Zinc resistance mechanisms in bacteria

REVIEWARTICLE ARTICLE REVIEW

Zinc resistance mechanisms in bacteria R. Choudhury and S. Srivastava* Department of Genetics, University of Delhi–South Campus, Benito Juarez Road, New Delhi 110 021, India

Zinc, though an essential metal ion, is toxic at higher concentrations. Various mechanisms are prevalent in the microbial world to overcome the toxicity of metals. These range from sequestration to active efflux and in some cases, both mechanisms can co-exist. Cell-wall modification and bioprecipitation are other mechanisms employed by the bacterial cells to reduce the toxic effect. Well-coordinated regulatory mechanisms exist, which allow the cells to retain essential amounts of zinc, while toxic concentrations are not allowed to build up. Few zinc-regulatory proteins and twocomponent regulatory systems comprising a ‘sensor’ and ‘response regulator’ are known for the regulation of the zinc resistance operon. METALS have formed an important constituent of the earth’s crust from the time it has evolved. Thus, even the early life has arisen in the presence of abundance of metals. Over the ages, all living systems have evolved to use some metals as vital constituents while they have learned to grapple with some others, which are toxic. The primary source of metals in all ecosystems is the underlying bedrock of the planet. Considerable variation in metal content at the surface can result from three major sources: differences in the underlying bedrock, i.e. concentration of ores, the atmosphere (mainly from volcanoes and forest fires) as well as pollution resulting due to human activities and the biosphere (resulting either from bioaccumulation or leaching of metals from soils)1. Though many metals are essential, all metal ions are toxic at some level. Heavy metal cations with high atomic masses tend to bind strongly to sulphide groups. The divalent cations of cobalt, nickel, copper and zinc are medium ‘sulphur lovers’ and these metals (known as trace elements) have essential functions at low concentrations but are toxic at high concentrations. The intracellular concentrations of these metals must be finely adjusted to avoid either metal deprivation or metal toxicity and careful homeostasis is necessary. In contrast, homeostasis of the purely toxic metals is simple; the cell must quickly eliminate them2. Of all life forms, bacteria are not only the oldest, but they also inhabit the greatest diversity of habitats, form a major proportion of the earth’s global biomass and have the greatest capacity to sorb metals from the solution, on a *For correspondence. (e-mail: [email protected]) 768

biomass to dry weight metal basis (µg/mg dry wt.). Thus they provide the ideal system to study the metal–microbe interactions. Like all other living organisms microbial communities adapt themselves to the metal concentrations encountered. Two major strategies adopted by the microorganisms to protect themselves against metal toxicity are avoidance and sequestration. These strategies are reflected in resistance phenotype to one or several metals1,3–10.

Mechanisms to evade metal toxicity The genesis of a given metal resistance mechanism is primarily dependent upon the interactions of the metal with the cell11. Essential metal ions enter the cell through specific and non-specific transporters. Of the two types of uptake systems12, the first is constitutively expressed, fast and generally driven by the chemiosmotic gradient across the cytoplasmic membrane of bacteria. These non-specific transporters bring in metal ions even during metal excess, a situation termed as ‘open gate’ which provides the basis as to why the heavy metal ions become toxic. The second uptake system is specific, relatively slow and is only expressed in times of need, starvation or special metabolic situation. These are, thus, inducible12,13. In yeast, Saccharomyces cerevisiae, two separate systems for zinc uptake exist. One system with high substrate affinity, is induced in the zinc-deficient cells and the second having a lower affinity is highly regulated by zinc status. The highaffinity transporter Zrt1p and low-affinity transporter Zrt2p are coded by ZRT1 and ZRT2 genes, respectively14,15. In order to understand the metal–microbe interaction, it is necessary to make a distinction between two types of heavy metals16: (i) heavy metals that are toxic per se; and (ii) metals which are essential for growth and maintenance but are toxic in excess. For resistance to metals which are physiologically required, survival is optimized by cooperation between the resistance mechanism and the normal cellular metal metabolism, allowing the cell to accumulate sufficient metal for the maintenance of metal-dependent activities whilst responding to supra-optimal metal concentrations11. The five mechanisms generally proposed for heavymetal resistance in bacteria and other microorganisms are: (a) exclusion of the metal by a permeability barrier3; (b) exclusion by active export of the metal from the cell4–6; (c) intracellular physical sequestration of metal by binding CURRENT SCIENCE, VOL. 81, NO. 7, 10 OCTOBER 2001

REVIEW ARTICLE proteins or other ligands to prevent it from damaging the metal-sensitive cellular targets10; (d) extracellular sequestration7–9; and (e) transformation and detoxification11,17–19.

