Molecular characterization of the mycobacterial SenX3–RegX3 two-component system: evidence for autoregulation

Sabine Himpens1, Camille Locht1 and Philip Supply1

INSERM U447, Institut Pasteur de Lille/Institut de Biologie de Lille, 1 rue du Professeur Calmette, F-59019 Lille Cedex, France1

Author for correspondence: Philip Supply. Tel: +33 3 20 87 11 54. Fax: +33 3 20 87 11 58. e-mail: philip.supply{at}pasteur-lille.fr


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Environmental regulation of bacterial gene expression is often mediated by two-component signal transduction systems, which are themselves tightly regulated. The response regulator RegX3 and the cytoplasmic portion of the histidine kinase SenX3 from Mycobacterium bovis BCG were overproduced in Escherichia coli and purified as N-terminally (His)6-tagged proteins. Phosphorylation assays demonstrated autophosphorylation of the cytoplasmic portion of SenX3 and a phosphotransfer from SenX3 to RegX3, involving conserved histidine and aspartate residues, respectively. In addition, as shown by electrophoretic mobility shift assays, (His)6RegX3 was able to specifically bind to the promoter region of the senX3–regX3 operon. Furthermore, operon fusion analyses indicated that the overexpression of the senX3–regX3 operon increases the activity of the senX3 promoter in Mycobacterium smegmatis. Together, these results indicate that the mycobacterial SenX3–RegX3 two-component system is positively autoregulated.

Keywords: two-component systems, autoregulation, phosphorylation, Mycobacterium tuberculosis

Abbreviations: DIG, digoxigenin


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Two-component systems provide a conserved mechanism for the coordinate regulation of gene expression in response to a wide variety of environmental input signals. Diverse processes such as chemotaxis, cell division, metabolic pathways, nitrogen fixation, stress responses and virulence are regulated by two-component systems (Stock et al., 1989 ). Several hundred such systems have been identified in bacteria. They have been found in all prokaryotes examined to date, and some have been recently discovered in eukaryotes. In most systems, the response regulator is a transcriptional activator (for reviews see Alex & Simon, 1994 ; Hoch & Silhavy, 1995 ). The core mechanism of two-component systems consists of an autophosphorylation on a conserved histidine residue in the cytoplasmic domain of the sensor, followed by the transfer of the phosphate group onto a conserved aspartate residue of the response regulator. This results in the activation of the regulator leading to the appropriate response (Parkinson & Kofoid, 1992 ).

Autoregulation is an important feature of many two-component systems. It allows amplification of the response to help the cell to rapidly modify target gene expression when the appropriate signals are encountered. For instance, many two-component systems controlling virulence, quorum sensing, envelope stress response or sporulation control their own upregulation (e.g. Soncini et al., 1995 ; Kleerebezem et al., 1997 ; Raivio et al., 1999 ). However, as shown for HilA, which regulates virulence factor genes in Salmonella typhimurium (Bajaj et al., 1996 ), autoregulation is not a universal rule.

The genome of Mycobacterium tuberculosis contains 30 genes encoding two-component-system proteins (Cole et al., 1998 ). Their functions, regulation and the signals sensed by these systems are not known, and only three of them, MtrA–MtrB, SenX3–RegX3 and TrcS–TrcR, have been partially characterized (Supply et al., 1997 ; Via et al., 1996 ; Haydel et al., 1999 ). The SenX3 sensor belongs to the PhoR and EnvZ subfamily and contains two hydrophobic, potential transmembrane regions in its N-terminal moiety, whereas the RegX3 response regulator belongs to the ROII subfamily, of which PhoB and OmpR are the prototypes (Supply et al., 1997 ). Here, we show that the cytoplasmic portion of SenX3 is able to autophosphorylate. The phosphate group is then transferred onto RegX3. This phosphorelay involves the conserved His-167 and Asp-52 residues of the transmitter domain of SenX3 and the receiver domain of RegX3, respectively. In addition, recombinant RegX3 is able to specifically bind the senX3 promoter region, and overproduction of RegX3 increases the expression of the senX3–regX3 operon in Mycobacterium smegmatis.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains.
All cloning steps were carried out in Escherichia coli XL-1 Blue (Stratagene). Recombinant SenX3 and RegX3 were expressed in E. coli M15 (Qiagen). Site-directed mutagenesis was performed in E. coli ES1301 mutS (Promega) followed by the amplification of the mutated plasmids in E. coli JM109 (Promega). The M. smegmatis mc2-155 strain (Jacobs et al., 1991 ) was used for electroporation of the pREP vectors.

