Heavy Metals in the Environment - Chapter 19 (end) pptx

In this chapter, we describe thebackground of bacterial interactions with heavy metals, and illustrate how thatinformation is being used in the development of biosensors for heavy metals

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

Society is learning to adapt to pollution by heavy metals in the environment, and

is now attempting to remediate, control, and minimize such pollution whereverpossible To do this, there is a need for methods of assessing the amount ofheavy metal pollution in the natural and industrial environments Although it isrelatively straightforward to use the techniques of analytical chemistry to detecttotal amounts of heavy metal in a given location, this rarely tells you how much

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 ple have been exposed to significant levels of toxic metals, although at leastsince Roman times heavy metals have been used in medicine and cosmetics (1)

peo-In contrast, microorganisms have always lived with ‘‘pollution’’ by heavymetals, as they have evolved to occupy ecological niches in which these toxicmetals naturally occur and in which they may be released by geochemicalprocesses Consequently, bacteria in particular, but also yeasts, fungi, andmany plants, have developed specific mechanisms to tolerate or detoxify heavymetals This chapter describes some of the ways in which we and others havebegun to exploit these biological mechanisms to determine the amount of ‘‘bio-available’’ heavy metal in natural and industrial environments These methodsare 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 tems with the high sensitivity of modern microelectronics in the development ofbiosensors

sys-Elsewhere in this book, you will find information on the occurrence ofheavy 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 inmany fields, such as environmental monitoring, clinical toxicology, wastewatertreatment, and industrial process monitoring Therefore, many spectroscopicmethods, including atomic absorption and emission spectroscopy (2), flameatomic 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 oftenrequire sophisticated instrumentation and trained personnel Electrochemicalmethods like ion selective electrodes, polarography, and other voltammetricmethods (5) are much simpler and require less complex instrumentation, but areoften unable to monitor at very low concentrations None of these techniquescan define or quantify the amount of heavy metal that is bioavailable and thereforelikely to be a risk to living organisms To achieve that, one needs a measurementthat 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 recognitionelement, responsible for the selectivity of the device, and a detection system (thetransducer) for quantifying the reaction between the biological component andthe target substance (analyte) to be monitored In this chapter, we describe thebackground of bacterial interactions with heavy metals, and illustrate how thatinformation is being used in the development of biosensors for heavy metals

2 BACTERIAL RESISTANCES TO HEAVY METALS

Heavy metals interact with living organisms in a variety of ways A number ofmetals (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 thepresence 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 heavymetals These transport or resistance systems may be inducible by the metal, andtherefore 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 inbacteria is given inTable 1.These include resistances to cations and to oxyanions

of metals in their most common physiological forms, and many of these resistancedeterminants 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-rangedeterminants, such as metallothioneins or phytochelatins Metallothioneins haverarely 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 proteinsconferring resistance have been predicted

The mechanisms of metal tolerance and resistance vary, but the majorityare due to efflux of the toxic metal from the cell (15) Some of these effluxsystems are part of the normal metal homeostasis systems of the bacterial celland the efflux pumps are encoded on the bacterial chromosome These can beconsidered 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 areP-type ATPases, with eight transmembrane helices, one of which contains theamino 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 aCys-X-X-Cys motif associated with heavy metal binding (where X is any aminoacid) (21) This N-terminal MerP-like region may be repeated and was thought

to confer metal specificity on the transporter

T ABLE 1 Some Heavy Metal Resistance Determinants in Bacteria

Hg Ps aeruginosa (and large number Uptake of HgIIand reduction to Plasmids and transposons 25

gram-positive genera)

cnr, Cd and Ni)

tem)

( pco system);

flux

Expression of the metal homeostasis proteins is usually regulated as part

of the mechanisms whereby the bacterial cell adjusts the intracellular tion of the individual metal ZntA and CopA are regulated by activator proteins

concentra-(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 act with the chromosomally encoded systems for metal homeostasis if the metal

inter-is also an essential nutrient For example, the copper resinter-istance 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 genescarried on the mercury-resistance plasmid (25)

