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Logo of actaf2this articlesearchopen accesssubscribesubmitActa Crystallographica. Section F, Structural Biology CommunicationsActa Crystallographica. Section F, Structural Biology Communications
Acta Crystallogr F Struct Biol Commun. 2015 August 1; 71(Pt 8): 946–950.
Published online 2015 July 28. doi:  10.1107/S2053230X15009577
PMCID: PMC4528921

Crystallographic studies of the extracytoplasmic function σ factor σJ from Mycobacterium tuberculosis


Mycobacterium tuberculosis has multiple σ factors which enable the bacterium to reprogram its transcriptional machinery under diverse environmental conditions. σJ, an extracytoplasmic function σ factor, is upregulated in late stationary phase cultures and during human macrophage infection. σJ governs the cellular response to hydrogen peroxide-mediated oxidative stress. σJ differs from other canonical σ factors owing to the presence of a SnoaL_2 domain at the C-terminus. σJ crystals belonged to the tetragonal space group I422, with unit-cell parameters a = b = 133.85, c = 75.08 Å. Diffraction data were collected to 2.16 Å resolution on the BM14 beamline at the European Synchrotron Radiation Facility (ESRF).

Keywords: σ factor, sigma factor, SnoaL_2 domain, potential drug target

1. Introduction  

Transcription in prokaryotes is primarily regulated at the initiation step. σ factors play a prominent role in transcription initiation as they govern both promoter recognition and promoter melting. Changes in gene expression are thus dependent on the reversible association of the σ factor with the RNA polymerase enzyme (Burgess et al., 1969  ). Mycobacterium tuberculosis has 13 σ factors. These include a principal σ factor, σA, a principal-like or group 2 σ factor, σB, a group 3 σ factor, σF, and ten group 4 or extracytoplasmic function (ECF) σ factors, σC, σD, σE, σG, σH, σI, σJ, σK, σL and σM (Cole et al., 1998  ; Rodrigue et al., 2006  ). ECF σ factors couple environmental changes with the transcriptional profile. The large number of ECF σ factors in M. tuberculosis suggests an elaborate mechanism to ensure the survival of this pathogen under diverse microenvironments in the host (Sachdeva et al., 2010  ).

ECF σ factors are typically two-domain proteins with a conserved domain 2 (σ2) that binds the Pribnow box element and domain 4 (σ4) that recognizes the −35 element of the promoter (Lonetto et al., 1994  ; Gruber & Gross, 2003  ). The activity of many ECF σ factors is post-translationally regulated by either a soluble anti-σ factor (in the case of σE and σH) or a membrane-bound anti-σ factor (regulating σD, σK, σL and σM) (Song et al., 2003  ; Hahn et al., 2005  ; Saïd-Salim et al., 2006  ; Dainese et al., 2006  ; Donà et al., 2008  ; Sklar et al., 2010  ). The release of the free ECF σ from an inactive σ–anti-σ complex is influenced by multiple mechanisms including phosphorylation, redox stimuli or specific degradation of the anti-σ domain by Clp proteases (Song et al., 2003  ; Park et al., 2008  ; Sklar et al., 2010  ; Jaiswal et al., 2013  ). Among ECF σ factors, σC, σG, σI and σJ are not associated with an anti-σ factor. Interestingly, these ECF σ factors have either N- or C-terminal polypeptide extensions that can influence the function of these transcription factors. While the N-terminal polypeptide is likely to play a role in the auto-regulation of σC, the role of the C-terminal domain in σG, σI and σJ has not been examined thus far. This C-terminal stretch of around 120 residues is conserved across orthologues (Fig. 1  ). The C-terminal polypeptide extension contains a putative SnoaL_2 domain in σG and σJ. In eukaryotes, proteins with SnoaL domains play a role in facilitating protein–protein interactions. This inference was based on the similarity of the SnoaL_2 domain to a region of nuclear transport factor 2 (NTF) that interacts with the Ras GTPase (Rodrigue et al., 2006  ).

Figure 1
Domain architecture of σJ. (a) σJ contains two DNA-binding domains (shown here as Sigma70_r2 and Sigma70_r4) preceding the SnoaL_2 domain. (b) Multiple sequence alignment of σJ homologues. The SnoaL_2 domain is conserved across ...

The expression of σJ is upregulated during the late stationary phase of growth and during human macrophage infection (Hu & Coates, 2001  ; Cappelli et al., 2006  ). Interestingly, σJ knockout mutants of M. tuberculosis have a comparable growth and survival ability to the wild-type strain in stationary-phase cultures. M. tuberculosis σJ deletion-mutant strains are more susceptible to killing by hydrogen peroxide compared with the wild type, suggesting a role for this σ factor in hydrogen peroxide resistance (Hu et al., 2004  ). In a study using an Escherichia coli two-plasmid system, σJ was shown to recognize a promoter upstream of the ECF σ factor σI, suggesting a direct role of σJ in the expression of σI (Homerova et al., 2008  ).

The unusual domain architecture of M. tuberculosis σJ and its role in hydrogen peroxide-mediated stress resistance and high expression levels during the late stationary phase makes this σ factor an interesting target for structure–function analysis. Here, we describe biochemical characterization and crystallization experiments on this protein

2. Materials and methods  

2.1. Macromolecule production  

The gene encoding σJ was PCR-amplified from M. tuberculosis H37Rv genomic DNA. The amplified product was cloned between the NheI and XhoI sites of a modified pET-15b vector (Novagen Inc.; Table 1  ). The clone was confirmed using single primer-based sequencing (Amnion Biosciences Pvt. Ltd). The expression clone was transformed into Escherichia coli BL21(DE3) strain (Novagen Inc.). A single colony was inoculated in Luria–Bertani (LB) medium containing ampicillin (100 µg ml−1). Cultures were grown at 310 K prior to induction with 0.2 mM IPTG. The cultures were grown for a further 12 h at 291 K. Cells were then spun at 7000g for 15 min. The pellet was resuspended in buffer A (50 mM Tris–HCl pH 8.0, 300 mM NaCl) containing 2 mM PMSF and EDTA-free protease-inhibitor tablets (Roche). The cells were lysed by sonication and the cell debris was removed by centrifugation at 30 000g for 45 min at 277 K. The supernatant was incubated with Ni–NTA resin (Sigma–Aldrich) for 1 h. The protein was then eluted using buffer A with a gradient of imidazole (10–200 mM). The protein was further purified by size-exclusion chromatography using a Sephacryl S200 HiPrep 16/60 column (GE Healthcare). The fractions containing the purified protein were concentrated to ~10 mg ml−1 for crystallization trials. The purity and molecular mass of the protein were verified using SDS–PAGE and mass spectrometry.

Table 1
Macromolecule-production information

2.2. Crystallization  

Initial crystallization trials were performed using screens from Hampton Research. The sparse-matrix conditions were examined using the microbatch method at 291 K. A crystallization drop consisted of 2 µl protein solution and 2 µl crystallization condition. Crystals were obtained in a condition consisting of 0.2 M potassium sodium tartrate, 0.1 M sodium citrate tribasic pH 5.6, 2 M ammonium sulfate (Fig. 2  ). Crystals were soaked in mother liquor containing 20% glycerol followed by flash-cooling in liquid nitrogen (Table 2  ).

Figure 2
Purification and crystallization of σJ. (a) 12% silver-stained SDS–PAGE. Lane 1, Bio-Rad dual colour protein markers (molecular masses are labelled in kDa). Lane 2, an analysis of σJ crystals suggests the crystallized species to ...
Table 2

2.3. Data collection and processing  

Diffraction data were collected on the BM14 beamline at the European Synchrotron Radiation Facility (ESRF) at 100 K. The diffraction data were collected using an oscillation of 0.5° per image. The diffraction data were processed using iMosflm (Battye et al., 2011  ) and were scaled (Table 3  ) using SCALA (Evans, 2006  ).

Table 3
Data collection and processing

3. Results and discussion  

The M. tuberculosis sigJ gene (Rv3328c) encoding the ECF σ factor σJ was cloned into a modified pET-15b expression vector (Novagen). The expression clone was confirmed using a single primer-based sequencing method (Sanger et al., 1977  ). The recombinant σJ protein could be purified to greater than 90% purity using a two-step purification process involving affinity and size-exclusion chromatography. The molecular mass of the recombinant protein was found to be 36 270 Da using liquid-chromatography electrospray ionization mass spectrometry (LC-ESI-MS). The oligomeric status of M. tuberculosis σJ was examined using analytical size-exclusion chromatography. These experiments, which were performed on a Superdex 200 column (GE Healthcare), suggested that this protein is a monomer in solution (Fig. 3  ). The protein could be concentrated to 10 mg ml−1. Tetragonal-shaped crystals of σJ could be obtained in approximately two weeks (Fig. 2  ). These crystals diffracted to a resolution of 2.16 Å on the BM14 beamline at ESRF, Grenoble. The diffraction data statistics are compiled in Table 3  . The Matthews coefficient (2.52 Å3 Da−1) suggests that this crystal form has one molecule of σJ in the asymmetric unit, with a solvent content of ~51% (Matthews, 1968  ). Analysis of the diffraction data using SFCHECK (Winn et al., 2011  ) suggested the absence of translational pseudo-symmetry or twinning. M. tuberculosis σJ has two DNA-binding domains, σJ 2 and σJ 4, that are likely to interact with the Pribnow box (−10 element) and the −35 element of the promoter. These domains are attached to a SnoaL_2 domain (Rodrigue et al., 2006  ). M. tuberculosis σJ shares very poor sequence similarity with other structurally characterized σ factors (~13%). This observation, as well as the unique domain organization of M. tuberculosis σJ, precludes the possibility of a straightforward molecular-replacement strategy using the structures of other characterized σ factors. The role of the SnoaL_2 domain in M. tuberculosis σJ activity or regulation remains to be determined. The crystal structure is likely to aid in this process as well as in the functional characterization of this transcription factor. Evaluation of heavy-atom derivatives of these crystals and efforts to obtain crystals of selenomethionine-labelled protein are in progress.

Figure 3
M. tuberculosis σJ is a monomer in solution. Freshly purified σJ (100 µl at 10 mg ml−1 concentration) was loaded onto a Superdex S200 column (GE Healthcare) in this size-exclusion chromatography ...


We acknowledge Dr Hassan Belrhali and Dr Babu A. Manjasetty for their help in data collection at beamline BM14 of the European Synchrotron Radiation Facility, Grenoble, France. This work was supported in part by a grant from the Department of Biotechnology, Government of India.


  • Battye, T. G. G., Kontogiannis, L., Johnson, O., Powell, H. R. & Leslie, A. G. W. (2011). Acta Cryst. D67, 271–281. [PMC free article] [PubMed]
  • Burgess, R. R., Travers, A. A., Dunn, J. J. & Bautz, E. K. (1969). Nature (London), 221, 43–46. [PubMed]
  • Cappelli, G., Volpe, E., Grassi, M., Liseo, B., Colizzi, V. & Mariani, F. (2006). Res. Microbiol. 157, 445–455. [PubMed]
  • Cole, S. T. et al. (1998). Nature (London), 393, 537–544. [PubMed]
  • Dainese, E., Rodrigue, S., Delogu, G., Provvedi, R., Laflamme, L., Brzezinski, R., Fadda, G., Smith, I., Gaudreau, L., Palù, G. & Manganelli, R. (2006). Infect. Immun. 74, 2457–2461. [PMC free article] [PubMed]
  • Donà, V., Rodrigue, S., Dainese, E., Palù, G., Gaudreau, L., Manganelli, R. & Provvedi, R. (2008). J. Bacteriol. 190, 5963–5971. [PMC free article] [PubMed]
  • Evans, P. (2006). Acta Cryst. D62, 72–82. [PubMed]
  • Gruber, T. M. & Gross, C. A. (2003). Annu. Rev. Microbiol. 57, 441–466. [PubMed]
  • Hahn, M. Y., Raman, S., Anaya, M. & Husson, R. N. (2005). J. Bacteriol. 187, 7062–7071. [PMC free article] [PubMed]
  • Homerova, D., Halgasova, L. & Kormanec, J. (2008). FEMS Microbiol. Lett. 280, 120–126. [PubMed]
  • Hu, Y. & Coates, A. R. (2001). FEMS Microbiol. Lett. 202, 59–65. [PubMed]
  • Hu, Y., Kendall, S., Stoker, N. G. & Coates, A. R. (2004). FEMS Microbiol. Lett. 237, 415–423. [PubMed]
  • Jaiswal, R. K., Prabha, T. S., Manjeera, G. & Gopal, B. (2013). Nucleic Acids Res. 41, 3414–3423. [PMC free article] [PubMed]
  • Lonetto, M. A., Brown, K. L., Rudd, K. E. & Buttner, M. J. (1994). Proc. Natl Acad. