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In vivo detection of molybdate-binding proteins using a competition assay with ModE in Escherichia coli
FEMS Microbiology Letters 218 (2003) 187^193
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In vivo detection of molybdate-binding proteins using a competition assay with ModE in Escherichia coli Jochen Kuper a;1 , Sonja Meyer zu Berstenhorst a;b;1 , Bernd Vo«disch b , Ralf R. Mendel a , Gu«nter Schwarz a; , David H. Boxer b b
a Department of Plant Biology, Technical University of Braunschweig, 38023 Braunschweig, Germany Division of Biological Chemistry and Molecular Microbiology, School of Life Science, University of Dundee, Dundee DD1 5EH, UK
Received 29 October 2002 ; received in revised form 6 November 2002; accepted 6 November 2002 First published online 29 November 2002
Abstract Molybdenum is an important trace element as it forms the essential part of the active site in all molybdenum-containing enzymes. We have designed an assay for the in vivo detection of molybdate binding to proteins in Escherichia coli. The assay is based on (i) the molybdate-dependent transcriptional regulation of the moa operon by the ModE protein, and (ii) the competition for molybdate between ModE and other molybdate-binding proteins in the cytoplasm of E. coli. We were able to verify in vivo molybdate binding to three different bacterial proteins that are known to bind molybdate. This sensitive in vivo system allows the testing of different proteins for molybdate binding under in vivo conditions and will facilitate the identification of other cellular factors needed for molybdate binding. As a first example, we examined the eukaryotic protein Cnx1 that is involved in the last step of molybdenum cofactor biosynthesis in plants, and show that it is able to compete with ModE for molybdate in a molybdopterin-dependent fashion. 9 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : ModE; Molybdenum; Molybdate binding ; Cnx1; Miller assay
1. Introduction Molybdenum is an essential trace element required for the activity of all molybdenum enzymes catalysing important redox reactions in the global C, N and S cycles [1]. In all these enzymes, except nitrogenase, molybdenum is activated and co-ordinated by the dithiolene moiety of a unique and conserved pterin, molybdopterin, thus forming the molybdenum cofactor [2]. Although the coordination state of molybdenum can vary [3] and di¡erent nucleotides can be attached to molybdopterin [1], the basic structure of the molybdenum cofactor consisting of molybdenum and molybdopterin is conserved throughout bacteria and eukaryotes. In Escherichia coli the mo loci are responsible for molybdenum transport and the synthesis of the molybdenum
* Corresponding author. Tel. : +49 (531) 391 5891 ; Fax : +49 (531) 391 8208. E-mail address : [email protected] (G. Schwarz). 1
Both authors contributed equally to this work.
cofactor [4]. The entire moa locus of E. coli is required for molybdopterin biosynthesis [5], while the modABCD operon encodes the high-a⁄nity ABC-type molybdate uptake system [6]. Strains defective in this locus are pleiotropically unable to synthesise active molybdenum enzymes. The supplementation of the growth medium with high levels of molybdate, however, fully restores all molybdenum enzyme activities in mod strains, indicating that molybdate can enter the bacterium by another route under such conditions [7]. Molybdate, as one major substrate for the biosynthesis of the molybdenum cofactor, regulates the expression of several operons in E. coli. ModE appears to be the main molybdate-dependent transcriptional regulator in the bacterium acting as a negative e¡ector of the operon modABCD [8] encoding the high-a⁄nity molybdate uptake system, and as a positive e¡ector for the transcription of moaABCDE [9] encoding enzymes responsible for the biosynthesis of molybdopterin. In vitro studies have demonstrated that the dimeric ModE binds two molybdate ions with a KD of 0.8 WM and that this molybdate^ModE complex binds to the promoter region of both mod and
0378-1097 / 02 / $22.00 9 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 1 1 2 1 - 7
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moa DNA [9,10]. The crystal structure of ModE has been determined and shows distinct DNA and molybdate-binding domains [11]. In strains lacking the ability both to synthesise functional molybdenum cofactor (moa) and to take up molybdate under normal growth conditions (mod), molybdate added to the growth medium exerts a powerful positive e¡ect on moa transcription that is absolutely dependent on ModE function [12]. Based on this ¢nding we have constructed moa L-galactosidase reporter strains lacking a functional high-a⁄nity uptake system (modC3 ). The lacZ gene under control of a truncated moa promoter was inserted either directly into the moa operon, resulting in the disruption of molybdopterin biosynthesis, or by lambda phage insertion without a¡ecting cofactor biosynthesis. In both strains, we titrated the molybdate-dependent activation of the moa operon by following L-galactosidase activity. Using these strains, we could show that the expression of di¡erent molybdate-binding proteins competes with ModE for molybdate binding, resulting in the reduction of moa expression. With this system we established the ¢rst in vivo molybdate-binding assay that can be used for testing di¡erent proteins for their ability to bind molybdate under in vivo conditions, thus allowing the identi¢cation of additional cellular factors required for molybdate binding. As a ¢rst example we demonstrated for the plant molybdenum cofactor biosynthetic protein Cnx1 molybdate binding and/or processing as Cnx1 is able to compete with ModE for molybdate in a molybdopterin-dependent fashion. The data presented here are a ¢rst step to establish a screening system for molybdate-binding proteins from eukaryotes, where almost nothing is known yet about the uptake, binding and processing of molybdate prior to its incorporation into the molybdenum cofactor.
2. Materials and methods 2.1. Bacterial strains, bacteriophages, plasmids and growing conditions All strains, bacteriophages and plasmids used in this work are listed in Table 1. With the exception of pQEcnx1 all other expression plasmids were generated using PCR cloning and the resulting constructs were sequenced after successful cloning. The C-terminal dimop domain of ModE (422 bp) was derived from pHW121 [10] and PstI/HindIII subcloned. ModA was ampli¢ed from pSJE350 [13], introducing BamHI/HindIII sites for subcloning. For modA (717 bp), the ¢rst 64 bp encoding the periplasmic target sequence were removed. MopII (207 bp) from pET15bMopII [14] was cloned via BamHI/HindIII sites. All PCR fragments were introduced into pQE80 (Qiagen, Hilden, Germany) using the speci¢ed restriction enzymes, resulting in the pQE constructs given in Table 1. Restriction enzymes used for cloning were obtained from Promega (Mannheim, Germany). Pwo polymerase for PCR was purchased from Peqlab (Erlangen, Germany). Oligonucleotides were obtained from Sigma (Taufkirchen, Germany). Cnx1 was cloned from pQE60cnx1 [15] by cloning the whole pQE60 expression cassette into pQE80 via EcoRI/HindIII. Bacteria were grown aerobically in Luria^Bertani medium at 37‡C with the addition of 50 Wg ml31 kanamycin and 125 Wg ml31 ampicillin where appropriate. For the modi¢ed Miller assay (see below) cells were grown in LeMaster minimal medium [16] and isopropyl-LD-thiogalactopyranoside (IPTG) was added for the induction of protein expression. Aerobic growth conditions were obtained by vigorous shaking on an orbital shaker (240^250 rpm).
Table 1 Strains, bacteriophages and plasmids Name
Genotype/insert
Source
DB1004 LA27 SE1597 SB117 VRS45 pQE80 pSJE350 pHW121 pEM101 pRS551 pET15bMopII pQE60cnx1 pQEdimop pQEmodA pQEcpmopII pQEcnx1
F araD139 v(argF-lac)U169 deoC1 £bB5201 gyrA 219 relA1 rpsL150 non-9 ptsF25 ; [P(moaB: :lacZ)4] As DB1004; modC118 zbh-623 : :Tn10 D(lacU)169 rpsL modC120 SE1597[P(moa: :lacZ)] del-lacZ blaP attP imm21 cloning vector source for E. coli modA source for E. coli dimop (dimop domain of ModE) source for E. coli moa promoter V cloning vector source for Clostridium pasteurianum mopII source for Arabidopsis thaliana cnx1 E. coli dimop in pQE80 E. coli modA (without N-terminal leader sequence) in pQE80 Clostridium pasteurianum mopII in pQE80 Arabidopsis thaliana cnx1 in pQE80
[28] [12] [29] This work [17] Qiagen [13] [10] [10] [17] [14] [15] This work This work This work This work
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2.2. Strain construction SB117 was constructed by V phage insertion as described [17] using pRS551 as homologous recombination vector for phage VRS45 and SE1597 as hosting strain [12]. The moa promoter fragment 117K (3121^+131) was derived from pEM101 [12] and subcloned into the EcoRI/ BamHI sites of pRS551 [17] using PCR. Fragment 117K uses the same start site as the previously described 117 fragment [12] but it ends immediately before the moaA start at position +131. After recombination, the presence of the prophage was con¢rmed via PCR using primers that bind between the start of 117K and the region directly in front of the lacZ gene from pRS551. 2.3. L-Galactosidase assay L-Galactosidase assays were performed with a microtitre plate system (Versa max, Molecular Devices, Sunnyvale, CA, USA) based on a protocol of Miller et al. [18]. Cells were grown overnight and 50 Wl of this culture was transferred to 10 ml fresh medium supplemented with the appropriate amounts of IPTG, molybdate and antibiotics and continued to grow for an additional 4^5 h. Cells were harvested (4‡C, 4300Ug, 15 min), resuspended in 700 Wl bu¡er Z ([18], 5 mM GSH instead of L-mercaptoethanol) and sonicated on ice two times each 10 s. Cell debris was removed by centrifugation (4‡C, 20 000Ug, 5 min). Protein content was determined using the Bradford protein assay [19]. Samples were diluted to 0.1 Wg Wl31 in bu¡er Z and 2^10 Wg total protein was applied to a 96-well microtitre plate (Sarstedt, Nu«mbrecht, Germany) in a ¢nal volume of 100 Wl. Reaction was started by addition of 50 Wl ortho-nitrophenol-L-D -galactoside (4 mg ml31 ) in bu¡er Z. The formation of o-nitrophenol was kinetically monitored, measuring the absorbance at 410 nm for 1 h, recording data points every 5 min. Values in the linear range of the reaction were used to calculate Vmax (vOD410 min31 ). L-Galactosidase activity is ¢nally given in Wmol o-nitrophenol Wg protein31 min31 .
3. Results 3.1. The principle of ModE competition for detecting of molybdate-binding proteins in E. coli We have previously shown that expression of moa genes, as monitored by L-galactosidase activity in a strain unable to acquire physiological amounts of molybdate from its growth medium (modC3 ), is restored following the addition of high amounts of molybdate (up to 10 mM) to the growth medium [12]. Under these conditions, internal molybdate levels are restored via molybdate uptake through less speci¢c anion uptake systems [4]. Careful titration of moa expression with di¡erent concentrations of
Fig. 1. Expression of a molybdate-binding protein can in£uence the molybdate-dependent expression of the E. coli moa operon. The moa promoter regulates expression of the moa operon that encodes proteins essential for the synthesis of molybdopterin in E. coli. ModE is a dimeric transcriptional regulator that binds molybdate anions on its dimop domain. This ModE^MoO24 complex activates the transcription of the moa operon. E. coli strain LA27 carries a lacZ reporter gene insertion in the moa operon in order to monitor moa-promoter activity. Heterologous expression of a molybdate-binding protein leads to internal competition for molybdate between ModE and the protein of interest, resulting in a reduction of moa expression as monitored by L-galactosidase activity.
molybdate in the growth media should result in internal conditions just su⁄cient to allow moa activation. Under these conditions, the expression of a molybdate-binding protein could alter the amount of the internal free molybdate by sequestering it to the newly expressed protein. Due to the competition for molybdate by the second molybdate-binding protein, less ModE^molybdate complex would be available and therefore moa expression would be reduced (Fig. 1). The magnitude of change in moa expression will be dependent on the ratio between the molybdate-binding capacity of the newly expressed protein and the molybdate-bu¡ering capacity of the interior of the bacterium. In this work, two di¡erent strains were used to accomplish the in vivo molybdate-binding detection. The modC3 mutant strain LA27 carries the reporter gene lacZ as chromosomal insertion in the moa operon and is therefore molybdenum cofactor-de¢cient because of the inactivation of the moa operon [12]. As a second approach, the moa promoter fragment 117K fused to the lacZ gene was inserted into the genome of the modC3 mutant strain SE1597 by lambda phage insertion. This resulted in strain SB117 (SE1597[P(moa: : lacZ)]) with the same features as LA27 except that it is still able to synthesise active molybdenum cofactor after addition of molybdate to the growth media [20,21]. 3.2. Sensitivity of moa expression to external molybdate in a modC3 strain L-Galactosidase activity of LA27 in the absence of ex-
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Fig. 2. Molybdate-dependent regulation of moa expression in the modCde¢cient strain LA27 and the parental strain DB1004. Shown are the average L-galactosidase activities in units (Vmax Wg protein31 ) of the parental strain DB1004 (black dots) and the reporter strain LA27 (grey rectangles) in response to increasing amounts of molybdate in the culture medium. Cultures were grown for 2.5 h, whereas for all other experiments (Figs. 3 and 4) cultures were grown for 4 h. Activities represent average values obtained from at least three di¡erent cultures with three di¡erent protein concentrations, performing kinetic assays on each culture, using only the linear range of the reaction for calculating the enzyme activity (Vmax ).
ing protein of the E. coli ABC-type molybdate transport system [24]. The cDNAs of the dimop domain, of MopII and of ModA cloned into pQE80 (Table 1) were transformed into LA27 and L-galactosidase activity was monitored at di¡erent molybdate concentrations in the medium (Fig. 3A) and protein expression levels (Fig. 3B). Expression of each protein (induced with 50 WM IPTG) completely abolished the molybdate-dependent restoration of moa expression, whereas the control strain (LA27/pQE80) already showed 60% restoration of moa expression with only 50 WM molybdate (Fig. 3A). These results con¢rm the molybdate binding of all three proteins under in vivo conditions and demonstrates their high a⁄nity for molyb-
ternal molybdate is below 0.5 units (Fig. 2). Supplementation of the growth medium with increasing amounts (0^1 mM) of sodium molybdate resulted in a restoration of moa expression as shown by a L-galactosidase activity up to 2.9 units. In comparison, DB1004, the strain isogenic to LA27 lacking the modC3 mutation, shows high-level moa expression with and without molybdate supplementation (about 3.0 units), indicating the presence of su⁄cient amounts of the ModE^molybdate complex. With 100 WM molybdate a signi¢cant restoration of moa expression (70% of DB1004) was achieved in LA27. Maximal moa expression in LA27 was found with 500 WM sodium molybdate, which was equivalent to the activity found in the positive control DB1004 (Fig. 2). Employing more molybdate (1 mM) led to a loss of activity in both wild-type and mutant, which is probably due to a toxic e¡ect of molybdate. 3.3. Expression of molybdate-binding proteins reduces molybdate-dependent moa activation in LA27 cells Does expression of a molybdate-binding protein reduce the pool of available molybdate and can this e¡ect be monitored in our reporter strains? We have chosen three di¡erent, well-characterised molybdate-binding proteins that were subjected to our assay. (i) The C-terminal dimop domain of E. coli ModE has been shown to bind molybdate with an a⁄nity similar to ModE [22], and the crystal structure of the ligand-bound form was determined [22]. (ii) MopII of Clostridium pasteurianum in its crystallised form binds eight molybdate ions per MopII hexamer [23]. (iii) ModA is the periplasmic high-a⁄nity molybdate-bind-
Fig. 3. In£uence of the expression of molybdate-binding proteins on molybdate-dependent moa expression in LA27 cells. A,B: Comparison of L-galactosidase activities of LA27 expressing the three molybdatebinding proteins E. coli dimop domain (Dimop), E. coli ModA and C. pasteurianum MopII as well as LA27 (control) and DB1004 containing pQE80. A: Cells were grown under di¡erent concentrations of sodium molybdate (0^500 WM) and protein expression was induced with 50 WM IPTG. 100% refers to the activity measured for the parental strain DB1004/pQE80 grown in molybdate-free medium. B: Protein expression was induced with di¡erent concentrations of IPTG (0^50 WM) in the presence of 100 WM sodium molybdate. 100% refers to the activity measured for the control strain grown in the absence of IPTG. C: Moa activity of LA27 expressing the plant protein Cnx1 in the presence (+) and absence (3) of 20 WM IPTG (IPTG) and 500 WM sodium molybdate (MoO23 4 ). 100% refers to the activity measured in the presence of molybdate and the absence of IPTG (black bar). Activities are average values derived from triplicate measurements as described in Fig. 2 and given in percentage of L-galactosidase activity units (Vmax Wg protein31 ).
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date because even in the presence of 500 WM molybdate no signi¢cant restoration of moa activity was found. As a next step we wanted to know whether or not the competitive binding of molybdate to those proteins is dependent on their expression level in LA27. The di¡erent LA27 strain transformans were grown in the presence of 100 WM sodium molybdate with di¡erent concentrations of IPTG (0^50 WM). In the absence of IPTG (with no or very low-level expression of molybdate-binding proteins), molybdate-restored moa expression in LA27 was similar to cells transformed with the control plasmid pQE80 (Fig. 3B, white bars). However, already 5 WM IPTG resulted in a signi¢cant reduction of the molybdate-restored L-galactosidase activity in cells expressing any of the molybdate-binding proteins. This e¡ect was IPTG-dependent (Fig. 3B), as the use of higher concentrations of IPTG resulted in an even stronger reduction down to 10% of the activity in the control strain (LA27/pQE80). Since the dimop domain has the same a⁄nity as host-endogenous ModE, the amount of expressed protein should be signi¢cantly higher as compared to the endogenous ModE.
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with the exception that the level of basal expression was slightly higher (25^30% of full induction), and 100 WM molybdate resulted in only a two-fold induction of moa expression. However, with 500 WM molybdate a four-fold higher L-galactosidase activity could be seen as compared to cells grown in the absence of molybdate (Fig. 4A). Expression of MopII from C. pasteurianum (induced with 50 WM IPTG) shows no induction of L-galactosidase activity at any molybdate concentration used in the assay indicating a very e¡ective binding of molybdate (Fig. 4A). We also analysed the dependence of MopII expression level on moa expression in SB117 (Fig. 4B). Here, a clear dose-dependent e¡ect of the reduction of L-galactosidase activity with increasing expression of MopII protein (5^50 WM IPTG) can be seen, whereas the control strain transformed with the empty pQE80 plasmid showed full activity at any IPTG concentration used. This dose-dependent
3.4. The plant protein Cnx1 does not a¡ect molybdatedependent moa activation in LA27 cells Molybdate-binding proteins are so far unknown in eukaryotes. However, the plant protein Cnx1 is involved in the last step of molybdenum cofactor biosynthesis, where it catalyses insertion of molybdenum into molybdopterin [25]. This protein is able to restore molybdenum cofactor biosynthesis in E. coli mogA, indicating its ability to process molybdate within the bacterial cells [26]. Therefore, a weak binding of molybdate was proposed [25]. However, when expressing Cnx1 (pQEcnx1, +50 WM IPTG) in LA27 cells no e¡ect on the molybdate-dependent (100 WM molybdate) restoration of moa expression was observed (Fig. 3C). Therefore, we concluded that under the experimental conditions used Cnx1 is not a¡ecting the molybdate pool in LA27 cells. 3.5. Expression of molybdate-binding proteins and Cnx1 in SB117 cells LA27 cells do not synthesise molybdopterin. Therefore one has to ask whether the absence of molybdopterin as substrate of Cnx1 could in£uence the ability of Cnx1 to bind or process molybdate. Magalon et al. [27] have recently shown that MogA and MoeA (the bacterial homologues of the two-domain protein Cnx1) interact with each other in a molybdopterin-dependent manner. This prompted us to study molybdate-dependent moa expression in a strain able to synthesise molybdopterin. Strain SB117 produces molybdopterin and was analysed for the molybdate-dependent restoration of moa expression. Molybdate-dependent L-galactosidase activity in SB117 carrying the control vector was comparable to that of LA27
Fig. 4. In£uence of the expression of molybdate-binding proteins on molybdate-dependent moa expression in SB117 cells. A,B : Comparison of L-galactosidase activity of SB117 expressing MopII from C. pasteurianum (MopII) with SB117 (control) transformed with the plasmid pQE80. A: Cells were grown with di¡erent concentrations of sodium molybdate (0^500 WM) in the presence of 50 WM IPTG. B: Protein expression was induced with di¡erent concentrations of IPTG (0^50 WM) in the presence of 100 WM sodium molybdate. 100% refers to the activity measured for control grown in molybdate-free medium (A) or in the absence of IPTG (B). C: Moa activity of SB117 expressing the plant protein Cnx1 in the presence (+) and absence (3) of 20 WM IPTG (IPTG) and 500 WM sodium molybdate (MoO23 4 ). 100% refers to the activity measured in the presence of molybdate and the absence of IPTG (black bar). Activities are average values derived from triplicate measurements as described in Fig. 2 and given in percentage of L-galactosidase activity units (Vmax Wg protein31 ).
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e¡ect of MopII in SB117 indicates a di¡erent ratio between the ModE^molybdate pool and the competing molybdate-binding protein as compared to LA27 (Fig. 3B). Finally, strain SB117 was used as host for expression of the plant protein Cnx1. In contrast to LA27, expression of Cnx1 in£uenced the ModE^molybdate pool in SB117 cells. The expression of Cnx1 in the presence of 20 WM IPTG resulted in a 25% reduction of L-galactosidase activity. This reduction was in the same range as the change of moa expression caused by MopII at low-level expression (5 WM IPTG). Therefore we conclude that Cnx1 is able to interfere with the internal molybdate pool, but this action is dependent on molybdopterin.