Zinc – An essential metal ion Zinc is an essential metal ion, but, excess of even this essential ion exerts toxic effects on the cell17,20. On the other hand, mechanisms to evade metal toxicity are also widespread in the microbial world11,13,17–21. Zinc plays an important role as an essential trace element in development, growth and differentiation of all living systems from bacteria to humans22. It occurs exclusively as the divalent cation Zn2+. With its completely filled d orbitals, the zinc cation is not able to undergo redox changes under biological conditions13. Zinc is a component of more than 200 enzymes isolated from different species where it is indispensable to their catalytic function and structural stability23,24. Besides these functions, zinc is also known to be the stabilizer of membranes and various macromolecules25–27. The myriad roles of zinc are reported in basic cellular functions such as DNA replication, transcription, cell division and cell activation. Zinc is a common constituent of DNA-binding proteins, where it exists in the form of ‘zinc-finger motif’. In this form, zinc is found in authentic transcription factors. Steroid receptors also contain zinc-binding consensus. The association between tRNA synthetase and tRNA is also Zn-dependent28. Apoptosis is potentiated by zinc deficiency. It also functions as an antioxidant26. Thus, zinc can be truly called as an element without which no life is possible13.

zinc, there is likely to be selection for inducible control (possibly linked to the expression of the normal homeostatic system) so as not to jeopardize metal homeostasis. This is because over-expression as well as underexpression of resistance can be both deleterious to the cell11. Resistance to toxic levels of zinc can be due to extracellular accumulation33, sequestration by metallothioneins (MT)34–36, intracellular physical sequestration, or efflux-based4–6,19 (Figure 1). The chemiosmotic, CDF and RND systems and P-type ATPase-based zinc effluxes are known13. While the P-type ATPases transport zinc only across the cytoplasmic membrane, the RND systems are thought to transport across the complete cell wall of Gram-negative bacteria13,37. Efflux by antiport system: The best studied zinc resistance mechanism is the Czc system operating in Ralstonia, a Gram-negative soil bacterium. The Czc system conferring resistance to cadmium, zinc and cobalt in R. eutrophus strain CH34, functions as cation/proton antiporter effluxing cations from the cells. The czc operon of the plasmid pMOL30 consists of three structural genes, czcC, czcB and czcA, whose products form the complex cation efflux pump4,38,39. Sequence homologies and topological similarities with outer-membrane factors (OMF) suggest CzcC to be an outer-membrane protein7, which efficiently transports across the outer membrane. CzcB, with its homology to membrane fusion proteins, appears to span the periplasmic space and funnel cations across it, thereby preventing release of free cation. CzcB also shows some homology with calphotin, which is a calcium-mobilizing protein found in sponges17. Based on predicted structure, CzcB is proposed to draw the outer membrane close to the

Toxicity of zinc Efflux pump

Zinc resistance mechanisms Mechanisms of resistance of essential metal ions like zinc are particularly of interest. It helps in the understanding of the homeostatic control which the cell exerts in taking in and maintaining the required amount of metal and managing the excess. As already discussed, zinc is associated with a number of processes essential for growth and metabolism, but at higher concentrations it becomes toxic. For a physiologically required metal, as in case of CURRENT SCIENCE, VOL. 81, NO. 7, 10 OCTOBER 2001

Uptake pump

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The safe limit of zinc concentration in drinking water is 5 µg/ml29. High concentrations of zinc are inhibitory, affecting many crucial functions. Zinc is known to be a potent inhibitor of the respiratory electron transport systems of bacteria and mitochondria25,30,31. Toxicity of zinc is found to be quite low compared to other metals like Hg, Cd, Cu, Ni, Co and Pb29,32.

Cytosol

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opl

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ic m

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Figure 1. Mechanisms of zinc resistance in bacteria. Extracellular binding; binding on the outer membrane; active export via an efflux pump (P-type ATPase or proton antiporter) after the zinc ions have been taken in by specific and non-specific pathways; sequestration by periplasmic and/or cytoplasmic proteins or other ligands like polyphosphate granules. 769

REVIEW ARTICLE CzcA antiporter, thereby connecting both the membranes in a flexible fashion40. CzcA, a chemiosmotic cation/H+ antiporter, composed of 12 transmembrane polypeptides with symmetry between the first and second halves and two large periplasmic domains, is an archetype of the RND superfamily41 (Figure 2). Deletion mutations in czcA result in the loss of export of all the three ions, whereas those in czcB result in complete loss of resistances to cadmium and zinc, with only partial loss to cobalt. Elimination of czcC led to full resistance to zinc, partial resistance to cobalt, but sensitivity to cadmium38. Thus both CzcB and CzcC function as substrate range modulators. The addition of CzcC to the CzcAB complex extends the substrate range to include cadmium and leads to efficient transport across the cytoplasmic membrane, periplasm and outer membrane13,17. A gene cluster, czr, involved in cadmium and zinc resistance was identified in P. aeruginosa CMG103. The predicted CzrC, CzrB, CzrA proteins encoded by czrCBA genes show remarkable similarities with the proteins of R. eutrophus CH34, leading to the prediction that the cation– antiporter efflux system is involved in Zn and Cd resistance in this organism42. Efflux by P-type ATPase: The integral membrane P-type ATPases are an important class of ion transport proteins that serve to maintain suitable ionic conditions. P-type ATPase consists of a single, large catalytic monomer of 70–200 kDa. The transfer of γ-phosphate of ATP to an aspartic acid residue results in formation of aspartyl-

phosphate intermediate19,43,44. Heavy metal ATPases contain elements common to all P-type ATPases as well as several unique features45. They carry putative heavy metal-binding sites in the polar amino-terminal region, have conserved intramembrane CPC, CPH or CPS motif (CPx motif) and thus are called CPx-type ATPases, contain conserved histidine–proline dipeptide (HP locus) 34 to 43 amino acids carboxy-terminal to the CPx motif, and possess unique number and topology of membranespanning domains. P-type ATPase-based resistance to zinc has been reported in E. coli and in P. putida strain S46,46. The ZntA P-type ATPase of E. coli has been predicted to be the first example of a zinc-specific transporter, based upon the close sequence homology of this ATPase to the CadA ATPase of Staphylococcus aureus and Helicobacter pylori enzymes, which affect the cellular zinc content47. The CadA ATPase (Figure 3) also confers resistance to zinc ions48 and promotes ATP-dependent zinc transport49. Mutants isolated both from E. coli K-12 as well as P. putida S4 result in higher intracellular accumulation of zinc and confer Zn-sensitive phenotype6,46,50. A potential gene in the E. coli genome, o732, is responsible for specific resistance to zinc and cadmium40. The sequence of the gene o732 is identical to that of zntA. The protein

Metal binding region

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Cytoplasm

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626

618 727 COOH

373 C SH

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Cytoplasmic membrane

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Figure 2. Czc efflux model. CzcA, a CDF protein, functions as a pump driven by a H+ gradient. CzcB, a membrane fusion protein, possibly acts to connect the inner and outer membranes and to facilitate the export of ions across both membranes without release in the periplasm. CzcC is an outer-membrane protein, which leads to an efficient transport across the outer membrane. CzcD is a ‘sensor’. 770

1

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C SH 4

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Outside Figure 3. Typical model for metal translocating P-type ATPase (Cad A of Staphylococcus aureus). It includes 6 membrane-spanning regions. (I) Metal-binding region: P-type ATPase starts with a metalbinding motif, including a vicinal Cys pair. It is intracellularly located. (II) Phosphatase domain: It is large domain of approximately 190 amino acids that functions as a transduction funnel involved in moving the trapped metal ions from their initial binding sites to the membrane surface. The conserved Thr-Gly-Glu-Ser tetrapeptide helps in removing the phosphate from its covalently-bound form in aspartate. The initially bound metal now binds to Cys 371 and Cys 373 residues deriving its energy from the phosphate cleaved by ATP. (III) Aspartyl kinase: This is a large (240 amino acid) intracellular domain having ATP-binding site (488-Lys-Gly-Ile-Val-492) and 7 amino acid kinase stretch (414– 421). ATP is bound between the tetrapeptide listed above and an αhelical loop (618–626) which is predicted to make a Mg-mediated salt bridge with γ-phosphate of ATP. CURRENT SCIENCE, VOL. 81, NO. 7, 10 OCTOBER 2001

REVIEW ARTICLE ZntA is vanadate-sensitive, suggesting an energy-dependent active efflux of zinc. A similar mechanism is likely to be important in humans as well, possibly by ZntA homologs40. Znt efflux protein can also be activated by cadmium, lead and silver, but not copper51. ZntA and CadA are multipurpose proteins also acting as Pb-exporting pumps. The zntA disrupted Pb-hypersensitive strain of E. coli could be complemented by cadA, indicating that both soft-metal translocating P-type ATPases are essential for lead resistance in bacteria52. Resistance to zinc and cadmium could be conferred on a sensitive zntA mutant of E. coli by H. pylori cadA gene53. It was reported that zntA gene encodes a transmembrane structural protein responsible for the efflux of zinc and cobalt ions in S. aureus54. Another well worked-out zinc-effluxing ATPase is the ZiaA P-type ATPase of the cyanobacterium, Synechocystis PCC6803 (ref. 55). A regulatory protein, ZiaR, has been identified and has been described later. Inducible efflux of zinc has also been reported from Pseudomonas sp. strain UDG26. This efflux mechanism serves the purpose of regulating the intracellular concentration of zinc5.

Zinc-binding proteins The biological functions of metals and their passage through cells and organisms are invariably linked to the existence of specific metal-binding macromolecules. The presence of metals in numerous enzymes and many nonenzymatic metalloproteins and other metal-binding biopolymers serves regulatory purposes or controls the metabolism of essential and non-essential metal ions themselves. The mode of metal binding in metalloproteins varies widely, yielding structures of divergent chemical and biological specificity56. Metallothioneins: Metallothionein (MT) is a stressinducible protein with antioxidant attributes, that may participate independently or in conjunction with glutathione (GSH) to protect cells against injurious agents57. MTs are characterized by extremely high metal and sulphur content, lack of aromatic amino acids, and occurrence of all cysteine residues in the reduced form that are coordinated to the metal ions through mercaptide bonds, giving rise to metal–thiolate complexes56. As a homeostatic mediator, MTs could also donate metal ions in the biosynthesis of zinc- and coppercontaining metalloproteins56. MTs were originally thought to be cadmium protein but are also known to bind zinc, cadmium, copper, mercury and silver in increasing order of affinity58. Though more common in eukaryotes, a unique prokaryotic MT was identified from the cyanobacterium, Synechococcus sp. This molecule could complex copper, cadmium and zinc and had a high thiol content34. The first well-defined bacterial MT from CURRENT SCIENCE, VOL. 81, NO. 7, 10 OCTOBER 2001

