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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: two-component systems, autoregulation, phosphorylation, Mycobacterium tuberculosis
Abbreviations: DIG, digoxigenin
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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, MtrAMtrB, SenX3RegX3 and TrcSTrcR, 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 senX3regX3 operon in Mycobacterium smegmatis.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 HindIIIBamHI fragment containing the senX3regX3 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 0·20·4. 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 0·70·8, 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 [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 pulsechase experiments, 10 µg (His)6SenX3 was incubated with 0·16 µM [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 [
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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 [
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.
|
|
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 [
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).
|
RegX3 specifically binds the senX3 promoter region
Previous results have indicated that the senX3regX3 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 52225 and encompassing the senX3 promoter region (Fig. 5a
).
|
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 107123 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 stemloop structure. A 42 bp double-stranded oligonucleotide corresponding to segment 124165 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 senX3regX3 operon is autoregulated.
Increase of the senX3 promoter activity by overexpression of senX3regX3 in M. smegmatis
In vitro binding of RegX3 to the senX3regX3 promoter region suggests that senX3regX3 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 senX3regX3 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 senX3regX3 operon is positively regulated by its own regulator.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 helixturnhelix 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 senX3regX3 operon itself. Taken together, these two results indicate that the SenX3RegX3 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 SenX3RegX3 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 SenX3RegX3 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 10100, 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 senX3regX3 operon compared to unphosphorylated (His)6RegX3 (not shown). These results suggest that phosphorylation of RegX3 may exert its effect predominantly on proteinprotein 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 proteinprotein interactions within proteinDNA 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 proteinprotein 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 |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alex, L. A. & Simon, M. I. (1994). Protein histidine kinases and signal transduction in prokaryotes and eukaryotes. Trends Genet 10, 133-138.[Medline]
Bajaj, V., Lucas, R. L., Hwang, C. & Lee, C. A. (1996). Co-ordinate regulation of the Salmonella typhimurium invasion genes by environmental and regulatory factors is mediated by control of hilA expression. Mol Microbiol 22, 703-714.[Medline]
Bird, T. H., Grimsley, J. K., Hoch, J. A. & Spiegelman, G. B. (1996). The Bacillus subtilis response regulator SpoOA stimulates transcription of the spoIIG operon through modification of RNA polymerase promoter complexes. J Mol Biol 256, 436-448.[Medline]
Boucher, P. E. & Stibitz, S. (1995). Synergistic binding of RNA polymerase and BvgA phosphate to the pertussis toxin promoter of Bordetella pertussis. J Bacteriol 177, 6486-6491.[Abstract]
Boucher, P. E., Menozzi, F. D. & Locht, C. (1994). The modular architecture of bacterial response regulators: insights into the activation mechanism of BvgA transactivator of Bordetella pertussis. J Mol Biol 241, 363-377.[Medline]
Boucher, P. E., Murakami, K., Ishihama, A. & Stibitz, S. (1997). Nature of DNA binding and RNA polymerase interaction of the Bordetella pertussis BvgA transcriptional activator at the fha promoter. J Bacteriol 179, 1755-1763.[Abstract]
Bowden, G. A. & Georgiou, G. (1990). Folding and aggregation of ß-lactamase in the periplasmic space of Escherichia coli. J Biol Chem 265, 16760-16766.
Cole, S., Brosch, R., Parkhill, J. & 39 other authors (1998). Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature393, 537544.[Medline]
Dahl, J. L., Wei, B. & Kadner, R. J. (1997). Protein phosphorylation affects binding of the Escherichia coli transcription activator UhpA to the uhpT promoter. J Biol Chem 272, 1910-1919.