Reporter gene construction.
The pREP7 vector containing the senX3lacZY fusion has been described previously (Supply et al., 1997 ). The pREP5 vector was constructed by inserting a 3·2 kb HindIII–BamHI fragment containing the senX3–regX3 operon from pRegX3Bc1 (Supply et al., 1997 ) into the unique ScaI site of pREP7.

ß-Galactosidase assays.
M. smegmatis was transformed with pREP5 or pREP7 as described by Kremer et al. (1995) and grown in Sauton medium supplemented with 25 µg kanamycin ml-1 until the OD600 reached 0·2–0·4. The cells were then lysed by sonication and the ß-galactosidase activity in the sonicate extracts was measured by the method of Miller (1992) as described previously (Supply et al., 1997 ). ß-Galactosidase units were calculated according to the formula U=1000x(OD420-1·75xOD550)/[t (min)xvol. (ml)xOD600].

Production of recombinant proteins.
To overproduce (His)6RegX3, the regX3 coding sequence was amplified by PCR from pRegX3Bc1 (Supply et al., 1997 ) using synthetic oligonucleotides that included 5'-terminal BamHI half-sites (underlined). The sequences of these oligonucleotides were 5'-TCCATGACCAGTGTGTTGATTGTGCA-3' and 5'-TCCGCCCTCGAGTTTGTAGCCCAC-3'. The PCR product was circularized by ligation, digested with BamHI to regenerate complete BamHI sites and cloned into the BamHI site of pQE30 (Qiagen). The stop codon, located 9 nt downstream of the regX3 coding sequence, was provided by the vector sequence after digestion of the plasmid by HindIII and SphI, T4 DNA polymerase treatment and religation. Therefore, the recombinant (His)6RegX3 contains an addition of three amino acids at its C-terminal end: Glu, Leu and Asn. To overproduce (His)6SenX3, the senX3 sequence encoding the cytoplasmic portion of SenX3 was amplified by PCR from pRegX3Bc1 using synthetic oligonucleotides that included 5'-terminal BamHI and HindIII restriction sites (underlined). The sequences of these oligonucleotides were 5'-GGGGATCCCGGTTGCTGAGCGAGGAAGA-3' and 5'-CCCAAGCTTTCATCGGCTCAGCTCTTCCT-3'. The PCR product was digested with BamHI/HindIII and cloned into pQE30. The two products encoded by pQE30-RegX3 and pQE30-SenX3, named (His)6RegX3 and (His)6SenX3 respectively, contained a tag composed of 6 histidine residues at their N termini.

The mutant proteins (His)6SenX3-H167Q and (His)6RegX3-D52N were obtained using the pAlter site-directed mutagenesis kit (Promega). The mutagenic oligonucleotides used had the following sequence: 5'-CGTCAGTCAAGAGCTCAAG-3' and 5'-AGCATCAGATTGAGCAGGA-3', respectively. Codons 167 CAC and 52 GAT were modified to CAA and AAT, respectively. The mutated sequences were used to replace the wild-type sequences in the expression vectors pQE30-SenX3 and pQE30-RegX3. All the constructs were verified by sequencing using the ABI system (Perkin Elmer).

