–10 mg/L 89 ase immobi- Hg 2ϩ 0.25–5 mg/L lized on differ- Cd 2ϩ 3–10 mg/L ent membranes, Pb 2ϩ 2–10 mg/L 0.02 M HEPES, 25°C, batch mode ISFET Inhibition of ure- Hg 2ϩ 1 µM90 ase immobi- Cu 2ϩ 3 µM lized in a Nafion film, 20°C Ammonia Inhibition of ure- Cu 2ϩ 0.25 ppm 0.4–0.7 ppm 91 sensor ase, cuvette test Hg 2ϩ 0.07 ppm 0.07–1 ppm with ammonia- Zn 2ϩ 50 ppm 50–70 ppm sensitive coat- Pb 2ϩ 100 ppm 100–350 ppm ing on the wall, 0.1 N maleate buffer pH 6 Copyright © 2002 Marcel Dekker, Inc. [...]... to the electrode and evaluating the resulting current transients, which are altered by any changes that affect the metal-binding proteins Thus, monitoring of heavy metals with this type of biosensor is based on the conformational change that occurs when the metal ions bind to the protein The conformational change results in change of the capacitance A schematic representation of the detection principle... protein immobilization methods were tested: (a) covalent coupling with 1-( 3-dimethylaminopropyl )-3 -ethyl-carbodiimide hydrochloride (EDC), (b) entrapment in a polymer (polyethylene-glycol-diglycidyl-ether), and (c) cross-linking with glutaraldehyde (30) The best results (highest sensitivity) were obtained by covalent coupling with EDC The sensitivity of the biosensors was found to be dependent on both the. .. measurements was plotted as the ratio of the bioluminescence observed in the presence of increasing lead concentrations to the bioluminescent signal obtained in the presence of MilliQ-treated water Copyright © 2002 Marcel Dekker, Inc FIGURE 6 Analysis of the soil with the AE2450 Pb-BIOMET sensor plotted against the total amount of Pb after CaCl 2 extraction procedure R 2, coefficient of linear regression metal... may be more relevant to the study of metal-protein and metal-cell interactions in the laboratory rather than as a realistic solution to quantitation of heavy metal salts in the environment ACKNOWLEDGMENTS Collaboration between the authors’ laboratories was supported by a contract with the European Commission (ENV4-CT9 5-0 141) and funding from the Swedish Institute NLB thanks the Biotechnology and Biological... Dekker, Inc 6 CONCLUSIONS As indicated at the beginning of this chapter, the development of biosensors for detecting heavy metals is still at an early stage compared with the development of biosensors for more orthodox biochemical compounds, and is certainly much less well developed than the detection of heavy metals by chemical methods However, the progress recently made in the development of whole-cell... and protein-based biosensors is encouraging and bodes well for the future One of the great difficulties is the simple fact that metals are rarely found in environmental or industrial situations in pure form Often other metals are present, or the metal salts are in a milieu of unusual pH or with high concentrations of interfering counterions Until more work is done to address these problems, the use of... tetracycline) β-Glycerophosphate was used in the medium to avoid lead precipitation An aliquot of 2 ml was Copyright © 2002 Marcel Dekker, Inc TABLE 3 Characterization of the Soil Samples by the BIOMET Lead Sensor and by Chemical Analysis Sample TNO ID Sample municipal ID 82252 3-0 01 82252 3-0 02 82252 3-0 03 82252 3-0 04 82252 3-0 05 82252 6-0 01 82252 6-0 02 82252 6-0 03 82252 6-0 04 82252 6-0 05 82252 6-0 06 82252 6-0 07... content 5.2 The Capacitance Biosensor To date members of two classes of metal-binding proteins have been tested for capacitance biosensor construction, namely, the synechococcal metallothionein, SmtA (14), and the mercury-resistance protein, MerR (25) 5.2.1 A GST-SmtA-Based Biosensor Metallothioneins are small proteins that sequester metal ions in a ‘‘cage’’ structure In animal metallothioneins there are... there are two domains, each of which can sequester three or four metal ions (65,66) Metal binding is associated with a large conformational change in the protein, as the sulfhydryl groups of cysteins coordinate the metal ions There have been two reported bacterial metallothioneins, one in Pseudomonas, a gram-negative soil organism (13), and another in the cyanobacterium Synechococcus (14) The former has... means that most of the lead was not available to the bacterial community As a consequence, none of the samples were toxic for our soil bacteria The relationship between the lead concentration found in the Na-acetateextractable fraction and the bioavailable concentration determined with the BIOMET lead sensor remains to be confirmed using other lead-polluted soil to generate more points in the region of high . be determined in a simple enzyme assay using the chromogenic substrate o-nitrophenol-β-d-galactose (46). This is of some use in the laboratory, but of little use in making a biosensor. The lux genes. detoxify heavy metals. This chapter describes some of the ways in which we and others have begun to exploit these biological mechanisms to determine the amount of ‘‘bio- available’’ heavy metal in. responsible for confer- ring resistance to heavy metals in bacterial systems. A list of some of the determinants of resistance to heavy metals found in bacteria is given in Table 1. These include resistances