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Annu. Rev. Microbiol. 1996. 50:753–89 Copyright c 1996 by Annual Reviews Inc.

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Annu. Rev. Microbiol. 1996.50:753-789. Downloaded from arjournals.annualreviews.org by UNIV.MASS.MED.SCH.LIB. on 05/20/05. For personal use only.

BACTERIAL HEAVY METAL RESISTANCE: New Surprises
Simon Silver
Department of Microbiology and Immunology, University of Illinois, College of Medicine, Chicago, Illinois 60612; e-mail: u20053@uicvm.uic.edu

Le T. Phung
Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637
KEY WORDS: plasmid resistances, mercury, cadmium, arsenic, copper, chromate, ATPases

ABSTRACT
Bacterial plasmids encode resistance systems for toxic metal ions including Ag+ , AsO− , AsO3− , Cd2+ , Co2+ , CrO2− , Cu2+ , Hg2+ , Ni2+ , Pb2+ , Sb3+ , 2 4 4 TeO2− , Tl+ , and Zn2+ . In addition to understanding of the molecular genetics 3 and environmental roles of these resistances, studies during the last few years have provided surprises and new biochemical mechanisms. Chromosomal determinants of toxic metal resistances are known, and the distinction between plasmid resistances and those from chromosomal genes has blurred, because for some metals (notably mercury and arsenic), the plasmid and chromosomal determinants are basically the same. Other systems, such as copper transport ATPases and metallothionein cation-binding proteins, are only known from chromosomal genes. The largest group of metal resistance systems function by energydependent efflux of toxic ions. Some of the efflux systems are ATPases and others are chemiosmotic cation/proton antiporters. The CadA cadmium resistance ATPase of gram-positive bacteria and the CopB copper efflux system of Enterococcus hirae are homologous to P-type ATPases of animals and plants. The CadA ATPase protein has been labeled with 32 P from γ -32 P-ATP and drives ATP-dependent Cd2+ uptake by inside-out membrane vesicles. Recently isolated genes defective in the human hereditary diseases of copper metabolism, Menkes syndrome and Wilson’s disease, encode P-type ATPases that are more similar to the bacterial CadA and CopB ATPases than to eukaryote ATPases that pump different cations. The arsenic resistance efflux system transports arsenite, using alternatively either a two-component (ArsA and ArsB) ATPase or a single polypeptide (ArsB) functioning as a chemiosmotic transporter. The third gene

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SILVER & PHUNG

Annu. Rev. Microbiol. 1996.50:753-789. Downloaded from arjournals.annualreviews.org by UNIV.MASS.MED.SCH.LIB. on 05/20/05. For personal use only.

in the arsenic resistance system, arsC, encodes an enzyme that converts intracellular arsenate [As (V)] to arsenite [As (III)], the substrate of the efflux system. The three-component Czc (Cd2+ , Zn2+ , and Co2+ ) chemiosmotic efflux pump of soil microbes consists of inner membrane (CzcA), outer membrane (CzcC), and membrane-spanning (CzcB) proteins that together transport cations from the cytoplasm across the periplasmic space to the outside of the cell. Finally, the first bacterial metallothionein (which by definition is a small protein that binds metal cations by means of numerous cysteine thiolates) has been characterized in cyanobacteria.

CONTENTS
INTRODUCTION AND OVERVIEW . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . BACTERIAL CADMIUM RESISTANCE AND GENES FOR HUMAN COPPER TRANSPORT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CadA Cd2+ ATPase of Gram-Positive Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Human Copper Transport Diseases: Menkes and Wilson’s . . . . . . . . . . . . . . . . . . . . . . . . Cadmium Resistance in Alcaligenes: A Three Polypeptide Chemiosmotic Antiporter . . . COPPER RESISTANCE DETERMINANTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CopA and CopB ATPases of Enterococcus hirae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cop of Pseudomonas syringae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pco and Cut of Escherichia coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . BACTERIAL ARSENIC RESISTANCE: MISSING GENES AND ENZYME ACTIVITIES The Molecular Genetics of Arsenic and Antimony Resistance . . . . . . . . . . . . . . . . . . . . . . Arsenite Efflux ATPase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Arsenate Reductase Enzyme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MERCURY AND ORGANOMERCURIAL RESISTANCES . . . . . . . . . . . . . . . . . . . . . . . . . Diversity of Operon Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mercuric Reductase and Organomercurial Lyase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulatory Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Resistance Genes in Polluted Environments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . METALLOTHIONEIN IN CYANOBACTERIA: A NEW SYSTEM . . . . . . . . . . . . . . . . . . . . OTHER SYSTEMS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chromate Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Silver, Thallous, and Other Cation Resistance Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . Tellurite Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . POSTSCRIPT: THE FUTURE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 754 756 756 758 760 762 762 764 765 766 766 768 769 770 770 773 774 776 777 778 780 780 780 781 781

INTRODUCTION AND OVERVIEW
Bacterial toxic metal ion resistance systems have been studied for many years. Nevertheless, with the exception of regulationof mercury resistance (3, 81, 108, 119), the overall effort has been rather limited, considering the range of toxic metal ions involved and the diversity of microbes within such systems. Bacterial plasmids have genes for resistances to many toxic ions of heavy metal elements, including Ag+ , AsO− , AsO3− , Cd2+ , Co2+ , CrO2− , Cu2+ , Hg2+ , 2 4 4

BACTERIAL HEAVY METAL RESISTANCES

755

Ni2+ , Sb3+ , TeO2− , Tl+ , and Zn2+ . There are reports for resistances to B4 O2− , 3 7 Pb2+ , and organo-tin compounds, although understanding of these systems has not been reached. Mostly, resistance systems have been found on plasmids, but, frequently, related systems are determined by chromosomal genes in other organisms (examples are mercury resistance in Bacillus, cadmium efflux mediated by a P-type ATPase also in Bacillus, and arsenic efflux by chromosomal Escherichia coli genes). In addition, bacterial chromosomes contain genes for resistances to many of the same heavy metal cations and oxyanions as do plasmids. The existence of some chromosomal systems had been unsuspected until recent genome sequencing efforts revealed them. Examples include the arsenic and antimony resistance system of E. coli K-12 that Sofia et al (106a) sequenced and that Carlin et al (12) and Diorio et al (27) showed to be functionally equivalent to plasmid systems and a similar ars operon found in the Bacillus subtilis strain 168 genome (110). The first complete bacterial genome sequence, 1.8 megabase pairs (Mbp) from Haemophilis influenzae (34), has listed homologs for arsC, merP, and merT that are discussed in the respective arsenic and mercury resistance sections below. Similarly the 1 Mbp sequence deposited from the cyanobacterium Synechocystis PCC6808 (51) includes a homolog to the Czc cadmium, zinc, and cobalt resistance system (see below) and other genes apparently involved in toxic metal ion transport, including some for arsenic and copper. Genes affecting copper tolerance may also be generally found in bacterial genomes. It is frequently thought that these resistances arose as a result of human pollution in recent centuries. It seems, however, more likely that toxic metal resistance systems arose soon after life began, in a world already polluted by volcanic activities and other geological sources. As with antibiotic resistance determinants, toxic heavy metal resistance determinants are preexistant to recent human activities that create polluted environments. The subject of bacterial plasmid-determined resistances to toxic inorganic cations and oxyanions has been frequently reviewed (104; in a special 1992 issue of Plasmid, in two 1995 issues of Journal of Industrial Microbiology, and in other reviews that are listed below ion by ion). Here we do not attempt to be comprehensive but instead focus on new findings, including several important surprises from the last few years. Three generalizations may be made: (a) The specificities of plasmid-determined metal resistances are similar to those for other genes affecting cell metabolism (i.e. highly specific). There is no general mechanism for resistance to all heavy metal ions. (b) Metal-ion resistance systems have been found on plasmids of every bacterial group tested. The absence of known resistance determinants in any group probably reflects insufficient effort. (c) The mechanisms of resistance are generally efflux “pumping” (removing toxic ions that entered the cell by systems involved in transport

Annu. Rev. Microbiol. 1996.50:753-789. Downloaded from arjournals.annualreviews.org by UNIV.MASS.MED.SCH.LIB. on 05/20/05. For personal use only.

1996.g. Among the recent surprises covered in this review are (a) the diversity of operon structures for bacterial mercury resistance (which were considered rather uniform because research efforts have been focused on a pair of closely related operons). Rev. Why it is easier to expend metabolic energy bringing in toxic ions and then more energy pumping them out than to exclude them totally requires an explanation. The mechanisms are frequently (but not always) the same in all bacterial types. Downloaded from arjournals. The Cd2+ ATPase has been found in soil THE CADA ATPASE .50:753-789.org by UNIV. and (d) the solid establishment of bacterial metallothioneins with cyanobacteria. Bio-sequestration may not solve the problem but only delay when it must be addressed.MED. giving rise to (a) the efflux ATPases in gram-positive bacteria. on 05/20/05. “bio-accumulation” or sequestration (either intracellular or at the cell surface) is a mechanism of resistance.LIB. Annu. the cadmium and copper ATPases of gram-positive and the arsenite ATPase of plasmids of gram-negative bacteria) or chemiosmotic (e. Microbiol.g.annualreviews. (c) a novel aspect of energy coupling for arsenic efflux so that the membrane transport protein functions by itself as a chemiosmotic porter or together with a second gene product as a tightly-coupled transport efflux ATPase. Occasionally. BACTERIAL CADMIUM RESISTANCE AND GENES FOR HUMAN COPPER TRANSPORT CadA Cd2+ ATPase of Gram-Positive Bacteria The 727 amino acid Cd2+ efflux ATPase from staphylococcal plasmid pI258 (Figure 1) was the first example of a system now widely found in gram-positive bacteria (49. 103). It may be that the metabolic “price” for having more specific uptake pumps is greater than the genetic cost of having plasmid genes in the population that can spread when needed. Efflux pumps are the major currently known group of resistance systems.SCH. and (c) the metallothionein of cyanobacteria. Unlike the mercury and arsenic resistance systems that are highly homologous in all bacteria studied. with both plasmid and chromosomal systems.MASS. cadmium resistance appears to have evolved at least three times. the divalent cation efflux systems of soil Alcaligenes and the arsenite efflux system of the chromosome of gram-negative bacteria and of plasmids in gram-positives). They can be either ATPases (e. Each will be considered section by section. For personal use only. (b) the unrelated chemiosmotic cation-proton antiporters of gram-negative bacteria. (b) the discovery of a new enzyme activity (arsenate reductase as a component of the arsenic resistance system).756 SILVER & PHUNG of nutrient cations or oxyanions) and enzymatic detoxification (generally redox chemistry) converting more toxic to less toxic or less available metal-ion species.

121)].MASS. animals. 80. and aspartyl kinase) are shown with key predicted amino acids. This motif (including a vicinal cysteine pair) is related to copper.LIB. The model for CadA (Figure 1) is typical of P-type ATPases. 105). Downloaded from arjournals. except that it starts with a metal-binding motif. Figure 1 The cadmium-resistance ATPase of S. on 05/20/05. The recent renaming of this family of proteins as P-type ATPases denotes that they are the only transport ATPases that have a covalent phospho-protein intermediate. Bacillus (45) and clinical Listeria (60). For personal use only.50:753-789. the protein is phosphorylated by ATP (112) probably at the invariant aspartate residue (Asp415 in CadA). Rev. Microbiol. and it includes a conserved Cys-Pro-Cys tripeptide (as shown in Figure 1) that is found in the Cd2+ ATPases and related divalent cation ATPases. The two intracellular domains common to all P-type ATPases are the aspartyl kinase domain and the phosphatase domain. This includes the six predicted membrane-spanning regions shown in Figure 1 [eight are predicted for the related copper ATPases (see below) (64. 103.annualreviews. The CadA cadmium ATPase is one of the few bacterial examples for which direct experimental evidence for phosphorylation is available (112). The fourth membrane span is thought to be part of the cation translocation pathway. membrane channel. Tsai et al (113) showed that vesicles containing the CadA protein carry out . Furthermore.and mercury-binding regions on efflux ATPases and other proteins (100. During the transport cycle. 1996.MED.org by UNIV. The protein is phosphorylated from ATP only in the presence of Cd2+ . and plants. A membrane ATPase region follows that is closely homologous to all P-type ATPases of bacteria. phosphatase. with permission).SCH.BACTERIAL HEAVY METAL RESISTANCES 757 Annu. (modified from 104. The predicted motifs (Cd2+ -binding. aureus.

32.MED. CadC. lysyl oxidase (needed for normal cross-linking of collagen and elastin). was recently identified by in vitro analysis with purified protein and purified operator-promoter DNA to be a DNA-binding transcriptional regulatory protein (30). indicating that the substrate specificity is Cd2+ or Zn2+ . For personal use only. These proteins all appear to function as metal-ion responding transcriptional repressors and contain homologous sequence regions thought to form a helix-turn-helix pattern with the metal-binding vicinal cysteines in one of these alpha helical regions. together with the arsenic system repressor ArsR (47. dopamine-β-hydroxylase. CADC REPRESSOR FAMILY The cadmium resistance operon is inducibly regulated by divalent cations (22. The first mutants available in the CadA ATPase have Cys23 or Cys26 (see Figure 1) altered to Ser or Gly. Annu. Therefore. Microbiol. personal communication). 127) and the cyanobacterial metallothionein repressor SmtB (31. It was known that copper was accumulated in the upper intestinal mucosa but failed to move across the serosal membrane layer into the blood. and cytochrome oxidase.LIB. Downloaded from arjournals. the product of the second gene of the cad operon (130). the best matches were with bacterial cadmium ATPase (see above) and mercury-binding proteins of bacterial mercury resistance (see below). Tsai & Linet (personal communication) have also shown ATP-dependent Zn2+ transport. there was no clear hypothesis for the nature of the gene product defective in Menkes patients. Soon afterwards. on 05/20/05.MASS.50:753-789.org by UNIV. suggesting that the cation-binding motif is involved but not essential in cation recognition (K-J Tsai & AL Linet. Human Copper Transport Diseases: Menkes and Wilson’s A major surprise came from the sequencing of candidate cDNAs for the human diseases of copper metabolism called Menkes syndrome and Wilson’s disease (10.758 SILVER & PHUNG ATP-dependent transport of cadmium. 103. These include superoxide dismutase (affecting oxygen damage). 131). and all copperrequiring enzymes are nonfunctional. all other body tissues are copper-starved. Menkes syndrome is a lethal X-chromosome hereditary disease of copper starvation (24). 93. bacterial copper-translocating ATPases (now more than six) were identified and found to be close homologs to those defective in Menkes and Wilson’s diseases.SCH. CadC binds specifically to the cad operator/promoter DNA in gel shift and DNase I–footprinting experiments. However. 121). At the time when the cDNAs were sequenced. 114).annualreviews. Cultured fibroblasts from Menkes patients show increased copper . Rev. Divalent cations stimulate operon activity in vivo (22) and alter protein binding in vitro (30). tyrosinase. 1996. Bacterial cells with these mutant ATPases have reduced but not zero transport activity. CadC is a member of a new family (4) of metal-binding repressor proteins. when the predicted protein product was scanned through available libraries.

BACTERIAL HEAVY METAL RESISTANCES 759 accumulation and reduced efflux.50:753-789. A mouse model for Menkes syndrome is available. The equivalent mouse cDNA has been sequenced.LIB. A major question is whether the Menkes defect is at the cytoplasmic membrane or in an intracellular vesicle (the preferred hypothesis). Menkes’ victims usually die before 3 years of age. including the invariant aspartate and the Cys-Pro-Cys tripeptide. The major difference between the bacterial CadA ATPase and the proposed human Menkes product is the presence Annu. It was also clear that the characteristics of this ATPase satisfactorily explain what was known about the human disease. personal communication). which hints at the primary cause of the disease and provides a basis for a specific radioactive Cu2+ -retention diagnostic test. with several mutations at the same X-chromosome locus and with copper deficiency symptoms. and mutants lacking mRNA (“dappled”) or with abnormal-length mRNA (“blotchy”) have been identified (65). For personal use only. frequently from neuro-degenerative defects or connective tissue disorders (24). very similar to that shown for the Cd2+ ATPase in Figure 1. metallothionein synthesis is induced by excess copper. Identification of the “Menkes gene” and of the “Wilson’s gene” will not provide a cure. J Camakaris & JFD Mercer.SCH. In the absence of copper homeostasis. now localized to an intracellular membrane. followed by Cu2+ sequestration with intracellular metallothionein (which does not occur with wild-type cells).org by UNIV. as with other hereditary diseases. An intracellular Cu2+ carrier protein that might carry copper to where it is needed for incorporation into enzymes was earlier postulated to be defective rather than a membrane pump. Because all copper-dependent enzymes and metabolism are affected. Consequently. The over-accumulation of copper by the Menkes fibroblasts. leads to a problem in intracellular copper movement. Rev. However. All of the listed or labeled components of the protein were found.MED. biosynthesis of elastin and collagen is defective. and once metallothionein binds copper. 1996. However. the current evidence points to a defect in copper efflux that is consistent with the finding that the Menkes gene determines a P-type ATPase. Consequently. the candidate cDNA sequence once available (121) clearly showed that the gene product defective in Menkes patients was a divalent cation efflux ATPase. excess copper sequestration by intracellular metallothionein).MASS. . While earlier hypotheses included direct defects in copper transport or more often indirect effects (for example. cellular copper is high but unavailable for metabolic functions. New immunofluorescence results using antibodies directed against the copper-binding region of the protein and copper-resistant Chinese hamster ovary cells (which over-express the Menkes copper ATPase) have localized the Menkes protein to the trans-Golgi network (11. Downloaded from arjournals.annualreviews. Microbiol. on 05/20/05. early diagnosis gives promise for a major reduction in the frequencies of these diseases. the copper is prevented from reaching the efflux site.

With Wilson’s disease.50:753-789. The rat equivalent to Wilson’s. which is synthesized in and exported from the liver. The candidate cDNA for the gene defective in Wilson’s disease was isolated and sequenced soon after the Menkes results became available. For personal use only. Once the patient attains normal copper levels. and in fetal liver (65). 105). and two . Rev. Otherwise the proteins are remarkably similar. on 05/20/05. continuing chelate treatment and limiting copper uptake by dietary control can maintain copper homeostasis for the patient (24). The symptoms in the LEC rat are somewhat different from human Wilson’s and include cirrhosis of the liver. neurological damage is extensive. and patients usually die in their teens. Wilson’s defect primarily affects the liver. 105). The Wilson’s ATPase functions primarily in the liver. but the two sequences are sufficiently different as to not cross-hybridize in DNA-DNA Southern blot analysis. Microbiol. one for chromate resistance. not starvation. the liver is the primary organ of copper accumulation and homeostatic regulation. Annu.760 SILVER & PHUNG of six metal-binding motifs at approximately 100 amino acid intervals (instead of the single one in CadA). so that the first predicted membrane segment starts at position 705 rather than 106 (as in Figure 1) (103. Wilson’s disease. In Wilson’s disease patients. Copper over-accumulation leads to liver necrosis and sudden uncontrolled releases of copper.MASS. Copper chelate therapy results in a copper adduct that can be rapidly excreted in the urine. differs significantly from Menkes syndrome in that effective treatment is available. The gene for the autosomal recessive Wilson’s disease maps to chromosome 13. however. The candidate Wilson’s disease gene and proposed ATPase product are highly homologous to those of Menkes syndrome (10. including three for mercury resistance.MED. and both liver-exit pathways (serum ceruloplasmin and bile excretion) are defective.annualreviews. the Menkes gene functions in most other body tissues. Downloaded from arjournals.LIB. Wilson’s disease is in a sense the opposite of Menkes syndrome: It is a disease of copper overload.SCH. In normal humans. Copper needed by other organs is shipped by the circulating carrier protein ceruloplasmin. 1996. Cadmium Resistance in Alcaligenes: A Three Polypeptide Chemiosmotic Antiporter The large plasmids of the soil chemilithotrophic autotroph Alcaligenes eutrophus have numerous heavy metal resistance determinants [in strain CH34. followed in surviving animals by eventual liver tumors. copper accumulates in the liver. recently cloned and sequenced from the defective LEC strain. encodes an ATPase closely related to human Wilson’s (126). Excess copper is accumulated by the liver and excreted from the body by the bile duct (24).org by UNIV. using PCR primers designed for the most conserved portions of the predicted Menkes gene product.

There is no evidence to distinguish whether Czc functions as a dimer complex (as shown in Figure 2) or as a complex consisting of one each of CzcA. Downloaded from arjournals. For personal use only. and cobalt efflux system functioning as proton/cation antiporter consisting of inner membrane (CzcA).SCH. outer membrane (CzcC). 76) show closely related systems with basically the same three proteins (Figure 2). zinc. Czc is an efflux pump (75) that functions as a chemiosmotic divalent cation/proton antiporter (74. Two or more regulatory Figure 2 Czc model for cadmium. 26). Microbiol. although there remains some uncertainty about its location (76). CzcC is thought to be an outer membrane protein (as shown in Figure 2) (26). 73. Indeed.MASS.LIB. The DNA sequences of czc (75) and cnr (62) have diverged sufficiently that Southern blot hybridization fails. the predicted protein amino acid sequences (26. .BACTERIAL HEAVY METAL RESISTANCES 761 Annu. 1996.annualreviews. and “membrane fusion” (CzcB) proteins functioning as a dimer (adapted from 26. and CzcB appears to be a “membrane fusion protein” that bridges the inner and outer cell membranes of gram-negative bacteria (references in 49.MED. for divalent cations called czc (for Cd2+ . and Co2+ resistances) and cnr (for Co2+ and Ni2+ resistances)]. Zn2+ . on 05/20/05. Rev. including members that efflux toxic cations or organic compounds (26). and CzcC polypeptides (as shown in the comparable figure in Ref.org by UNIV. 76). CzcA is the basic inner membrane transport protein (Figure 2). The proteins involved have become the prototype for a new family of three-component chemiosmotic exporters. CzcB. 29). Transcriptional regulation of the czc and cnr operons is currently under active study.50:753-789. mutations of the Cnr system give additional Zn2+ resistance. 26). again showing that the two systems are fundamentally the same. Nevertheless.

which allows for separate gene regulation. Bacterial mutants lacking CopB become copper hypersensitive (107).LIB. the PacS (50) efflux ATPase is located in the thylakoid photosynthetic membrane (51). and therefore that monovalent Cu+ may be the substrate rather than divalent Cu2+ (as shown in Figure 3). Solioz & Odermatt (106) isolated inside out–subcellular membrane vesicles from E. In contrast. . The Enterococcus Cop system is regulated in response to both copper starvation (when the CopA uptake ATPase is needed) and copper-excess (when the CopB efflux ATPase is needed) (78). The substrate for CopB is therefore thought to be Cu+ (Figure 3) rather than Cu2+ . hirae cells. but there is no agreement about their functions (26. in cyanobacterium Synechococcus PCC7942. the two genes. the genes for copper uptake and efflux ATPases are unlinked (50. and with a gene for a potential transcriptional regulator between the pacS genes and in the opposite orientation (51). Remarkably.MASS.annualreviews.” in contrast to PacL. If this hypothesis proves correct. which is a calcium efflux ATPase. Microbiol. then there should be a cell-surface copper reductase associated with copper transport (106). 76). see above). copA and copB. 87). The CopB copper efflux ATPase of E. For personal use only. a characteristic that suggests that Ag+ may also be a substrate for CopA. Downloaded from arjournals. Rev. suggesting a role for CopA in uptake. Interestingly. for “larger. CopA and CopB ATPases of Enterococcus hirae The best understood copper transport and resistance system today is that of the gram-positive pathogen Enterococcus hirae (previously called Streptococcus faecalis) (107).50:753-789. hirae is one of two bacterial cation efflux ATPases for which subcellular transport data are available (the other is CadA. 1996. Two new version of Czc-family efflux systems have recently become available.SCH. one for a Czc system (57a) and the other for a homolog involved in mammalian host cell cytopathicity by Legionella pneumophila (64a). on 05/20/05.MED. as is the case for Saccharomyces cerevisiae (3a).” The quite different cyanobacterium Synechocystis PCC8303 has two genes homologous to pacS.org by UNIV. the first evidence that CopB is an efflux ATPase. and the vesicles required ATP and reducing conditions in order to accumulate 64 Cu and 110m Ag.762 SILVER & PHUNG genes are involved. COPPER RESISTANCE DETERMINANTS Annu. CopA mutants are also somewhat resistant to Ag+ . 73. that determine respectively uptake and efflux P-type ATPases are found in a single operon (80) (Figure 3). PacS was named as “smaller. E. downstream from and in the same orientation as a czcAB complex. hirae mutants lacking the CopA uptake ATPase become somewhat copper resistant and require higher levels of medium copper for growth. whereas the CtaA copper uptake ATPase of cyanobacteria is thought to be located on the cellular membrane.

CopY was shown to bind DNA in gel shift and DNase I–footprinting experiments in a region upstream of copY and that includes a diad symmetry. converting it to a DNA-binding repressor (78). A moderate level of intracellular Cu+ binds to CopY. hirae.org by UNIV.50:753-789. Transcription of copA and copB is governed by the upstream repressor (copY) and antirepressor (copZ) gene products.SCH. Figure 3 Transport and regulation of copper in E.BACTERIAL HEAVY METAL RESISTANCES 763 Annu. typical of operator/promoter regions. and CopZ-Cu+ binds CopY-Cu+ . The start point for transcription has also been directly identified by reverse transcriptase primer extension experiments (A Odermatt & M Solioz.annualreviews.MASS.MED. . the CopZ antirepressor binds Cu+ . The CopY apo-repressor is thought to be inactive and fails to bind to the operator/promoter DNA in the absence of intracellular Cu+ . rather than eight times as proposed by Odermatt et al (79.forming an inactive (i. Rev. personal communication).LIB. Microbiol. The copA and copB determine copper uptake and efflux P-type ATPases respectively. on 05/20/05. non-DNA-binding) complex. 1996. At higher intracellular Cu+ levels. The ATPases are shown passing six times across the membrane. For personal use only. transcription needs to be regulated for both low and high copper concentrations . The model in Figure 3 explains the simultaneous induction of synthesis of both ATPases by 20 µM Ag+ or 2 mM Cu2+ (77). 80). Regulation of the cop operon is governed by the products of the first two genes in the operon: copY (which determines a 145 amino acid repressor protein) and copZ (which determines a smaller antirepressor) (Figure 3). With genes for uptake and efflux ATPases as part of the same operon (Figure 3).e. Downloaded from arjournals.

MED. The DNA-binding responder protein.. Rev. Downloaded from arjournals. 21) and contain the same genes. the outer membrane protein CopB. Hypothesized chromosomal uptake and efflux membrane transporters are shown. 8). hirae (above) leads to confusion. Microbiol. CopA and CopC are blue copper- Annu. The four structural proteins determining copper resistance are the inner membrane protein CopD. and the four structural genes copABCD (Figure 4). For Pseudomonas sp.MASS. and two periplasmic proteins CopA and CopC (Figure 4. The sensor protein CopS is found in the membrane and probably can be labeled by an auto-kinase activity at a specific conserved histidine residue with 32 P from γ -32 P-ATP. 20. 1996.SCH. Xanthomonas (61a). 20. 8).org by UNIV.50:753-789. 21). coli (7. . coli genes are called pcoABCD and pcoRS (1. on 05/20/05. The CopR and CopS proteins are the only current examples among the metal resistance systems of transcriptional regulation by a classical two-component regulatory system (42a). and this along with the different use of cop for chromosomal genes of E. CopR. and E. For personal use only. the two regulatory genes are called copR and copS. the products of the copper resistance system of P. The comparable E. Figure 4 Copper transport and resistance in Pseudomonas. as well as intracellular copper-binding protein (8) and plasmid-encoded CopABCD. These systems are highly homologous (8. is thought to be transphosphorylated on a specific aspartate residue by 32 P-labeled CopS (8. syringae (21). 21).annualreviews.LIB.764 SILVER & PHUNG Cop of Pseudomonas syringae Bacterial resistance to copper conferred by plasmids has been described in Pseudomonas (21).