Synechococcus 6301 is coded by the locus smt consisting of smtA and divergently transcribed smtB. Deletion of the smt locus reduces zinc/cadmium tolerance. SmtA represents a class II MT and smtB functions as repressor of smtA transcription34–36,59. Znu proteins: The Znu proteins belong to the recently defined new family of binding proteins. Znu ABC constitutes a high-affinity periplasmic binding proteindependent transport system for zinc in E. coli 60. Post-efflux binding: Several authors have emphasized upon the operation of a post-efflux mechanism to avoid the re-entry of effluxed ions6–8,13,19. This may consist of precipitation or binding to a protein or a cell component. Ralstonia sp. CH34 shows an interesting phenomenon of precipitation as a post-efflux management of metal ions. The pH of the growth medium increases as a function of increase in the metal ion concentration, suggesting that the progressive alkalinization of the growth medium is related to the proton influx during the Czc-mediated efflux of cations. The effluxed metal is finally precipitated in the form of bicarbonates and hydroxides. The avoidance or the minimization of re-entry of toxic metals is crucial under all oligotropic conditions, where all czc bacteria are generally found7. Choudhury and Srivastava6,50 reported post-efflux periplasmic binding of zinc ions. Since Zn is widely used in various metabolic functions, effluxed Zn2+ may be stored and utilized later on6. Periplasmic storage of effluxed Zn2+ has been postulated in Synechocystis PCC 6803 also55. This dual strategy of efflux and binding can be very effective. While efflux can be used as a quick and immediate response to protect the intracellular targets from metal poisoning, binding/storage can be employed for long-term handling in resistance, as also reported in P. syringae and E. coli61,62.

Regulation of expression of zinc resistance genes Though ubiquitous, environmental factors can greatly influence the abundance and availability of many metals, changing their effective concentrations in different locations. To survive, microorganisms must constantly monitor their environment and control the import of metals into their cytoplasm, so that they can acquire sufficient amounts of essential metals and yet avoid accumulation of toxic levels. This response includes the expression of high-affinity uptake systems, the expression of genes that export metals present in excess and the down-regulation of genes encoding proteins which require metals that are not available63,64. In S. cerevisiae, the genes ZRT1 and ZRT2 are regulated by zap1p according to zinc availability in the cell. When zinc-replete cells become zinc-limited, zap1p, that acts both as ‘sensor’ and 771

REVIEW ARTICLE as a transcriptional activator65,66 is induced, which in turn increases the expression of other target genes. For toxic but essential metals, expression of a resistance system must not only be specific, but carefully regulated, such that it does not deplete the cell of target metal. A simple model for copper management and utilization consisting of uptake-binding-efflux (and regulation) has been proposed16. When the concentration of metal in the cells is too high, there is increased expression of resistance, whereas when the same is too low, uptake and export are adjusted in such a way that cells do not die of copper starvation. The same logic can be applied for other essential ions like zinc. All known heavy metal resistances are, therefore, regulated by heavy metal/s at the level of transcription2. Many operons are regulated at the level of transcription initiation, often by trans-acting regulatory proteins, in response to environmental signals67. Metalloregulatory proteins sense the intracellular levels of specific metal ions and mediate a transcriptional or translational response68. A few such zinc-specific proteins have been studied in detail. Smt B: This is a trans-acting repressor encoded by a divergently transcribed gene, smtB, of a MT locus, smt, from the cyanobacteria Synechococcus PCC7942 and PCC6301. SmtB is required for zinc-responsive expression of the MT gene smtA35, in a way that the latter is induced when the SmtB repressor dissociates from the operator in response to zinc35,36,55,64,68–70. Besides zinc, a number of additional metals like copper, cadmium, cobalt, nickel and chromium inhibit SmtB from binding to the operator/ promoter69. Smt B has four zinc-binding sites per dimer and like ArsR and CadC contains conserved Cys residues associated with the putative DNA-binding helix-turn-helix motifs71. Mutation of Cys residues to serine does not abolish inducer recognition or SmtB-mediated repression of expression from smtA operator/promoter. Inducer recognition by SmtB has been proposed to involve the formation of metal-thiolate bonds to Cys-61 (ref. 72). Znt R: Encoded by zntR gene, ZntR regulates zntA in E. coli. It belongs to the growing family of MerR-like prokaryotic transcriptional regulators, introducing changes in the DNA conformation, which apparently make the promoter a better substrate for RNA polymerase72. It acts as a direct Zn sensor and catalyses transcriptional activation of zinc efflux gene. Unlike mer system, binding of ZntR to the promoter (pzntA) does not actively repress transcription. A hybrid regulatory protein containing the N-terminal region of MerR and the C-terminal region of ZntR showed that the ZntR-C terminal region is capable of recognizing zinc and participating in a MerR-like activation mechanism, when attached to a heterologous DNA-binding domain73. Twenty-five micromolar zinc caused complete dissociation of the bound repressor from the znt operator and higher concentration (100 µM) can 772

