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The response regulator Spo0A from Bacillus subtilis is efficiently phosphorylated in Escherichia coli
FEMS Microbiology Letters 223 (2003) 153^157
www.fems-microbiology.org
The response regulator Spo0A from Bacillus subtilis is e⁄ciently phosphorylated in Escherichia coli Joanne C. Ladds a , Katar|¤na Muchova¤ b , Dus›an Blas›kovic› b , Richard J. Lewis c , James A. Brannigan a , Anthony J. Wilkinson a , Imrich Bara¤k b; a
Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5YW, UK Institute of Molecular Biology, Slovak Academy of Sciences, Du¤bravska¤ cesta 21, 84251 Bratislava, Slovak Republic Laboratory of Molecular Biophysics, The Rex Richards Building, University of Oxford, South Parks Road, Oxford OX1 3QU, UK b
c
Received 27 February 2003; received in revised form 6 April 2003 ; accepted 9 April 2003 First published online 28 May 2003
Abstract The response regulator proteins of two-component systems mediate many adaptations of bacteria to their ever-changing environment. Most response regulators are transcription factors that alter the level of transcription of specific sets of genes. Activation of response regulators requires their phosphorylation on a conserved aspartate residue by a cognate sensor kinase. For this reason, expression of a recombinant response regulator in the absence of the requisite sensor kinase is expected to yield an unphosphorylated product in the inactive state. For Spo0A, the response regulator controlling sporulation in Bacillus subtilis however, we have found that a significant fraction of the purified recombinant protein is phosphorylated. This phosphorylated component is dimeric and binds to Spo0A recognition sequences in DNA. Treatment with the Spo0A-specific phosphatase, Spo0E, leads to dissociation of the dimers and loss of DNA binding. It is therefore necessary to pre-treat recombinant Spo0A preparations with the cognate phosphatase, to generate the fully inactive state of the molecule. 8 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Spo0A; Phosphorylation; Response regulator; Histidine kinase ; Bacillus subtilis
1. Introduction Spo0A plays a crucial role in the regulation of gene expression during sporulation, a developmental process triggered in Bacillus subtilis as a response to a deteriorating environment and starvation. Extracellular signals are monitored by a set of sensor kinases and transduced, through an expanded two-component system called a phosphorelay, into a chemical event, the phosphorylation of Spo0A [1]. Phosphorylated Spo0A directly or indirectly in£uences the transcription of hundreds of genes as sporulation commences [2]. Spo0A consists of a phosphoacceptor or receiver domain (N-Spo0A) and a DNA binding or e¡ector (CSpo0A) domain. It becomes active as a transcription factor after phosphorylation of the receiver domain on a * Corresponding author. Tel. : +421 (2) 59307418; Fax : +421 (2) 59307416. E-mail addresses : [email protected] (I. Bara¤k), [email protected] (I. Bara¤k).
conserved aspartate [3,4]. Dephosphorylation, catalysed by the Spo0AVP-speci¢c phosphatase, Spo0E, reverses this activation [5,6]. The capacity of fragments of Spo0A encompassing the e¡ector domain to bind to DNA and activate transcription [7] together with the constitutive activity of Spo0A mutants, carrying large deletions in the receiver domain [3], leads to the conclusion that in intact Spo0A, the receiver domain inhibits the e¡ector domain and phosphorylation overcomes this inhibition. It is also clear that, upon phosphorylation, Spo0A forms dimers and that these dimers represent the activated form [8]. The crystal structure of N-Spo0A from Bacillus stearothermophilus has been solved using recombinant protein puri¢ed from Escherichia coli. To our surprise the active site aspartate was phosphorylated indicating that heterologous phosphorylation of the protein had taken place in E. coli [9]. Here we show that heterologous phosphorylation extends to the better characterised Spo0A from B. subtilis and demonstrate that heterologously phosphorylated Spo0A forms dimers and binds to cognate DNA sequences.
0378-1097 / 03 / $22.00 8 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S0378-1097(03)00321-5
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J.C. Ladds et al. / FEMS Microbiology Letters 223 (2003) 153^157
2. Materials and methods
3. Results and discussion
2.1. Spo0A isolation and puri¢cation
To investigate the level of phosphorylation in preparations of B. subtilis Spo0A isolated from E. coli (Spo0AEc), we used gel ¢ltration chromatography and native polyacrylamide gel electrophoresis assays. Gel ¢ltration chromatograms of Spo0AEc and Spo0A subsequently phosphorylated by phosphoramidate (Spo0AVP) are shown in Fig. 1. Spo0AEc eluted from the Pharmacia Superdex 75 HR10/30 gel ¢ltration column as two distinct peaks with elution volumes of 9.4 ml and 10.7 ml, corresponding to the dimeric and monomeric forms of Spo0A, respectively [8]. Spo0AVP eluted from this column with an elution volume of 9.5 ml, corresponding to the dimeric form. The earlier eluting peak in the Spo0AEc chromatogram therefore suggests that a signi¢cant fraction of this protein is phosphorylated. This is con¢rmed by the observation that treatment of Spo0AEc with the Spo0AVP-speci¢c phosphatase, Spo0E94, leads to loss of the fast eluting peak, and enhancement of the slower migrating peak which corresponds to the monomer. Mild tryptic digestion of Spo0A produces two fragments, N-Spo0A and C-Spo0A [10,12], which can be easily resolved by electrophoresis in polyacrylamide gels under non-denaturing conditions [8]. In these gels, N-Spo0AVP dimers resulting from digestion of Spo0AVP with trypsin migrate as a single band with an Rf value (relative to bromophenol blue) of 0.48 (Fig. 2, lane 3). Pre-treatment of Spo0AVP with Spo0E94, prior to tryptic cleavage and gel analysis, produces a faster migrating species with Rf = 0.56 (Fig. 2, lane 4). N-Spo0AEc resulting from the tryptic digestion of Spo0AEc migrates as two distinct bands, corresponding to phosphorylated dimers (Rf = 0.48) and small amount of unphosphorylated monomers (Rf = 0.56) (Fig. 2, lane 1). After dephosphorylation
Spo0A was overexpressed and puri¢ed as described previously [8]. Spo0A samples (20^200 WM) were phosphorylated in a bu¡er containing 20 mM Tris^HCl pH 7.5, 20 mM MgCl2 , 1 mM dithiothreitol (DTT) and 50 mM phosphoramidate over a period of 30 min at 37‡C. Spo0AVP samples were dephosphorylated by incubation in the presence of 5 WM of a hyperactive mutant form of Spo0E, Spo0E94, for 30 min at 37‡C [5,8]. To prepare N-Spo0A fragments, samples of Spo0A were incubated with trypsin at a molar ratio of 2500:1 for 30 min at 16‡C [10] and the products were analysed immediately by non-denaturing 12.5% polyacrylamide gel electrophoresis. 2.2. Preparation of DNA 100 ng (11 pmol) of the oligonucleotide ABRBTOP (5PGGATTTTGTCGAATAATGACGAAGAAAAAT-3P) was heated for 5 min at 95‡C, and rapidly cooled in an ice bath prior to its 5P end labelling with polynucleotide kinase and 27 WCi (3.86 pmol) [Q-32 P]ATP (ICN, 7000 Ci mmol31 ) in a total volume of 25 Wl. The radiolabelled ABRBTOP was incubated with an equimolar amount of ABRBBOT (5P-ATTTTTCTTCGTCATTATTCGACAAAATCC-3P) and the oligonucleotide pair was annealed by heating for 5 min at 95‡C and 20 min at 65‡C before slow cooling to 20‡C. Unincorporated radionucleotides were removed from duplex DNA by passage through a Sephadex G-50 microtip column. A non-speci¢c competitor DNA was prepared by annealing equimolar amounts of the ATNESCC (5P-TTAATATTCCTTATATAT-3P) and ATNESGG (5P-ATATATAAGGAATATTAA-3P) oligonucleotides. 2.3. Electrophoretic mobility shift assay Gel mobility shift assays were performed essentially as described previously [11]. Samples of Spo0A (100 ng) were incubated with 43 000 cpm (0.19 ng) 32 P-labelled abrB-speci¢c DNA in the absence or presence of the competitor DNA (unlabelled abrB-speci¢c DNA) for 15 min at 37‡C in a solution containing 40 mM Tris-acetate pH 8.0, 40 mM sodium acetate, 10 mM magnesium acetate, 0.1 mM DTT, 10 Wg ml31 bovine serum albumin (BSA), 1 mM EDTA, and 5% (v/v) glycerol in a total volume of 20 Wl. The binding mix was loaded onto an 8% polyacrylamide gel prepared in 1UTAE bu¡er (40 mM Tris, 20 mM acetic acid, 1 mM EDTA pH 7.9, containing 2% (v/v) glycerol). The gel was run at room temperature and 150 V in 1UTAE bu¡er until the bromophenol blue dye had migrated to within 2 cm of the bottom of the gel. The gel was then dried and analysed by autoradiography.
Fig. 1. Sephadex 75HR 10/30 gel ¢ltration chromatograms of B. subtilis Spo0A isolated from E. coli. The column was equilibrated in a bu¡er of 20 mM Tris^HCl pH 7.5, 250 mM NaCl, 2 mM EDTA, 2 mM DTT. The continuous line represents Spo0A protein puri¢ed from E. coli (Spo0AEc), dashed line Spo0AVP (Spo0AEc after incubation at 37‡C for 30 min with 50 mM phosphoramidate). The samples contained Spo0A at a concentration of 70 WM. The dotted line is the chromatogram for a mixture of molecular weight standards, BSA (67 kDa), ovalbumin (43 kDa), chymotrypsinogen (25 kDa) and ribonuclease A (14 kDa). Blue dextran (2 000 Da) was used in order to determine the void volume of the column.
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J.C. Ladds et al. / FEMS Microbiology Letters 223 (2003) 153^157
Fig. 2. Examination of the oligomeric state of Spo0A using oligomeric state using non-denaturing 12.5% gel electrophoresis. Each lane contains 8 Wg of protein. Analysis of Spo0A fragments resulting from proteolysis of intact Spo0AEc with trypsin in a ratio 2500:1. Lanes: (1) Spo0AEc, (2) Spo0AEc+0E (Spo0AEc after incubation with Spo0A-speci¢c phosphatase Spo0E94), (3) Spo0AVP (Spo0AEc after incubation with 50 mM phosphoramidate), (4) Spo0AVP+0E (Spo0AEc after phosphoramidate treatment as above followed by dephosphorylation with Spo0E94). Note that C-Spo0A stays stuck at the top of the gel.
of Spo0AEc with the phosphatase Spo0E, this sample (Spo0AEc+0E) was completely converted to the dephosphorylated monomeric form (Fig. 2, lane 2). These experiments reinforce the conclusion (i) that there is a system in E. coli which heterologously phosphorylates Spo0A and (ii) that Spo0AVP is very stable with a half-life extending over the several days that it takes to purify the protein to homogeneity. Analysis of samples of N-Spo0AVP ‘aged’ in 20 mM Tris^HCl pH 7.5, 20 mM MgCl2 , 1 mM DTT at room temperature over 5 days, in similar native gel electrophoresis assays to those described above, con¢rms the latter conclusion (data not shown). To assay whether Spo0AEc is able to bind site-speci¢cally to DNA, we performed DNA gel shift experiments using a synthetic duplex DNA derived from the abrB promoter region and containing two 0A-box sequences (TGNCGAA). These experiments reveal that 0.166 WM Spo0AEc is able to cause retardation of a signi¢cant proportion of the abrB promoter fragment when present at 0.45 nM (Fig. 3, lane 1); moreover, the extent of the gel mobility shifts is similar to that mediated by Spo0A that has been phosphorylated using phosphoramidate (Fig. 3, lane 4). Treatment of both Spo0AEc and Spo0AVP with Spo0E abolished DNA binding completely (Fig. 3, lane 7). The intensities of the bands in the autoradiograph are consistent with slightly weaker binding of Spo0AEc in comparison to the in vitro phosphorylated Spo0AVP, consistent with the incomplete phosphorylation of the former. Previous reports suggested that unphosphorylated Spo0A binds detectably to the 0A-box regions of a set of promoters and that the extent of binding is increased V20-fold by phosphorylation [7,13^15]. Our ¢nding here
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is that unphosphorylated Spo0A does not bind to abrB promoter DNA. An obvious explanation for the di¡erent observations is that a proportion of the Spo0A that was assumed to be unphosphorylated in the earlier studies was in fact heterologously phosphorylated. If this explanation is correct the reported V20 increase in DNA-binding activity attributable to phosphorylation in Spo0A would be an underestimate. We estimate from our own experiments that DNA binding is stimulated 50^100-fold by phosphorylation. The experiments reported here show that Spo0A expressed in E. coli is heterologously phosphorylated and functional in DNA-binding. This phosphorylation may result from the action of sensor kinases present in E. coli, such as EnvZ, CheA or NRII [16,17] or by the action of small molecule phosphodonors [18]. It has been shown that Spo0A can be phosphorylated e¡ectively in vitro with an N-terminally truncated form of phosho-EnvZ [16]. The phosphotransferase domains of EnvZ and Spo0B, the phosphotransferase that mediates phosphotransfer to Spo0A in B. subtilis, have similar structures [19,20]. There are about 100 molecules EnvZ in an E. coli cell [21], so signi¢cant phosphorylation of highly expressed Spo0A would require a high turnover number. However, other histidine kinases may be involved. The surfaces of B. subtilis response regulators that contact the sensor kinase phosphotransferase domains have been analysed in detail [22,23]. In Fig. 4, we have compared the amino acid res-
Fig. 3. Gel mobility shift assays of Spo0A binding to abrB promoter DNA. Speci¢ed Spo0A preparations and competitor DNAs were added as: Spo0AEc (lane 1), Spo0AEc in the presence of speci¢c competitor (lane 2), Spo0AEc in the presence of non-speci¢c competitor (lane 3), Spo0AVP (lane 4), Spo0AVP in the presence of speci¢c competitor (lane 5), Spo0AVP in the presence of non-speci¢c competitor (lane 6), Spo0AEc+0E (lane 7), Spo0AEc+0E in the presence of speci¢c competitor (lane 8), Spo0AEc+0E in the presence of non-speci¢c competitor (lane 9). Each lane contains 100 ng of protein and 0.19 ng of abrB promoter DNA.