The proteins required for metal homeostasis or metal resistance are oftenexpressed by the bacteria in response to metal ion concentration (26) For ex-ample, many resistance determinants are expressed only in the presence of thespecific metal ions at high subtoxic concentration (8) This involves specificregulatory proteins, either repressors or activators, that bind the metal ion andalter transcription of the structural genes responsible for metal sequestration,transport, or modification Metal-resistance determinants and the chromosomaldeterminants of metal homeostasis contain metal-responsive genetic elementsresponsible for expression of structural gene products that bind and/or transportthe metal ions These regulatory elements, the regulatory proteins, or the products

of the structural genes could be used in the construction of metal-specific sors

biosen-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 inFigure 1.Three structuralgenes, encoding (a) a small periplasmic protein, MerP, (b) an inner membranetransport protein, MerT, and (c) the enzyme mercuric reductase, are expressedunder the regulation of the activator protein, MerR, which binds Hg(II) andactivates 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 bacterialresistance determinants for other metals in the development of biosensors Someexamples of the creation of such biosensors are given below

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 proteinhave all been used in the development of biosensors for Hg(II)

A number of heavy-metal-regulated bacterial systems not directly related toheavy-metal-resistance mechanisms have been described Those systems aremainly involved in the intracellular regulation of essential transition metals ionssuch as iron, nickel, molybdenum, and magnesium

The transport and regulation of iron concentration in bacteria has been ied in detail This essential metal for cellular metabolism is needed as a cofactorfor a large number of enzymes, but is not easily available to microorganisms inaerobic environments Therefore, most aerobic bacteria produce and secrete low-molecular-weight compounds termed siderophores to capture Fe3⫹from the extra-cellular medium The iron uptake has to be very well regulated to maintain theintracellular concentration of the metal between desirable limits, since too high

stud-an intracellular concentration of iron cstud-an catalyze Fenton reactions stud-and generatetoxic 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 tion with target DNA sequences, and the involvement of Fur in many cell func-

interac-tions indicate that the Fur protein performs more like a general regulator than aspecific repressor (34) The cooperative binding of the Fur protein in extendedpromoter 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, 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

de-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 A znuA-lacZ

operon fusion was repressed by 5 µM zinc and showed a more than 20-foldincrease inβ-galactosidase activity when zinc was bound to a zinc chelator; this

was under the control of the zur (zinc uptake regulator) gene High-affinity65Zn

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 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 betweenthese proteins even more complex

More-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 dependent repressor proteins (39) SirR functions as a divalent cation-dependent transcriptional repressor and is widespread among the staphylo-

metal-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 bacteriarather than with iron transport The Fur, Zur, SirR, and PerR are all proteins thatcan be used as potential biological components of biosensor devices

Other nonspecific metal-regulated genes related to global stress responseshave 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 moters for dnaK and grpE were fused to the lux genes of Vibrio fischeri, and it

pro-has been suggested that biosensors constructed in this manner have potential forenvironmental monitoring (42)

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 theseshould show high sensitivity and high specificity, and should work robustly incomplex matrices, such as soil, water, and biological material The selectivity of

a biosensor depends on the biological component, and its sensitivity depends onthe response of that component and the ease with which this can be transducedinto a measurable signal A large variety of biological components and transduc-ers that can be used for heavy metal sensing are summarized inTable 2,showingtheir main analytical characteristics, such as limit of detection (LOD), dynamicrange (DR), and selectivity As can be seen, these characteristics are highly de-pendent on the type of biological molecule and the transducer used for biosensordesign and construction The various biosensors also display different stability,and those based on immobilized enzymes are characterized by a low operatingperiod

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 alterthe 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 toxiccompounds 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 ‘‘reportersystems’’ 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 encodesβ-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 ofthe reaction (47) The light that is emitted can be measured

T ABLE 2 Heavy Metal Biosensors and Their Properties

Transducer Operating

Whole cell Mosses Sphagnum sp. Stripping differ- Acetate pH 6.0, Pb 2⫹ 2 ng/ml 5–125 ng/ml 82

ential pulse IS 0.7, 10%

voltammetry moss, carbon

paste trodes

elec-E coli ⫹ mer pro- Optical detection Bioluminescence Hg2⫹ 0.1 µM 20 nM–4 µM 83

genes from V fi- 28 °C, 30 min

under aeration

E coli ⫹ lux genes Optical detection Hg 2⫹ 0.1 µM 84

R silverii ⫹ lux op- Optical detection 23°C, 0.2% ace- Cu2⫹ 2 µM 2–40 µM 43,50,

scheri MOPS, pH 7.0, Cd 2⫹ 5 µM 5–200 µM

20 µg/ml tet- Cr 6⫹ 1 µM 1 µM–40 µM racycline Pb 2⫹ 1 µM 1 µM–40 µM

Tl ⫹

Ni 2⫹

Bacteria R silverii ⫹ lux op- Optical detection Microorganisms Cu2⫹ 1 µM 1 86

eron from V fi- immobilized in

matri-ces, 25°C

E coli ⫹ lux operon Optical detection 30 °C, M9 Hg 2⫹ 10 nM 52

medium Cu 2⫹ 1 µM

E coli⫹ firefly lucif- Optical detection Luminescence is Hg 2⫹ 0.1 fM 0.1 fM–0.1 µM 87

microtiter plate after 60 min at 30°C

T ABLE 2 Continued

Transducer Operating

Staph aureus⫹ Optical detection Luminescence is AsO 43 1 µM 1–5 µM 49 firefly luciferase measured in Cd 2⫹ 1 µM 1–20 µM

counter after

60 min

Staphy aureus⫹ Optical detection Luminescence is Cd2⫹ 10 nM–1 µM 88

ase immobi- Hg 2⫹ 0.25–5 mg/L lized on differ- Cd 2⫹ 3–10 mg/L ent membranes, Pb 2⫹ 2–10 mg/L 0.02 M HEPES,

25 °C, batch mode

ase immobi- Cu 2⫹ 3 µM lized in a Nafion

film, 20 °C Ammonia Inhibition of ure- Cu 2⫹ 0.25 ppm 0.4–0.7 ppm 91 sensor ase, cuvette test Hg 2⫹ 0.07 ppm 0.07–1 ppm

with ammonia- Zn2⫹ 50 ppm 50–70 ppm sensitive coat- Pb 2⫹ 100 ppm 100–350 ppm ing on the wall,

0.1 N maleate

buffer pH 6

Ammonia Enzyme reactor Hg 0–15 nM 92 sensor with urease in-

hibited by cury, enzyme immobilized

mer-on glass beads

ase immobi- Hg 2⫹ 2 ppb lized with thy-

mol blue covalently bound to aminopropyl glass at the tip of

an optical fiber Conductometric Enzyme on inter- Hg 2⫹ Cu 2⫹ 1–50 µM 94 detection digitated gold Cd2⫹ 2–100 µM

electrodes, re- Pb 2⫹ 5–200 µM sidual activity of Co 2⫹ 0.02–5 mM

sured, 5 mM Tris-HNO 3 pH 7.4, 50 mM urea Conductometric Inhibition of ure- Hg2⫹ 20 ppb 95 detection ase is moni-

tored with a standing acoustic wave device Fluorimetric de- Flow system, en- Hg 2⫹ 0.5–100 ng/ml 96 tection at 340/ zyme immobi-

485 nm lized on

con-trolled pore glass, 0.005 M phosphate buffer pH 6.5

T ABLE 2 Continued

Transducer Operating

Fluorescence de- Flow system, in- Hg 2⫹ 2 ppb 97 tection at 340/ hibition of ure-

M Tris-HCl, pH 8.3

Carbonic anhydrase Fluorescence an- Enzyme labeled Cu 2⫹ pM 99

isotropy de- with deriva- Co 2⫹ pM tection tives of benzo- Zn2⫹ pM

xadiazole fonamide

sul-L -Lactate dehydro- Amperometric Enzyme coim- Hg 2⫹ 1 µM 100 genase detection mobilized with Cu 2⫹ 10 µM

