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The iron dependent regulatory protein IdeR (DtxR) of Rhodococcus equi
FEMS Microbiology Letters 191 (2000) 1^5
www.fems-microbiology.org
The iron dependent regulatory protein IdeR (DtxR) of Rhodococcus equi Clara A. Boland, Wim G. Meijer * Department of Industrial Microbiology, Conway Institute of Biomolecular and Biomedical Research, National University of Ireland, Dublin, Dublin 4, Ireland Received 26 June 2000 ; accepted 24 July 2000
Abstract This paper reports the presence of an ideR gene, which encodes an iron-dependent regulatory protein, in Rhodococcus erythropolis and in the intracellular pathogen Rhodococcus equi. The ideR gene of the latter encoded a protein of 230 amino acids with a molecular mass of 25 619. The K-helices forming the helix-turn-helix motif of the R. equi protein were identical to those of the DtxR protein of Corynebacterium diphtheriae, which is an IdeR homologue. This indicates that the two proteins bind to the same DNA binding site. This was confirmed following expression of IdeR in Escherichia coli, which showed that the IdeR protein could repress transcription of the tox promoter of C. diphtheriae in an iron dependent manner. An open reading frame specifying a 283-amino acid polypeptide similar to galE encoding UDP-galactose 4-epimerase was present downstream of the ideR gene. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : IdeR; DtxR; Iron repressor ; GalE; Rhodococcus equi
1. Introduction A number of pathogenic bacteria are able to grow within macrophages, which represent the ¢rst line of defence against invading micro-organisms. Examples of these are the actinomycetes Mycobacterium tuberculosis and the closely related Rhodococcus equi, which are mycolic acidcontaining, Gram-positive bacteria sharing many physiological and pathological features [1]. Although originally identi¢ed as one of the most important pathogens of foals, R. equi has more recently been recognised as a pathogen of immunocompromised humans [1]. Virulent strains of R. equi harbour a large plasmid, which is essential for survival and growth in alveolar macrophages [2^4]. Virulence genes are usually expressed in response to changing environmental parameters which signal entry into the host. For example, expression of VapA, a small surface protein
* Corresponding author. Tel. : +353 (1) 706-1512; Fax: +353 (1) 706-1183; E-mail: [email protected]
which is encoded on the virulence plasmid of R. equi, is controlled by pH and temperature [5]. Following uptake by their hosts, pathogens encounter extremely low concentrations of free iron, which is largely due to iron binding proteins such as lactoferrin and transferrin [6]. It is therefore not surprising that the expression of a large number of virulence factors by pathogenic bacteria are controlled by iron. Regulation of gene expression by iron in M. tuberculosis is controlled by the global iron regulator IdeR, which is a homologue of the diphtheria toxin repressor DtxR of Corynebacterium diphtheriae [7,8]. Following binding of Fe2 , these proteins bind to their target DNA sequences as a homodimer, resulting in repression of gene expression [8]. In addition to controlling genes involved in iron uptake, IdeR also controls expression of virulence factors in C. diphtheriae and M. tuberculosis [8^10]. Although R. equi is an important veterinary pathogen, virtually nothing is known about the regulation of gene expression in this bacterium. Since Rhodococcus species are closely related to Mycobacterium and Corynebacterium, it is likely that they employ an iron repressor protein related to IdeR to control iron-dependent gene expression. This paper reports the presence of ideR in Rhodococcus erythropolis and R. equi. The ideR gene of the latter was cloned and characterised.