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Fig. 4. Comparison of interacting residues in Spo0A with the residues at the similar positions in all known response regulators in E. coli. Residues are numbered as in Spo0A. Conserved residues are in black boxes; similar residues are in capital bold letters and all other residues are in small letters.
idues in Spo0A inferred to be in contact with Spo0B with the residues at the corresponding positions in all the response regulators found in E. coli. It has been proposed that residues corresponding to Phe88, Gly89 and Gln90 in Spo0A are important in de¢ning the speci¢city of the interaction with cognate sensor kinases [23]. Our comparison does not obviously reveal an E. coli response regulator with a speci¢city-de¢ning region closely similar to that in Spo0A, and we cannot easily point to a particular sensor kinase as a candidate agent for the heterologous phosphorylation we have observed. The studies described here show that it is necessary to pre-treat recombinant Spo0A preparations with the cognate phosphatase, to generate the authentic inactive, unphosphorylated state of the molecule. It seems unlikely that heterologous phosphorylation of two-component system response regulators in E. coli will be con¢ned to Spo0A. However, the much shorter half-lives of the phosphorylated forms of most other response regulators (seconds to minutes) relative to that of Spo0A (hours to days) means that the phosphorylated form is less likely to persist through the steps of protein puri¢cation. In general however, it should not be assumed that puri¢ed recombinant response regulator proteins are fully unphosphorylated.
Acknowledgements We would like to thank Dusan Perecko for technical
assistance with electrophoretic mobility shift assays. We thank Marta Perego for providing us with a clone directing overexpression of Spo0Ev94. This work was supported by The Wellcome Trust Grants, the Grant 2/1004/21 from the Slovak Academy of Sciences and The Royal Society International Exchanges Project. R.J.L. is currently the recipient of a Wellcome Trust Research Career Development Fellowship.
References [1] Hoch, J.A. (1993) The phosphorelay signal transduction pathway in the initiation of Bacillus subtilis sporulation. J. Cell. Biochem. 51, 55^61. [2] Fawcett, P., Eichenberger, P., Losick, R. and Youngman, P. (2000) The transcriptional pro¢le of early to middle sporulation in Bacillus subtilis. Proc. Natl. Acad. Sci. USA 97, 8063^8068. [3] Ireton, K., Rudner, D.Z., Siranosian, K.J. and Grossman, A.D. (1993) Integration of multiple developmental signals in Bacillus subtilis through the Spo0A transcription factor. Genes Dev. 7, 283^294. [4] Green, B.D., Olmedo, G. and Youngman, P. (1991) A genetic analysis of spo0A structure, function. Res. Microbiol. 142, 825^830. [5] Ohlsen, K.L., Grimsley, J.K. and Hoch, J.A. (1994) Deactivation of the sporulation transcription factor Spo0A by the Spo0E protein phosphatases. Proc. Natl. Acad. Sci. USA 91, 1756^1760. [6] Perego, M. (2001) A new family of aspartyl phosphates targeting the sporulation transcription factor Spo0A of Bacillus subtilis. Mol. Microbiol. 42, 133^143. [7] Rowe-Magnus, D.A. and Spiegelman, G.B. (1998) Contributions of the domains of the Bacillus subtilis response regulator Spo0A to transcription stimulation of the spoIIG operon. J. Biol. Chem. 273, 25818^25824.