L -lactate oxi- Zn 2⫹ 25 µM dase on the

top of an gen electrode Glycerophosphate Oxygen elec- Inactivation of Hg 2⫹ µmolar 20–500 µM 101

metal ions, zyme immobi- lized by reticu- lation in gelatin film or covalent bind- ing on a mem- brane

en-Pyruvate oxidase Oxygen elec- Hg 10 nM 101

trode Cholinesterase Voltammetric de- Flow system, en- Pb 2⫹ 5 µM 102

tection zyme immobi- Cu2⫹ 50 nM

lized on nitro- Cd 2⫹ 5 µM cellulose film

with dehyde Alkaline phospha- Spectrophoto- Chemilumines- Zn 2⫹ 0.17 ppm 103 tase metric de- cence from en-

glutaral-tection

zyme-cata-lyzed hydrolysis of a phosphate derivative of 1,2-dioxetane

is measured Horseradish peroxi- Spectrophoto- Inhibition of en- Hg2⫹ 0.1 pptr 4 orders of 104

tection lized on solid

supports is measured Invertase Amperometric Inhibition of en- Hg 2⫹ 1 ng/ml 105

detection zyme

immobi-lized on a membrane is measured Acetylcho- Amperometric Inhibition of en- Cu 2⫹ 0.01 pM 106 linesterase detection zyme by metal Cd 2⫹ 1 pM

ions is mea- Fe 2⫹ 10 pM

Apoenzyme Alkaline phospha- Calorimetric de- Enzyme immobi- Zn2⫹ 0.01–1.0 mM 107

ide acrylic beads, 100

mM TRIS-HCl

pH 8.0

T ABLE 2 Continued

Transducer Operating

Spectrophoto- Flow injection Zn 2⫹ sub- µ 0.1–10 µM 108, 109

tection change in

ab-sorbance at

405 nm is sured Potentiometric Flowthrough IS- Zn 2⫹ 0.01–1.0 mM 110 detection FET, pH shift

mea-detected Optical detection Chemilumines- Zn 2⫹ 0.5 ppb 0.5–50 ppb 103

cence from zyme-cata- lyzed hydrolysis of a phosphate derivative of 1,2-dioxetane

en-is measured Ascorbate oxidase Calorimetric de- Flow system, en- Cu 2⫹ 1–50 µM 111

tection zyme

immobi-lized on rous glass beads Spectrophoto- Absorbance at Cu 2⫹ 0.1–10 µM 112 metric de- 265 nm is mea-

po-tection sured Amperometric Polarographic Cu 2⫹ 0.5–2 µM 113 detection oxygen elec-

trode is used

Carbonic anhydrase Calorimetric de- Flow system, en- Zn 25–250 µM 114, 115

tection zyme immobi- Co 2⫹ 50–200 µM

lized on rous glass beads Optical detection Recognition of Zn 2⫹ 40–1000 nM 99,116

po-at 326/460 and metal ion by

560 nm apoenzyme

transduced by the dansylam- ide fluorescent probe Galactose oxidase Calorimetric de- Cu 2⫹ 5–20 mM 117

tection Amperometric Detection with Cu 2⫹ 0.1–10 mM 113 detection oxygen elec-

trode Alkaline phospha- Amperometric Enzymes coim- Cu2⫹ 2–100 µM 118 tase ⫹ ascorbate detection mobilized on a Zn 2⫹ 2–200 µM

membrane attached to a polarographic oxygen elec- trode

T ABLE 2 Continued

Transducer Operating

MerR-LacZα:M15 Spectrophoto- Microtiter plates Hg2⫹ ppb level 121 complex metric de- coated with

tection

BSA-divinyls-ulfone- tathion were treated with

glu-Hg 2⫹ trations and after washing the protein was bound to it

spectrophoto- one is used metric

Cd-EDTA com- metric de- were coated

Cd-EDTA-BSA gate and then the antibody was added, HEPES buffer

conju-pH 7.0–7.2

The principle of whole-cell biosensors is simple (43,44) The biologicalcomponent is a viable bacterial cell that has been modified to contain, say, the

lux genes under the control of a metal-responsive promoter, together with the

regulatory proteins required to express that promoter in the presence of metal

A stylized system is shown in Figure 2 The lux ‘‘reporter gene’’ is only pressed, and therefore, the lux gene products are only produced, in the presence

ex-of metal Therefore, a calibrated system can detect the presence ex-of metal by theemission of light As virtually all metal homeostasis genes and all metal resistance

F IGURE 2 Diagram of the lux transposon in whole-cell biosensors for heavy

metals Luciferase is expressed from the gene fusion with a metal-regulatedpromoter (Pr) under the control of the regulatory gene (Re)