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Nitric oxide releases intracellular zinc from prokaryotic metallothionein in Escherichia coli
FEMS Microbiology Letters 213 (2002) 121^126
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Nitric oxide releases intracellular zinc from prokaryotic metallothionein in Escherichia coli Marie R.B. Binet a , Hugo Cruz-Ramos a , Jay Laver a , Martin N. Hughes b , Robert K. Poole a; a
Department of Molecular Biology and Biotechnology, Firth Court, Western Bank, University of She⁄eld, She⁄eld S10 2TN, UK b Department of Chemistry, King’s College London, Strand, London WC2R 2LS, UK Received 20 March 2002; received in revised form 30 May 2002; accepted 30 May 2002 First published online 22 June 2002
Abstract Nitric oxide (NO) has a broad spectrum of signalling and regulatory functions and multiple molecular targets. Recently, the intrabacterial toxicity of NO and mechanisms for NO resistance have been intensively investigated. Here we report for the first time that NO elicits release of zinc from a bacterial protein. Using the zinc-responsive expression of zntA (encoding a Zn-exporting P-type ATPase) fused to lacZ, i.e. x(zntA-lacZ), to monitor intracellular zinc, and SmtA (the Synechococcus metallothionein) as zinc store, we have shown that the NO donors NOC-5 and NOC-7 elicit zinc ejection. No increase in x(zntA-lacZ) activity was observed in a zntR mutant, indicating the specificity of the zntA promoter response to zinc ions. 6 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Nitric oxide; Bacterial metallothionein ; Zinc homeostasis ; Zinc ATPase ; Escherichia coli
1. Introduction Zinc is an essential element in living organisms. It is a key structural component of a large number of proteins in all living systems and plays an important catalytic role in numerous enzymes [1,2]. Zinc also has a very important role in regulatory gene expression being involved in zinc ¢nger motifs of proteins that bind DNA or RNA. Several zinc-responsive transcription factors are known to mediate zinc homeostasis in vivo [3^7]. Thus, ZntR regulates the ZntA zinc export system in Escherichia coli by direct interaction between zinc and ZntR and the promoter region of zntA [8,9]. Similarly, Zur regulates the ZnuABC Zn import system in E. coli [10^12] and SmtB the metallothionein SmtA in Synechococcus spp. [13]. Nitric oxide (NO) has a broad spectrum of signalling functions in physiological and pathophysiological process-
es. These activities are due to the participation of NO in numerous chemical reactions, particularly (i) reaction with superoxide anion to form peroxynitrite, (ii) S-nitrosation in the presence of oxidising agents, and (iii) nitrosylation (reactions with transition metals). Indeed, NO has recently been shown to interact with metallothioneins and cause release of bound zinc or cadmium [14]. Because of the potential implications of this process in microbial zinc homeostasis and NO sensing, and for further understanding of NO action in bacteria, we were interested to determine whether NO could release zinc in E. coli and whether the prokaryotic metallothionein SmtA was sensitive to NO.