4. Discussion Here we describe an in vivo system for the detection of molybdate binding to proteins based on the competition for intracellular molybdate between the molybdate-dependent transcriptional regulator ModE and a heterologously expressed protein in E. coli. By monitoring the molybdateloaded state of ModE the competitive binding of molybdate to the protein of interest was measurable. This was achieved by ensuring two prerequisites : (i) the internal molybdate pool was controlled by using a mutant strain defective in high-a⁄nity molybdate uptake and supplementation of the growth medium with molybdate; (ii) the molybdate-loaded state of ModE was monitored using a moa promoter fusion to lacZ allowing the detection of moa expression via L-galactosidase activity. To analyse a large number of conditions we modi¢ed the originally described Miller assay [18] to a more reproducible and easier to handle microtitre plate-based kinetic assay that allows a high sample throughput. Both reporter strains used in this study showed a similar behaviour regarding the suppression of the modC3 phenotype and subsequent moa activation. Suppression of the modC3 phenotype was observed when molybdate concentrations around 100 WM were present in the growth medium. This re£ects the alternative molybdate uptake by the sulfate transport system in E. coli [4]. In LA27, the higha⁄nity binding of molybdate to three di¡erent proteins could be con¢rmed in vivo. In addition, in SB117 we detected a dose-dependent response of moa expression with C. pasteurianum MopII. Hence this in vivo assay should be suitable to examine the ability of other proteins to bind molybdate, and we used it for the analysis of plant Cnx1. The eukaryotic Cnx1 protein catalyses the last step of molybdenum cofactor biosynthesis in plants, the insertion of molybdenum into molybdopterin [25]. Cnx1 binds molybdopterin with high a⁄nity [15], but the binding of molybdate as its second proposed substrate has not been observed yet. Therefore, the e¡ect of Cnx1 expression on the intracellular molybdate pool was studied under in vivo conditions using the above assay system. First we used
LA27 because it showed a strong e¡ect at even low concentrations of any competing protein used for calibration. However, Cnx1 expression did not a¡ect the ModE-dependent moa expression suggesting no binding of molybdate to Cnx1 under the experimental conditions. Even a metabolic processing of molybdate by Cnx1 as it is assumed to occur during molybdenum cofactor biosynthesis would in£uence the molybdate pool (as a secondary e¡ect) resulting in the reduction of moa expression. However, LA27 does not contain molybdopterin and therefore Cnx1 cannot form molybdenum cofactor. Magalon et al. [27] described a molybdopterin-dependent in vivo interaction between MoeA and MogA, the two bacterial homologues of both Cnx1 domains. Based on this and our negative result obtained in LA27 we propose a molybdopterin-dependent molybdate binding to and/or processing by Cnx1. If this assumption is correct a molybdopterin-producing reporter strain should respond to Cnx1 expression. In contrast to LA27, strain SB117 is able to form molybdopterin that is converted to molybdenum cofactor when growing the cells in the presence of molybdate. Feedback inhibition of moa expression by molybdenum cofactor [12] is suppressed in SB117 because the responsible element is located further downstream of the truncated moa promoter fragment 117K (D.H. Boxer, unpublished results). The expression of Cnx1 in SB117 resulted in a reproducible reduction of moa activity. Two conclusions can be drawn from this result: (i) molybdate is sequestered to Cnx1 where it either stays bound to it or is subsequently processed and incorporated into molybdopterin; (ii) since the observed e¡ect of Cnx1 is molybdopterin-dependent we suppose that molybdate binding and/ or processing by Cnx1 takes place only when molybdopterin as the other substrate of Cnx1 is present. Taken together, the described in vivo system for the sensitive detection of molybdate binding to proteins provides a cellular environment that could give clues as to which other factors might be needed for molybdate binding. Finally, this assay provides the basis for screening expression libraries in order to identify novel molybdatebinding proteins in eukaryotes.
Acknowledgements Financial support by the Deutsche Forschungsgemeinschaft and the Fritz-Thyssen-Stiftung is gratefully acknowledged.
References [1] Hille, R. (1996) The mononuclear molybdenum enzymes. Chem. Rev. 96, 2757^2816. [2] Rajagopalan, K.V. and Johnson, J.L. (1992) The pterin molybdenum cofactors. J. Biol. Chem. 267, 10199^10202.
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J. Kuper et al. / FEMS Microbiology Letters 218 (2003) 187^193 [3] Romao, M.J., Archer, M., Moura, I., Moura, J.J., LeGall, J., Engh, R., Schneider, M., Hof, P. and Huber, R. (1995) Crystal structure of the xanthine oxidase-related aldehyde oxidoreductase from D. gigas. Science 270, 1170^1176. [4] Self, W.T., Grunden, A.M., Hasona, A. and Shanmugam, K.T. (2001) Molybdate transport. Res. Microbiol. 152, 311^321. [5] Rivers, S.L., McNairn, E., Blasco, F., Giordano, G. and Boxer, D.H. (1993) Molecular genetic analysis of the moa operon of Escherichia coli K-12 required for molybdenum cofactor biosynthesis. Mol. Microbiol. 8, 1071^1081. [6] Maupin-Furlow, J.A., Rosentel, J.K., Lee, J.H., Deppenmeier, U., Gunsalus, R.P. and Shanmugam, K.T. (1995) Genetic analysis of the modABCD (molybdate transport) operon of Escherichia coli. J. Bacteriol. 177, 4851^4856. [7] Glaser, J.H. and DeMoss, J.A. (1971) Phenotypic restoration by molybdate of nitrate reductase activity in chlD mutants of Escherichia coli. J. Bacteriol. 108, 854^860. [8] Grunden, A.M., Ray, R.M., Rosentel, J.K., Healy, F.G. and Shanmugam, K.T. (1996) Repression of the Escherichia coli modABCD (molybdate transport) operon by ModE. J. Bacteriol. 178, 735^744. [9] McNicholas, P.M., Rech, S.A. and Gunsalus, R.P. (1997) Characterization of the ModE DNA-binding sites in the control regions of modABCD and moaABCDE of Escherichia coli. Mol. Microbiol. 23, 515^524. [10] Anderson, L.A., Palmer, T., Price, N.C., Bornemann, S., Boxer, D.H. and Pau, R.N. (1997) Characterisation of the molybdenum-responsive ModE regulatory protein and its binding to the promoter region of the modABCD (molybdenum transport) operon of Escherichia coli. Eur. J. Biochem. 246, 119^126. [11] Hall, D.R., Gourley, D.G., Leonard, G.A., Duke, E.M., Anderson, L.A., Boxer, D.H. and Hunter, W.N. (1999) The high-resolution crystal structure of the molybdate-dependent transcriptional regulator (ModE) from Escherichia coli: a novel combination of domain folds. EMBO J. 18, 1435^1446. [12] Anderson, L.A., McNairn, E., Lu«bke, T., Pau, R.N. and Boxer, D.H. (2000) ModE-dependent molybdate regulation of the molybdenum cofactor operon moa in Escherichia coli. J. Bacteriol. 182, 7035^7043. [13] Johann, S. and Hinton, S.M. (1987) Cloning and nucleotide sequence of the chlD locus. J. Bacteriol. 169, 1911^1916. [14] Harrison, J.A., Schuttelkopf, A.W., Boxer, D.H. and Hunter, W.N. (2001) Crystallization and X-ray di¡raction measurements on recombinant molbindin, MopII, from Clostridium pasteurianum. Acta Crystallogr. D Biol. Crystallogr. 57, 1715^1717. [15] Schwarz, G., Boxer, D.H. and Mendel, R.R. (1997) Molybdenum cofactor biosynthesis. The plant protein Cnx1 binds molybdopterin with high a⁄nity. J. Biol. Chem. 272, 26811^26814. [16] Hendrickson, W.A., Horton, J.R. and LeMaster, D.M. (1990) Selenomethionyl proteins produced for analysis by multiwavelength anomalous di¡raction (MAD) : a vehicle for direct determination of three-dimensional structure. EMBO J. 9, 1665^1672.