Forst, S., Delgado, J. & Inouye, M. (1989). Phosphorylation of OmpR by the osmosensor EnvZ modulates expression of the ompF and ompC genes in Escherichia coli. Proc Natl Acad Sci USA 86, 6052-6056.[Abstract]
Fujitaki, J. M. & Smith, R. A. (1984). Techniques in the detection and characterization of phosphoramidate-containing proteins. Methods Enzymol 107, 23-36.[Medline]
Haydel, S. E., Dunlap, N. E. & Benjamin, W. H.Jr (1999). In vitro evidence of two-component system phosphorylation between the Mycobacterium tuberculosis TcR/TcrS proteins. Microb Pathog 26, 195-206.[Medline]
Hess, J. F., Bourret, R. B. & Simon, M. I. (1988). Histidine phosphorylation and phosphoryl group transfer in bacterial chemotaxis. Nature 336, 139-143.[Medline]
Hoch, J. A. & Silhavy, T. J. (1995). Two-Component Signal Transduction. Washington, DC: American Society for Microbiology.
Igo, M. M. & Silhavy, T. J. (1988). EnvZ, a transmembrane environmental sensor of Escherichia coli K-12, is phosphorylated in vitro. J Bacteriol 170, 5971-5973.[Medline]
Igo, M. M., Ninfa, A. J., Stock, J. B. & Silhavy, T. J. (1989). Phosphorylation and dephosphorylation of a bacterial transcriptional activator by a transmembrane receptor. Genes Dev 3, 1725-1734.[Abstract]
Jacobs, W. B.Jr, Kalpana, G. V., Cirillio, J. D., Pascopella, L., Snapper, S. B., Udani, R. A., Jones, W., Barletta, R. G. & Bloom, B. R. (1991). Genetic systems for mycobacteria. Methods Enzymol 204, 537-555.[Medline]
Kleerebezem, M., Quadri, L. E., Kuipers, O. P. & De Vos, W. M. (1997). Quorum sensing by peptide pheromones and two-component signal-transduction systems in Gram-positive bacteria. Mol Microbiol 25, 895-904.
Kondo, H., Nakagawa, A., Nishihira, J., Nishimura, Y., Mizuno, T. & Tanaka, I. (1997). Escherichia coli positive regulator OmpR has a large loop structure at the putative RNA polymerase interaction site. Nat Struct Biol 4, 28-31.[Medline]
Kremer, L., Baulard, A., Estaquier, J., Poulain-Godefroy, O. & Locht, C. (1995). Green fluorescent protein as a new expression marker in mycobacteria. Mol Microbiol 17, 913-922.[Medline]
Li, J., Kustu, S. & Stewart, V. (1994). In vitro interaction of nitrate-responsive regulatory protein NarL with DNA target sequences in the fdnG, narG, narK and frdA operon control regions of Escherichia coli K-12. J Mol Biol 241, 150-165.[Medline]
Liu, W. & Hulett, F. M. (1997). Bacillus subtilis PhoP binds to the phoB tandem promoter exclusively within the phosphate starvation-inducible promoter. J Bacteriol 179, 6302-6310.[Abstract]
Lynch, A. S. & Lin, E. C. C. (1996). Transcriptional control mediated by the ArcA two-component response regulator protein of Escherichia coli: characterization of DNA binding at target promoters. J Bacteriol 178, 6238-6249.[Abstract]
Makino, K., Shinagawa, H., Amemura, M., Kawamoto, T., Yamada, M. & Nakata, A. (1989). Signal transduction in the phosphate regulon of Escherichia coli involves phosphotransfer between PhoR and PhoB proteins. J Mol Biol 210, 551-559.[Medline]
Martinez-Hackert, E. & Stock, A. M. (1997). Structural relationships in the OmpR family of winged-helix transcription factors. J Mol Biol 269, 301-312.[Medline]
Meyer, M., Dimroth, P. & Bott, M. (1997). In vitro binding of the response regulator CitB and of its carboxy-terminal domain to A+T-rich DNA target sequences in the control region of the divergent citC and citS operons of Klebsiella pneumoniae. J Mol Biol 269, 719-731.[Medline]
Miller, J. H. (1992). A Short Course in Bacterial Genetics: a Laboratory Manual and Handbook for Escherichia coli and Related Bacteria. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Mukhopadhyay, A. (1997). Inclusion bodies and purification of proteins in biologically active forms. Adv Biochem Eng Biotechnol 56, 61-109.[Medline]
Nakashima, K., Kanamaru, K., Aiba, H. & Mizuno, T. (1991). Signal transduction and osmoregulation in Escherichia coli. J Biol Chem 266, 10775-10780.
Ninfa, A. J. & Bennet, R. L. (1991). Identification of the site of autophosphorylation of the bacterial protein kinase/phosphatase NRII. J Biol Chem 266, 6888-6893.
Parkinson, J. S. & Kofoid, E. C. (1992). Communication modules in bacterial signalling proteins. Annu Rev Genet 26, 71-112.[Medline]
Perego, M., Hanstein, C., Welsh, K. M., Djavakhishvli, T., Glaser, P. & Hoch, J. A. (1994). Multiple protein-aspartate phosphatases provide a mechanism for the integration of diverse signals in the control of development in B. subtilis. Cell 79, 1047-1055.[Medline]
Porter, S. C., North, A. K., Wedel, A. B. & Kustu, S. (1993). Oligomerization of NtrC at the glnA enhancer is required for transcriptional activation. Genes Dev 7, 2258-2273.[Abstract]
Raivio, T. L., Popkin, D. L. & Silhavy, T. J. (1999). The Cpx envelope stress response is controlled by amplification and feedback inhibition. J Bacteriol 181, 5263-5272.
Roberts, D. L., Bennett, D. W. & Forst, S. A. (1994). Identification of the site of phosphorylation on the osmosensor, EnvZ, of Escherichia coli. J Mol Biol 269, 8728-8733.
Soncini, F. C., Vescovi, E. G. & Groisman, E. A. (1995). Transcriptional autoregulation of the Salmonella typhimurium phoPQ operon. J Bacteriol 177, 4364-4371.[Abstract]
Steffen, S., Goyard, P. & Ullmann, A. (1996). Phosphorylated BvgA is sufficient for transcriptional activation of virulence-regulated genes in Bordetella pertussis. EMBO J 15, 102-109.[Abstract]
Stock, J. B., Ninfa, A. J. & Stock, A. M. (1989). Protein phosphorylation and regulation of adaptive responses in bacteria. Microbiol Rev 53, 450-490.
Stock, J. B., Surette, M. G., Levit, M. & Park, P. (1995). Two-component signal transduction systems: structure-function relationships and mechanisms of catalysis. In Two-Component Signal Transduction , pp. 25-52. Edited by J. A. Hoch & T. J. Silhavy. Washington, DC:American Society for Microbiology.
Supply, P., Magdalena, J., Himpens, S. & Locht, C. (1997). Identification of novel intergenic repetitive units in a mycobacterial two-component system operon. Mol Microbiol 26, 991-1003.[Medline]
Swanson, R. V., Alex, L. A. & Simon, M. I. (1994). Histidine and aspartate phosphorylation: two-component systems and the limits of homology. Trends Biochem Sci 19, 485-490.[Medline]
Via, L. E., Curcic, R., Mudd, M. H., Dhandayuthapani, S., Ulmer, R. J. & Deretic, V. (1996). Elements of signal transduction in Mycobacterium tuberculosis: in vitro phosphorylation and in vivo expression of the response regulator MtrA. J Bacteriol 178, 3314-3321.[Abstract]
Weiss, V., Claverie-Martin, F. & Magasanik, B. (1992). Phosphorylation of nitrogen regulator I of Escherichia coli induces strong cooperative binding to DNA essential for activation of transcription. Proc Natl Acad Sci USA 89, 5088-5092.[Abstract]
Wyman, C., Rombel, I., North, A. K., Bustamante, C. & Kustu, S. (1997). Unusual oligomerization required for activity of NtrC, a bacterial enhancer-binding protein. Science 275, 1658-1661.
Received 3 March 2000;
revised 4 August 2000;
accepted 31 August 2000.