Formation of a Phosphorylated Enzyme Intermediate by the cadA Cd2+-ATPase

Tsai, Kan-Jen; Linet, A. L.

doi:10.1006/abbi.1993.1421

Cited by 44 publications

(30 citation statements)

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“…Previous studies showed that expression of the cadmium resistance system is tightly regulated (3,5,20,25,26), but the trans-acting regulatory protein was not identified. The vector plasmid used by Yoon et al (25,26) contained an unrecognized copy of the cadC gene, which precluded determination of the role of the cadC gene in reporter gene expression experiments (25).…”

Section: Discussionmentioning

confidence: 99%

“…The CadA ATPase protein itself is sufficient for resistance and decreased cadmium uptake in Bacillus subtilis cells (10) and for ATP-dependent Cd 2ϩ uptake by inside-out subcellular membranes (21), equivalent to efflux by intact cells. The CadA ATPase can be labelled with 32 P from [ 32 P]ATP (20), the characteristic of P-type ATPases that distinguishes them from other classes of membrane transport ATPases.…”

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confidence: 99%

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CadC, the transcriptional regulatory protein of the cadmium resistance system of Staphylococcus aureus plasmid pI258

1995

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, and Pb 2؉ caused the release of CadC from DNA in gel retardation assays. DNase I footprinting measurements showed that the CadC protein specifically associated with and protected a region of operator/promoter DNA from nucleotide positions ؊7 to ؉14 relative to the start point of mRNA synthesis. Runoff transcription assays with the operator/promoter region of DNA (plus the first 69 nucleotides of the cadC gene) and purified E. coli RNA polymerase gave an mRNA product of the predicted size. Added CadC protein inhibited transcription in vitro.The cadmium resistance system of Staphylococcus aureus plasmid pI258 was identified by Novick and Roth (9) and subsequently mapped by deletion analysis (8). The cadA system results in reduced net uptake of Cd 2ϩ because of increased energy-dependent efflux of the toxic cation (22). Cloning and DNA sequence analysis identified two genes called cadC and cadA (10). The cadA gene product is a P-type ATPase, a member of the large family of related cation transport ATPases found in plant and animal cells as well as in microbes (16, While the properties of CadA satisfactorily explain cadmium resistance, the role of cadC and the nature of the trans-acting component of the carefully regulated CadA system (3, 25) remain unclear. The cadC gene is involved in full cadmium resistance; cadC can be provided in trans and need not be present on the same plasmid as cadA (26). The inducible nature of transcription was established, and the initiation site for cad mRNA synthesis was identified by reverse transcriptase primer extension (25) to a position 28 nucleotides upstream of the ATG start codon of the cadC gene (Fig. 1). Operon-length mRNA of 2.6 kb, including both cadC and cadA, was identified by Northern blot DNA-RNA hybridization (25). Canonical Ϫ10 and Ϫ35 Escherichia coli-like RNA polymerase recognition sites (Fig. 1) were identified in the sequence (25). A 7-nucleotide inverted repeat segment, including the first nucleotides transcribed into mRNA, was proposed as the operator site for binding of a then-elusive trans-acting DNA-binding regulatory protein (25).Cation-inducible gene function was studied by measurements of Cd 2ϩ efflux by whole cells (25) and subcellular membranes (21) and through induction of the reporter genes ␤-lactamase (25) and luciferase (3). However, the presumed transacting regulatory protein was not identified (25). Because of the need for cadC in gene regulation (21) and the sequence homologies (1, 26) among the predicted CadC protein product, the ArsR regulatory protein (12,14,15,23) for the arsenic resistance system, and the SmtB regulatory protein for cyanobacterial metallothionein synthesis (6), we undertook a direct test of the hypothesis that CadC is the regulatory protein for cadA operon transcription. By in vitro assays with CadC protein and the appropriate operator/promoter DNA, CadC has been shown to be a DNA-binding, negatively acting regulatory protein. MATERIALS AND METHODSAbbreviations. DTT, dithiothreitol; IPTG, isopropyl-␤-D-thiogalactopyranosid...

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Section: Discussionmentioning

confidence: 99%

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CadC, the transcriptional regulatory protein of the cadmium resistance system of Staphylococcus aureus plasmid pI258

1995

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“…CadA had been shown to catalyze ATP-coupled, vanadatesensitive Cd(II) transport (7) and to form a phosphoenzyme intermediate (27). ZntA has been shown to transport both Zn(II) and Cd(II) in an ATP-requiring, vanadate-sensitive reaction (6).…”

Section: Discussionmentioning

confidence: 99%

“…First, transport requires ATP and is inhibited by orthovanadate, a classical inhibitor of P-type ATPases (5). Second, P-type ATPases form a ␤-acylphosphate intermediate, and several soft metal pumps have been shown to form these intermediates (27,28).…”

Section: Discussionmentioning

confidence: 99%

Pb(II)-translocating P-type ATPases

Rensing

Sun

Mitra

et al. 1998

Journal of Biological Chemistry

182

169

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The cad operon of Staphylococcus aureus plasmid pI258, which confers cadmium resistance, encodes a transcriptional regulator, CadC, and CadA, an ATP-coupled Cd(II) pump that is a member of the superfamily of cation-translocating P-type ATPases. The Escherichia coli homologue of CadA, termed ZntA, is a Zn(II)/Cd(II) pump. The results described in this paper support the hypothesis that ZntA and CadA are Pb(II) pumps. First, CadC is a metal-responsive repressor that responds to soft metals in the order Pb>Cd>Zn. Second, both CadA and ZntA confer resistance to Pb(II). Third, transport of 65 Zn(II) in everted membrane vesicles of E. coli catalyzed by either of these two P-type ATPase superfamily members is inhibited by Pb(II).Exposure to environmental sources of lead is a serious public health concern. In humans chronic lead exposure produces neurotoxicity, anemia, and kidney damage, and acute lead toxicity can be fatal. Neither the specific lead transporters nor the regulatory elements that control the expression of the transporter genes have been identified. As models for human metal toxicity, we have been characterizing transporters for toxic metals and their genetic regulation (1, 2) and report here the identification of two P-type ATPases that are responsible for Pb(II) extrusion and resistance in bacteria.Bacterial metal ion resistance probably arose early in evolution due to widespread geological occurrence of metals. Bacterial cells have chromosomally and plasmid-encoded mechanisms for extrusion of antimicrobial substances, including toxic soft metals (3). While the ionic forms of some of these metals such as zinc and copper are essential for all organisms, all of these ions are toxic in excess. ZntA from Escherichia coli and CadA from plasmid pI258 of Staphylococcus aureus are both members of the superfamily of P-type cation-translocating ATPases but belong to a subgroup of soft metal transporters that includes CopA, a Cu(I) pump from Enterococcus hirae, and eukaryotic Cu(I) homeostasis proteins such as the Menkes and Wilson disease-associated proteins (1, 4, 5). ZntA has been shown to catalyze ATP-dependent transport of Zn(II) and Cd(II) (6), and CadA has been shown to transport Cd(II) (7). Both have been shown to confer resistance to cadmium and zinc ions (8 -10). The pI258 cadCA operon is regulated at the transcriptional level by the product of the cadC gene, which encodes the 122-residue CadC repressor (11-13).In this report, we show that CadC repression of the cad promoter is relieved upon addition of soft metals, with the order of effectiveness Pb(II) Ͼ Cd(II) Ͼ Zn(II). In E. coli Zn(II) responsiveness could be observed only in a zntA-disrupted strain. The zntA-disrupted strain of E. coli exhibited hypersensitivity to Pb(II) that was complemented by cadA, indicating that both soft metal-translocating P-type ATPases are essential for Pb(II) resistance in bacteria. Everted membrane vesicles from cells expressing either zntA or cadA exhibited ATPdependent 65 Zn(II) accumulation. Since no radioisotopes of ...

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“…There are two main genes involved in Cd resistance, namely, cadA and cadC [45]. CadA codes for a Cd efflux ATPase and cadC are said to be a transcriptional regulatory protein and a repressor of the cad operon [46]. In the presence of Cd ions, the repressor detaches itself from the DNA molecule, thus, allowing the transcription of the operon [47].…”

Section: Resultsmentioning

confidence: 99%

Bioremediation of heavy metals from aquatic environment through microbial processes: A potential role for probiotics?

2017

J App Biol Biotech

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Heavy metals can cause disastrous effects on any living organisms. Probiotics have the ability to reduce metal toxicity. Bioremediation of polluted waters using these bacteria could be an alternative to conventional remediation methods. The aim of this study was to isolate and characterize heavy metal resistant probiotics. Soil samples were collected for the isolation of probiotics. Morphological, biochemical, and molecular characterization was performed for the isolates. In addition, a preliminary heavy metal minimum inhibitory concentration test was done followed by atomic absorption spectrometry (AAS) analysis analysis. Four Enterococci (BT1, BT2, MC1, and MC2) and Bacillus acidiproducens (SM1 and SM2) were isolated. Moreover, all the isolates demonstrated probiotic characteristics. BT1 and BT2 were able to tolerate mercury (Hg), cadmium (Cd), lead (Pb), and chromium (Cr) but they demonstrated poor Hg removal abilities (0.75-1.42%). MC1 and MC2 isolates could grow in medium supplemented with Cd, Pb, and Cr, respectively. MC1 showed the highest level of Pb removal (43.00% ± 0.776%) and Cd removal (46.19% ± 7.651%) from broth media. Yet, SM1 and SM2 isolates tolerated only Pb and Cr. SM2 had the ability to remove the highest amount of Cr (43.06% ± 7.991%). These reasonable heavy metal removal abilities could be further studied for efficient use in bioremediation.

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Formation of a Phosphorylated Enzyme Intermediate by the cadA Cd2+-ATPase

Cited by 44 publications

References 0 publications

CadC, the transcriptional regulatory protein of the cadmium resistance system of Staphylococcus aureus plasmid pI258

CadC, the transcriptional regulatory protein of the cadmium resistance system of Staphylococcus aureus plasmid pI258

Pb(II)-translocating P-type ATPases

Bioremediation of heavy metals from aquatic environment through microbial processes: A potential role for probiotics?

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