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[17] Simons, R.W., Houman, F. and Kleckner, N. (1987) Improved single and multicopy lac-based cloning vectors for protein and operon fusions. Gene 53, 85^96. [18] Miller, J.H. (1972) Experiments in molecular genetics Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [19] Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248^254. [20] Stewart, V. and MacGregor, C.H. (1982) Nitrate reductase in Escherichia coli K-12: involvement of chlC, chlE, and chlG loci. J. Bacteriol. 151, 788^799. [21] Joshi, M.S., Johnson, J.L. and Rajagopalan, K.V. (1996) Molybdenum cofactor biosynthesis in Escherichia coli mod and mog mutants. J. Bacteriol. 178, 4310^4312. [22] Gourley, D.G., Schuttelkopf, A.W., Anderson, L.A., Price, N.C., Boxer, D.H. and Hunter, W.N. (2001) Oxyanion binding alters conformation and quaternary structure of the C-terminal domain of the transcriptional regulator mode. Implications for molybdate-dependent regulation, signaling, storage, and transport. J. Biol. Chem. 276, 20641^20647. [23] Schuttelkopf, A.W., Harrison, J.A., Boxer, D.H. and Hunter, W.N. (2002) Passive acquisition of ligand by the MopII molbindin from Clostridium pasteurianum : structures of apo and oxyanion-bound forms. J. Biol. Chem. 277, 15013^15020. [24] Rech, S., Wolin, C. and Gunsalus, R.P. (1996) Properties of the periplasmic ModA molybdate-binding protein of Escherichia coli. J. Biol. Chem. 271, 2557^2562. [25] Schwarz, G., Schulze, J., Bittner, F., Eilers, T., Kuper, J., Bollmann, G., Nerlich, A., Brinkmann, H. and Mendel, R.R. (2000) The molybdenum cofactor biosynthetic protein Cnx1 complements molybdate-repairable mutants, transfers molybdenum to the metal binding pterin, and is associated with the cytoskeleton. Plant Cell 12, 2455^ 2472. [26] Stallmeyer, B., Nerlich, A., Schiemann, J., Brinkmann, H. and Mendel, R.R. (1995) Molybdenum co-factor biosynthesis: the Arabidopsis thaliana cDNA cnx1 encodes a multifunctional two-domain protein homologous to a mammalian neuroprotein, the insect protein Cinnamon and three Escherichia coli proteins. Plant J. 8, 751^762. [27] Magalon, A., Frixon, C., Pommier, J., Giordano, G. and Blasco, F. (2002) In vivo interactions between gene products involved in the ¢nal stages of molybdenum cofactor biosynthesis in Escherichia coli. J. Biol. Chem. 7, 7. [28] Baker, K.P. and Boxer, D.H. (1991) Regulation of the chlA locus of Escherichia coli K12: involvement of molybdenum cofactor. Mol. Microbiol. 5, 901^907. [29] Rosentel, J.K., Healy, F., Maupin-Furlow, J.A., Lee, J.H. and Shanmugam, K.T. (1995) Molybdate and regulation of mod (molybdate transport), fdhF, and hyc (formate hydrogenlyase) operons in Escherichia coli. J. Bacteriol. 177, 4857^4864.
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In vivo detection of molybdate-binding proteins using a competition assay with ModE in Escherichia coli Jochen Kuper a;1 , Sonja Meyer zu Berstenhorst a;b;1 , Bernd Vo«disch b , Ralf R. Mendel a , Gu«nter Schwarz a; , David H. Boxer b b
a Department of Plant Biology, Technical University of Braunschweig, 38023 Braunschweig, Germany Division of Biological Chemistry and Molecular Microbiology, School of Life Science, University of Dundee, Dundee DD1 5EH, UK
Received 29 October 2002 ; received in revised form 6 November 2002; accepted 6 November 2002 First published online 29 November 2002
Abstract Molybdenum is an important trace element as it forms the essential part of the active site in all molybdenum-containing enzymes. We have designed an assay for the in vivo detection of molybdate binding to proteins in Escherichia coli. The assay is based on (i) the molybdate-dependent transcriptional regulation of the moa operon by the ModE protein, and (ii) the competition for molybdate between ModE and other molybdate-binding proteins in the cytoplasm of E. coli. We were able to verify in vivo molybdate binding to three different bacterial proteins that are known to bind molybdate. This sensitive in vivo system allows the testing of different proteins for molybdate binding under in vivo conditions and will facilitate the identification of other cellular factors needed for molybdate binding. As a first example, we examined the eukaryotic protein Cnx1 that is involved in the last step of molybdenum cofactor biosynthesis in plants, and show that it is able to compete with ModE for molybdate in a molybdopterin-dependent fashion. 9 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : ModE; Molybdenum; Molybdate binding ; Cnx1; Miller assay
1. Introduction Molybdenum is an essential trace element required for the activity of all molybdenum enzymes catalysing important redox reactions in the global C, N and S cycles [1]. In all these enzymes, except nitrogenase, molybdenum is activated and co-ordinated by the dithiolene moiety of a unique and conserved pterin, molybdopterin, thus forming the molybdenum cofactor [2]. Although the coordination state of molybdenum can vary [3] and di¡erent nucleotides can be attached to molybdopterin [1], the basic structure of the molybdenum cofactor consisting of molybdenum and molybdopterin is conserved throughout bacteria and eukaryotes. In Escherichia coli the mo loci are responsible for molybdenum transport and the synthesis of the molybdenum
* Corresponding author. Tel. : +49 (531) 391 5891 ; Fax : +49 (531) 391 8208. E-mail address : [email protected] (G. Schwarz). 1
Both authors contributed equally to this work.
cofactor [4]. The entire moa locus of E. coli is required for molybdopterin biosynthesis [5], while the modABCD operon encodes the high-a⁄nity ABC-type molybdate uptake system [6]. Strains defective in this locus are pleiotropically unable to synthesise active molybdenum enzymes. The supplementation of the growth medium with high levels of molybdate, however, fully restores all molybdenum enzyme activities in mod strains, indicating that molybdate can enter the bacterium by another route under such conditions [7]. Molybdate, as one major substrate for the biosynthesis of the molybdenum cofactor, regulates the expression of several operons in E. coli. ModE appears to be the main molybdate-dependent transcriptional regulator in the bacterium acting as a negative e¡ector of the operon modABCD [8] encoding the high-a⁄nity molybdate uptake system, and as a positive e¡ector for the transcription of moaABCDE [9] encoding enzymes responsible for the biosynthesis of molybdopterin. In vitro studies have demonstrated that the dimeric ModE binds two molybdate ions with a KD of 0.8 WM and that this molybdate^ModE complex binds to the promoter region of both mod and
0378-1097 / 02 / $22.00 9 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 1 1 2 1 - 7
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moa DNA [9,10]. The crystal structure of ModE has been determined and shows distinct DNA and molybdate-binding domains [11]. In strains lacking the ability both to synthesise functional molybdenum cofactor (moa) and to take up molybdate under normal growth conditions (mod), molybdate added to the growth medium exerts a powerful positive e¡ect on moa transcription that is absolutely dependent on ModE function [12]. Based on this ¢nding we have constructed moa L-galactosidase reporter strains lacking a functional high-a⁄nity uptake system (modC3 ). The lacZ gene under control of a truncated moa promoter was inserted either directly into the moa operon, resulting in the disruption of molybdopterin biosynthesis, or by lambda phage insertion without a¡ecting cofactor biosynthesis. In both strains, we titrated the molybdate-dependent activation of the moa operon by following L-galactosidase activity. Using these strains, we could show that the expression of di¡erent molybdate-binding proteins competes with ModE for molybdate binding, resulting in the reduction of moa expression. With this system we established the ¢rst in vivo molybdate-binding assay that can be used for testing di¡erent proteins for their ability to bind molybdate under in vivo conditions, thus allowing the identi¢cation of additional cellular factors required for molybdate binding. As a ¢rst example we demonstrated for the plant molybdenum cofactor biosynthetic protein Cnx1 molybdate binding and/or processing as Cnx1 is able to compete with ModE for molybdate in a molybdopterin-dependent fashion. The data presented here are a ¢rst step to establish a screening system for molybdate-binding proteins from eukaryotes, where almost nothing is known yet about the uptake, binding and processing of molybdate prior to its incorporation into the molybdenum cofactor.
2. Materials and methods 2.1. Bacterial strains, bacteriophages, plasmids and growing conditions All strains, bacteriophages and plasmids used in this work are listed in Table 1. With the exception of pQEcnx1 all other expression plasmids were generated using PCR cloning and the resulting constructs were sequenced after successful cloning. The C-terminal dimop domain of ModE (422 bp) was derived from pHW121 [10] and PstI/HindIII subcloned. ModA was ampli¢ed from pSJE350 [13], introducing BamHI/HindIII sites for subcloning. For modA (717 bp), the ¢rst 64 bp encoding the periplasmic target sequence were removed. MopII (207 bp) from pET15bMopII [14] was cloned via BamHI/HindIII sites. All PCR fragments were introduced into pQE80 (Qiagen, Hilden, Germany) using the speci¢ed restriction enzymes, resulting in the pQE constructs given in Table 1. Restriction enzymes used for cloning were obtained from Promega (Mannheim, Germany). Pwo polymerase for PCR was purchased from Peqlab (Erlangen, Germany). Oligonucleotides were obtained from Sigma (Taufkirchen, Germany). Cnx1 was cloned from pQE60cnx1 [15] by cloning the whole pQE60 expression cassette into pQE80 via EcoRI/HindIII. Bacteria were grown aerobically in Luria^Bertani medium at 37‡C with the addition of 50 Wg ml31 kanamycin and 125 Wg ml31 ampicillin where appropriate. For the modi¢ed Miller assay (see below) cells were grown in LeMaster minimal medium [16] and isopropyl-LD-thiogalactopyranoside (IPTG) was added for the induction of protein expression. Aerobic growth conditions were obtained by vigorous shaking on an orbital shaker (240^250 rpm).
Table 1 Strains, bacteriophages and plasmids Name
Genotype/insert
Source
DB1004 LA27 SE1597 SB117 VRS45 pQE80 pSJE350 pHW121 pEM101 pRS551 pET15bMopII pQE60cnx1 pQEdimop pQEmodA pQEcpmopII pQEcnx1
F araD139 v(argF-lac)U169 deoC1 £bB5201 gyrA 219 relA1 rpsL150 non-9 ptsF25 ; [P(moaB: :lacZ)4] As DB1004; modC118 zbh-623 : :Tn10 D(lacU)169 rpsL modC120 SE1597[P(moa: :lacZ)] del-lacZ blaP attP imm21 cloning vector source for E. coli modA source for E. coli dimop (dimop domain of ModE) source for E. coli moa promoter V cloning vector source for Clostridium pasteurianum mopII source for Arabidopsis thaliana cnx1 E. coli dimop in pQE80 E. coli modA (without N-terminal leader sequence) in pQE80 Clostridium pasteurianum mopII in pQE80 Arabidopsis thaliana cnx1 in pQE80
[28] [12] [29] This work [17] Qiagen [13] [10] [10] [17] [14] [15] This work This work This work This work
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2.2. Strain construction SB117 was constructed by V phage insertion as described [17] using pRS551 as homologous recombination vector for phage VRS45 and SE1597 as hosting strain [12]. The moa promoter fragment 117K (3121^+131) was derived from pEM101 [12] and subcloned into the EcoRI/ BamHI sites of pRS551 [17] using PCR. Fragment 117K uses the same start site as the previously described 117 fragment [12] but it ends immediately before the moaA start at position +131. After recombination, the presence of the prophage was con¢rmed via PCR using primers that bind between the start of 117K and the region directly in front of the lacZ gene from pRS551. 2.3. L-Galactosidase assay L-Galactosidase assays were performed with a microtitre plate system (Versa max, Molecular Devices, Sunnyvale, CA, USA) based on a protocol of Miller et al. [18]. Cells were grown overnight and 50 Wl of this culture was transferred to 10 ml fresh medium supplemented with the appropriate amounts of IPTG, molybdate and antibiotics and continued to grow for an additional 4^5 h. Cells were harvested (4‡C, 4300Ug, 15 min), resuspended in 700 Wl bu¡er Z ([18], 5 mM GSH instead of L-mercaptoethanol) and sonicated on ice two times each 10 s. Cell debris was removed by centrifugation (4‡C, 20 000Ug, 5 min). Protein content was determined using the Bradford protein assay [19]. Samples were diluted to 0.1 Wg Wl31 in bu¡er Z and 2^10 Wg total protein was applied to a 96-well microtitre plate (Sarstedt, Nu«mbrecht, Germany) in a ¢nal volume of 100 Wl. Reaction was started by addition of 50 Wl ortho-nitrophenol-L-D -galactoside (4 mg ml31 ) in bu¡er Z. The formation of o-nitrophenol was kinetically monitored, measuring the absorbance at 410 nm for 1 h, recording data points every 5 min. Values in the linear range of the reaction were used to calculate Vmax (vOD410 min31 ). L-Galactosidase activity is ¢nally given in Wmol o-nitrophenol Wg protein31 min31 .