Purification of recombinant proteins.
E. coli M15 cells containing the different expression vectors were grown in 500 ml LB medium containing 100 µg ampicillin ml-1 and 25 µg kanamycin ml-1. When the OD600 reached 0·7–0·8, expression of the genes encoding the recombinant proteins was induced with 1 mM IPTG for 3 h 30 min. The cells were then harvested by centrifugation, resuspended in 5 ml buffer B (8 M urea, 0·1 M sodium phosphate, 0·01 M Tris/HCl, pH 8·0) per gram fresh weight and gently stirred for 1 h. After centrifugation at 10000 g for 15 min at 4 °C the supernatant was loaded onto a 4 ml Ni/NTA column (Qiagen). The column was first washed with buffer C (8 M urea, 0·1 M sodium phosphate, 0·01 M Tris/HCl, pH 6·3) until the OD280 became below 0·01, and then with 10 ml buffer D (8 M urea, 0·1 M sodium phosphate, 0·01 M Tris/HCl, pH 5·9). The proteins were eluted with 10 ml buffer E (8 M urea, 0·1 M sodium phosphate, 0·01 M Tris/HCl, pH 4·5). Two millilitre fractions were collected and analysed by SDS-PAGE and Coomassie blue staining. The purified proteins were dispensed in aliquots and stored at -20 °C. For small-scale preparations of the proteins, recombinant M15 cells containing the expression vectors were grown in 10 ml cultures and induced as described above. After centrifugation, the cells were resuspended in 2 ml buffer B. Three hundred microlitres of a 50% slurry of Ni/NTA resin was added to the supernatant after centrifugation at 10000 g for 15 min at 4 °C and the mixture was stirred for 30 min at room temperature. The resin was harvested by centrifugation at 15000 g for 10 s, washed three times with 1·5 ml buffer C, resuspended in 400 µl buffer C containing 100 mM EDTA and gently mixed for 2 min at room temperature. This suspension was then centrifuged at 15000 g for 10 s. The supernatant was carefully removed and stored at -20 °C.

The purified proteins were renatured by two successive rounds of dialysis at 4 °C. Approximately 100 µg proteins were first dialysed for 24 h against 150 ml PBS, 0·4 M sucrose, 0·4 M L-arginine, 1 mM EDTA (pH 8·0), and then against 200 ml PBS, 10% (v/v) glycerol (pH 8·0). The purified proteins were dispensed in aliquots and stored at -20 °C.

Phosphorylation assays.
For phosphotransfer assays, 2 µg renatured (His)6SenX3 was incubated for 20 min at 25 °C with 10 µCi of [{gamma}32P]ATP [6000 Ci mmol-1 (222 TBq mmol-1); Amersham] in a total volume of 20 µl containing 10 mM MgCl2, 25 mM Tris/HCl (pH 7·6). When indicated, 10 µg purified (His)6RegX3 was added to the reaction mixture and incubated for 15 min at 37 °C. The reaction was stopped by the addition of 10 µl 3xloading buffer (150 mM Tris/HCl, pH 6·8, 6% SDS, 0·3% bromophenol blue, 3% 2-mercaptoethanol, 30%, v/v, glycerol). The samples were loaded onto a 12% SDS-polyacrylamide gel. After electrophoresis the proteins were transferred onto a nitrocellulose membrane. The membrane was dried and then exposed for autoradiography to an X-ray film.

For pulse–chase experiments, 10 µg (His)6SenX3 was incubated with 0·16 µM [{gamma}32P]ATP as described above, then 50 µg purified (His)6RegX3 and 320 µM unlabelled ATP were simultaneously added to the reaction mixture. The standard phosphorylation reaction was performed in 100 µl from which 20 µl aliquots were removed 0, 1, 5, 10 and 30 min after the addition of (His)6RegX3 and unlabelled ATP. For each aliquot, the reaction was immediately stopped by the addition of 10 µl 3x loading buffer. Alternatively, 10 µg purified (His)6RegX3 and 0·16 µM [{gamma}32P]ATP were simultaneously added to the phosphorylation reaction mixture containing (His)6SenX3 preincubated with 320 µM unlabelled ATP.

To test the sensitivity of the phosphorylated proteins to acidic and basic conditions, 2·5 µl 10% SDS and 2·5 µl 1 M HCl or 10 M NaOH were added to 20 µl of the phosphorylation reaction mixture and then incubated for 15 min at 37 °C. The samples were then dialysed against 1 M Tris/HCl (pH 6·8) before SDS-PAGE.

Electrophoretic mobility shift assays.
DNA-binding activities of renatured (His)6RegX3 were assessed by using digoxigenin (DIG)-labelled double-stranded DNA. Oligonucleotides 5'-GGGGTACCTTGTTTGAGATCCCACCTGC-3' and 5'-G GGGTACCAAGGAAAATCCTACAAATCCGGTGA-3' were used to amplify a 189 bp DNA fragment upstream of the senX3 gene. These oligonucleotides included terminal KpnI sites (underlined) to increase the efficiency of DIG labelling by the terminal transferase. The PCR product named 5'senX3-189 was digested with KpnI and labelled at its 3' ends with DIG-dideoxy-UTP using the DIG Oligonucleotide 3' End Labelling Kit (Boehringer Mannheim) as recommended by the supplier. The following DNA fragments were used as competitors in electrophoretic mobility shift assays: (1) an 85 bp fragment (5'senX3-85) corresponding to the 3' region of the 5'senX3-189 probe (obtained by PCR using the pair of oligonucleotides 5'-TGGCGTAGTGTGTGACTTGTC-3' and 5'-GGGGTACCAAGGAAAATCCTACAAATCCGGTGA-3'); (2) a 122 bp fragment (5'senX3-122) corresponding to the 5' region of the 5'senX3-189 probe (obtained by PCR using the pair of oligonucleotides 5'-GGGGTACCTTGTTTGAGATCCCACCTGC-3' and 5'-AAGTCACACACTACGCCACAG-3'); and (3) a 42 bp double-stranded oligonucleotide (5'senX3-42) with the sequence 5’-ATGTGAACGGTAACCGAACAGCTGTGGCGTAGTGTGTGACTT-3' corresponding to an internal segment of the 5'senX3-189 probe.

Purified renatured (His)6RegX3 was incubated for 20 min at room temperature with 5 ng DIG-labelled 5'senX3-189 (1·5 fmol) and 2 µl 5xbinding buffer (10 mM Tris/HCl, pH 8·0, 2 mM MgCl2, 50 mM KCl, 1 mM DTT, 50%, v/v, glycerol, 0·05%, v/v, Nonidet P40) in a total volume of 10 µl. The samples were then loaded onto a 12% (w/v) polyacrylamide/45 mM Tris-borate/1 mM EDTA (pH 8·0) native gel. After electrophoresis, the DNA was blotted onto a positively charged nylon membrane (Boehringer Mannheim), fixed under UV light and developed using the Boehringer Mannheim Bioluminescence kit. The membrane was exposed for 20 min to 2 h for autoradiography to an X-ray film. When the effect of phosphorylation on binding was tested, (His)6RegX3 was phosphorylated in the same phosphorylation buffer as above containing 500 µM unlabelled ATP.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Synthesis and purification of histidine-tagged RegX3 and SenX3
Full length RegX3 and the cytoplasmic portion of SenX3 were overproduced and purified as N-terminally (His)6-tagged proteins. The cytoplasmic portion of SenX3 was chosen to avoid insolubility due to hydrophobic amino acid stretches and to thereby facilitate purification and biochemical analysis of this protein. After induction with IPTG, two major proteins of 31 kDa and 33 kDa were detected in crude lysates of the E. coli strains containing the RegX3 and the SenX3 expression vectors, respectively (Fig. 1). These proteins correspond to (His)6RegX3 and (His)6SenX3, as they were recognized by anti-His-tag antibodies (data not shown). The apparent molecular masses were somewhat higher than expected from the sequences (26·8 kDa and 32·1 kDa, respectively). This could at least partly be due to the presence of the polyhistidine tag, which may increase the apparent molecular mass by several kilodaltons.



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Fig. 1. Overproduction and purification of (His)6RegX3 and (His)6SenX3. (His)6RegX3 and (His)6SenX3 were overproduced in E. coli and purified on a Ni/NTA column as described in Methods. Crude lysates and purified fractions were subjected to SDS-PAGE and stained with Coomassie blue. (a) RegX3 purification. Lane 1, crude lysate of uninduced cells; lane 2, crude lysate of IPTG-induced cells; lane 3, 5 µg purified (His)6RegX3. (b). SenX3 purification. Lane 1, 5 µg purified (His)6SenX3; lane 2, crude lysate of uninduced cells; lane 3, crude lysate of IPTG-induced cells. The positions of (His)6RegX3 and (His)6SenX3 are indicated by the arrows. Molecular mass markers are indicated on the left.