0378-1097 / 00 / $20.00 ß 2000 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 0 ) 0 0 3 5 4 - 2
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2. Materials and methods 2.1. Bacterial strains and plasmids Escherichia coli DH5K (Bio-Rad), E. coli BL21 DE3 (pLysE) (Novagen), R. erythropolis SQ1 [11], R. equi ATCC 33701, pBluescript II KS (Stratagene), pDA71 [12], pET3b (Novagen), pWKS129 [13], pWSK130 [13]. 2.2. Media and growth conditions E. coli DH5K, E. coli BL21 DE3 (pLysE) and R. equi were grown on Luria^Bertani (LB) medium at 37³C. The chelator 2,2-dipyridyl was added to the medium to achieve low Fe2 growth conditions. When appropriate the following supplements were added: ampicillin, 50 Wg ml31 ; chloramphenicol, 34 Wg ml31 ; isopropyl-L-D-thiogalactoside (IPTG), 0.1 mM; X-Gal, 20 Wg ml31 . 2.3. DNA manipulations Plasmid DNA was isolated with the alkaline lysis method [14] or by using the Wizard Plus SV miniprep (Promega). Chromosomal DNA was isolated as described [15]. DNA modifying enzymes were used according to the manufacturer's recommendations (Roche). An internal fragment of the ideR gene was ampli¢ed by PCR with Taq polymerase as recommended by the manufacturer (Promega) using the oligonucleotides 5P-GAYACNACNGARATGTA-3P (IDER1) and 5P-CTCATNACRTGYTCCCA-3P (IDER2). The reaction mixture was incubated at 95³C for 5 min and was subsequently subjected to 30 cycles of 95³C for 45 s, 37³C for 45 s, 74³C for 45 s, followed by an incubation at 74³C for 7 min. Dideoxy sequencing reactions were done with the CEQ DCTS kit as described by the manufacturer (Beckman). The nucleotide sequence was determined using a Beckman CEQ2000 automatic sequencer. The nucleotide sequence data were compiled and analysed using the Wisconsin Package Version 8 (Genetics Computer Group). 2.4. Southern hybridisation Chromosomal DNA of R. equi was digested with PstI, EcoRI, and BamHI, and transferred to a positively charged nylon membrane as recommended by the manufacturer (Roche) following agarose gel electrophoresis. DNA used as a probe, was labelled with digoxigenin 11dUTP using the DIG High Prime kit (Roche). Prehybridisation, hybridisation and detection of the labelled probe were done according to the manufacturer's recommendations. 2.5. Expression of ideR in E. coli The 5P-end of ideR was ampli¢ed by PCR from
pIDE214 (Fig. 1), which harbours a 1.2-kb SacI fragment of pIDE21 in the SacI site of pBluescript II KS. The oligonucleotides used in the PCR reactions were : 5P-GTAAAACGACGGCCAGT-3P and 5P-GCGCATATGAAGGATCTGTCGACACCACGG-3P, which introduces an NdeI restriction site in the initiation codon of ideR. The resulting PCR fragment was cloned into the EcoRV site of pBluescript II KS, yielding pIDE2141. A 1.6-kb SacI fragment of pIDE21 containing the 3P end of ideR was cloned into the SacI site of pIDE2141 and the complete ideR gene was subsequently cloned as a NdeI^BamHI fragment into NdeI^BamHI-digested pET3b yielding pIDE2143. E. coli BL21 DE3 (pLysE) harbouring pIDE2143 was grown at 30³C until OD660 = 0.5. Expression of ideR from the T7 promoter of pIDE2143 was induced by adding IPTG (1 mM) to the culture medium ; growth was allowed to proceed for an additional 3 h. Cells were harvested by centrifugation, and lysed by sonication. To facilitate low-level expression of the ideR gene, a XbaI^ BamHI fragment of pIDE2143 harbouring the complete ideR gene and the ribosome binding site of pET3b was cloned into the XbaI^BamHI sites of pWKS129 and pWSK130, yielding respectively pIDE2144a and pIDE2144b. 2.6. Enzyme assays Cell-extracts were prepared by sonication. L-Galactosidase activity was determined according to Miller [16]. Protein was determined according to Bradford using bovine serum albumin as standard [17]. 3. Results and discussion 3.1. Cloning of the ideR gene Alignment of IdeR proteins revealed the presence of two strongly conserved amino acid sequences, which were used to design two oligonucleotides, IDER1 and IDER2 (Fig. 2). A PCR reaction using these oligonucleotides and chromosomal DNA isolated from R. erythropolis and R. equi yielded 236-bp DNA fragments. Nucleotide sequencing of the PCR products (R. erythropolis accession number is GenBank AF277296) revealed a high degree of similarity with IdeR from M. tuberculosis, which con¢rmed that the 236-bp PCR product represents an internal fragment of the ideR gene (Fig. 2). The presence of ideR in two Rhodococcus species suggest that this gene may be common in this genus. Southern hybridisation of chromosomal DNA of R. equi using the 236-bp PCR product as heterologous probe showed that the ideR gene was present on a 4.6-kb PstI fragment (data not shown). A mini-library of PstI fragments ranging in size between 4 and 5 kb was constructed in the PstI site of pDA71; the mini-library was subsequently screened by PCR using oligonucleotides
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3.2. Nucleotide sequence of ideR
Fig. 1. Restriction map of the PstI insert of pIDE21. Arrows indicate the position and direction of transcription of the genes encoding IdeR and GalE
IDER1 and IDER2. This resulted in the isolation of a pDA71 derivative with a 4.6-kb PstI insert, which was subcloned in pBluescript II KS, yielding pIDE21. The ideR gene was localised on pIDE21 by PCR using IDER1 and IDER2 in conjunction with oligonucleotides complementary to lacZ, in which the presence and length of the PCR product indicates the position and direction of transcription of ideR on pIDE21 (Fig. 1).