coli copper resistance system (8). These motifs are related to copper-binding motifs of the blue copper enzyme ascorbate oxidase. or binding. However.MASS. Microbiol. Gupta et al (39) showed that cutF is identical to nlpE. syringae (21). 60%. For example. Similar motifs are found in the CopA and PcoA protein sequences. 1996. efflux. these results are confusing. as well as specific . An additional seventh gene (of unknown function but regulated by available copper level) called pcoE lies distal. 61%.g. the previously assigned role for CutE was as an intracellular copper-binding protein (8). 38%. Figure 4 lacks such assignments. and gene names cutABCDEF have been tentatively assigned (8). down stream from the regulatory genes (gene order pcoABCDRSE) (7). proposed to be involved in copper binding (8).LIB. As noted above. indicating a role for CopD in copper uptake by the cell. The PcoB/CopB protein contains four imperfect repeats of the octapeptide AspHis-Ser-Gln/Lys-Met-Gln-Gly-Met. those of Xanthomonas and E. Given the closely homologous gene products. 8 and earlier papers quoted there). Storage of excess copper in the periplasmic space is considered to protect the cell from toxic copper. For personal use only. efflux. Furthermore. Rev. However. a mutant operon containing copD but not all three of the other genes confers hypersensitivity and hyperaccumulation of intercellular copper (18). and 30% identical amino acids respectively. Tentative models for specific genes and their functions have changed from year to year (e. However. How the membrane proteins CopD and CopB are involved in movement of copper across the outer and inner membranes is not understood. coli cells require control of uptake and efflux of copper. but CutE appears to actually be the apolipoprotein N-acyltransferase.50:753-789. Downloaded from arjournals. coli turn brown and show no sign of periplasmic copper storage (8). on 05/20/05. and intracellular copper binding.MED. 55%. 21). coli pco system appeared recently (7).BACTERIAL HEAVY METAL RESISTANCES 765 Annu. the gene encoding an outer membrane lipoprotein that might affect copper permeability. How basically the same gene products in different bacteria result in differing phenotypes needs to be understood. coli. binding proteins containing 11 and 1 Cu2+ ions respectively (17.org by UNIV. the PcoABCDR and S gene products are closely related to the Cop products of P.annualreviews. In addition. Colonies of the copper resistant Pseudomonas turn bright blue when grown in high copper-containing media. with 76%. Pco and Cut of Escherichia coli The primary paper on the genes of the E. Mutations affecting these three steps have been isolated in E. there is preliminary evidence for copper efflux (not uptake) associated with the E. Two conclusions are clear: (a) E.SCH. Chromosomal genes also affect copper transport and resistance. because newer biochemical data challenge earlier assignments of specific mutants to either uptake. Figure 4 shows three hypothesized chromosomal gene functions in copper uptake. as well as the chromosomally determined CutE sequence. which attaches a fatty acid to apo-NlpE (CutF).

MASS. which is encoded by basically the same plasmid genes (47. 1996. 96) and the most thoroughly studied ars operon (91. BACTERIAL ARSENIC RESISTANCE: MISSING GENES AND ENZYME ACTIVITIES The Molecular Genetics of Arsenic and Antimony Resistance Understanding of bacterial arsenic resistance systems has recently progressed rapidly. These include the plasmid R773 system. Downloaded from arjournals. The R773 and R46 ars systems contain two genes. 92. However. that converts intracellular arsenate to arsenite. that are missing from the recently identified E. on 05/20/05.SCH. however.annualreviews. Both gram-negative and -positive bacteria use the same biochemical mechanism. The DNA and the protein sequences of R773 and R46 are closely related (Figure 5). The gene order is arsR [determining the transcriptional repressor protein (93. Rev. arsD [a second regulatory gene whose product appears to function as a “throttle” setting an upper limit on operon function (128)].g. and therefore gene products are needed for these roles. and five of these are shown in Figure 5. Three gram-negative ars operons are diagrammed in Figure 5. coli chromosomal arsenic resistance operon (Figure 5. arsB (the gene for the membrane protein that functions alone as a chemiosmotic arsenite transport protein and as the binding site for ArsA). the number of genes can vary and the details of their functions differ. the substrate for ArsB transport). Six ars operons have been sequenced. its presence or absence might have little effect on the resistance level. 49).LIB. cutE and cutF) and other processes may have secondary effects on copper tolerance. arsA (the determinant of a membrane-associated oxyanion-stimulated ATPase protein). Because the ArsD protein is a secondary regulator of ars operon transcription.MED. and (b) genes whose primary products determine cell surface properties (e. which are unrelated (9)]. converts the ArsB chemiosmotic carrier (6) into an ATPase complex (25). 106a) and the staphylococcal plasmids. The existence of a membrane transporter than can be either chemiosmotic or ATPase and that can be converted from one form to the other (by addition of or removal of the arsA gene) is novel and has not been seen elsewhere in microbial biochemistry and molecular biology.org by UNIV. arsenate reductase. An earlier-studied but newly sequenced operon from plasmid R46 (9) is very similar. Annu. Microbiol. 12.50:753-789.766 SILVER & PHUNG intracellular copper-binding proteins to protect the cytoplasm from copperrelated redox damage. This topic of bacterial copper research is only starting. 27. arsA and arsD. For personal use only. This capability to switch . with the protein products between 85% and 93% identical [with the interesting exception of the 25-amino acid–interdomain “hinge regions” in the ArsAs (see below). 102). 127)]. the first sequenced (19. and arsC (the determinant of the small soluble enzyme. The ArsA protein.

All other membrane transporters are either chemiosmotic. Rev. 94) and from plasmid pI258 (the classic penicillinase plasmid from clinical Staphylococcus aureus). Microbiol. A third ars operon was recently found in another low-percent G+C gram-positive microbe.MASS.50:753-789.annualreviews. 1996. i.BACTERIAL HEAVY METAL RESISTANCES 767 Annu. Alignment and functions (below) of arsenic resistance genes (boxes) with amino acid (aa) sizes of predicted products (above and below genes) and percent identities between amino acid products. Bacillus subtilis strain 168 has a chromosomal ars operon (110) in a 48–kilobase pair (kb) region that intervenes in the middle of the gene determining a sporulation-specific sigma factor (component of RNA polymerase). For personal use only.SCH. The two staphylococcal systems shown in Figure 5 are from a plasmid of Staphylococcus xylosus (a microbe used in the meat processing industry.org by UNIV. on 05/20/05. secondary transporters. This putative ars operon may be the same as the determinant for arsenate resistance mapped to a similar chromosomal location nearly 25 . energy-coupling modes is a major surprise from recent efforts on arsenic resistance operons.MED. Figure 5 Genes and products for arsenic resistance in gram-positive and gram-negative bacteria. The two systems share about 93% identical amino acids of their putative products (Figure 5).e. This region loops out by site-specific recombination during sporulation and appears to be the remnant of an ancestral temperate phage.LIB. Downloaded from arjournals. or primary (frequently ATP-driven) pumps.

LIB.and antimonite-stimulated ATPase (5. (19) and each appears to form separate domains. 53. Whether the trigonal complex of As(III) with three cysteines involves the comparable C113 and C422 residues from the two halfmolecules of one polypeptide as shown in the Figure 6 (91) or occur with residues from different subunits of the active ArsA dimer is not known. 54) that is part of an aggregate with the membrane-bound ArsB protein (111. a low level of sequence identity of putative ArsB product (much lower than the values given in Figure 5).2–2. and Cys422 (Figure 6).MASS. 129).MED. means that the sequence of Takemaru et al (110) requires further analysis and direct experimental studies of phenotypes of the proposed genes. ArsA energizes the arsenite efflux pump by ATP hydrolysis (25. Downloaded from arjournals. The Bacillus chromosomal ars operon consists of arsR.org by UNIV.and carboxyl-half molecule sequences relative to the ATP-binding sites) result in reduced affinity for arsenite and antimony in vitro and reduced Vmax (but normal K m ) for ATP hydrolysis. However. 91). For personal use only. they can interact and function both in vitro and in vivo (54).50:753-789. the aggregate contains four ATP-binding sites (Figure 6). Rev. The amino-terminal and carboxyl-terminal halves of ArsA are closely homologous in sequence. showing that each domain contains an independent ATP-binding site. which is consistent with ArsA sitting on the ArsB membrane protein as a dimer. Cys113. The ArsA protein is an arsenite. 91). Cys172. arsB. The conclusion is that the three cysteines fold closely together in the three-dimensional protein structure so as to form the As-S bonds ˚ of 2. Annu. Microbiol. because mutations eliminating any of these three cysteines eliminate resistance to oxyanions in vivo. Mutants with altered aminoor carboxyl-half defects in ArsA can complement in vivo. and arsC genes homologous to those of the staphylococcal systems. on 05/20/05. plus the presence of an additional small open reading frame (ORF) between arsR and arsB. 1996. . When the two halfArsA molecules are produced as separate polypeptides.9 A length (5). A photo-adduct of ArsA with 32 P-ATP involves the phosphate-binding region near amino acid residue 10 and adenine ring-binding in the adduct near amino acid position 290. Mutagenesis experiments showed that both the amino and carboxyl ATPbinding sites of ArsA are required for transport and resistance in vivo and for ATPase activity in vitro (52. at the end of the first ArsA domain and at the start of the linker region.SCH.768 SILVER & PHUNG years ago (1). As(III) or Sb(III) activation of the ArsA ATPase appears to involve a tricoordinate complex with three cysteine residues. Those mutations eliminating the first and third of these cysteine residues (which are similarly placed in the amino. both of which contain a recognizable ATP-binding region.annualreviews. Arsenite Efflux ATPase Biochemical studies have been carried out with each ars gene product. Because ArsA sits on the membrane as a homodimer.