 
The recombinant proteins were purified by using a nickel-chelate resin in the presence of 8 M urea. As shown in Fig. 1, purified (His)6RegX3 and (His)6SenX3 appeared as single bands on Coomassie blue-stained gels after electrophoresis in the presence of SDS. The recombinant proteins were then renatured by dialysis against a buffer containing 0·4 M sucrose and 0·4 M L-arginine to avoid proline cistrans isomerization and to facilitate correct folding of the proteins (Bowden & Georgiou, 1990 ; Mukhopadhyay, 1997 ). L-Arginine and sucrose were subsequently eliminated by a second dialysis step.

Phosphotransfer between SenX3 and RegX3
The transduction of sensory signals by two-component systems generally occurs through a phosphorylation cascade. The (His)6SenX3 protein was therefore tested for its capacity to be phosphorylated and to transfer the phosphate group to (His)6RegX3. As shown in Fig. 2, (His)6SenX3 was readily autophosphorylated in the presence of [{gamma}32P]ATP. When (His)6RegX3 was added to the SenX3 phosphorylation reaction mixture both proteins were phosphorylated. In the absence of (His)6SenX3, no detectable (His)6RegX3 phosphorylation occurred.



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Fig. 2. Phosphorylation of (His)6RegX3 and (His)6SenX3. The purified proteins were incubated with [{gamma}32P]ATP in 10 mM MgCl2, 25 mM Tris/HCl (pH 7·6), as described in Methods. Lane 1, no protein; lane 2, 2 µg (His)6SenX3; lane 3, 10 µg (His)6RegX3; lanes 4–7, 2 µg (His)6SenX3 and 10 µg (His)6RegX3. After incubation, the proteins were treated in with 1 M HCl (lane 5), 0·1 M HCl (lane 6) or 1 M NaOH (lane 7), as described in Methods.

 
To determine whether RegX3 was phosphorylated directly from phosphorylated SenX3 or from the free nucleotide pool, (His)6SenX3 was preincubated with 0·16 µM [{gamma}32P]ATP for 20 min, after which (His)6RegX3 was added and incubation continued for various lengths of time. Phosphorylation of (His)6RegX3 coincided with dephosphorylation of (His)6SenX3 and the maximal level was reached within 5 min (Fig. 3c). Incorporation of 32P into (His)6RegX3 was not inhibited by the simultaneous addition of excess unlabelled ATP (Fig. 3b). In contrast, when (His)6SenX3 was first incubated for 20 min with 320 µM unlabelled ATP, followed by the addition of (His)6RegX3 and 0·16 µM [{gamma}32P]ATP, efficient labelling of (His)6SenX3 and (His)6RegX3 was not detected (Fig. 3a). The competition by excess unlabelled ATP in the preincubation mixture indicates that the incorporation of label into (His)6RegX3 depends on prior incorporation into (His)6SenX3. Consistent with this result, the chase of label from (His)6SenX3 to (His)6RegX3 was not competed by excess unlabelled ATP, indicating that (His)6RegX3 is phosphorylated by phosphotransfer from (His)6SenX3 and not from the free nucleotide pool. These results also indicate that the cytoplasmic portion of SenX3 is sufficient for autophosphorylation and phosphotransfer to RegX3. Interestingly, both the (His)6SenX3 and the (His)6RegX3 phosphorylation were resistant to an excess of unlabelled ATP for at least up to 30 min (Fig. 3b), indicating that their phosphatidyl bond is rather stable.



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Fig. 3. Phosphotransfer from (His)6SenX3 to (His)6RegX3. (a) (His)6SenX3 was preincubated for 20 min with 320 µM unlabelled ATP. (His)6RegX3 was then added together with 0·16 µM [{gamma}32P]ATP and incubation was continued for 0 (lane 1), 5 (lane 2), 10 (lane 3) or 30 min (lane 4). (b) (His)6SenX3 was preincubated for 20 min with 0·16 µM [{gamma}32P]ATP. (His)6RegX3 was then added together with 320 µM unlabelled ATP, and incubation was continued for 0 (lane 1), 1 (lane 2), 5 (lane 3), 10 (lane 4) or 30 min (lane 5). (c) (His)6SenX3 was preincubated for 20 min with 0·16 µM [{gamma}32P]ATP. (His)6RegX3 was then added, and incubation was continued for 0 (lane 1), 1 (lane 2), 5 (lane 3), 10 (lane 4) or 30 min (lane 5).

 
Residues involved in the phosphotransfer reaction
Phosphorylation of acidic (aspartate, glutamate) residues can be distinguished from that of basic (arginine, histidine, lysine) residues by treatment with acid or base (Fujitaki & Smith, 1984 ). The phosphatidyl bonds of (His)6SenX3 and (His)6RegX3 were therefore tested for their chemical stability under acidic and basic conditions (Fig. 2, lanes 5–7). The phosphatidyl bond of (His)6SenX3 was acid labile and base stable, suggesting that phosphorylation occurred at a basic residue, which in two-component sensor proteins is typically a histidine. The phosphatidyl bond of (His)6RegX3 was base labile but stable under moderate acidic conditions, suggesting that phosphorylation occurred at an acidic residue, which in other regulators is typically an aspartate.

By analogy to other sensors and regulators, the conserved His-167 in (His)6SenX3 and Asp-52 in (His)6RegX3 (Swanson et al., 1994 ; Supply et al., 1997 ) were chosen to be altered by mutagenesis, and the recombinant mutant proteins (His)6SenX3-H167Q and (His)6RegX3-D52N were purified. When (His)6SenX3-H167Q was incubated with [{gamma}32P]ATP no autophosphorylation occurred (Fig. 4, lane 3), and the protein could not serve as phosphodonor for (His)6RegX3 (Fig. 4, lane 7). (His)6RegX3-D52N could not be phosphorylated, even in the presence of wild-type (His)6SenX3 (Fig. 4, lane 8).



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Fig. 4. Lack of phosphorylation of SenX3-H167Q and RegX3-D52N. (His)6SenX3 or (His)6SenX3-H167Q was incubated with [{gamma}32P]ATP. (His)6RegX3 or (His)6RegX3-D52N was then added as described in Methods. Lane 1, no protein; lane 2, 2 µg (His)6SenX3; lane 3, 2 µg (His)6SenX3-H167Q; lane 4, 10 µg (His)6RegX3; lane 5, 10 µg (His)6RegX3-D52N; lane 6, 2 µg (His)6SenX3 and 10 µg (His)6RegX3; lane 7, 2 µg (His)6SenX3-H167Q and 10 µg (His)6RegX3; lane 8, 2 µg (His)6SenX3 and 10 µg (His)6RegX3-D52N.

 
These results indicate that His-167 and Asp-52 are the sites of phosphorylation in SenX3 and RegX3, respectively, in agreement with the data obtained for other systems (Hess et al., 1988 ; Igo & Silhavy, 1988 ; Ninfa & Bennett, 1991 ).

RegX3 specifically binds the senX3 promoter region
Previous results have indicated that the senX3–regX3 genes are expressed as a polycistronic operon, under the control of a promoter region upstream of the senX3 gene (Supply et al., 1997 ). The ability of (His)6RegX3 to bind to this region was assessed by electrophoretic mobility shift assays using a 189 bp DIG-labelled PCR fragment containing nucleotides 52–225 and encompassing the senX3 promoter region (Fig. 5a).



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Fig. 5. Binding activity of RegX3 to the senX3 promoter region. (a) The sequence of the senX3 promoter region. The 189 bp DIG-labelled DNA fragment corresponds to region 52–225. The 85 bp fragment is shown by a dotted line, the 122 bp fragment by a dashed line and the 42 bp fragment by a continuous line. The putative Shine–Dalgarno (SD) and -10 box sequences are underlined. The inverted arrows indicate a palindromic sequence. (b) Electrophoretic mobility shift assays. One and a half femtomoles of 5’senX3-189 was mixed with purified (His)6RegX3 in 10 µl binding buffer as described in Methods. The binding mixture contained 15 pmol (His)6RegX3 alone, or with a 50x excess of specific unlabelled 5’senX3-189 (S) or a 100x excess of poly(dIdC) nonspecific DNA (NS) (lanes 2, 3 and 4, respectively). Lane 1 contained no protein. The positions of the well (W), the complexed DNA (C) and the free DNA (F) are indicated on the left.

 
The data presented in Fig. 5 demonstrate specific DNA-binding activity of (His)6RegX3 to this DNA fragment. Purified (His)6RegX3 retarded the labelled probe (Fig. 5b). DNA binding was specific for the senX3 promoter region, because the shifted band disappeared only in the presence of excess unlabelled DNA with the same sequence, but not in the presence of non-related DNA. Although the senX3and regX3 genes are most likely polycistronic, the two genes are separated by a rather long intercistronic region composed of a class of duplicated sequences named mycobacterial interspersed repetitive units (MIRUs; Supply et al., 1997 ), which could be conceivably involved in the regulation of the downstream gene. Therefore, electrophoretic mobility shift assays were carried out to test whether this intercistronic region could be bound by RegX3. This region was not retarded by (His)6RegX3 (not shown), which suggests that it does not contain regulatory sequences recognized by RegX3.

Several additional competition assays with sequences internal to the 189 bp PCR fragment were carried out in order to define more precisely the (His)6RegX3-binding site. A 122 bp fragment containing the 5' part of this region (from position 52 to 166) displaced the regulator from its labelled target, whereas an 85 bp fragment composed of the 3' part of this region (from 148 to 225) did not (Fig. 5a). Region 107–123 between the upstream pgm gene and senX3 corresponds to a short palindromic sequence which might function as a transcriptional terminator of the pgm gene. Therefore, we reasoned that the (His)6RegX3-binding site might lie 3' of this potential stem–loop structure. A 42 bp double-stranded oligonucleotide corresponding to segment 124–165 was thus tested and found to compete for the 189 bp labelled target sequence, indicating that this 42 bp fragment contains the RegX3 binding site (Fig. 5a). In addition to indicating specificity of the (His)6RegX3-binding activity these results also suggest that the senX3–regX3 operon is autoregulated.

Increase of the senX3 promoter activity by overexpression of senX3–regX3 in M. smegmatis
In vitro binding of RegX3 to the senX3–regX3 promoter region suggests that senX3–regX3 is autoregulated. To assess whether this system is positively or negatively autoregulated, the complete operon was cloned into a mycobacterial shuttle vector together with the senX3 promoter region fused to the promoterless lacZ reporter gene (yielding pREP5), and introduced into M. smegmatis. After growth in liquid medium, ß-galactosidase activities measured in extracts of cells containing pREP5 were reproducibly more than two-fold higher than those obtained in extracts of cells containing pREP7, a plasmid that bears the senX3 promoter region fused with lacZ in the absence of the senX3–regX3 operon (the mean values±standard deviation of ß-galactosidase activities of four independent assays were 39±5 units, compared to 17±4 units, respectively). These results indicate that the senX3–regX3 operon is positively regulated by its own regulator.


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The general mechanism of communication in two-component systems involves successive phosphorylation of the two partners. Sensors autophosphorylate on a conserved histidine residue using ATP as the phosphodonor. The phosphate group is then transferred onto a conserved aspartate residue of the response regulator (Parkinson & Kofoid, 1992 ; Stock et al., 1995 ). Our results indicate that the same mechanism prevails for the mycobacterial SenX3–RegX3 system. A truncated form of the SenX3 sensor containing the cytoplasmic C-terminal portion of the protein was found to be sufficient to carry out the autophosphorylation reaction, as has been observed for other sensors (Roberts et al., 1994 ). The cytoplasmic domain of SenX3 contains His-167 found to be critical for phosphorylation, as well as the typical motifs of the kinase domain (Parkinson & Kofoid, 1992 ).

Sequence similarity analysis ties RegX3 to the ROII subfamily of response regulators (Parkinson & Kofoid, 1992 ), which are presumed or demonstrated transcriptional regulators. The C terminus of RegX3 contains a putative helix–turn–helix DNA-binding motif, similar to that recently identified in the OmpR structure (Kondo et al., 1997 ; Martinez-Hackert & Stock, 1997 ). Electrophoretic mobility shift assays showed that RegX3 specifically binds the senX3 promoter region. Furthermore, operon fusion analyses indicated that activity of the senX3 promoter is significantly increased by the expression of the senX3–regX3 operon itself. Taken together, these two results indicate that the SenX3–RegX3 system is positively autoregulated and suggest that the response mediated by this system is subject to autoamplification.

In addition to their autokinase activity using their partner sensors as phosphodonors, some response regulators also possess an autophosphatase activity, resulting in half-lives of their phosphate group of a few seconds or minutes (Hess et al., 1988 ; Makino et al., 1989 ). In other systems, it is the cytoplasmic transmitter domain of the sensor that exerts phosphatase activity on the cognate response regulator. In that case, the dephosphorylation of the regulator is stimulated by the presence of ATP (Aiba et al., 1989 ; Igo et al., 1989 ). Autoactivation combined with the phosphatase activities allows for a temporary amplification of the response. Such a mechanism may be important for systems controlling responses to transient stresses and properly timed expression of the target genes (Raivio et al., 1999 ). As far as SenX3–RegX3 is concerned, we found that after rapid initial phosphotransfer from phosphorylated SenX3 to RegX3, the phosphate group remained stably attached to RegX3 for at least 30 min, even in the presence of excess ATP. From these observations we infer that neither RegX3 or SenX3 contain a strong phosphatase activity, at least under the experimental conditions used here. This suggests that the modulation of the adaptive response of the SenX3–RegX3 system may be rather slow, which is perhaps related to the general slow growth rate of mycobacteria. Alternatively, RegX3 dephosphorylation may be catalysed by auxiliary phosphatases as has been shown for the Bacillus subtilis Spo system (Perego et al., 1994 ).

For some response regulators, phosphorylation appears to be an absolute requirement for detectable binding to specific DNA targets (Boucher & Stibitz, 1995 ; Li et al., 1994 ). In many other cases, phosphorylation increases the affinity for the target DNA by a factor of 10–100, although the unphosphorylated response regulator retains some DNA-binding activity (Aiba et al., 1989 ; Makino et al., 1989 ; Forst et al., 1989 ; Nakashima et al., 1991 ; Boucher et al., 1994 ; Hoch & Silhavy, 1995 ; Lynch & Lin, 1996 ; Dahl et al., 1997 ; Meyer et al., 1997 ). Our results indicate that phosphorylation is not essential for RegX3 binding. The observed binding of the non-phosphorylatable mutant (His)6RegX3-D52N rules out effect of phosphorylation of recombinant RegX3 from non-cognate sensors in E. coli. Moreover, we observed no detectable effect of (His)6RegX3 phosphorylation on binding to the senX3–regX3 operon compared to unphosphorylated (His)6RegX3 (not shown). These results suggest that phosphorylation of RegX3 may exert its effect predominantly on protein–protein interactions stimulating transcription, rather than on DNA binding. For several two-component systems it is known that, in addition to its effect on DNA binding, phosphorylation may act by favouring protein–protein interactions within protein–DNA transcription complexes (Weiss et al., 1992 ; Porter et al., 1993 ; Bird et al., 1996 ; Wyman et al., 1997 ; Boucher et al., 1997 ). Interestingly, a predominant role of phosphorylation on protein–protein interaction has also been proposed for PhoP of B. subtilis, which is a close homologue of RegX3. This protein also binds its DNA targets regardless of its phosphorylation state, and phosphorylation only marginally affects DNA-binding affinity (Liu & Hulett, 1997 ). Alternatively, the above observations may reflect differences in affinities between the promoter of the two-component system operon and the promoters of other members of the regulon for the phosphorylated regulator, as shown for the Bordetella pertussis BvgA regulator (Steffen et al., 1996 ). In that case, different effects of phosphorylation on binding to various target promoters may offer a wider range of regulation levels and potentially allow for differential expression of the regulon members during the pathway activation (Raivio et al., 1999 ).


   ACKNOWLEDGEMENTS
 
We gratefully acknowledge E. Fort for photography and E. Clément for help in sequencing. The work was supported by INSERM, Institut Pasteur de Lille, Région Nord-Pas de Calais and Ministère de la Recherche. S.H. held a fellowship of Région Nord-Pas de Calais. P.S. is a Chercheur of the CNRS.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 3 March 2000; revised 4 August 2000; accepted 31 August 2000.