The nucleotide sequence of ideR and its upstream and downstream regions were determined, and was deposited in GenBank under accession number AF277002. Two open reading frames were identi¢ed, which were preceded by plausible ribosome binding sites and initiated with a GTG instead of an ATG codon. The open reading frames, which were separated by 45 bp, could encode proteins of respectively 25 619 and 30 069 Da. The smaller protein was 75% and 57% identical to the IdeR proteins of M. tuberculosis and C. diphtheriae, respectively. The highest degree of similarity is in the N-terminal DNA-binding domain of the protein, which contains a helix-turn-helix motif [18]. The two K-helices of this motif (residues 29^35 and 40^52, R. equi numbering) are identical to those in other IdeR proteins (Fig. 2). Another highly conserved region of the
Fig. 2. Sequence alignment of IdeR proteins of Streptomyces lividans (JC4512), Streptomyces pilosus (JC4513), Mycobacterium smegmatis (U14190), M. tuberculosis (Z96072), R. equi, R. erythropolis, C. diphtheriae (A35968) and B. lactofermentum (AAA91345). The conserved regions used to design the degenerate oligonucleotides IDER1 and IDER2 are indicated in bold, italicised font. The bars above the alignment indicate the position of the helix-turn-helix motif of IdeR. The residues making up metal binding sites 1 and 2 are indicated with `1' and `2', respectively. Identical amino acid residues are indicated by an asterisk.
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protein contains the metal binding sites: the residues making up metal binding sites I (His81 , Glu85 , His100 ) and II (Cys104 , Glu107 , His108 ) [19] are conserved in the R. equi protein. The larger protein encoded downstream of the putative ideR gene displayed a high degree of similarity to UDP-galactose 4-epimerase (GalE) of C. diphtheriae (67% identity). Although the R. equi IdeR protein is more related to the M. tuberculosis than to the C. diphtheriae protein, the genetic organisation is similar to the latter [20]. The presence of galE downstream from ideR is also encountered in Brevibacterium lactofermentum where the two genes constitute a dicistronic operon [18]. Considering the small intergenic region (45 bp) between ideR and galE in R. equi, it is likely that these too are cotranscribed. 3.3. Expression of ideR in E. coli
Fig. 3. Expression of IdeR in E. coli BL21 DE3 (pLysE). E. coli harbouring pET3b and pIDE2143 were grown on LB medium and induced with IPTG. Cell extracts were subsequently analysed on a 15% denaturing polyacrylamide gel and stained with Coomassie brilliant blue. The arrow indicates the position of the IdeR protein. Lane 1, molecular mass markers : 203, 116, 83, 49, 33, 28, 21 and 8 kDa; lane 2, pET3b ; lane 3, pIDE2143.
Analysis of the nucleotide sequence of ideR showed that the gene has a poor ribosome binding site and starts with a GTG initiation codon. In order to facilitate expression of ideR in E. coli, the initiation codon was replaced by ATG. The resulting mutant ideR was cloned into the expression vector pET3b. Analysis of cell extracts by denaturing acrylamide gel electrophoresis showed that a protein of apparent molecular mass of 29 kDa was expressed in E. coli harbouring the ideR expression vector, but not in the control (Fig. 3). The observed molecular mass of this protein is in agreement with the predicted molecular mass of IdeR. The promoter of the diphtheria toxin gene of C. diphtheriae is controlled by the IdeR homologue DtxR. In order to establish that the IdeR protein of R. equi is functional, the ability of IdeR to repress expression of lacZ fused to the tox promoter of C. diphtheriae (pQFtox) was examined [21]. E. coli harbouring pQFtox was transformed with compatible low copy plasmids containing ideR (pIDE2144a, pIDE2144b). The presence of the ideR gene completely abolished expression of lacZ from the tox promoter. When the experiment was repeated in the presence of the iron chelator 2,2-dipyridyl, repression was partially alleviated (Table 1). This shows that the ideR gene of R. equi encodes an active IdeR protein which is able to bind to the tox promoter in an iron dependent manner.
The DtxR protein of C. diphtheriae recognises a conserved palindromic sequence (5P-TWAGGTTAGSCTAACCTWA-3P) present in for example the tox promoter of C. diphtheriae [22]. Recognition of this sequence is mediated by the helix-turn-helix motif of DtxR, which is conserved in IdeR protein of R. equi (Fig. 2). Furthermore, IdeR of R. equi is able to control expression of the tox promoter, which strongly suggests that the R. equi protein interacts with the same sequence as the DtxR protein. This information will greatly facilitate identi¢cation of genes on the virulence plasmid of R. equi which may be controlled by the concentration of Fe2 . This will enhance our understanding of the mechanisms by which this pathogen establishes itself in the host.
Table 1 L-Galactosidase activities in E. coli harbouring pQFtox in the presence or absence of the ideR gene following overnight growth in LB medium
[1] Mosser, D.M. and Hondalus, M.K. (1996) Rhodococcus equi: an emerging opportunistic pathogen. Trends Microbiol. 4, 29^33. [2] Takai, S., Sekizaki, T., Ozawa, T., Sugawara, T., Watanabe, Y. and Tsubaki, S. (1991) Association between a large plasmid and 15- to 17-kilodalton antigens in virulent Rhodococcus equi. Infect. Immun. 59, 4056^4060. [3] Hondalus, M.K. and Mosser, D.M. (1994) Survival and replication of Rhodococcus equi in macrophages. Infect. Immun. 62, 4167^4175. [4] Giguere, S., Hondalus, M.K., Yager, J.A., Darrah, P., Mosser, D.M. and Prescott, J.F. (1999) Role of the 85-kilobase plasmid and plasmid-encoded virulence-associated protein A in intracellular survival and virulence of Rhodococcus equi. Infect. Immun. 67, 3548^3557. [5] Takai, S., Iie, M., Watanabe, Y., Tsubaki, S. and Sekizaki, T. (1992) Virulence-associated 15- to 17-kilodalton antigens in Rhodococcus
Plasmid
2,2-Dipyridyl (300 WM)
L-Galactosidase activity nmol (min mg protein)31
pQFtox+pWSK129 pQFtox+pIDE2144a pQFtox+pIDE2144b pQFtox+pIDE2144a pQFtox+pIDE2144b
3 3 3 + +
41 0 0 8 10
The iron chelator 2,2-dipyridyl was added for low Fe2 growth conditions.
Acknowledgements The authors thank R. van der Geize for a gift of chromosomal DNA of R. erythropolis, and E.R. Dabbs and M. Schmitt for making pDA71 and pQFtox available. This study was supported by the President's Research Award and Enterprise Ireland, Grant SC/1998/303
References
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equi: temperature-dependent expression and location of the antigens. Infect. Immun. 60, 2995^2997. Wooldridge, K.G. and Williams, P.H. (1993) Iron uptake mechanisms of pathogenic bacteria. FEMS Microbiol Rev. 12, 325^348. Schmitt, M.P., Predich, M., Doukhan, L., Smith, I. and Holmes, R.K. (1995) Characterization of an iron-dependent regulatory protein (IdeR) of Mycobacterium tuberculosis as a functional homolog of the diphtheria toxin repressor (DtxR) from Corynebacterium diphtheriae. Infect. Immun. 63, 4284^4289. Tao, X., Schiering, N., Zeng, H.Y., Ringe, D. and Murphy, J.R. (1994) Iron, DtxR, and the regulation of diphtheria toxin expression. Mol. Microbiol. 14, 191^197. Manabe, Y.C., Saviola, B.J., Sun, L., Murphy, J.R. and Bishai, W.R. (1999) Attenuation of virulence in Mycobacterium tuberculosis expressing a constitutively active iron repressor. Proc. Natl. Acad. Sci. USA 96, 12844^12848. Dussurget, O., Rodriguez, M. and Smith, I. (1996) An ideR mutant of Mycobacterium smegmatis has derepressed siderophore production and an altered oxidative-stress response. Mol. Microbiol. 22, 535^ 544. Powell, J.A. and Archer, J.A. (1998) Molecular characterisation of a Rhodococcus ohp operon. Antonie van Leeuwenhoek 74, 175^188. Dabbs, E.R., Gowan, B., Quan, S., Anderson, S.J. (1995) Development of improved Rhodococcus plasmid vectors and their use in cloning genes of potential commercial and medical importance. Biotekhnologiya 7^8, 129^135. Wang, R.F. and Kushner, S.R. (1991) Construction of versatile lowcopy-number vectors for cloning, sequencing and gene expression in Escherichia coli. Gene 100, 195^199. Birnboim, H.C. and Doly, J. (1979) A rapid alkaline extraction procedure for screening of recombinant plasmid DNA. Nucleic Acids Res. 7, 1513^1523.
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[15] Nagy, I., Schoofs, G., Compernolle, F., Proost, P., Vanderleyden, J. and De Mot, R. (1995) Degradation of the thiocarbamate herbicide EPTC (S-ethyl dipropylcarbamothioate) and biosafening by Rhodococcus sp. strain NI86/21 involve an inducible cytochrome P-450 system and aldehyde dehydrogenase. J. Bacteriol. 177, 676^687. [16] Miller, J.H. (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [17] 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. 73, 248^254. [18] Oguiza, J.A., Marcos, A.T., Malumbres, M. and Martin, J.F. (1996) The galE gene encoding the UDP-galactose 4-epimerase of Brevibacterium lactofermentum is coupled transcriptionally to the dmdR gene. Gene 177, 103^107. [19] Schiering, N., Tao, X., Zeng, H., Murphy, J.R., Petsko, G.A. and Ringe, D. (1995) Structures of the apo- and the metal ion-activated forms of the diphtheria tox repressor from Corynebacterium diphtheriae. Proc. Natl. Acad. Sci. USA 92, 9843^9850. [20] Boyd, J., Oza, M.N. and Murphy, J.R. (1990) Molecular cloning and DNA sequence analysis of a diphtheria tox iron-dependent regulatory element (dtxR) from Corynebacterium diphtheriae. Proc. Natl. Acad. Sci. USA 87, 5968^5972. [21] Schmitt, M.P. and Holmes, R.K. (1994) Cloning, sequence, and footprint analysis of two promoter/operators from Corynebacterium diphtheriae that are regulated by the diphtheria toxin repressor (DtxR) and iron. J. Bacteriol. 176, 1141^1149. [22] Tao, X. and Murphy, J.R. (1994) Determination of the minimal essential nucleotide sequence for diphtheria tox repressor binding by in vitro a¤nity selection. Proc. Natl. Acad. Sci. USA 91, 9646^ 9650.
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The iron dependent regulatory protein IdeR (DtxR) of Rhodococcus equi Clara A. Boland, Wim G. Meijer * Department of Industrial Microbiology, Conway Institute of Biomolecular and Biomedical Research, National University of Ireland, Dublin, Dublin 4, Ireland Received 26 June 2000 ; accepted 24 July 2000
Abstract This paper reports the presence of an ideR gene, which encodes an iron-dependent regulatory protein, in Rhodococcus erythropolis and in the intracellular pathogen Rhodococcus equi. The ideR gene of the latter encoded a protein of 230 amino acids with a molecular mass of 25 619. The K-helices forming the helix-turn-helix motif of the R. equi protein were identical to those of the DtxR protein of Corynebacterium diphtheriae, which is an IdeR homologue. This indicates that the two proteins bind to the same DNA binding site. This was confirmed following expression of IdeR in Escherichia coli, which showed that the IdeR protein could repress transcription of the tox promoter of C. diphtheriae in an iron dependent manner. An open reading frame specifying a 283-amino acid polypeptide similar to galE encoding UDP-galactose 4-epimerase was present downstream of the ideR gene. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : IdeR; DtxR; Iron repressor ; GalE; Rhodococcus equi
1. Introduction A number of pathogenic bacteria are able to grow within macrophages, which represent the ¢rst line of defence against invading micro-organisms. Examples of these are the actinomycetes Mycobacterium tuberculosis and the closely related Rhodococcus equi, which are mycolic acidcontaining, Gram-positive bacteria sharing many physiological and pathological features [1]. Although originally identi¢ed as one of the most important pathogens of foals, R. equi has more recently been recognised as a pathogen of immunocompromised humans [1]. Virulent strains of R. equi harbour a large plasmid, which is essential for survival and growth in alveolar macrophages [2^4]. Virulence genes are usually expressed in response to changing environmental parameters which signal entry into the host. For example, expression of VapA, a small surface protein
* Corresponding author. Tel. : +353 (1) 706-1512; Fax: +353 (1) 706-1183; E-mail: [email protected]
which is encoded on the virulence plasmid of R. equi, is controlled by pH and temperature [5]. Following uptake by their hosts, pathogens encounter extremely low concentrations of free iron, which is largely due to iron binding proteins such as lactoferrin and transferrin [6]. It is therefore not surprising that the expression of a large number of virulence factors by pathogenic bacteria are controlled by iron. Regulation of gene expression by iron in M. tuberculosis is controlled by the global iron regulator IdeR, which is a homologue of the diphtheria toxin repressor DtxR of Corynebacterium diphtheriae [7,8]. Following binding of Fe2 , these proteins bind to their target DNA sequences as a homodimer, resulting in repression of gene expression [8]. In addition to controlling genes involved in iron uptake, IdeR also controls expression of virulence factors in C. diphtheriae and M. tuberculosis [8^10]. Although R. equi is an important veterinary pathogen, virtually nothing is known about the regulation of gene expression in this bacterium. Since Rhodococcus species are closely related to Mycobacterium and Corynebacterium, it is likely that they employ an iron repressor protein related to IdeR to control iron-dependent gene expression. This paper reports the presence of ideR in Rhodococcus erythropolis and R. equi. The ideR gene of the latter was cloned and characterised.
0378-1097 / 00 / $20.00 ß 2000 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 0 ) 0 0 3 5 4 - 2
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2. Materials and methods 2.1. Bacterial strains and plasmids Escherichia coli DH5K (Bio-Rad), E. coli BL21 DE3 (pLysE) (Novagen), R. erythropolis SQ1 [11], R. equi ATCC 33701, pBluescript II KS (Stratagene), pDA71 [12], pET3b (Novagen), pWKS129 [13], pWSK130 [13]. 2.2. Media and growth conditions E. coli DH5K, E. coli BL21 DE3 (pLysE) and R. equi were grown on Luria^Bertani (LB) medium at 37³C. The chelator 2,2-dipyridyl was added to the medium to achieve low Fe2 growth conditions. When appropriate the following supplements were added: ampicillin, 50 Wg ml31 ; chloramphenicol, 34 Wg ml31 ; isopropyl-L-D-thiogalactoside (IPTG), 0.1 mM; X-Gal, 20 Wg ml31 . 2.3. DNA manipulations Plasmid DNA was isolated with the alkaline lysis method [14] or by using the Wizard Plus SV miniprep (Promega). Chromosomal DNA was isolated as described [15]. DNA modifying enzymes were used according to the manufacturer's recommendations (Roche). An internal fragment of the ideR gene was ampli¢ed by PCR with Taq polymerase as recommended by the manufacturer (Promega) using the oligonucleotides 5P-GAYACNACNGARATGTA-3P (IDER1) and 5P-CTCATNACRTGYTCCCA-3P (IDER2). The reaction mixture was incubated at 95³C for 5 min and was subsequently subjected to 30 cycles of 95³C for 45 s, 37³C for 45 s, 74³C for 45 s, followed by an incubation at 74³C for 7 min. Dideoxy sequencing reactions were done with the CEQ DCTS kit as described by the manufacturer (Beckman). The nucleotide sequence was determined using a Beckman CEQ2000 automatic sequencer. The nucleotide sequence data were compiled and analysed using the Wisconsin Package Version 8 (Genetics Computer Group). 2.4. Southern hybridisation Chromosomal DNA of R. equi was digested with PstI, EcoRI, and BamHI, and transferred to a positively charged nylon membrane as recommended by the manufacturer (Roche) following agarose gel electrophoresis. DNA used as a probe, was labelled with digoxigenin 11dUTP using the DIG High Prime kit (Roche). Prehybridisation, hybridisation and detection of the labelled probe were done according to the manufacturer's recommendations. 2.5. Expression of ideR in E. coli The 5P-end of ideR was ampli¢ed by PCR from
pIDE214 (Fig. 1), which harbours a 1.2-kb SacI fragment of pIDE21 in the SacI site of pBluescript II KS. The oligonucleotides used in the PCR reactions were : 5P-GTAAAACGACGGCCAGT-3P and 5P-GCGCATATGAAGGATCTGTCGACACCACGG-3P, which introduces an NdeI restriction site in the initiation codon of ideR. The resulting PCR fragment was cloned into the EcoRV site of pBluescript II KS, yielding pIDE2141. A 1.6-kb SacI fragment of pIDE21 containing the 3P end of ideR was cloned into the SacI site of pIDE2141 and the complete ideR gene was subsequently cloned as a NdeI^BamHI fragment into NdeI^BamHI-digested pET3b yielding pIDE2143. E. coli BL21 DE3 (pLysE) harbouring pIDE2143 was grown at 30³C until OD660 = 0.5. Expression of ideR from the T7 promoter of pIDE2143 was induced by adding IPTG (1 mM) to the culture medium ; growth was allowed to proceed for an additional 3 h. Cells were harvested by centrifugation, and lysed by sonication. To facilitate low-level expression of the ideR gene, a XbaI^ BamHI fragment of pIDE2143 harbouring the complete ideR gene and the ribosome binding site of pET3b was cloned into the XbaI^BamHI sites of pWKS129 and pWSK130, yielding respectively pIDE2144a and pIDE2144b. 2.6. Enzyme assays Cell-extracts were prepared by sonication. L-Galactosidase activity was determined according to Miller [16]. Protein was determined according to Bradford using bovine serum albumin as standard [17]. 