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J.C. Ladds et al. / FEMS Microbiology Letters 223 (2003) 153^157 [8] Lewis, R.J., Scott, D.J., Brannigan, J.A., Ladds, J.C., Cervin, M.A., Spiegelman, G.B., Hoggett, J.G., Bara¤k, I. and Wilkinson, A.J. (2002) Dimer formation, transcription activation in the sporulation response regulator Spo0A. J. Mol. Biol. 316, 235^245. [9] Lewis, R.J., Brannigan, J.A., Muchova¤, K., Bara¤k, I. and Wilkinson, A.J. (1999) Phosphorylated aspartate in the structure of a response regulator protein. J. Mol. Biol. 294, 9^15. [10] Muchova¤, K., Lewis, R.J., Brannigan, J.A., O¡en, W.A., Brown, D.P., Bara¤k, I., Youngman, P. and Wilkinson, A.J. (1999) Crystallisation of the regulatory and e¡ector domains of the key sporulation response regulator Spo0A. Acta Crystallogr. Sect. D 55, 671^676. [11] Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.O., Seidman, J.S., Smith, J.A. and Struhl, K. (1987) Current Protocols in Molecular Biology. Wiley, New York. [12] Grimsley, J.K., Tjalkens, R.B., Strauch, M.A., Bird, T.H., Spiegelman, G.B., Hostomsky, Z., Whiteley, J.M. and Hoch, J.A. (1994) Subunit composition and domain structure of the Spo0A sporulation factor of Bacillus subtilis. J. Biol. Chem. 269, 16977^16982. [13] Asayama, M., Yamamoto, A. and Kobayashi, Y. (1995) Dimer form of phosphorylated Spo0A, a transcriptional regulator, stimulates the spo0F transcription at the initiation of sporulation in Bacillus subtilis. J. Mol. Biol. 250, 11^23. [14] Baldus, J.M., Green, B.D., Youngman, P. and Moran Jr., C.P. (1994) Phosphorylation of Bacillus subtilis transcription factor Spo0A stimulates transcription from the spoIIG promoter by enhancing binding to weak 0A boxes. J. Bacteriol. 176, 296^306. [15] Satola, S., Kirchman, P.A. and Moran Jr., C.P. (1991) Spo0A binds
[16]
[17]
[18]
[19]
[20] [21] [22] [23]
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to a promoter used by cA RNA polymerase during sporulation of Bacillus subtilis. Proc. Natl. Acad. Sci. USA 88, 4533^4537. Asayama, M. and Kobayashi, Y. (1993) Signal transduction, sporulation in Bacillus subtilis : heterologous phosphorylation of Spo0A, a sporulation initiation gene product. J. Biochem. 114, 385^388. Olmedo, G., Ninfa, E.G., Stock, J. and Youngman, P. (1990) Novel mutations that alter the regulation of sporulation in Bacillus subtilis Evidence that phosphorylation of regulatory protein Spo0A controls the initiation of sporulation. J. Mol. Biol. 215, 359^372. McCleary, W.R. and Stock, J.B. (1994) Acetyl phosphate and the activation of two-component response regulators. J. Biol. Chem. 269, 31567^31572. Tomomori, C., Tanaka, T., Dutta, R., Park, H., Saha, S.K., Zhu, Y., Ishima, R., Liu, D., Tong, K.I. and Kurosawa, H. (1999) Solution structure of the homodimeric core domain of Escherichia coli histidine kinase EnvZ. Nat. Struct. Biol. 6, 729^734. Varughese, K.I. (2002) Molecular recognition of bacterial phosphorelay proteins. Curr. Opin. Microbiol. 5, 142^148. Cai, S.J. and Inouye, M. (2002) EnvZ-OmpR interaction and osmoregulation in Escherichia coli. J. Biol. Chem. 277, 24155^24161. Hoch, J.A. and Varughese, K.I. (2001) Keeping signals straight in phosphorelay signal transduction. J. Bacteriol. 183, 4941^4949. Zapf, J., Sen, U., Madhusudan, Hoch, J.A. and Varughese, K.I. (2000) A transient interaction between two phosphorelay proteins trapped in a crystal lattice reveals the mechanism of molecular recognition and phosphotransfer in signal transduction. Structure 8, 851^862.
FEMSLE 10991 16-6-03
www.fems-microbiology.org
The response regulator Spo0A from Bacillus subtilis is e⁄ciently phosphorylated in Escherichia coli Joanne C. Ladds a , Katar|¤na Muchova¤ b , Dus›an Blas›kovic› b , Richard J. Lewis c , James A. Brannigan a , Anthony J. Wilkinson a , Imrich Bara¤k b; a
Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5YW, UK Institute of Molecular Biology, Slovak Academy of Sciences, Du¤bravska¤ cesta 21, 84251 Bratislava, Slovak Republic Laboratory of Molecular Biophysics, The Rex Richards Building, University of Oxford, South Parks Road, Oxford OX1 3QU, UK b
c
Received 27 February 2003; received in revised form 6 April 2003 ; accepted 9 April 2003 First published online 28 May 2003
Abstract The response regulator proteins of two-component systems mediate many adaptations of bacteria to their ever-changing environment. Most response regulators are transcription factors that alter the level of transcription of specific sets of genes. Activation of response regulators requires their phosphorylation on a conserved aspartate residue by a cognate sensor kinase. For this reason, expression of a recombinant response regulator in the absence of the requisite sensor kinase is expected to yield an unphosphorylated product in the inactive state. For Spo0A, the response regulator controlling sporulation in Bacillus subtilis however, we have found that a significant fraction of the purified recombinant protein is phosphorylated. This phosphorylated component is dimeric and binds to Spo0A recognition sequences in DNA. Treatment with the Spo0A-specific phosphatase, Spo0E, leads to dissociation of the dimers and loss of DNA binding. It is therefore necessary to pre-treat recombinant Spo0A preparations with the cognate phosphatase, to generate the fully inactive state of the molecule. 8 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Spo0A; Phosphorylation; Response regulator; Histidine kinase ; Bacillus subtilis
1. Introduction Spo0A plays a crucial role in the regulation of gene expression during sporulation, a developmental process triggered in Bacillus subtilis as a response to a deteriorating environment and starvation. Extracellular signals are monitored by a set of sensor kinases and transduced, through an expanded two-component system called a phosphorelay, into a chemical event, the phosphorylation of Spo0A [1]. Phosphorylated Spo0A directly or indirectly in£uences the transcription of hundreds of genes as sporulation commences [2]. Spo0A consists of a phosphoacceptor or receiver domain (N-Spo0A) and a DNA binding or e¡ector (CSpo0A) domain. It becomes active as a transcription factor after phosphorylation of the receiver domain on a * Corresponding author. Tel. : +421 (2) 59307418; Fax : +421 (2) 59307416. E-mail addresses : [email protected] (I. Bara¤k), [email protected] (I. Bara¤k).