We assume this extraordinary difference reflects the failure 4 of the in vitro assay conditions to adequately model the in vivo conditions. coli arsenate reductases differ by 10. 48) and R773 (36) have been purified and studied.MASS. The K m s of the staphylococcal and E. ArsC is an enzyme that reduces less toxic arsenate [As(V)] to more toxic arsenite [As(III)] (48). 46).SCH. presumedly by recycling oxidized cystine to two reduced cysteine residues (of the four found in the sequence. arsenate reductase of plasmid R773 uses glutaredoxin (but not Annu.000-fold in vitro: the staphylococcal enzyme has a K m of 1 µM AsO3− (46). and oxyanion activation of ATPase activity is shown as involving As(III) binding to three cysteine residues. In contrast. Rev. Energy coupling with the gram-positive and gram-negative arsenate reductases also differ. For personal use only. and if there were not directly demonstrated enzyme activities. but ArsC activity is closely coupled with efflux from the cells (48) so that intracellular arsenite never accumulates. Downloaded from arjournals.LIB.annualreviews.50:753-789. their sequences are less than 20% identical (Figure 5). two on the amino-half domain (Cys113 and Cys172) and the other on the carboxyl-half domain (Cys422) of the same polypeptide [after data of Rosen et al (91)]. The staphylococcal arsenate reductase derives reducing power from thioredoxin (glutaredoxin will not work). on 05/20/05. each of which has an ATP binding site (four for the complex). coli R773 enzyme has a K m of 4 10 mM AsO3− (36). 1996. The domains of the ArsA polypeptide are shown connected by a flexible linker region (91). shown as a homodimer of two subunits.BACTERIAL HEAVY METAL RESISTANCES 769 Arsenate Reductase Enzyme The final gene product of the ars operon has also presented a surprise. . Figure 6 Model for structure of ArsA ATPase. the substrate for the ArsB transport protein.org by UNIV. each of which contains homologous amino-half and carboxyl-half domains. Microbiol. one might argue whether the proteins are even homologous. and the E. Although both enzymes reduce arsenate and confer resistance to arsenate [As(V)].MED. Arsenate reductases from plasmids pI258 (46. Conversion of a less toxic compound to a more toxic form seems counter-productive.

Downloaded from arjournals. 101. whose product is a unique positively acting activator protein that twists and bends the operator DNA region in the presence of Hg2+ . there are one to three genes whose products are involved in transport of toxic Hg2+ across the cell membrane to the intracellular detoxifying enzyme. 105). together with functional merC transport genes and nonfunctional partial merA genes (44).annualreviews. transregulation by MerR is known but the location of the merR gene is not known. the order and approximate number and functions of the genes are similar. MERCURY AND ORGANOMERCURIAL RESISTANCES Progress in understanding the bacterial mercury resistance system has been reviewed repeatedly (49. However. merR is transcribed separately and in the opposite direction from the remaining mer genes. conferring resistances to As(III). For the Thiobacillus system. coli. 108). 108.org by UNIV.770 SILVER & PHUNG thioredoxin). and Sb(III) and utilizing the same biochemical mechanism. 59. The results with ArsC are novel.50:753-789. with the current absence of more direct data such as protein structural analysis. and we touch here only on new findings and insights. Diversity of Operon Structure Closely related systems for resistance to inorganic mercury have been found on plasmids of gram-negative and gram-positive bacteria. In the Hammersmith Hospital collection of some 800 antibiotic-resistance plasmids that had been mobilized from various gram-negative bacteria into E. which have merR as the first gene on the multi-gene mer operon (not shown in Figure 7. 1996. As (V). occurs widely in gram-negative and grampositive bacteria (15. and probably only one of the two cysteines found in the protein sequence is required (63). In the first five mer systems of gram-negative bacteria shown in Figure 7. Plasmid R100 (with transposon Tn21) was the first example with the . Annu. two functional merR genes occur separately from the mer operon. it is clear that basically the same ars operon. which is quite different from the impression given by detailed studies concentrating on the mer operons of R100 (Tn21) and Tn501 (104. see below). 27. allowing RNA polymerase to synthesize mRNA (81. 25% carried mercury resistance (98). 102).SCH.MED. For the new pMERPH gram-negative plasmid system (85). we expect more surprises and changes of models. Microbiol.LIB. 85). 100. In most cases. After the operator/promoter site. Rev. allowing tighter control of the mer operon than possible with gram-positive bacteria. For personal use only. merR. All but two known mer systems of gram-negatives start with a regulatory gene. The new overall message (from information shown in Figure 7) is that mer operons are not all the same (8a. mercuric reductase. 84. 124). Figure 7 shows the currently known range of mer operons from gram-negative bacteria.MASS. on 05/20/05.

see text. but more recently several other merC genes have been sequenced (Figure 7). merC gene (which can function alone as a mercury transport system. merB occurs rarely (98). For personal use only. Figure 7 Genes and proteins of mercury resistance systems of gram-negative bacteria.SCH.MASS. Genes are shown by gray boxes and operator/promoter transcriptional start sites by open boxes. In gram-negative bacteria.and methylmercury are called “broad spectrum. The merA gene determining mercuric reductase is found after the transport genes in each case. Microbiol. Downloaded from arjournals. and systems with merA but not merB are called “narrow spectrum” because of their limited range of resistances to organomercurials.LIB. The arrows indicate the directions and lengths of mRNA transcripts.org by UNIV. The wiggly line for Thiobacillus indicates a lack of linkage between the two gene clusters.” in contrast. Systems with merB and conferring resistances to phenyl.BACTERIAL HEAVY METAL RESISTANCES 771 Annu.MED. For details and references to specific plasmids. The merA gene is followed immediately by merB (which encodes the enzyme organomercurial lyase that breaks the carbon-mercury bond in toxic substrates such as phenylmercury acetate) in only one of the seven mer systems diagrammed in Figure 7.annualreviews.50:753-789. Rev. 1996. 58). on 05/20/05. .

the functional significance of which we do not understand. in the related Bacillus mer operon. unpublished data). . merT and merP. on 05/20/05. The entire staphylococcal mer operon (merR ORF3 ORF4 merT merA merB) is transcribed on a common mRNA (106). except for an additional gene of unknown function that has been called merF (Figure 7).MED.LIB. and in addition a 181-codon open reading frame (between merA and merB). strain K-62.8-kb gap was reported between merA and merB (70. merA and merB are contiguous and cotranscribed. has recently been shown to have two separate merB genes for organomercurial lyase and phenylmercury resistance (55. the merB gene is found between merR and merT. We have recently determined that a second merR and a second merB gene occur in that gap (L Chakravarty & S Silver. 104). aureus. These genes. A 6-kb region containing the mercury-resistance operon was sequenced (M Kiyono & H Pan-Hou. Still another recently sequenced mer operon from plasmid pMER419 (42) and transposon Tn 5053 (57) contains the same genes and 92% identical nucleotides as Tn 501.50:753-789. Most mer operons from gram-negative bacteria end with the gene merD. a 1. On exposure of Streptomyces to mercury.772 SILVER & PHUNG In one recently studied example from Pseudomonas (not shown in Figure 7 because the sequence is incomplete). the first mercury resistant strain studied (25 years ago).SCH. the mer region of DNA amplifies so that 20 copies are found per chromosomal equivalent (99). but the regulatory and transport genes are transcribed separately and in the opposite direction (99. Pseudomonas spp. Rev.MASS. personal communication) and contains the same genes (as shown in Figure 7) for plasmid pDU1358. Clearly there are variations in gene order and structure among mer operons. 104). but its physiological significance has not been studied.annualreviews. For personal use only. 1996. 124). Whether both versions of merR and merB are transcribed and function (or not) is yet to be determined. Another distinctive characteristic of the Streptomyces mer determinant is the absence of the N-terminal MerA Hg2+ –binding domain that has been ascribed a role in mercury binding (see below). In the most-studied mer system from a gram-positive microbe. Microbiol. which in addition has determinants of mercury transport activity.org by UNIV. the predicted product of which lacks homology to known proteins. In the gram-positive mer determinant from high G + C Streptomyces lividans. One of the two Pseudomonas K-62 organomercurial resistance determinants was mapped on a 26-kb plasmid (55). together with an extra operator/promoter site (88). merB follows merA on plasmid pI258 of S. However. determine hyperaccumulation of Hg2+ but not of CH3 Hg+ (56). 56). While the other reductases have a single copy of this binding determinant. MerD appears to be a minor regulatory protein that down-regulates mer operon expression (68. a result that suggests that methylmercury does not use the inorganic mercury uptake system. which encodes a secondary regulatory protein that binds to the same DNA site as MerR (68). Downloaded from arjournals. the Bacillus MerA Annu.

org by UNIV. The first vicinal cysteine pair Cys24 and Cys25 of MerT is required for mercury resistance in the presence of mercuric reductase or mercury hypersensitivity of cells lacking reductase activity (67).MED. who replaced each of the two cysteines of MerP and four cysteines of MerT individually with serine residues. Japan.BACTERIAL HEAVY METAL RESISTANCES 773 protein has a tandem duplication of this binding region (124).MASS. Although both MerT and MerP are required for full resistance and wild-type mercury volatilization rates. a conclusion confirmed by Morby et al (67). Rev. For personal use only. The glove model has been repeated. Microbiol. which allows rapid exchanges from dithiol to dithiol (120). the second cysteine pair of MerT and the entire MerP protein were not essential for mercury resistance. however. Direct Hg2+ -binding experiments by Hamlett et al (41) showed that cells lacking either MerP. two alpha helical segments. Transport Findings of hyperaccumulation of mercuric cations and hypersensitivity to mercuric cations (69) associated with the two proximal gene products MerT and MerP 17 years ago led to the so-called “baseball glove” model. Transport of Hg2+ by MerT itself is consistent with Hg2+ transport by the alternative membrane Annu. Downloaded from arjournals. the role of MerT was more crucial. on 05/20/05. Although Hg2+ binding by protein and small soluble thiol compounds is of very high affinity. discussed. The structure consists of four antiparallel beta sheets. The baseball glove model consists of binding of Hg2+ initially by a pair of vicinal cysteines in MerP. followed by sequential passing of Hg2+ from glove to glove (cysteine pair to cysteine pair. directly related data were few until recently. A defective MerP protein with Cys36Ser resulted in greater sensitivity to mercury compared to greater resistance with a deletion mutant lacking MerP (67). The PCR product size indicating a duplicated Hg2+ binding domain was found in all Minamata Bay.LIB. or both. lacked the transport-specific binding activity first studied by Nakahara et al (69). The Streptomyces MerR sequence is surprisingly less closely homologous to other known MerR proteins than it is to the repressors CadC and ArsR of the cadmium and arsenic resistance systems. mercury resistant Bacilli (70). and the cysteine pair in a region without secondary structure that extends like a thumb from the edge of the core protein. without free cationic mercury intermediates) in MerT. the binding is kinetically very active.50:753-789. Hamlett et al (41) constructed deletion and frame shift mutations in merP and merT. However. as proposed by Hamlett et al (41) and Morby et al (67). This structure allows direct protein-protein docking of MerP with the inner membrane protein MerT. MerT. Sahlman & Sk¨ rfstad (95) demonstrated the need for both cysteine residues a of MerP for specific mercury binding in vitro. and accepted. and finally to the active site of MerA.SCH.annualreviews. the mercuric reductase enzyme. Eriksson & Sahlman (33) then constructed a secondary structure model for MerP from proton NMR analysis. 1996. .

mercuric reductase is a roughly globular dimer.MASS. the active site dithiolate Cys207 Cys212 is reduced and Cys212 forms a charge-transfer complex with the flavin. As expected from the close sequence homology to human glutathione reductase. Nevertheless. This mobility of the N-terminal domain is consistent with the proposed role for this mercuric reductase region in carrying Hg2+ from the MerT protein at the inner membrane surface to the substrate binding determinant at the carboxyl-terminus of the enzyme. 1996. so that the tetrahedral “cage” around the divalent cation consists of two cysteines and two tyrosine residues (97). Cys207 moves to a position closer to the carboxyl-terminal thiol pair Cys628 Cys629 (which is conserved in all mercuric reductases) of the second subunit. After passage of bound mercury from MerP to the two cysteine pairs of MerT.LIB. Downloaded from arjournals.50:753-789. with each active site requiring cooperation from both subunits. The N-terminal region of mercuric reductase was recognized early as closely homologous to MerP in sequence and assumed therefore to have a binding function. The duplicate MerP-like N-terminal 160 amino acids of Bacillus mercuric reductase do not have fixed positions in crystals of mercuric reductase and therefore were excluded from the structure of the protein solved by X-ray methods (97). protein MerC (58) in the absence of MerP and the newly found heterogeneity of mer operons with regard to mercury transport proteins. The bound FAD and the soluble cofactor NADPH are located in equivalent positions in human glutathione reductase and in bacterial mercuric reductase. on 05/20/05. However. except for 30 amino acids at the hinge just before the X-ray structure of the Bacillus reductase starts. and not four cysteines. 66d). whose X-ray structure had been solved earlier. On binding of NADPH. mutagenesis studies changing the four cysteines and two tyrosine residues have shown that all four cysteine residues are necessary for enzymatic activity (28. 66c. the model becomes more uncertain.MED. Tyr264 and Tyr605 (one from each subunit) are involved in metal cation binding cooperatively at the inter-dimer face of the crystal structure. 66b). Rev.annualreviews. For personal use only. as expected.774 SILVER & PHUNG Annu. Microbiol. Changing the two tyrosines individually to alanines affected the K m of the enzyme either not at all and the K cat only sixfold (Tyr605Phe) or had more dramatic effects but still did not .org by UNIV. then to the amino terminal cysteine pair of mercuric reductase.SCH. so as to form a candidate for a four cysteine Hg2+ binding site (28). The mercuric reductase sequences of the best studied Tn21 and Tn501 systems are also highly homologous (more than 90% identical). Mercuric Reductase and Organomercurial Lyase The major progress in understanding of mercuric reductase comes from solution of the crystal structure of the enzyme from Bacillus by X-ray diffraction (97) and kinetic analysis with mutant enzymes in the laboratories of CT Walsh and CH Williams (66a. In this reduced complex.

BACTERIAL HEAVY METAL RESISTANCES 775 completely eliminate enzyme activity (Tyr264Phe) (89). the homology to other metal binding domains and the conservation of this region in all mercuric reductases (except that for Streptomyces. 1996. so that when the first site contains a reduced NADPH. enzymatic cleavage of mercuric reductase that removes this region has no effect on in vitro enzyme activity (66e). and second (after the reduced NADPH+ is in place). However. 99) make it likely that this region has functional significance in natural environments. However. . to bind mercury more tightly than other cellular proteins (perhaps in a tri. Rev. Annu. Completing a long effort.MASS. forming an enzymeFADH− complex (66a). which is involved in metal binding in the crystal structure) had a much greater effect on in vitro enzyme activity than did elimination of Cys559 (equivalent to Cys629.org by UNIV. on 05/20/05. mercuric reductase differs in an important way from glutathione reductase in that the cysteines position the Hg2+ that is then reduced directly by the FADH− (23). Mutational changing of the N-terminal cysteines of the Tn501 mercuric reductase did not alter resistance levels either.or tetra-amino acid coordinated site). A flavin C (4a)-thiolate involving Cys140 of the Tn 21 enzyme (which corresponds to Cys212 of the Bacillus enzyme) is formed next. These results add uncertainty to any function for this region of the protein. which is not directly involved in metal binding) (66d). Downloaded from arjournals. showing that both cysteines are essential in vivo. The difficulty in obtaining laboratory conditions for measuring the function of the proposed mercury-binding region may be related to the same difficulty in demonstrating a MerP phenotype with mutants (67).annualreviews.MED. elimination of either cysteine resulted in hypersensitivity to Hg2+ by growing cells (66d). Whereas our model of passing the bound Hg2+ from the membrane protein MerT dithiolate to mercuric reductase involves the N-terminal domain that is homologous to the binding protein MerP. to have mercury in a labile position (probably as a dithiolate complex) so that it can be reduced by FADH− . In glutathione reductase it is the reduced cysteine thiolates that reduce the substrate. Spectroscopic studies with a mutant form that lacks the ability to bind Hg2+ demonstrated that NADPH reduces the flavin of FAD. elimination of Cys558 of the Tn501 reductase (equivalent to Bacillus position Cys628.LIB. Similarly. Kinetic analysis (66b) supports a model where the two pyridine-binding sites of the mercuric reductase dimer function in an alternating manner. Microbiol. the second site contains an oxidized NADP+ that cannot function in Hg2+ reduction.SCH.50:753-789. at least after cloning into a high copy number plasmid (66e). Miller et al (66b) propose that this alternating site model will achieve the two energetically incompatible needs for an enzyme with mercury as a substrate: first. For personal use only. Rugh et al (94a) succeeded in transforming the model plant Arabidopsis thaliana so that it makes bacterial mercuric reductase.

Downloaded from arjournals. that leads to the simultaneous formation of a methane from methylmercury or benzene from phenylmercury plus inorganic mercury covalently bound to two lyase cysteine thiolates. Subsequently. In addition to the thoroughly studied MerR regulator from the transposons Tn21 and Tn501 of gram-negative bacteria. organomercurial lyase has low turnover numbers relative to mercuric reductase. the binding of Hg2+ at one position affects the binding to the DNA involving different protein residues) metalloregulatory protein (81. Microbiol. aureus plasmid pI258 (19a.e.org by UNIV. dimeric transcriptional activator with a carboxyl-terminal presumed DNA-binding helix-turn-helix motif and an amino terminal Hg2+ binding region. which is longer by 2 bp than canonical bacterial promoters in distance between the − 35 and − 10 RNA polymerase binding sites and achieves a functional fit to the polymerase by bending and twisting (3). with the result that the toxic product inorganic Hg2+ does not accumulate in the cell.LIB. In contrast to mercuric reductase. 108). TV O’Halloran. The other major consideration is the DNA promoter site itself. Both in vivo and in vitro. By mutational .SCH.annualreviews. AO Summers. excess soluble thiol in vivo (or we think perhaps the N-terminal thiolate pair of mercuric reductase in vivo) releases the mercury from the organomercurial lyase and make it available for mercuric reductase.MASS.776 SILVER & PHUNG Annu. Rev. which is the best understood allosterically functioning (i. First is the MerR protein itself. For personal use only. probably by a histidine-derived proton. Regulatory Genes Understanding of the molecular details of how the positively acting regulatory protein MerR interacts with both Hg2+ cations and its cognate DNA operator/promoter site is the most sophisticated by far of those for any heavy metal resistance system. a somewhat different MerR protein (only 35% amino acid identities) is found with the mercuric resistance system of Bacillus (41a) and the S. Most progress has come from efforts of NL Brown. and their coworkers working on the closely related Tn501 and Tn21 MerR proteins.MED. organomercurial lyase is a small (just over 200 amino acids) monomeric protein.50:753-789. 1996. 59). The initial step is binding of an organomercurial-thiolate complex to an active site cysteine thiolate. The transgenic plants are resistant to mercury and volatilize mercury. MerR is a 144–amino acid (monomer length). Three of the four cysteines in MerR appear to be involved in binding of a single Hg2+ cation. followed by attack of the Hg-S bond. There are two rather novel aspects of MerR regulation. making the possibility of “phytoremediation” of mercury-polluted soils closer to reality. Organomercurial lyase requires a soluble thiol compound but lacks bound metal ions or other cofactors (4c). Its organometallic bond as substrate is unusual. on 05/20/05. Kinetic studies with differing substrates have led to the model of a concerted SE 2 protonolytic cleavage mechanism for the enzyme (123).

] Resistance Genes in Polluted Environments The diversity of mer operon structures shown in Figure 7 prompts the question of how these assort into different environments and how their nature and abundance reflect environmental selection. Transcription rate increases with cooperative kinetics as if binding of one Hg2+ cation facilitates the binding of another leading to activation. but at the moment only strong evidence that ambient available mercury selects for higher frequencies of mercury-resistant bacteria.g. as occurs in vivo. analysis. about 10−8 M Hg2+ both in vitro and in vivo (81.SCH. R Gourse (personal communication) has obtained evidence of positive enhancement of the regulation of the mercury resistance operon of Tn21 by an upstream − 40 to − 60 DNA element that apparently interacts with the carboxyl-end of the alpha subunit of RNA polymerase. There are no solid answers with concern to specific genes. 108). so how this sharp response curve occurs is not mechanistically clear. [For more information on MerR.MED. Remarkably.annualreviews. this volume). on 05/20/05. 70). A striking feature of MerR activation is that it occurs abruptly (or cooperatively) as a function of added Hg2+ . For personal use only.org by UNIV. The half-activation concentration is very low. Helmann et al (41a) demonstrated convincingly that three of the four cysteines present in Bacillus MerR (and these are in equivalent positions in the plasmid pI258 and transposon Tn21 and Tn501 versions of MerR) were needed for high-affinity in vitro Hg2+ binding. with Cys79 present on one subunit and Cys114 and Cys123 present on the other subunit. Rev. However. inactive mutant homodimers could dissociate and reassociate into active mutant heterodimers. The merB gene is needed for organomercurial resistance and is found in all organomercurial resistant Bacillus (70) from the .LIB. Recently. 81). but also the human intestinal tract with release of mercury from dental amalgams (109). even above that obtained by the complex interaction between MerR with the −10 to −35 region of the promoter (twisting and unbending). much as this occurs for transcription of ribosomal RNA operons (see 36a.50:753-789. Mercury-polluted environments include not only the familiar settings for industrial pollution (e. only a single Hg2+ binds per MerR dimer (41a. The MerR-binding DNA operator/promoter region for the mer operons of transposons of gram-negative bacteria has an unusual non-optimal 19-bp distance between the −35 and −10 RNA polymerase binding sites.MASS. see end of chapter (Addition in Proof). 1996. Downloaded from arjournals. Microbiol. Binding of Hg2+ -bound MerR both twists and shortens that distance. so that the sites are oriented about 70◦ out of phase on the DNA surface.BACTERIAL HEAVY METAL RESISTANCES 777 Annu. The heterodimers both repressed (in the absence of mercury) and activated transcription in the presence of Hg2+ in vitro. forming both a greater distance than can be bridged and a twist. The interaction between the alpha subunit of polymerase and DNA assures a high level of transcription.

778 SILVER & PHUNG Annu. coli (98) but was present in about 50% of Pseudomonas mer operons (19b). The order of preference of cation binding for cyanobacterial metallothionein is Zn2+ > Cd2+ . the merC gene itself was found more often in brackish water than in fresh water samples (4b) and still less in marine environments (4a). We do not know why.LIB.MASS. polluted site of Minamata Bay. The organomercurial lysase (merB) activity was found rarely in E. thiolate-rich metal binding proteins of animal systems (approximately 60 amino acids long with one-third of these as cysteines). have not been widely reported. Like the cysteines in animal metallothioneins. Within that group of bacteria. including yeast and Neurospora. Cells that lack metallothionein have reduced uptake of Zn2+ and reduced tolerance to high Zn2+ concentrations (although Cu2+ and Hg2+ tolerances are unchanged. This small 56 amino acid-long polypeptide product of the smtA gene contains nine cysteine residues. Microbiol. and lower eukaryotes. homologous at the protein and functional levels to the small. For personal use only. and missing in Pseudomonas and other gram-negative genera (35a).MED. The alternative transport gene merC is the major difference between the best studied mer operons of transposons Tn21 (plasmid R100) and Tn501 (Figure 7). with very low binding affinity for Cu2+ (unlike metallothioneins of eukaryotes). Yet merC was found commonly in E. Downloaded from arjournals. To date. on 05/20/05. higher plants.org by UNIV. 115). different restriction nuclease polymorphisms were more abundant in some Bacillus species than in others (70) but with no known functional correlate. This may be related to the effects of Na+ ions on the transport of Hg2+ and induction of the mer operon (99a).50:753-789. Rev. In spite of promising but not reproducible reports of metallothionein in Pseudomonas. Metallothioneins of apparently separate evolutionary origin have been found in most animals. these probably bind divalent cations independently in N-terminal and C-terminal clusters. coli. Cells selected by growth on increasing high levels of Cd2+ contain multiple copies of the gene producing metallothionein and are Cd2+ resistant (38). clustered in two groups of four and five. respectively. no functional difference in resistance or cellular uptake of Hg2+ has been found with merC present. Whereas mer operon DNA homologous to that of Tn21 and Tn501 is found associated with mercury pollution and selection (4a). METALLOTHIONEIN IN CYANOBACTERIA: A NEW SYSTEM Bacterial metallothioneins. to date the only prokaryotic cells with well-studied metallothionein are two cyanobacterial strains in the genus Synechococcus (114).annualreviews. Japan. . 1996.SCH.

B. Microbiol.BACTERIAL HEAVY METAL RESISTANCES 779 Annu. Inducible wild-type system with divergently transcribed smtA (metallothionein) and smtB (regulatory) genes (represented as open boxes).SCH. Deletion of 352 nucleotides from the first to the fourth palindrome (from the left as shown) leading to constitutive synthesis of metallothionein. 115). . 115). between the − 10 position for the smtA transcript and the start of the smtB gene. on 05/20/05. Small vertical arrows mark the locations of seven highly iterated palindromic sequences (5 GCGATCGC3 ). 114. For personal use only. Three complexes of SmtB protein bound specifically to the smtA operator/promoter have been identified with differing electrophoretic mobilities (31. Arrows under the genes indicate the direction and extent of transcription. This figure summarizes results from Ref.MED. metallothionein (SmtA) synthesis is negatively regulated by the repressor protein SmtB which binds (Figure 8) at an imperfect inverted repeat TGAAACA-GT-TATTCA between the transcriptional initiation site and the ribosome binding site for smtA (31. 114 and its references. The first appears to correspond to the binding at a site downstream from the transcriptional start site. 1996.annualreviews. First. The synthesis of metallothionein is regulated at the transcriptional level in three ways. 115). Downloaded from arjournals. With reporter gene fusions smtA-lacZ and smtA-lux (32. A. metal-dependent transcription from the smtA promoter has been demonstrated. SmtB is a predicted helixturn-helix protein in the same family as ArsR and CadC (see above) and appears to bind at the operator region as a dimer or higher multimer form (31. 115).LIB.MASS. Rev. the smtB gene is specifically and irreversibly inactivated by removal of 352 bp between the first and Figure 8 The metallothionein metal-binding system of Synechococcus. and the other two to the binding at sites upstream.org by UNIV.50:753-789. In a second level of regulation in response to high Zn2+ or other metals.

eutrophus (72) chromate resistance systems share homologous chrA genes. As far as we are aware. The other sequence (GenBank protein S41228) is of a fragment that encodes only 74 amino acids.50:753-789. Plasmid-determined chromate resistance re4 sults from reduced uptake of CrO2− by the resistant cells (82). that was proposed to be responsible for the inducibility of the resistance (72).MED. Recently. 82). For personal use only. locus SSU20224) sequenced a DNA open reading frame from the large plasmid of the cyanobacterium Synechococcus PCC7942 that encodes a protein closer to ChrA of Alcaligenes eutrophus (67% amino acid identities) than our original Pseudomonas ChrA sequence (only 28% identical amino acids). The DNA sequences of the Pseudomonas aeruginosa (14) and A. Microbiol. Downloaded from arjournals. Preliminary work indicates that reduction of Ag+ to Ag0 (which occurs with both resistant and sensitive cells) is not the mechanism of resistance. Rev. two chromate-resistance–related sequences have been recognized in sequence libraries. Multiple tandem copies of the smtA gene are found (38). eutrophus chromate-resistance determinant contains an additional upstream gene.annualreviews. which encode membrane proteins. 1996. OTHER SYSTEMS Annu. Nor has . but with a high similarity (39% amino acid identities) to the N-terminal region of the cyanobacterial ChrA. however. The P.org by UNIV. Chromate Resistance Resistance to chromate governed by bacterial plasmids appears to have nothing to do with chromate reduction (13. Nicholson and the late DE Laudenbach (71.780 SILVER & PHUNG fourth of seven repeated sequences in this region (Figure 8) (37). 16. 72). aeruginosa determinant as cloned lacks both the chrB gene and inducibility. GenBank accession U20224. no direct studies have been done with either.MASS. Cyanobacterial metallothionein synthesis is regulated at a third level by gene amplification.SCH. and Other Cation Resistance Systems Highly specific Ag+ resistance was reported more than 10 years ago (40). which occurs when cells are gradually adapted to high Cd2+ . Silver. but we have 4 been unable to determine whether there is chromate efflux (as in other resistance mechanisms) or a direct block on uptake. Thallous. chrB. it is not clear whether the chromate reduction ability found with several bacterial isolates confers resistance to CrO2− (83). There have been no direct studies of bacterial chromate resistance since our earlier efforts on Alcaligenes and Pseudomonas (14. The A. on 05/20/05. Furthermore. suggesting a functional role in divalent oxyanion transport. but no start has been made on the molecular genetic and biochemical basis.LIB. This sequence occurs in a region involved in sulfate metabolism.