3. Results and discussion 3.1. Cloning of the ideR gene Alignment of IdeR proteins revealed the presence of two strongly conserved amino acid sequences, which were used to design two oligonucleotides, IDER1 and IDER2 (Fig. 2). A PCR reaction using these oligonucleotides and chromosomal DNA isolated from R. erythropolis and R. equi yielded 236-bp DNA fragments. Nucleotide sequencing of the PCR products (R. erythropolis accession number is GenBank AF277296) revealed a high degree of similarity with IdeR from M. tuberculosis, which con¢rmed that the 236-bp PCR product represents an internal fragment of the ideR gene (Fig. 2). The presence of ideR in two Rhodococcus species suggest that this gene may be common in this genus. Southern hybridisation of chromosomal DNA of R. equi using the 236-bp PCR product as heterologous probe showed that the ideR gene was present on a 4.6-kb PstI fragment (data not shown). A mini-library of PstI fragments ranging in size between 4 and 5 kb was constructed in the PstI site of pDA71; the mini-library was subsequently screened by PCR using oligonucleotides
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3.2. Nucleotide sequence of ideR
Fig. 1. Restriction map of the PstI insert of pIDE21. Arrows indicate the position and direction of transcription of the genes encoding IdeR and GalE
IDER1 and IDER2. This resulted in the isolation of a pDA71 derivative with a 4.6-kb PstI insert, which was subcloned in pBluescript II KS, yielding pIDE21. The ideR gene was localised on pIDE21 by PCR using IDER1 and IDER2 in conjunction with oligonucleotides complementary to lacZ, in which the presence and length of the PCR product indicates the position and direction of transcription of ideR on pIDE21 (Fig. 1).
The nucleotide sequence of ideR and its upstream and downstream regions were determined, and was deposited in GenBank under accession number AF277002. Two open reading frames were identi¢ed, which were preceded by plausible ribosome binding sites and initiated with a GTG instead of an ATG codon. The open reading frames, which were separated by 45 bp, could encode proteins of respectively 25 619 and 30 069 Da. The smaller protein was 75% and 57% identical to the IdeR proteins of M. tuberculosis and C. diphtheriae, respectively. The highest degree of similarity is in the N-terminal DNA-binding domain of the protein, which contains a helix-turn-helix motif [18]. The two K-helices of this motif (residues 29^35 and 40^52, R. equi numbering) are identical to those in other IdeR proteins (Fig. 2). Another highly conserved region of the
Fig. 2. Sequence alignment of IdeR proteins of Streptomyces lividans (JC4512), Streptomyces pilosus (JC4513), Mycobacterium smegmatis (U14190), M. tuberculosis (Z96072), R. equi, R. erythropolis, C. diphtheriae (A35968) and B. lactofermentum (AAA91345). The conserved regions used to design the degenerate oligonucleotides IDER1 and IDER2 are indicated in bold, italicised font. The bars above the alignment indicate the position of the helix-turn-helix motif of IdeR. The residues making up metal binding sites 1 and 2 are indicated with `1' and `2', respectively. Identical amino acid residues are indicated by an asterisk.
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protein contains the metal binding sites: the residues making up metal binding sites I (His81 , Glu85 , His100 ) and II (Cys104 , Glu107 , His108 ) [19] are conserved in the R. equi protein. The larger protein encoded downstream of the putative ideR gene displayed a high degree of similarity to UDP-galactose 4-epimerase (GalE) of C. diphtheriae (67% identity). Although the R. equi IdeR protein is more related to the M. tuberculosis than to the C. diphtheriae protein, the genetic organisation is similar to the latter [20]. The presence of galE downstream from ideR is also encountered in Brevibacterium lactofermentum where the two genes constitute a dicistronic operon [18]. Considering the small intergenic region (45 bp) between ideR and galE in R. equi, it is likely that these too are cotranscribed. 3.3. Expression of ideR in E. coli
Fig. 3. Expression of IdeR in E. coli BL21 DE3 (pLysE). E. coli harbouring pET3b and pIDE2143 were grown on LB medium and induced with IPTG. Cell extracts were subsequently analysed on a 15% denaturing polyacrylamide gel and stained with Coomassie brilliant blue. The arrow indicates the position of the IdeR protein. Lane 1, molecular mass markers : 203, 116, 83, 49, 33, 28, 21 and 8 kDa; lane 2, pET3b ; lane 3, pIDE2143.