conserved aspartate [3,4]. Dephosphorylation, catalysed by the Spo0AVP-speci¢c phosphatase, Spo0E, reverses this activation [5,6]. The capacity of fragments of Spo0A encompassing the e¡ector domain to bind to DNA and activate transcription [7] together with the constitutive activity of Spo0A mutants, carrying large deletions in the receiver domain [3], leads to the conclusion that in intact Spo0A, the receiver domain inhibits the e¡ector domain and phosphorylation overcomes this inhibition. It is also clear that, upon phosphorylation, Spo0A forms dimers and that these dimers represent the activated form [8]. The crystal structure of N-Spo0A from Bacillus stearothermophilus has been solved using recombinant protein puri¢ed from Escherichia coli. To our surprise the active site aspartate was phosphorylated indicating that heterologous phosphorylation of the protein had taken place in E. coli [9]. Here we show that heterologous phosphorylation extends to the better characterised Spo0A from B. subtilis and demonstrate that heterologously phosphorylated Spo0A forms dimers and binds to cognate DNA sequences.
0378-1097 / 03 / $22.00 8 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S0378-1097(03)00321-5
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J.C. Ladds et al. / FEMS Microbiology Letters 223 (2003) 153^157
2. Materials and methods
3. Results and discussion
2.1. Spo0A isolation and puri¢cation
To investigate the level of phosphorylation in preparations of B. subtilis Spo0A isolated from E. coli (Spo0AEc), we used gel ¢ltration chromatography and native polyacrylamide gel electrophoresis assays. Gel ¢ltration chromatograms of Spo0AEc and Spo0A subsequently phosphorylated by phosphoramidate (Spo0AVP) are shown in Fig. 1. Spo0AEc eluted from the Pharmacia Superdex 75 HR10/30 gel ¢ltration column as two distinct peaks with elution volumes of 9.4 ml and 10.7 ml, corresponding to the dimeric and monomeric forms of Spo0A, respectively [8]. Spo0AVP eluted from this column with an elution volume of 9.5 ml, corresponding to the dimeric form. The earlier eluting peak in the Spo0AEc chromatogram therefore suggests that a signi¢cant fraction of this protein is phosphorylated. This is con¢rmed by the observation that treatment of Spo0AEc with the Spo0AVP-speci¢c phosphatase, Spo0E94, leads to loss of the fast eluting peak, and enhancement of the slower migrating peak which corresponds to the monomer. Mild tryptic digestion of Spo0A produces two fragments, N-Spo0A and C-Spo0A [10,12], which can be easily resolved by electrophoresis in polyacrylamide gels under non-denaturing conditions [8]. In these gels, N-Spo0AVP dimers resulting from digestion of Spo0AVP with trypsin migrate as a single band with an Rf value (relative to bromophenol blue) of 0.48 (Fig. 2, lane 3). Pre-treatment of Spo0AVP with Spo0E94, prior to tryptic cleavage and gel analysis, produces a faster migrating species with Rf = 0.56 (Fig. 2, lane 4). N-Spo0AEc resulting from the tryptic digestion of Spo0AEc migrates as two distinct bands, corresponding to phosphorylated dimers (Rf = 0.48) and small amount of unphosphorylated monomers (Rf = 0.56) (Fig. 2, lane 1). After dephosphorylation