Downloaded from arjournals. 122. but basically nothing is known about the mechanism.MED. POSTSCRIPT: THE FUTURE The last few years have seen enormous progress in our understanding of the four most thoroughly studied heavy metal resistance systems. 116. Because of the relationship in cation size and electron orbitals. Rev. A new plasmid resistance to tributyltin (used as antifouling compounds for ship hulls) has recently been reported (66). A relationship between silver transport and copper transport may be involved (35.SCH. the number of genes differs and the sequences of their protein products are not homologous nor recognizably related to any other protein sequences of known function (49. other than common genes in the two IncHI2 determinants pMER610 and R478. and we do not understand the mechanism of tellurite resistance (49. and R478. 64. We must wait for clarification of tellurite resistance mechanisms. cadmium. that several systems seem different and second. 125). 125). reduced uptake. For personal use only. convincing data for any mechanistic hypothesis have been lacking. pHH1508a. Thalium resistance determined by a plasmid system has been reported (99a). 116. 1996. 106b). on 05/20/05.MASS. MECHANISMS AND RELATIONSHIP TO TELLURIUM REDUCTION Several mechanisms of tellurite resistance have been proposed. Yet. coli (43).org by UNIV. Therefore. 122.LIB. the two unexpected conclusions from sequencing studies of plasmid-determined tellurite resistance operons are first. Tellurite Resistance RANGE OF GENETIC DETERMINANTS There are well-studied and sequenced determinants of plasmid-governed tellurite resistance. and this homology provides a working hypothesis involving resistance by altered thallous cation uptake or efflux. and enhanced efflux (43. that the predicted amino acid sequences have not led to a mechanistic model for resistance. There is even a chromosomal determinant of tellurite resistance in E. those for mercury. 117).annualreviews. However. 117) in any case. 122). arsenic. 77.BACTERIAL HEAVY METAL RESISTANCES 781 Annu. 64. Unfortunately. including enzymatic reduction. and a “cryptic” determinant on IncPα plasmid RK2 that was activated by mutation (43. including those from IncH plasmids pMER610. Tl+ is a K+ transport homolog (23a).50:753-789. and copper resistances. Microbiol. Tellurite resistance determined by plasmids does not appear to involve reduction to black metallic Te (0)—which occurs with many bacterial colonies if resistance allows cell growth (43. It differs in sequence and gene number from the plasmid systems. silver efflux been demonstrated as the basis for this resistance. the fundamental mechanism of resistance or gene regulation at the molecular level is not understood for any .

annualreviews. . if the next few years are like the last half dozen. 108). sometimes to wild-type inducible levels. MerR binds to the promoter DNA region. Downloaded from arjournals. Analysis of specific promoter positions by mutations together with reporter gene transcription and DNA footprinting (61. but quadruple mutants result in constitutive transcription. on 05/20/05. unexpected enzymes and mechanisms..MED. Although the emphasis in the published literature on the mercury regulatory protein MerR has led to a sense that the topic is well developed. 86) has defined the contact positions between the promoter DNA and the MerR protein. The next few years should see steady progress and mechanistic understanding from both protein and mutagenic approaches. allowing contact with RNA polymerase at both the −35 and the −10 region (3. then we expect more surprises.782 SILVER & PHUNG Annu. those unable to repress the basic (low) level of transcription in the absence of Hg2+ and those that can repress but not activate when Hg2+ is added (108). For personal use only. This “open” complex is transcribed. One interesting result from these studies of MerR of Tn501 was the involvement of six negatively charged amino acids (that occur in three vicinal pairs in the middle of the sequence) in transcription regulation by apo(non-Hg-containing) MerR. There is no sign of movement of MerR along the DNA. An additional double mutant A89V S131L also showed constitutive transcription (86a). In the absence of Hg2+ . Comess et al (19c) constructed a synthetic merR gene to facilitate further mutational analysis. Upon the addition of H2+ . Mutations affecting one amino acid pair have little effect on repression. Aside from the single example of mercuric reductase from Bacillus sp. and the initial characterization of the physiological basis for some metal resistance systems that are listed now–but without meaningful information on mechanism. although in a nonfunctional complex (3). causing a binding of the DNA and facilitating binding of RNA polymerase.org by UNIV. Firstly. 81. Rev. which is unsuited for productive association with RNA polymerase and transcription to an “open” form with RNA polymerase to DNA contacts and that carries out transcription (3. 81). we have given the contrary view that the surface (of understanding) is barely exposed. MerR mutants come in two classes. 81. Microbiol. there are no solved protein structures for the gene products involved.LIB.MASS. or even much changed affinity of the protein for the DNA in the presence of Hg2+ .50:753-789.SCH. What happens instead is a radical change in the bent MerR-DNA complex. the DNA straightens and untwists. apoMerR and Hg2+ -bound MerR bind to the same DNA region. Addition in Proof Studies of the promoter site where MerR binds have built a detailed understanding with several unique features (3. 108). case. 1996. In addition to progress on developed systems. 41b.

Barkay T. Walsh CT. The ars operon of Escherichia coli confers arsenical and antimonial resistance. Sci. Appl. Acids Res. Plasmid 27:65–71 Cha J-S. Camakaris J. A Sigel. Petris MJ.org by UNIV. Bacterial transport of and resistance to copper. Bacteriol. Rosen BP. Biol. 175:3480–85 7. Environ. pp. 1994. 12. Rosen BP. 1990.MED. Cooksey DA. Shi W. 1995. Microbiol. 1995. Pearson AJ. J. Genetics 74:197–213 2. Strike P. 15. 16. 1991. isolation. Proc. 19. Nucleotide sequence of the structural genes for an anion pump: the plasmid-encoded arsenical resistance operon. 14. Plasmid chromate resistance and chromate reduction.MASS. 10:246–52 Camakaris J. 1996. Adams. Microbiol. Misra TK. Roberto F. Microbiol. Li J. Ecol. 1995. 59:1671–74 Chen CM. Liebert C. Ram´rez JL. Plasmiddetermined resistance to chromate in Pseudomonas aeruginosa. Hybridization of DNA probes with whole community genome for detection of genes that encode microbial responses to pollutants: mer genes and Hg2+ resistance. 10. Annu. 4:605–12 Bruhn DF. Silı ver S. 56:173–76 Cervantes C. 1986. O’Halloran TV. 177:981–86 Cervantes C. Barkay T. Liebert C. Transposition of the arsenate resistance locus of Bacillus subtilis strains 23 and 168. Ohtake H.annualreviews. Brown NL. nucleotide sequence. Shen P. DNA-bend modulation in a repressor-to-activator switching mechanism. Lett. Bhattacharjee H. USA 88:8915–19 Cha J-S. 1995. Molecular biology of iron acquisition in Saccharomyces cerevisiae.SCH. Biol. FEMS Microbiol. New York: Dekker 8a. Vol. Askwith CC. Genetic di- 783 9. Silver S. Nucl. Resistance to arsenic compounds in microorganisms. Bacteriol. Bairoch A. Li J. and characterization. Environ. Ansari AZ. Appl. 270:1–6 6. Mol. o o Arsenic efflux governed by the arsenic resistance determinant of Staphylococcus aureus plasmid pI258. 1994. Rosen BP. 1989. Ji G. 1995. A possible mechanism for metal-ion induced DNA-protein dissociation in a family of prokaryotic transcriptional regulators. FEMS Microbiol. 17:1153–66 8. et al. Chem. 13. Rosen BP. 1993. Barrett SR. 11. 1996. Ksenzenko MY. on 05/20/05. 21:2515 4a. A. 55:1574–77 4c. Rouch DA. Cloning. ATP7A) P-type ATPase gene of CHO cells is associated with copper resistance and enhanced copper efflux. Role of cysteinyl residues in metalloactivation of the oxyanion-translocating ArsA ATPase. Nature 374:371–75 3a. Gillman M.LIB. Environ. 1992. In press Bull PC. 55:1196–1202 4b. Microbiol. For personal use only. Ritchie DA. Lett. Begley TP. Chem. 1996. Walts AE. Copper resistance in Pseudomonas syringae mediated by periplasmic and outer membrane proteins. 1995. Gene amplification of the Menkes (MNK. 1993. Natl. J. Trends Genet.BACTERIAL HEAVY METAL RESISTANCES Literature Cited 1. Kaplan J. Lee BTO. Rev. Br¨ er A. Bailey L. Br¨ er S. Appl. 1993. 261:15030– 38 . Bacteriol. Molecular genetics and transport analysis of the copper-resistance determinant (pco) from Escherichia coli plasmid pRJ1004. Lee BTO. J. Misra TK. Silver S. Mol. Brown NL. Deleted in proof 3. 18. J. 1989. 17. 20:27–34 4. Mol. de Silva D. DW. Silver S. 1973. Gillman M.50:753-789. Dey S. In Metal Ions in Biological Systems. versity within mer genes directly amplified from communities of noncultivated soil and sediment bacteria. Osborn AM. Hum. Lockhart P. Bruce KD. Arsenic resistance operon of IncN plasmid R46. J. Environmental significance of the potential for mer(Tn21)-mediated reduction of Hg2+ to Hg0 in natural waters. Acad. Ohtake H. and expression of the chromate resistance determinant of Pseudomonas aeruginosa plasmid pUM505. Silver S. Mol. 4:2117–23 Carlin A. Downloaded from arjournals. 1994. Cox. Silver S. 30. Rev. Biochemistry 25:7186–92 5. Copper hypersensitivity and uptake in Pseudomonas syringae containing cloned components of the copper resistance operon. Microbiol. Microbiol. H Sigel. Genet. ed. Wilson disease and Menkes disease: new handles on heavy metal transport. 1988. FEMS Microbiol. Cooksey DA. Chu L. Bradner JE. Bacterial organomercurial lyase: overproduction. 172:287–91 Cervantes C. 405–34. 15:355–67 Cervantes C. Silver S. 1986. Ji G.

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