3. Results 3.1. The principle of ModE competition for detecting of molybdate-binding proteins in E. coli We have previously shown that expression of moa genes, as monitored by L-galactosidase activity in a strain unable to acquire physiological amounts of molybdate from its growth medium (modC3 ), is restored following the addition of high amounts of molybdate (up to 10 mM) to the growth medium [12]. Under these conditions, internal molybdate levels are restored via molybdate uptake through less speci¢c anion uptake systems [4]. Careful titration of moa expression with di¡erent concentrations of
Fig. 1. Expression of a molybdate-binding protein can in£uence the molybdate-dependent expression of the E. coli moa operon. The moa promoter regulates expression of the moa operon that encodes proteins essential for the synthesis of molybdopterin in E. coli. ModE is a dimeric transcriptional regulator that binds molybdate anions on its dimop domain. This ModE^MoO24 complex activates the transcription of the moa operon. E. coli strain LA27 carries a lacZ reporter gene insertion in the moa operon in order to monitor moa-promoter activity. Heterologous expression of a molybdate-binding protein leads to internal competition for molybdate between ModE and the protein of interest, resulting in a reduction of moa expression as monitored by L-galactosidase activity.
molybdate in the growth media should result in internal conditions just su⁄cient to allow moa activation. Under these conditions, the expression of a molybdate-binding protein could alter the amount of the internal free molybdate by sequestering it to the newly expressed protein. Due to the competition for molybdate by the second molybdate-binding protein, less ModE^molybdate complex would be available and therefore moa expression would be reduced (Fig. 1). The magnitude of change in moa expression will be dependent on the ratio between the molybdate-binding capacity of the newly expressed protein and the molybdate-bu¡ering capacity of the interior of the bacterium. In this work, two di¡erent strains were used to accomplish the in vivo molybdate-binding detection. The modC3 mutant strain LA27 carries the reporter gene lacZ as chromosomal insertion in the moa operon and is therefore molybdenum cofactor-de¢cient because of the inactivation of the moa operon [12]. As a second approach, the moa promoter fragment 117K fused to the lacZ gene was inserted into the genome of the modC3 mutant strain SE1597 by lambda phage insertion. This resulted in strain SB117 (SE1597[P(moa: : lacZ)]) with the same features as LA27 except that it is still able to synthesise active molybdenum cofactor after addition of molybdate to the growth media [20,21]. 3.2. Sensitivity of moa expression to external molybdate in a modC3 strain L-Galactosidase activity of LA27 in the absence of ex-
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Fig. 2. Molybdate-dependent regulation of moa expression in the modCde¢cient strain LA27 and the parental strain DB1004. Shown are the average L-galactosidase activities in units (Vmax Wg protein31 ) of the parental strain DB1004 (black dots) and the reporter strain LA27 (grey rectangles) in response to increasing amounts of molybdate in the culture medium. Cultures were grown for 2.5 h, whereas for all other experiments (Figs. 3 and 4) cultures were grown for 4 h. Activities represent average values obtained from at least three di¡erent cultures with three di¡erent protein concentrations, performing kinetic assays on each culture, using only the linear range of the reaction for calculating the enzyme activity (Vmax ).
ing protein of the E. coli ABC-type molybdate transport system [24]. The cDNAs of the dimop domain, of MopII and of ModA cloned into pQE80 (Table 1) were transformed into LA27 and L-galactosidase activity was monitored at di¡erent molybdate concentrations in the medium (Fig. 3A) and protein expression levels (Fig. 3B). Expression of each protein (induced with 50 WM IPTG) completely abolished the molybdate-dependent restoration of moa expression, whereas the control strain (LA27/pQE80) already showed 60% restoration of moa expression with only 50 WM molybdate (Fig. 3A). These results con¢rm the molybdate binding of all three proteins under in vivo conditions and demonstrates their high a⁄nity for molyb-
ternal molybdate is below 0.5 units (Fig. 2). Supplementation of the growth medium with increasing amounts (0^1 mM) of sodium molybdate resulted in a restoration of moa expression as shown by a L-galactosidase activity up to 2.9 units. In comparison, DB1004, the strain isogenic to LA27 lacking the modC3 mutation, shows high-level moa expression with and without molybdate supplementation (about 3.0 units), indicating the presence of su⁄cient amounts of the ModE^molybdate complex. With 100 WM molybdate a signi¢cant restoration of moa expression (70% of DB1004) was achieved in LA27. Maximal moa expression in LA27 was found with 500 WM sodium molybdate, which was equivalent to the activity found in the positive control DB1004 (Fig. 2). Employing more molybdate (1 mM) led to a loss of activity in both wild-type and mutant, which is probably due to a toxic e¡ect of molybdate. 3.3. Expression of molybdate-binding proteins reduces molybdate-dependent moa activation in LA27 cells Does expression of a molybdate-binding protein reduce the pool of available molybdate and can this e¡ect be monitored in our reporter strains? We have chosen three di¡erent, well-characterised molybdate-binding proteins that were subjected to our assay. (i) The C-terminal dimop domain of E. coli ModE has been shown to bind molybdate with an a⁄nity similar to ModE [22], and the crystal structure of the ligand-bound form was determined [22]. (ii) MopII of Clostridium pasteurianum in its crystallised form binds eight molybdate ions per MopII hexamer [23]. (iii) ModA is the periplasmic high-a⁄nity molybdate-bind-
Fig. 3. In£uence of the expression of molybdate-binding proteins on molybdate-dependent moa expression in LA27 cells. A,B: Comparison of L-galactosidase activities of LA27 expressing the three molybdatebinding proteins E. coli dimop domain (Dimop), E. coli ModA and C. pasteurianum MopII as well as LA27 (control) and DB1004 containing pQE80. A: Cells were grown under di¡erent concentrations of sodium molybdate (0^500 WM) and protein expression was induced with 50 WM IPTG. 100% refers to the activity measured for the parental strain DB1004/pQE80 grown in molybdate-free medium. B: Protein expression was induced with di¡erent concentrations of IPTG (0^50 WM) in the presence of 100 WM sodium molybdate. 100% refers to the activity measured for the control strain grown in the absence of IPTG. C: Moa activity of LA27 expressing the plant protein Cnx1 in the presence (+) and absence (3) of 20 WM IPTG (IPTG) and 500 WM sodium molybdate (MoO23 4 ). 100% refers to the activity measured in the presence of molybdate and the absence of IPTG (black bar). Activities are average values derived from triplicate measurements as described in Fig. 2 and given in percentage of L-galactosidase activity units (Vmax Wg protein31 ).
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date because even in the presence of 500 WM molybdate no signi¢cant restoration of moa activity was found. As a next step we wanted to know whether or not the competitive binding of molybdate to those proteins is dependent on their expression level in LA27. The di¡erent LA27 strain transformans were grown in the presence of 100 WM sodium molybdate with di¡erent concentrations of IPTG (0^50 WM). In the absence of IPTG (with no or very low-level expression of molybdate-binding proteins), molybdate-restored moa expression in LA27 was similar to cells transformed with the control plasmid pQE80 (Fig. 3B, white bars). However, already 5 WM IPTG resulted in a signi¢cant reduction of the molybdate-restored L-galactosidase activity in cells expressing any of the molybdate-binding proteins. This e¡ect was IPTG-dependent (Fig. 3B), as the use of higher concentrations of IPTG resulted in an even stronger reduction down to 10% of the activity in the control strain (LA27/pQE80). Since the dimop domain has the same a⁄nity as host-endogenous ModE, the amount of expressed protein should be signi¢cantly higher as compared to the endogenous ModE.