Analysis of the nucleotide sequence of ideR showed that the gene has a poor ribosome binding site and starts with a GTG initiation codon. In order to facilitate expression of ideR in E. coli, the initiation codon was replaced by ATG. The resulting mutant ideR was cloned into the expression vector pET3b. Analysis of cell extracts by denaturing acrylamide gel electrophoresis showed that a protein of apparent molecular mass of 29 kDa was expressed in E. coli harbouring the ideR expression vector, but not in the control (Fig. 3). The observed molecular mass of this protein is in agreement with the predicted molecular mass of IdeR. The promoter of the diphtheria toxin gene of C. diphtheriae is controlled by the IdeR homologue DtxR. In order to establish that the IdeR protein of R. equi is functional, the ability of IdeR to repress expression of lacZ fused to the tox promoter of C. diphtheriae (pQFtox) was examined [21]. E. coli harbouring pQFtox was transformed with compatible low copy plasmids containing ideR (pIDE2144a, pIDE2144b). The presence of the ideR gene completely abolished expression of lacZ from the tox promoter. When the experiment was repeated in the presence of the iron chelator 2,2-dipyridyl, repression was partially alleviated (Table 1). This shows that the ideR gene of R. equi encodes an active IdeR protein which is able to bind to the tox promoter in an iron dependent manner.
The DtxR protein of C. diphtheriae recognises a conserved palindromic sequence (5P-TWAGGTTAGSCTAACCTWA-3P) present in for example the tox promoter of C. diphtheriae [22]. Recognition of this sequence is mediated by the helix-turn-helix motif of DtxR, which is conserved in IdeR protein of R. equi (Fig. 2). Furthermore, IdeR of R. equi is able to control expression of the tox promoter, which strongly suggests that the R. equi protein interacts with the same sequence as the DtxR protein. This information will greatly facilitate identi¢cation of genes on the virulence plasmid of R. equi which may be controlled by the concentration of Fe2 . This will enhance our understanding of the mechanisms by which this pathogen establishes itself in the host.
Table 1 L-Galactosidase activities in E. coli harbouring pQFtox in the presence or absence of the ideR gene following overnight growth in LB medium
[1] Mosser, D.M. and Hondalus, M.K. (1996) Rhodococcus equi: an emerging opportunistic pathogen. Trends Microbiol. 4, 29^33. [2] Takai, S., Sekizaki, T., Ozawa, T., Sugawara, T., Watanabe, Y. and Tsubaki, S. (1991) Association between a large plasmid and 15- to 17-kilodalton antigens in virulent Rhodococcus equi. Infect. Immun. 59, 4056^4060. [3] Hondalus, M.K. and Mosser, D.M. (1994) Survival and replication of Rhodococcus equi in macrophages. Infect. Immun. 62, 4167^4175. [4] Giguere, S., Hondalus, M.K., Yager, J.A., Darrah, P., Mosser, D.M. and Prescott, J.F. (1999) Role of the 85-kilobase plasmid and plasmid-encoded virulence-associated protein A in intracellular survival and virulence of Rhodococcus equi. Infect. Immun. 67, 3548^3557. [5] Takai, S., Iie, M., Watanabe, Y., Tsubaki, S. and Sekizaki, T. (1992) Virulence-associated 15- to 17-kilodalton antigens in Rhodococcus
Plasmid
2,2-Dipyridyl (300 WM)
L-Galactosidase activity nmol (min mg protein)31
pQFtox+pWSK129 pQFtox+pIDE2144a pQFtox+pIDE2144b pQFtox+pIDE2144a pQFtox+pIDE2144b
3 3 3 + +
41 0 0 8 10
The iron chelator 2,2-dipyridyl was added for low Fe2 growth conditions.
Acknowledgements The authors thank R. van der Geize for a gift of chromosomal DNA of R. erythropolis, and E.R. Dabbs and M. Schmitt for making pDA71 and pQFtox available. This study was supported by the President's Research Award and Enterprise Ireland, Grant SC/1998/303
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