lead to complete derepression of transcription of znt operon in vitro. In this report among ArsR/SmtB family of metal-inducible metal resistance systems, the operator– repressor DNA complex is dissociated in the presence of a near physiological concentration of the metal under in vitro conditions74. Unlike ArsR/SmtB metalloregulatory proteins, ZntR does not contain any cysteine residue and lacks the characteristic metal-binding box54,74. However, it has two histidine-rich regions, one at the C-terminus and the other near the N-terminus. Similar histidine-rich regions have been reported for zinc and cobalt transporters which are thought to be domains for zinc-binding ions2,54. Zia R: The ziaA operator–promoter of zia divergon (zinc ATPase) in Synechocystis PCC6803 was specifically induced by Zn but not by Cd, Cu, Ag and Co. ZiaR is a zinc responsive repressor of ziaA and a member of ArsR family of regulators. Zinc sensing by ZiaR involves: (i) zinc thiolate bond formation at one or both of a pair of Cys residues (cys-71 and 73) located adjacent to the predicted DNA binding site and (ii) zinc imidazole bond formation at His-116 located towards the C-terminus of ZiaR55. Interestingly, Synechocystis PCC6803 and Synechococcus PCC7942 possess closely-related zinc sensors, ZiaR and SmtB, respectively, but they regulate very different structural proteins with different consequences for the cell biology of zinc. The former triggers the expulsion of excess zinc via ZiaA-mediated efflux into the periplasm and the latter internal sequestration by MT, SmtA55,68. Some mutants of ziaR retained the ability to bind to DNA in vitro and to repress transcription from ziaA operator–promoter, but failed to respond to zinc in vivo55. Zur: This is a metalloregulatory protein, isolated from Bacillus subtilis related to ferric uptake repressor (Fur) family of regulators. This protein is required for zincspecific repression of two operons involved in the expression of zinc homeostasis: (i) ycdH operon, encoding a putative high-affinity zinc transport system, and (ii) yciC operon, encoding an integral membrane protein68.

Two-component regulatory systems In bacteria, signal transduction in response to a wide variety of environmental stimuli is mediated by pairs of proteins that communicate to each other by a conserved mechanism involving protein phosphorylation2,75. In response to varying metal concentration in nature, a cell has to adjust its regulatory system in such a way so as to enable it to acquire them in the required concentration. This makes it a fit case of a two-component regulatory CURRENT SCIENCE, VOL. 81, NO. 7, 10 OCTOBER 2001

REVIEW ARTICLE system. The two-component regulatory system has several special features2. Basically it consists of a protein pair called a ‘sensor’ and a ‘response regulator’. Signal input leads to phosphorylation of a histidine residue of the sensor protein and the phosphate residue is transferred to an aspartate residue of the response regulator protein. A given signal intensity leads to a corresponding level of phosphorylation of the response regulator. In the DNAbinding response regulators, the phosphorylation level determines the affinity of the response regulator as repressor or activator. The signal output of the DNAbinding response regulators is reflected in the frequency of initiation of transcription. Of the metal-responsive bacterial systems, the products of copR and copS genes of P. syringae76 and pcoR and pcoS genes of E. coli61 both involved in copper resistance, are examples of two-component regulatory systems2.

Czc regulatory genes Possible czc regulatory genes are arranged in regions upstream and downstream of structural genes czcCBA. The downstream regulatory region contains the genes czcD, czcR and czcS, with czcS (histidine kinase) and czcR (response regulator) forming the two-component regulatory system2,77. The upstream regulatory region contains czcN and czcI and another ORF, ORF69a4. The knockout experiments proved that CzcRS is not essential for Czc control and CzcR perhaps acts only on czcNp; CzcRS may therefore control the differential expression of czcN rather than other czc genes78. CzcN is probably membrane-bound, with four to five transmembrane αhelices78, but does not seem to be essential for slow nonspecific Czc regulation4,78. Also, no sigma factor has yet been identified for czc transcription. It has been suggested that any of the regulatory genes whose functions are unknown might encode products which together with the membrane-bound CzcD, the extracellular cation sensor, could be responsible for the control of a sigma factor by periplasmic (czcI ) signals in a system similar to Rse system78,79. Thus, CzcR and CzcD may form two-component regulatory system4,12,75. CzcD belongs to ‘cation diffusion facilitator’ (CDF) family2,12,80. The other members of this family include ZRC-1 protein, which mediates zinc resistance in S. cerevisiae81 and Znt-1 and Znt-2 (refs 82, 83), which confer zinc resistance in mammals2. However, there is no sequence homology between CzcD and typical sensors of the classical two-component systems. CzcD is also not essential for the activation of czc by CzcR and shares 34% homology on the amino acid level, with the ZRC1 protein4,12 (zinc resistance conferring) which confers resistance to zinc, cadmium and cobalt in S. cerevisiae81. This homology could mean that CzcD acts as a slow zinc permease essential for activation of czc. CzcD–CzcR probably does not function via phosphoCURRENT SCIENCE, VOL. 81, NO. 7, 10 OCTOBER 2001

rylation, but it could represent a novel category of twocomponent regulatory systems4. The predicted CzrS and CzrR proteins, reported from P. aeruginosa CMG103, show a significant similarity to the sensor and regulatory protein, respectively of the two-component regulatory systems such as Cu-resistance regulating CopS/CopR and PcoS/PcoR and CzcS/CzcR involved in czc regulation2,42,61,76.

Other mechanisms Extracellular accumulation: P. stutzeri RS34, isolated from the industrially-polluted soil in New Delhi has been reported to resist Zn by accumulating high levels of zinc on its outer membrane33. The accumulation brings about a number of morphological and ultrastructural changes that makes the strain an efficient accumulator of zinc84. The utility of the strain in removing Zn from solutions, lowgrade ores and ore-tailings has been shown85,86. Reduced uptake: Reduced uptake-based zinc resistance in Azospirillum brasiliense sp.7 was reported3. The zincsensitive variants of A. brasiliense sp.7 took up more zinc compared to the wild type. The cells of this organism also expressed a unique response towards Zn, when they were converted into enlarged, non-motile pigmented (melanized) structures. These were termed as encapsulated forms, perhaps, preceding encystation87.

Conclusion Varied mechanisms of zinc resistance are found in the microbial world. These mechanisms range from reduced uptake to uptake and efflux, external and internal sequestration and in some cases transformation of metals to less toxic forms11,13,18,19,21. All these mechanisms aim at reducing the intracellular concentration of a metal so as to protect the cellular targets. Often, this may mean reducing the intracellular concentration of free metal ions as they are likely to be more toxic than the bound ones. The resistance mechanisms for essential metal ions are intricately interwoven with metal homeostasis mechanisms so as to ensure the cell’s survival, both under metalexcess and metal-depleted conditions. Studies on transport, genetics and mechanisms of toxic ion resistance may help in basic understanding of the physiology of the cell and in enhancing the ability of microorganisms to extract deleterious ions from the environment. Heavy metal pollution of the environment is a problem for which bioremediation by microorganisms is a natural, viable and economic solution. 1. Tomsett, A. B., Stress Tolerance of Fungi (ed. Jennines, D. H.), Marcel Dekker, NY, 1994. 773