Spo0A was overexpressed and puri¢ed as described previously [8]. Spo0A samples (20^200 WM) were phosphorylated in a bu¡er containing 20 mM Tris^HCl pH 7.5, 20 mM MgCl2 , 1 mM dithiothreitol (DTT) and 50 mM phosphoramidate over a period of 30 min at 37‡C. Spo0AVP samples were dephosphorylated by incubation in the presence of 5 WM of a hyperactive mutant form of Spo0E, Spo0E94, for 30 min at 37‡C [5,8]. To prepare N-Spo0A fragments, samples of Spo0A were incubated with trypsin at a molar ratio of 2500:1 for 30 min at 16‡C [10] and the products were analysed immediately by non-denaturing 12.5% polyacrylamide gel electrophoresis. 2.2. Preparation of DNA 100 ng (11 pmol) of the oligonucleotide ABRBTOP (5PGGATTTTGTCGAATAATGACGAAGAAAAAT-3P) was heated for 5 min at 95‡C, and rapidly cooled in an ice bath prior to its 5P end labelling with polynucleotide kinase and 27 WCi (3.86 pmol) [Q-32 P]ATP (ICN, 7000 Ci mmol31 ) in a total volume of 25 Wl. The radiolabelled ABRBTOP was incubated with an equimolar amount of ABRBBOT (5P-ATTTTTCTTCGTCATTATTCGACAAAATCC-3P) and the oligonucleotide pair was annealed by heating for 5 min at 95‡C and 20 min at 65‡C before slow cooling to 20‡C. Unincorporated radionucleotides were removed from duplex DNA by passage through a Sephadex G-50 microtip column. A non-speci¢c competitor DNA was prepared by annealing equimolar amounts of the ATNESCC (5P-TTAATATTCCTTATATAT-3P) and ATNESGG (5P-ATATATAAGGAATATTAA-3P) oligonucleotides. 2.3. Electrophoretic mobility shift assay Gel mobility shift assays were performed essentially as described previously [11]. Samples of Spo0A (100 ng) were incubated with 43 000 cpm (0.19 ng) 32 P-labelled abrB-speci¢c DNA in the absence or presence of the competitor DNA (unlabelled abrB-speci¢c DNA) for 15 min at 37‡C in a solution containing 40 mM Tris-acetate pH 8.0, 40 mM sodium acetate, 10 mM magnesium acetate, 0.1 mM DTT, 10 Wg ml31 bovine serum albumin (BSA), 1 mM EDTA, and 5% (v/v) glycerol in a total volume of 20 Wl. The binding mix was loaded onto an 8% polyacrylamide gel prepared in 1UTAE bu¡er (40 mM Tris, 20 mM acetic acid, 1 mM EDTA pH 7.9, containing 2% (v/v) glycerol). The gel was run at room temperature and 150 V in 1UTAE bu¡er until the bromophenol blue dye had migrated to within 2 cm of the bottom of the gel. The gel was then dried and analysed by autoradiography.
Fig. 1. Sephadex 75HR 10/30 gel ¢ltration chromatograms of B. subtilis Spo0A isolated from E. coli. The column was equilibrated in a bu¡er of 20 mM Tris^HCl pH 7.5, 250 mM NaCl, 2 mM EDTA, 2 mM DTT. The continuous line represents Spo0A protein puri¢ed from E. coli (Spo0AEc), dashed line Spo0AVP (Spo0AEc after incubation at 37‡C for 30 min with 50 mM phosphoramidate). The samples contained Spo0A at a concentration of 70 WM. The dotted line is the chromatogram for a mixture of molecular weight standards, BSA (67 kDa), ovalbumin (43 kDa), chymotrypsinogen (25 kDa) and ribonuclease A (14 kDa). Blue dextran (2 000 Da) was used in order to determine the void volume of the column.
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Fig. 2. Examination of the oligomeric state of Spo0A using oligomeric state using non-denaturing 12.5% gel electrophoresis. Each lane contains 8 Wg of protein. Analysis of Spo0A fragments resulting from proteolysis of intact Spo0AEc with trypsin in a ratio 2500:1. Lanes: (1) Spo0AEc, (2) Spo0AEc+0E (Spo0AEc after incubation with Spo0A-speci¢c phosphatase Spo0E94), (3) Spo0AVP (Spo0AEc after incubation with 50 mM phosphoramidate), (4) Spo0AVP+0E (Spo0AEc after phosphoramidate treatment as above followed by dephosphorylation with Spo0E94). Note that C-Spo0A stays stuck at the top of the gel.