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with the exception that the level of basal expression was slightly higher (25^30% of full induction), and 100 WM molybdate resulted in only a two-fold induction of moa expression. However, with 500 WM molybdate a four-fold higher L-galactosidase activity could be seen as compared to cells grown in the absence of molybdate (Fig. 4A). Expression of MopII from C. pasteurianum (induced with 50 WM IPTG) shows no induction of L-galactosidase activity at any molybdate concentration used in the assay indicating a very e¡ective binding of molybdate (Fig. 4A). We also analysed the dependence of MopII expression level on moa expression in SB117 (Fig. 4B). Here, a clear dose-dependent e¡ect of the reduction of L-galactosidase activity with increasing expression of MopII protein (5^50 WM IPTG) can be seen, whereas the control strain transformed with the empty pQE80 plasmid showed full activity at any IPTG concentration used. This dose-dependent
3.4. The plant protein Cnx1 does not a¡ect molybdatedependent moa activation in LA27 cells Molybdate-binding proteins are so far unknown in eukaryotes. However, the plant protein Cnx1 is involved in the last step of molybdenum cofactor biosynthesis, where it catalyses insertion of molybdenum into molybdopterin [25]. This protein is able to restore molybdenum cofactor biosynthesis in E. coli mogA, indicating its ability to process molybdate within the bacterial cells [26]. Therefore, a weak binding of molybdate was proposed [25]. However, when expressing Cnx1 (pQEcnx1, +50 WM IPTG) in LA27 cells no e¡ect on the molybdate-dependent (100 WM molybdate) restoration of moa expression was observed (Fig. 3C). Therefore, we concluded that under the experimental conditions used Cnx1 is not a¡ecting the molybdate pool in LA27 cells. 3.5. Expression of molybdate-binding proteins and Cnx1 in SB117 cells LA27 cells do not synthesise molybdopterin. Therefore one has to ask whether the absence of molybdopterin as substrate of Cnx1 could in£uence the ability of Cnx1 to bind or process molybdate. Magalon et al. [27] have recently shown that MogA and MoeA (the bacterial homologues of the two-domain protein Cnx1) interact with each other in a molybdopterin-dependent manner. This prompted us to study molybdate-dependent moa expression in a strain able to synthesise molybdopterin. Strain SB117 produces molybdopterin and was analysed for the molybdate-dependent restoration of moa expression. Molybdate-dependent L-galactosidase activity in SB117 carrying the control vector was comparable to that of LA27
Fig. 4. In£uence of the expression of molybdate-binding proteins on molybdate-dependent moa expression in SB117 cells. A,B : Comparison of L-galactosidase activity of SB117 expressing MopII from C. pasteurianum (MopII) with SB117 (control) transformed with the plasmid pQE80. A: Cells were grown with di¡erent concentrations of sodium molybdate (0^500 WM) in the presence of 50 WM IPTG. B: Protein expression was induced with di¡erent concentrations of IPTG (0^50 WM) in the presence of 100 WM sodium molybdate. 100% refers to the activity measured for control grown in molybdate-free medium (A) or in the absence of IPTG (B). C: Moa activity of SB117 expressing the plant protein Cnx1 in the presence (+) and absence (3) of 20 WM IPTG (IPTG) and 500 WM sodium molybdate (MoO23 4 ). 100% refers to the activity measured in the presence of molybdate and the absence of IPTG (black bar). Activities are average values derived from triplicate measurements as described in Fig. 2 and given in percentage of L-galactosidase activity units (Vmax Wg protein31 ).
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e¡ect of MopII in SB117 indicates a di¡erent ratio between the ModE^molybdate pool and the competing molybdate-binding protein as compared to LA27 (Fig. 3B). Finally, strain SB117 was used as host for expression of the plant protein Cnx1. In contrast to LA27, expression of Cnx1 in£uenced the ModE^molybdate pool in SB117 cells. The expression of Cnx1 in the presence of 20 WM IPTG resulted in a 25% reduction of L-galactosidase activity. This reduction was in the same range as the change of moa expression caused by MopII at low-level expression (5 WM IPTG). Therefore we conclude that Cnx1 is able to interfere with the internal molybdate pool, but this action is dependent on molybdopterin.
4. Discussion Here we describe an in vivo system for the detection of molybdate binding to proteins based on the competition for intracellular molybdate between the molybdate-dependent transcriptional regulator ModE and a heterologously expressed protein in E. coli. By monitoring the molybdateloaded state of ModE the competitive binding of molybdate to the protein of interest was measurable. This was achieved by ensuring two prerequisites : (i) the internal molybdate pool was controlled by using a mutant strain defective in high-a⁄nity molybdate uptake and supplementation of the growth medium with molybdate; (ii) the molybdate-loaded state of ModE was monitored using a moa promoter fusion to lacZ allowing the detection of moa expression via L-galactosidase activity. To analyse a large number of conditions we modi¢ed the originally described Miller assay [18] to a more reproducible and easier to handle microtitre plate-based kinetic assay that allows a high sample throughput. Both reporter strains used in this study showed a similar behaviour regarding the suppression of the modC3 phenotype and subsequent moa activation. Suppression of the modC3 phenotype was observed when molybdate concentrations around 100 WM were present in the growth medium. This re£ects the alternative molybdate uptake by the sulfate transport system in E. coli [4]. In LA27, the higha⁄nity binding of molybdate to three di¡erent proteins could be con¢rmed in vivo. In addition, in SB117 we detected a dose-dependent response of moa expression with C. pasteurianum MopII. Hence this in vivo assay should be suitable to examine the ability of other proteins to bind molybdate, and we used it for the analysis of plant Cnx1. The eukaryotic Cnx1 protein catalyses the last step of molybdenum cofactor biosynthesis in plants, the insertion of molybdenum into molybdopterin [25]. Cnx1 binds molybdopterin with high a⁄nity [15], but the binding of molybdate as its second proposed substrate has not been observed yet. Therefore, the e¡ect of Cnx1 expression on the intracellular molybdate pool was studied under in vivo conditions using the above assay system. First we used
LA27 because it showed a strong e¡ect at even low concentrations of any competing protein used for calibration. However, Cnx1 expression did not a¡ect the ModE-dependent moa expression suggesting no binding of molybdate to Cnx1 under the experimental conditions. Even a metabolic processing of molybdate by Cnx1 as it is assumed to occur during molybdenum cofactor biosynthesis would in£uence the molybdate pool (as a secondary e¡ect) resulting in the reduction of moa expression. However, LA27 does not contain molybdopterin and therefore Cnx1 cannot form molybdenum cofactor. Magalon et al. [27] described a molybdopterin-dependent in vivo interaction between MoeA and MogA, the two bacterial homologues of both Cnx1 domains. Based on this and our negative result obtained in LA27 we propose a molybdopterin-dependent molybdate binding to and/or processing by Cnx1. If this assumption is correct a molybdopterin-producing reporter strain should respond to Cnx1 expression. In contrast to LA27, strain SB117 is able to form molybdopterin that is converted to molybdenum cofactor when growing the cells in the presence of molybdate. Feedback inhibition of moa expression by molybdenum cofactor [12] is suppressed in SB117 because the responsible element is located further downstream of the truncated moa promoter fragment 117K (D.H. Boxer, unpublished results). The expression of Cnx1 in SB117 resulted in a reproducible reduction of moa activity. Two conclusions can be drawn from this result: (i) molybdate is sequestered to Cnx1 where it either stays bound to it or is subsequently processed and incorporated into molybdopterin; (ii) since the observed e¡ect of Cnx1 is molybdopterin-dependent we suppose that molybdate binding and/ or processing by Cnx1 takes place only when molybdopterin as the other substrate of Cnx1 is present. Taken together, the described in vivo system for the sensitive detection of molybdate binding to proteins provides a cellular environment that could give clues as to which other factors might be needed for molybdate binding. Finally, this assay provides the basis for screening expression libraries in order to identify novel molybdatebinding proteins in eukaryotes.
Acknowledgements Financial support by the Deutsche Forschungsgemeinschaft and the Fritz-Thyssen-Stiftung is gratefully acknowledged.
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