2. Materials and methods 2.1. Bacterial strains
* Corresponding author. Tel. : +44 (114) 222 4447 ; Fax : +44 (114) 272 8697. E-mail address : r.poole@she⁄eld.ac.uk (R.K. Poole). Abbreviations : LB, Luria broth; IPTG, isopropyl L-D-thiogalactopyranoside
The E. coli K12 strains used were RKP2910 carrying a single copy x(zntA-lacZ) fusion [15] and RKP2997 which carries the same fusion in a zntR background (zntR : :kan) [15]. Strain RKP3017 was constructed by transformation of RKP2910 with the isopropyl L-D-thiogalactopyranoside
0378-1097 / 02 / $22.00 6 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 8 0 3 - 0
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(IPTG)-inducible plasmid pET29a-SmtA, kindly provided by N. Robinson [13]. In the presence of IPTG and zinc ions, pET29a-SmtA over-expresses the Synechococcus metallothionein SmtA. Strain RKP3017 was checked by PCR and by endonuclease analysis as in Poole et al. [16]. Cells were grown in Luria broth (LB) medium. Kanamycin and IPTG were used at ¢nal concentrations of 50 Wg ml31 and 1 mM, respectively. 2.2. Culture conditions LB medium (200 ml) was inoculated with 2% of the culture volume with overnight pre-cultures and shaken at 200 rpm at 37‡C in 1-l £asks. After growth to OD600nm 0.5, IPTG and ZnSO4 (Sigma, ‘101% pure’) were added to give ¢nal concentrations of 1 mM and 0.5 mM, respectively, and cells were grown for a further 4 h. Cells treated with IPTG and zinc are referred to as ‘preinduced’ and have elevated SmtA levels ; cells not so treated are ‘noninduced’. Cells were harvested at 5000Ug for 10 min at 4‡C and washed three times in 10 mM HEPES bu¡er pH 7.5 containing 10 mM EDTA and then three times in the same bu¡er without EDTA. The cell pellet was ¢nally resuspended in a small volume of 10 mM HEPES, pH 7.5 and the thick suspension was used to inoculate 1 ml LB to obtain OD600nm 0.5. Cultures were challenged with zinc or a mixture of NOC-5 (half-time 25 min) and NOC-7 (half-time 5 min at 37‡C) and incubated at 37‡C with shaking. NOC-5 and NOC-7 (Calbiochem) stock solutions (60 mM) were prepared in 10 mM Tris, pH 10 and added to cultures to give the ¢nal concentrations shown. Aliquots were taken at 0.5, 1 or 2 h and cells were pelleted and frozen at 320‡C, prior to L-galactosidase assay. 2.3. L-Galactosidase assay Assays were carried out at room temperature as described before [16]. Cell pellets were suspended in 200 Wl of bu¡er and kept on ice. L-Galactosidase activity was measured in CHCl3 - and sodium dodecyl sulfate-permeabilised cells by monitoring the hydrolysis of o-nitrophenyl L-D-galactopyranoside. Activities are expressed in terms of the OD600 of cell suspensions using the formula of Miller [16]. Each culture was assayed in triplicate and results were con¢rmed in at least two independent experiments.
3. Results 3.1. Zinc-mediated induction of the zntA promoter To determine whether NO releases intracellular zinc from proteins in E. coli, we used zinc-responsive expression of the x(zntA-lacZ) fusion. In monocopy, this fusion carried in strain RKP2910 responds to the presence of zinc, cadmium and lead ions added to cultures [9,15].
Fig. 1. E¡ects of NOC-5 and NOC-7 (1^5 mM) and zinc ions (0.5 mM) in x(zntA-lacZ) activity in non-induced cells (i.e. cells not treated with IPTG and zinc). Shaded bars, strain RKP2910 (x(zntA-lacZ)); open bars, strain RKP3017 (same but with pET29a-SmtA). Standard deviations of the mean are shown, but in some cases are within the line widths.
We expected that metal ions released from the interaction of nitric oxide with Zn-containing proteins would activate zntA expression reported by L-galactosidase activity. We used in parallel strains RKP2910 (x(zntA-lacZ)) and RKP3017 which additionally carries plasmid pET29aSmtA. To investigate NO-mediated e¡ects, NO-releasing compounds provide, in a controllable and easy way, a source of NO throughout several hours of culture. Polyamine/NO complexes such as NOC-5 and NOC-7 spontaneously release NO under physiological conditions with de¢ned generation rates. Fig. 1 shows the response of x(zntA-lacZ) activity in non-induced cells to di¡erent concentrations of NO releaser. Under such conditions (without zinc or IPTG during the 1-h incubation), low levels of zntA expression were measured as described by Binet and Poole [15] even at up to 5 mM NOC. These results suggest that exogenously generated NO does not substantially increase the intracellular zinc pool that is detectable by the x(zntA-lacZ) fusion. However, 0.5 mM zinc added to media e¡ectively stimulated reporter activity in both strains. We attempted to increase intracellular Zn loading and thus susceptibility to NO by over-expressing SmtA, one molecule of which binds four Zn atoms, and addition of zinc to the growth medium. Cells of the x(zntA-lacZ) strain pre-grown in the presence of 0.5 mM zinc (and IPTG, although this was expected to be without e¡ect in this strain) exhibited x(zntA-lacZ) activity that was 6-fold higher when grown without zinc than in non-induced cells (compare ¢lled bars for 0 mM zinc in Figs. 1 and 2). Preinduction caused a greater increase for the strain harbouring pET29a-SmtA. Thus even after six washes of the cells following pre-induction (three with EDTA, three with water), remaining intracellular zinc was sensed by the x(zntA-lacZ) fusion. Subsequent growth of the preinduced cells with 0.5^1.5
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Fig. 2. E¡ects of di¡erent concentrations of zinc on x(zntA-lacZ) activity in preinduced cells (i.e. previously grown with IPTG and zinc to elevate SmtA levels). After induction, cells were washed repeatedly to remove excess zinc. Shaded bars, strain RKP2910 (x(zntA-lacZ)); open bars, strain RKP3017 (same but with pET29a-SmtA). Standard deviations of the mean are shown.
mM zinc further increased x(zntA-lacZ) activity. Highest activity was observed with the strain over-expressing SmtA challenged with 1 mM zinc (Fig. 2) in which L-galactosidase activity was 30-fold higher than the basal level shown in Fig. 1. At the highest concentration of zinc (1.5 mM) added to preinduced cells, L-galactosidase activities were reduced particularly in the strain over-expressing SmtA (Fig. 2); this presumably re£ects zinc toxicity. These experiments show that cells expressing high levels of x(zntA-lacZ) activity following pre-induction remain responsive to further challenges with extracellular zinc and are therefore useful for determining whether NO elicits intracellular zinc release. 3.2. NO-mediated induction of zntA promoter Fig. 3 shows the e¡ect of increasing concentrations of NOC-5 and NOC-7 on x(zntA-lacZ) in strains RKP2910 and RKP3017. Cells were preinduced with zinc and IPTG, then washed six times as before to remove external zinc, and incubated for 1 h with NOC-5 and NOC-7. NO is released when the alkaline stock solutions of the NOC compounds are diluted into the culture at a pH near 7. After treatments, cells were harvested and L-galactosidase activity was determined. A NOC mixture at a ¢nal concentration of 1 mM was
su⁄cient to increase measurably expression of the fusion above the control. x(zntA-lacZ) activity increased as the NOC concentration increased, reaching a maximum at 5 mM NOC, then declined at higher concentrations, presumably due to toxicity from the NO added to the cultures. The toxicity is not due to the zinc that NO releases since L-galactosidase activity declines irrespective of whether cells contain SmtA. Strain RKP2910 lacking pET29a-SmtA was much less responsive to NOC addition than was strain RKP3017 over-expressing the metallothionein, suggesting that most of the zinc derives from SmtA. The L-galactosidase activity in the over-expressing strain at 5 mM NOC (Fig. 3) is close to that observed when cells are treated instead with 1 mM zinc (Fig. 2). To investigate whether the increases in L-galactosidase activities report sensing of intracellular zinc by ZntR, the metal-responsive regulator of zntA, experiments were performed on a zntR mutant (RKP2997) carrying x(zntAlacZ). No induction of the fusion could be obtained even at the optimal NOC concentration of 5 mM (not shown). This con¢rms the speci¢city of the response of the zntA promoter to NO-mediated zinc release, rather than response to NO per se. The experiments shown in Fig. 3 were also carried out after incubation for 0.5 and 2 h (not shown). After 30 min incubation, the response to NOC was low compared to the results shown in Fig. 3; at 2 h, the results were similar to
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Fig. 3. E¡ects of di¡erent concentrations of the NO releaser mixture (NOC-5 and -7) on x(zntA-lacZ) activity in preinduced cells (i.e. previously grown with IPTG and zinc to elevate SmtA levels). Shaded bars, strain RKP2910 (x(zntA-lacZ)); open bars, strain RKP3017 (same but with pET29a-SmtA). Standard deviations of the mean are shown.
those at 1 h but with a decrease in activities probably due to the degradation of L-galactosidase.
4. Discussion NO elicits zinc release from transcription factors possessing zinc ¢ngers [17,18] in di¡erent eukaryotic cell lines [19] and in eukaryotic metallothioneins by destroying zinc^sulfur clusters [14,20,21]. These observations have signi¢cant implications for gene expression patterns in response to NO and for the possible role of metallothioneins in NO signalling [14,19]. The present results show for the ¢rst time the release of zinc in vivo mediated by nitric oxide in E. coli. We measured the intracellular release of zinc during NO treatment by following the expression of the E. coli zinc-responsive promoter of zntA. A monocopy x(zntA-lacZ) fusion was su⁄ciently sensitive for detection of intracellular zinc and L-galactosidase activity was easily accessible to measurement during growth. To enhance the zinc release phenomenon, the metallothionein SmtA from Synechococcus [13] was employed. The ¢nding that both strains, either lacking or over-expressing SmtA, respond similarly to extracellular zinc (Fig. 2) but not to NOC compounds strongly suggests that SmtA is the main
source of released zinc. That the response is due to released metal ion is shown by use of the zntR mutant, but since this regulator senses not only zinc but also cadmium and lead [15], we cannot rule out the possibility that some of the x(zntA-lacZ) up-regulation is due to cadmium or lead. However, only zinc was used in the pre-induction protocol found to increase x(zntA-lacZ) activity (Fig. 2) and the metal content of SmtA re£ects primarily which metal ions are added in excess during culture [13]. NO plays an important regulatory role in cellular gene expression [22^24] and inhibits DNA-binding activities of transcription factors containing one or several cysteines within or near their DNA-binding domains [25]. This could explain the decrease of L-galactosidase activities at very high NOC concentrations (7 and 10 mM; Fig. 3) as ZntR, the transcription regulator of zntA, binds zinc via cysteines (and probably histidines as well) which could be nitrosated by NO. Outten and O’Halloran [26] have demonstrated that in E. coli the free zinc concentration is around femtomolar, i.e. there is essentially no free zinc in normal cells. The intracellular £uxes of zinc between metalloenzymes and metal sensor, storage and transport proteins are likely to involve direct transfer of the metal ion between proteins. The present ¢nding that NO ejects zinc from SmtA, de-
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tectable as the activation of transcription of zntA by ZntR, suggests that any hypothetical ‘chaperone’, or low a⁄nity labile zinc site, which transfers the released zinc to ZntR has not been damaged by NO. The NO-sensitive metal binding of SmtA may suggest an interplay between the functions of such prokaryotic metallothioneins with the regulatory and cytotoxic e¡ects of both zinc and NO in prokaryotes. For example, bacterial metallothioneins might confer protection not only from zinc [13] but also from NO by reacting with it, thus reducing cytotoxicity, as proposed for animal metallothionein [27]. SmtA deletion mutants appear not to have been tested for sensitivity to NO or its congeners. SmtA might also function as a transducer between NO and zinc metabolism. The recently solved solution structure of SmtA [28] reveals a Zn4 Cys9 His2 cluster; the two ZnCys3 His sites and one of the ZnCys4 sites readily exchange with added Cd, whereas the other is inert. The topology of this inert site resembles that of zinc ¢nger portions of the GATA eukaryotic transcription proteins. Thus, the susceptibility to NO of di¡erent metal atoms within SmtA should be explored; in mouse metallothionein [29], metals are released selectively from the aminoterminal domain. Bacteria are known to be exposed to NO in diverse environments, particularly in soils and aqueous environments where denitri¢cation occurs. Furthermore, NO is a central component of innate immunity and an e¡ective antimicrobial agent [30]. Reactive nitrogen intermediates e¡ectively kill pathogenic bacteria and E. coli [31], although resistance mechanisms, exempli¢ed by the £avohaemoglobins [32] provide e¡ective enzymic removal of NO. The present work reveals that a previously unrecognised e¡ect of NO on bacteria is liberation of intracellular zinc, which might augment the direct e¡ects of NO on cell function that occur via, for example, nitrosation and nitrosylation.
Acknowledgements This work was supported by BBSRC grant P10354 and by a Nu⁄eld Foundation Undergraduate Bursary held by J.L. We thank an anonymous referee for useful comments on SmtA structure and function.
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Nitric oxide releases intracellular zinc from prokaryotic metallothionein in Escherichia coli Marie R.B. Binet a , Hugo Cruz-Ramos a , Jay Laver a , Martin N. Hughes b , Robert K. Poole a; a
Department of Molecular Biology and Biotechnology, Firth Court, Western Bank, University of She⁄eld, She⁄eld S10 2TN, UK b Department of Chemistry, King’s College London, Strand, London WC2R 2LS, UK Received 20 March 2002; received in revised form 30 May 2002; accepted 30 May 2002 First published online 22 June 2002
Abstract Nitric oxide (NO) has a broad spectrum of signalling and regulatory functions and multiple molecular targets. Recently, the intrabacterial toxicity of NO and mechanisms for NO resistance have been intensively investigated. Here we report for the first time that NO elicits release of zinc from a bacterial protein. Using the zinc-responsive expression of zntA (encoding a Zn-exporting P-type ATPase) fused to lacZ, i.e. x(zntA-lacZ), to monitor intracellular zinc, and SmtA (the Synechococcus metallothionein) as zinc store, we have shown that the NO donors NOC-5 and NOC-7 elicit zinc ejection. No increase in x(zntA-lacZ) activity was observed in a zntR mutant, indicating the specificity of the zntA promoter response to zinc ions. 6 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Nitric oxide; Bacterial metallothionein ; Zinc homeostasis ; Zinc ATPase ; Escherichia coli
1. Introduction Zinc is an essential element in living organisms. It is a key structural component of a large number of proteins in all living systems and plays an important catalytic role in numerous enzymes [1,2]. Zinc also has a very important role in regulatory gene expression being involved in zinc ¢nger motifs of proteins that bind DNA or RNA. Several zinc-responsive transcription factors are known to mediate zinc homeostasis in vivo [3^7]. Thus, ZntR regulates the ZntA zinc export system in Escherichia coli by direct interaction between zinc and ZntR and the promoter region of zntA [8,9]. Similarly, Zur regulates the ZnuABC Zn import system in E. coli [10^12] and SmtB the metallothionein SmtA in Synechococcus spp. [13]. Nitric oxide (NO) has a broad spectrum of signalling functions in physiological and pathophysiological process-
es. These activities are due to the participation of NO in numerous chemical reactions, particularly (i) reaction with superoxide anion to form peroxynitrite, (ii) S-nitrosation in the presence of oxidising agents, and (iii) nitrosylation (reactions with transition metals). Indeed, NO has recently been shown to interact with metallothioneins and cause release of bound zinc or cadmium [14]. Because of the potential implications of this process in microbial zinc homeostasis and NO sensing, and for further understanding of NO action in bacteria, we were interested to determine whether NO could release zinc in E. coli and whether the prokaryotic metallothionein SmtA was sensitive to NO.
2. Materials and methods 2.1. Bacterial strains
* Corresponding author. Tel. : +44 (114) 222 4447 ; Fax : +44 (114) 272 8697. E-mail address : r.poole@she⁄eld.ac.uk (R.K. Poole). Abbreviations : LB, Luria broth; IPTG, isopropyl L-D-thiogalactopyranoside
The E. coli K12 strains used were RKP2910 carrying a single copy x(zntA-lacZ) fusion [15] and RKP2997 which carries the same fusion in a zntR background (zntR : :kan) [15]. Strain RKP3017 was constructed by transformation of RKP2910 with the isopropyl L-D-thiogalactopyranoside
0378-1097 / 02 / $22.00 6 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 8 0 3 - 0
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(IPTG)-inducible plasmid pET29a-SmtA, kindly provided by N. Robinson [13]. In the presence of IPTG and zinc ions, pET29a-SmtA over-expresses the Synechococcus metallothionein SmtA. Strain RKP3017 was checked by PCR and by endonuclease analysis as in Poole et al. [16]. Cells were grown in Luria broth (LB) medium. Kanamycin and IPTG were used at ¢nal concentrations of 50 Wg ml31 and 1 mM, respectively. 2.2. Culture conditions LB medium (200 ml) was inoculated with 2% of the culture volume with overnight pre-cultures and shaken at 200 rpm at 37‡C in 1-l £asks. After growth to OD600nm 0.5, IPTG and ZnSO4 (Sigma, ‘101% pure’) were added to give ¢nal concentrations of 1 mM and 0.5 mM, respectively, and cells were grown for a further 4 h. Cells treated with IPTG and zinc are referred to as ‘preinduced’ and have elevated SmtA levels ; cells not so treated are ‘noninduced’. Cells were harvested at 5000Ug for 10 min at 4‡C and washed three times in 10 mM HEPES bu¡er pH 7.5 containing 10 mM EDTA and then three times in the same bu¡er without EDTA. The cell pellet was ¢nally resuspended in a small volume of 10 mM HEPES, pH 7.5 and the thick suspension was used to inoculate 1 ml LB to obtain OD600nm 0.5. Cultures were challenged with zinc or a mixture of NOC-5 (half-time 25 min) and NOC-7 (half-time 5 min at 37‡C) and incubated at 37‡C with shaking. NOC-5 and NOC-7 (Calbiochem) stock solutions (60 mM) were prepared in 10 mM Tris, pH 10 and added to cultures to give the ¢nal concentrations shown. Aliquots were taken at 0.5, 1 or 2 h and cells were pelleted and frozen at 320‡C, prior to L-galactosidase assay. 2.3. L-Galactosidase assay Assays were carried out at room temperature as described before [16]. Cell pellets were suspended in 200 Wl of bu¡er and kept on ice. L-Galactosidase activity was measured in CHCl3 - and sodium dodecyl sulfate-permeabilised cells by monitoring the hydrolysis of o-nitrophenyl L-D-galactopyranoside. Activities are expressed in terms of the OD600 of cell suspensions using the formula of Miller [16]. Each culture was assayed in triplicate and results were con¢rmed in at least two independent experiments.
3. Results 3.1. Zinc-mediated induction of the zntA promoter To determine whether NO releases intracellular zinc from proteins in E. coli, we used zinc-responsive expression of the x(zntA-lacZ) fusion. In monocopy, this fusion carried in strain RKP2910 responds to the presence of zinc, cadmium and lead ions added to cultures [9,15].
Fig. 1. E¡ects of NOC-5 and NOC-7 (1^5 mM) and zinc ions (0.5 mM) in x(zntA-lacZ) activity in non-induced cells (i.e. cells not treated with IPTG and zinc). Shaded bars, strain RKP2910 (x(zntA-lacZ)); open bars, strain RKP3017 (same but with pET29a-SmtA). Standard deviations of the mean are shown, but in some cases are within the line widths.
We expected that metal ions released from the interaction of nitric oxide with Zn-containing proteins would activate zntA expression reported by L-galactosidase activity. We used in parallel strains RKP2910 (x(zntA-lacZ)) and RKP3017 which additionally carries plasmid pET29aSmtA. To investigate NO-mediated e¡ects, NO-releasing compounds provide, in a controllable and easy way, a source of NO throughout several hours of culture. Polyamine/NO complexes such as NOC-5 and NOC-7 spontaneously release NO under physiological conditions with de¢ned generation rates. Fig. 1 shows the response of x(zntA-lacZ) activity in non-induced cells to di¡erent concentrations of NO releaser. Under such conditions (without zinc or IPTG during the 1-h incubation), low levels of zntA expression were measured as described by Binet and Poole [15] even at up to 5 mM NOC. These results suggest that exogenously generated NO does not substantially increase the intracellular zinc pool that is detectable by the x(zntA-lacZ) fusion. However, 0.5 mM zinc added to media e¡ectively stimulated reporter activity in both strains. We attempted to increase intracellular Zn loading and thus susceptibility to NO by over-expressing SmtA, one molecule of which binds four Zn atoms, and addition of zinc to the growth medium. Cells of the x(zntA-lacZ) strain pre-grown in the presence of 0.5 mM zinc (and IPTG, although this was expected to be without e¡ect in this strain) exhibited x(zntA-lacZ) activity that was 6-fold higher when grown without zinc than in non-induced cells (compare ¢lled bars for 0 mM zinc in Figs. 1 and 2). Preinduction caused a greater increase for the strain harbouring pET29a-SmtA. Thus even after six washes of the cells following pre-induction (three with EDTA, three with water), remaining intracellular zinc was sensed by the x(zntA-lacZ) fusion. Subsequent growth of the preinduced cells with 0.5^1.5
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Fig. 2. E¡ects of di¡erent concentrations of zinc on x(zntA-lacZ) activity in preinduced cells (i.e. previously grown with IPTG and zinc to elevate SmtA levels). After induction, cells were washed repeatedly to remove excess zinc. Shaded bars, strain RKP2910 (x(zntA-lacZ)); open bars, strain RKP3017 (same but with pET29a-SmtA). Standard deviations of the mean are shown.
mM zinc further increased x(zntA-lacZ) activity. Highest activity was observed with the strain over-expressing SmtA challenged with 1 mM zinc (Fig. 2) in which L-galactosidase activity was 30-fold higher than the basal level shown in Fig. 1. At the highest concentration of zinc (1.5 mM) added to preinduced cells, L-galactosidase activities were reduced particularly in the strain over-expressing SmtA (Fig. 2); this presumably re£ects zinc toxicity. These experiments show that cells expressing high levels of x(zntA-lacZ) activity following pre-induction remain responsive to further challenges with extracellular zinc and are therefore useful for determining whether NO elicits intracellular zinc release. 3.2. NO-mediated induction of zntA promoter Fig. 3 shows the e¡ect of increasing concentrations of NOC-5 and NOC-7 on x(zntA-lacZ) in strains RKP2910 and RKP3017. Cells were preinduced with zinc and IPTG, then washed six times as before to remove external zinc, and incubated for 1 h with NOC-5 and NOC-7. NO is released when the alkaline stock solutions of the NOC compounds are diluted into the culture at a pH near 7. After treatments, cells were harvested and L-galactosidase activity was determined. A NOC mixture at a ¢nal concentration of 1 mM was
su⁄cient to increase measurably expression of the fusion above the control. x(zntA-lacZ) activity increased as the NOC concentration increased, reaching a maximum at 5 mM NOC, then declined at higher concentrations, presumably due to toxicity from the NO added to the cultures. The toxicity is not due to the zinc that NO releases since L-galactosidase activity declines irrespective of whether cells contain SmtA. Strain RKP2910 lacking pET29a-SmtA was much less responsive to NOC addition than was strain RKP3017 over-expressing the metallothionein, suggesting that most of the zinc derives from SmtA. The L-galactosidase activity in the over-expressing strain at 5 mM NOC (Fig. 3) is close to that observed when cells are treated instead with 1 mM zinc (Fig. 2). To investigate whether the increases in L-galactosidase activities report sensing of intracellular zinc by ZntR, the metal-responsive regulator of zntA, experiments were performed on a zntR mutant (RKP2997) carrying x(zntAlacZ). No induction of the fusion could be obtained even at the optimal NOC concentration of 5 mM (not shown). This con¢rms the speci¢city of the response of the zntA promoter to NO-mediated zinc release, rather than response to NO per se. The experiments shown in Fig. 3 were also carried out after incubation for 0.5 and 2 h (not shown). After 30 min incubation, the response to NOC was low compared to the results shown in Fig. 3; at 2 h, the results were similar to
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Fig. 3. E¡ects of di¡erent concentrations of the NO releaser mixture (NOC-5 and -7) on x(zntA-lacZ) activity in preinduced cells (i.e. previously grown with IPTG and zinc to elevate SmtA levels). Shaded bars, strain RKP2910 (x(zntA-lacZ)); open bars, strain RKP3017 (same but with pET29a-SmtA). Standard deviations of the mean are shown.
those at 1 h but with a decrease in activities probably due to the degradation of L-galactosidase.
4. Discussion NO elicits zinc release from transcription factors possessing zinc ¢ngers [17,18] in di¡erent eukaryotic cell lines [19] and in eukaryotic metallothioneins by destroying zinc^sulfur clusters [14,20,21]. These observations have signi¢cant implications for gene expression patterns in response to NO and for the possible role of metallothioneins in NO signalling [14,19]. The present results show for the ¢rst time the release of zinc in vivo mediated by nitric oxide in E. coli. We measured the intracellular release of zinc during NO treatment by following the expression of the E. coli zinc-responsive promoter of zntA. A monocopy x(zntA-lacZ) fusion was su⁄ciently sensitive for detection of intracellular zinc and L-galactosidase activity was easily accessible to measurement during growth. To enhance the zinc release phenomenon, the metallothionein SmtA from Synechococcus [13] was employed. The ¢nding that both strains, either lacking or over-expressing SmtA, respond similarly to extracellular zinc (Fig. 2) but not to NOC compounds strongly suggests that SmtA is the main
source of released zinc. That the response is due to released metal ion is shown by use of the zntR mutant, but since this regulator senses not only zinc but also cadmium and lead [15], we cannot rule out the possibility that some of the x(zntA-lacZ) up-regulation is due to cadmium or lead. However, only zinc was used in the pre-induction protocol found to increase x(zntA-lacZ) activity (Fig. 2) and the metal content of SmtA re£ects primarily which metal ions are added in excess during culture [13]. NO plays an important regulatory role in cellular gene expression [22^24] and inhibits DNA-binding activities of transcription factors containing one or several cysteines within or near their DNA-binding domains [25]. This could explain the decrease of L-galactosidase activities at very high NOC concentrations (7 and 10 mM; Fig. 3) as ZntR, the transcription regulator of zntA, binds zinc via cysteines (and probably histidines as well) which could be nitrosated by NO. Outten and O’Halloran [26] have demonstrated that in E. coli the free zinc concentration is around femtomolar, i.e. there is essentially no free zinc in normal cells. The intracellular £uxes of zinc between metalloenzymes and metal sensor, storage and transport proteins are likely to involve direct transfer of the metal ion between proteins. The present ¢nding that NO ejects zinc from SmtA, de-
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tectable as the activation of transcription of zntA by ZntR, suggests that any hypothetical ‘chaperone’, or low a⁄nity labile zinc site, which transfers the released zinc to ZntR has not been damaged by NO. The NO-sensitive metal binding of SmtA may suggest an interplay between the functions of such prokaryotic metallothioneins with the regulatory and cytotoxic e¡ects of both zinc and NO in prokaryotes. For example, bacterial metallothioneins might confer protection not only from zinc [13] but also from NO by reacting with it, thus reducing cytotoxicity, as proposed for animal metallothionein [27]. SmtA deletion mutants appear not to have been tested for sensitivity to NO or its congeners. SmtA might also function as a transducer between NO and zinc metabolism. The recently solved solution structure of SmtA [28] reveals a Zn4 Cys9 His2 cluster; the two ZnCys3 His sites and one of the ZnCys4 sites readily exchange with added Cd, whereas the other is inert. The topology of this inert site resembles that of zinc ¢nger portions of the GATA eukaryotic transcription proteins. Thus, the susceptibility to NO of di¡erent metal atoms within SmtA should be explored; in mouse metallothionein [29], metals are released selectively from the aminoterminal domain. Bacteria are known to be exposed to NO in diverse environments, particularly in soils and aqueous environments where denitri¢cation occurs. Furthermore, NO is a central component of innate immunity and an e¡ective antimicrobial agent [30]. Reactive nitrogen intermediates e¡ectively kill pathogenic bacteria and E. coli [31], although resistance mechanisms, exempli¢ed by the £avohaemoglobins [32] provide e¡ective enzymic removal of NO. The present work reveals that a previously unrecognised e¡ect of NO on bacteria is liberation of intracellular zinc, which might augment the direct e¡ects of NO on cell function that occur via, for example, nitrosation and nitrosylation.
Acknowledgements This work was supported by BBSRC grant P10354 and by a Nu⁄eld Foundation Undergraduate Bursary held by J.L. We thank an anonymous referee for useful comments on SmtA structure and function.
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