of Spo0AEc with the phosphatase Spo0E, this sample (Spo0AEc+0E) was completely converted to the dephosphorylated monomeric form (Fig. 2, lane 2). These experiments reinforce the conclusion (i) that there is a system in E. coli which heterologously phosphorylates Spo0A and (ii) that Spo0AVP is very stable with a half-life extending over the several days that it takes to purify the protein to homogeneity. Analysis of samples of N-Spo0AVP ‘aged’ in 20 mM Tris^HCl pH 7.5, 20 mM MgCl2 , 1 mM DTT at room temperature over 5 days, in similar native gel electrophoresis assays to those described above, con¢rms the latter conclusion (data not shown). To assay whether Spo0AEc is able to bind site-speci¢cally to DNA, we performed DNA gel shift experiments using a synthetic duplex DNA derived from the abrB promoter region and containing two 0A-box sequences (TGNCGAA). These experiments reveal that 0.166 WM Spo0AEc is able to cause retardation of a signi¢cant proportion of the abrB promoter fragment when present at 0.45 nM (Fig. 3, lane 1); moreover, the extent of the gel mobility shifts is similar to that mediated by Spo0A that has been phosphorylated using phosphoramidate (Fig. 3, lane 4). Treatment of both Spo0AEc and Spo0AVP with Spo0E abolished DNA binding completely (Fig. 3, lane 7). The intensities of the bands in the autoradiograph are consistent with slightly weaker binding of Spo0AEc in comparison to the in vitro phosphorylated Spo0AVP, consistent with the incomplete phosphorylation of the former. Previous reports suggested that unphosphorylated Spo0A binds detectably to the 0A-box regions of a set of promoters and that the extent of binding is increased V20-fold by phosphorylation [7,13^15]. Our ¢nding here
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is that unphosphorylated Spo0A does not bind to abrB promoter DNA. An obvious explanation for the di¡erent observations is that a proportion of the Spo0A that was assumed to be unphosphorylated in the earlier studies was in fact heterologously phosphorylated. If this explanation is correct the reported V20 increase in DNA-binding activity attributable to phosphorylation in Spo0A would be an underestimate. We estimate from our own experiments that DNA binding is stimulated 50^100-fold by phosphorylation. The experiments reported here show that Spo0A expressed in E. coli is heterologously phosphorylated and functional in DNA-binding. This phosphorylation may result from the action of sensor kinases present in E. coli, such as EnvZ, CheA or NRII [16,17] or by the action of small molecule phosphodonors [18]. It has been shown that Spo0A can be phosphorylated e¡ectively in vitro with an N-terminally truncated form of phosho-EnvZ [16]. The phosphotransferase domains of EnvZ and Spo0B, the phosphotransferase that mediates phosphotransfer to Spo0A in B. subtilis, have similar structures [19,20]. There are about 100 molecules EnvZ in an E. coli cell [21], so signi¢cant phosphorylation of highly expressed Spo0A would require a high turnover number. However, other histidine kinases may be involved. The surfaces of B. subtilis response regulators that contact the sensor kinase phosphotransferase domains have been analysed in detail [22,23]. In Fig. 4, we have compared the amino acid res-
Fig. 3. Gel mobility shift assays of Spo0A binding to abrB promoter DNA. Speci¢ed Spo0A preparations and competitor DNAs were added as: Spo0AEc (lane 1), Spo0AEc in the presence of speci¢c competitor (lane 2), Spo0AEc in the presence of non-speci¢c competitor (lane 3), Spo0AVP (lane 4), Spo0AVP in the presence of speci¢c competitor (lane 5), Spo0AVP in the presence of non-speci¢c competitor (lane 6), Spo0AEc+0E (lane 7), Spo0AEc+0E in the presence of speci¢c competitor (lane 8), Spo0AEc+0E in the presence of non-speci¢c competitor (lane 9). Each lane contains 100 ng of protein and 0.19 ng of abrB promoter DNA.
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Fig. 4. Comparison of interacting residues in Spo0A with the residues at the similar positions in all known response regulators in E. coli. Residues are numbered as in Spo0A. Conserved residues are in black boxes; similar residues are in capital bold letters and all other residues are in small letters.
idues in Spo0A inferred to be in contact with Spo0B with the residues at the corresponding positions in all the response regulators found in E. coli. It has been proposed that residues corresponding to Phe88, Gly89 and Gln90 in Spo0A are important in de¢ning the speci¢city of the interaction with cognate sensor kinases [23]. Our comparison does not obviously reveal an E. coli response regulator with a speci¢city-de¢ning region closely similar to that in Spo0A, and we cannot easily point to a particular sensor kinase as a candidate agent for the heterologous phosphorylation we have observed. The studies described here show that it is necessary to pre-treat recombinant Spo0A preparations with the cognate phosphatase, to generate the authentic inactive, unphosphorylated state of the molecule. It seems unlikely that heterologous phosphorylation of two-component system response regulators in E. coli will be con¢ned to Spo0A. However, the much shorter half-lives of the phosphorylated forms of most other response regulators (seconds to minutes) relative to that of Spo0A (hours to days) means that the phosphorylated form is less likely to persist through the steps of protein puri¢cation. In general however, it should not be assumed that puri¢ed recombinant response regulator proteins are fully unphosphorylated.
Acknowledgements We would like to thank Dusan Perecko for technical
assistance with electrophoretic mobility shift assays. We thank Marta Perego for providing us with a clone directing overexpression of Spo0Ev94. This work was supported by The Wellcome Trust Grants, the Grant 2/1004/21 from the Slovak Academy of Sciences and The Royal Society International Exchanges Project. R.J.L. is currently the recipient of a Wellcome Trust Research Career Development Fellowship.
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