REVIEW ARTICLE 2. Nies, D. H. and Brown, N., Metal Ions in Gene Regulation (eds Silver, S. and Walden, W.), Chapman and Hall, NY, 1998. 3. Gowri P. Mangala and Srivastava, S., Curr. Sci., 1996, 71, 139– 142. 4. Nies, D. H., J. Bacteriol., 1992, 174, 8102–8110. 5. Mago, R. and Srivastava, S., Appl. Environ. Microbiol., 1994, 60, 2367–2370. 6. Choudhury, R. and Srivastava, S., World J. Microbiol. Biotechnol., 2001, 17, 149–153. 7. Diels, L., Dong, Q., van der Lelie, D., Baeyens, W. and Mergeay, M., J. Ind. Microbiol., 1995, 14, 142–153. 8. Saxena, D. and Srivastava, S., World J. Microbiol. Biotechnol., 1998, 14, 291–293. 9. Nies, D. H., Extremophiles, 2000, 2, 77–82. 10. Gilotra, U. and Srivastava, S., Curr. Microbiol., 1997, 34, 378– 381. 11. Rouch, D. A., Lee, B. T. O. and Morby, A. P., J. Ind. Microbiol, . 1995, 14, 132–141. 12. Nies, D. H. and Silver, S., J. Ind. Microbiol., 1995, 14, 186– 199. 13. Nies, D. H., Appl. Microbiol. Biotechnol., 1999, 31, 730–750. 14. Zhao, H. and Eide, D., Proc. Natl. Acad. Sci. USA, 1996, 93, 2454–2458. 15. Zhao, H. and Eide, D., J. Biol. Chem., 1996, 271, 23203–23210. 16. Lee, B. T. O., Brown, N. L., Rogers, S., Bergemann, A., Camakaris, J. and Rouch, D. A., NATO ASI Ser., 1990, G23, 625– 632. 17. Ji, G. and Silver, S., J. Ind. Microbiol., 1995, 14, 61–75. 18. Srivastava, S., Mago, R. and Bhagat, R., Microbes: For Health, Wealth and Sustainable Environment (ed. Varma, A.), MPH, New Delhi, 1998. 19. Srivastava, S., Choudhury, R. and Joshi, N., Advances in Microbial Biotechnology (eds Tewari, J. P. et al.), APH Publishing Corporation, New Delhi, 1999. 20. Silver, S. and Walderhaug, M., Encyclopaedia of Microbiology (ed. Lederberg, J.), Academic Press, San Diego, 1992, vol. 2. 21. Silver, S., J. Ind. Microbiol. Biotechnol., 1998, 20, 1–12. 22. Ohnesorge, F. K. and Wilhelm, M., Metals and their Compounds in the Environment (ed. Mariam, E.), VGH, Germany, 1991. 23. Vallee, B. L., Zinc Enzymes – Metal Ions in Biology (ed. Spiro, T. G.), Series 5, John Wiley and Sons, NY, 1983. 24. Eide, D., Metal Ions in Gene Regulation (ed. Silver, S. and Walden, W.), Chapman and Hall, NY, 1998. 25. Chvapil, M., Life Sci., 1973, 13, 1041–1049. 26. Sugarman, B., Rev. Infect. Dis., 1983, 5, 137–147. 27. Shankar, A. H. and Prasad, A. S., Am. J. Clin. Nutr., 1998, 68, 447–463. 28. Glasfield, E. and Schimmel, P., Biochemistry, 1997, 36, 6739– 6744. 29. Iqbal, M., Srivastava, P. S. and Siddiqi, T. O., Environmental Hazards – Plants and People (eds Iqbal, M., Srivastava, P. S. and Siddiqi, T. O.), CBS Publishers and Distributors, New Delhi, 2000. 30. Beard, S. J., Hughes, M. N. and Poole, R. K., FEMS Microbiol Lett., 1995, 131, 205–210. 31. Kasahara, M. and Anraku, Y., J. Biochem., 1974, 76, 967–976. 32. Duxbury, T., FEMS Microbiol. Lett., 1981, 11, 217–220. 33. Bhagat, R., Indian J. Exp. Biol., 1993, 31, 590–594. 34. Olafson, R. W., McCubbin, W. D. and Kay, C. M., Biochem. J., 1988, 251, 691–699. 35. Morby. A. P., Turner, J. S., Huckle, J. W. and Robinson, N. J., Nucleic Acids Res., 1993, 21, 921–925. 36. Robinson, N. J., Bird, A. J. and Turner, J. S., Metal Ions in Gene Regulation (eds Silver, S. and Walden, W.), Chapman and Hall, NY, 1998. 37. Paulsen, I. T. and Saier, M. H. Jr., J. Membr. Biol., 1997, 156, 99– 103. 774

38. Nies, D. H., Nies, A., Chu, L. and Silver, S., Proc Natl. Acad. Sci. USA, 1989, 86, 7351–7355. 39. Nies, D. H., J. Bacteriol., 1995, 177, 2454– 2458. 40. Rensing, C., Mitra, B. and Rosen, B. P., Proc. Natl. Acad. Sci. USA, 1997, 94, 14326–14331. 41. Goldberg, M., Pribyl, T., Juhake, S. and Nies, D. H., J. Biol. Chem., 1999, 274, 26065–26070. 42. Hassan, M., van Der Lelie, D., Springael, D., Romling, U., Ahmed, N. and Mergeay, M., Gene, 1999, 239, 417–425. 43. Pederson, P. L. and Carafoli, E., Trends Biochem. Sci., 1987, 12, 146–150. 44. Solioz, M and Vulpe, C., TIBS, 1996, 21, 237–241. 45. Lutsenko, S. and Kaplan, J. H., Biochemistry, 1995, 34, 15607– 15613. 46. Beard, S. J., Hashim, R., Membrillo-Hernandez, J., Hughes, M. N. and Poole, R. K., Mol. Microbiol., 1997, 25, 883–891. 47. Silver, S., Gene, 1996, 179, 9–19. 48. Novick, R. P. and Roth, C., J. Bacteriol., 1968, 95, 1335–1342. 49. Silver, S. and Phung, L., Annu. Rev. Microbiol., 1996, 50, 753– 789. 50. Choudhury, R. and Srivastava, S., Curr. Microbiol., 2001 (in press). 51. Okkeri, J. and Haltia, T., Biochemistry, 1999, 38, 14109–14116. 52. Rensing, C., Sun, Y., Mitra, B. and Rosen, B. P., J. Biol. Chem., 1998, 273, 32614–32617. 53. Hermann, L., Schwan, D., Garner, R., Mobley, H. L. T., Haas, R., Schafer, K. P. and Melchers, K., Mol. Microbiol., 1999, 33, 12775–12779. 54. Xiong, A. and Jayaswal, R. K., J. Bacteriol., 1998, 180, 4024– 4029. 55. Thelwell, C., Robinson, N. J. and Turner-Cavet, J. S., Proc. Natl. Acad. Sci. USA, 1998, 95, 10728–10733. 56. Kagi, J. H. R. and Schaffer, A., Perspectives in Biochemistry (ed. Neurath, H.), 1989, vol. I, pp. 46–52. 57. Lazo, J. S., Kuo, S. M., Woo, E. S. and Pitt, B. R., Chem. Biol. Interact., 1998, 111–112, 255–262. 58. Brady, F. O., TIBS, 1982, 7, 143–145. 59. Shi, J., Lindsay, W. P., Huckle, J. W., Morby, A. P. and Robinson, N. J., FEBS Lett., 1992, 303, 159–163. 60. Patzer, S. I. and Hantke, K., Mol. Microbiol., 1998, 28, 1199– 1210. 61. Brown, N. L., Barrett, S. R., Camakaris, J., Lee, B. T. O. and Rouch, D. A., Mol. Microbiol., 1995, 17, 1153–1156. 62. Cervantes, C. and Silver, S., Molecular Biology of Pseudomonads (eds Nakazawa, T. et al.), ASM Press, Washington DC, 1996. 63. Westenberg, D. J. and Guerinot, M. L., Adv. Genet., 1997, 36, 187–238. 64. Xu, C. and Rosen, B. P., Metals and Genetics (ed. Sarkar, B.), Kluwer Academic/Plenum Publishers, NY, 1998. 65. Zhao, H. and Eide, D., Mol. Cell Biol., 1997, 17, 5044–5052. 66. Eide, D., Zhao, H., Butler, E. and Rodgers, J., Metals and Genetics (ed. Sarkar, B.), Kluwer Academic, NY, 1998. 67. Helmann, J. D., Wang, Y., Mahler, I. and Walsh, C. T., J. Bacteriol., 1989, 171, 222–229. 68. Gaballa, A. and Helmann, J. D., J. Bacteriol., 1998, 180, 5815– 5821. 69. Erbe, J. L., Taylor, K. B. and Hall, L. M., Nucleic Acids Res., 1995, 23, 2472–2478. 70. Turner, J. S., Glands, P. D., Samson, A. C. R. and Robinson, N. J., Nucleic Acids Res., 1996, 24, 3714–3721. 71. Cook, W. J., Kar, S. R., Taylor, K. B. and Hall, L. M., J. Mol. Biol., 1998, 275, 337–346. 72. Outten, C. E., Outten, F. W. and O’ Halloran, T. V., J. Biol. Chem., 1999, 274, 37517–37524. 73. Brocklehurst, K. R., Hobman, J. L., Lawley, B., Blank, L., Marshall, S. J., Brown, N. J. and Morby, A. P., Mol. Microbiol., 1999, 31, 893–902. CURRENT SCIENCE, VOL. 81, NO. 7, 10 OCTOBER 2001

REVIEW ARTICLE 74. Singh, V. K., Xiong, A, Usgaard, T. R., Chakrabarti, S, Deora, R., Misra, T. K. and Jayaswal, R. K., Mol. Microbiol., 1999, 33, 200– 207. 75. Albright, L. M., Huala, E. and Ausubel, F. M., Annu. Rev. Genet., 1989, 23, 311–336. 76. Mills, S. D., Jasalvich, C. A. and Cooksey, D. A., J. Bacteriol., 1993, 175, 1656–1664. 77. Van der Lelie, D., Schwuchow, T., Schweidetzky, U., Wuertz, S., Baeyens, W., Mergeay, M. and Nies, D. H., Mol. Microbiol., 1997, 23, 493–503. 78. Große, C. et al., J. Bacteriol., 1999, 181, 2385–2393. 79. De Las Penas, Connolly, L. and Gross, C. A., Mol. Microbiol., 1997, 24, 373–385. 80. Anton, A., Große, C., Reißmann, J., Pribyl, T. and Nies, D. H., J. Bacteriol., 1999, 181, 6876–6881. 81. Kamizono, A., Nishizawa, M., Teranishi, Y, Murata, K and Kimura, A., Mol. Gen. Genet., 1989, 219, 161–167.

CURRENT SCIENCE, VOL. 81, NO. 7, 10 OCTOBER 2001

82. Palmiter, R. D., Cole, T. B. and Findley, S. D., EMBO J., 1996, 15, 1784–1791. 83. Palmiter, R. D. and Findley, S. D., EMBO J., 1995, 14, 639–649. 84. Bhagat, R. and Srivastava, S., J. Gen. Appl. Microbiol., 1994, 40, 265–270. 85. Bhagat, R. and Srivastava, S., Biohydrometallurgical Technologies (eds Torma, A. E., Apel, M. L. and Brierley, C. L.), The Minerals, Metals and Materials Society, Pennsylvania, 1993, vol. II. 86. Bhagat, R. and Srivastava, S., Environmental Biotechnology: Principles and Applications (eds Moo-Young, W. A. and Chakravarty, A. M.), Kluwer Academic Publishers, the Netherlands, 1995. 87. Gowri, P. Mangala and Srivastava, S., World J. Microbiol. Biotechnol., 1996, 12, 319–322. Received 8 May 2001; revised accepted 27 July 2001

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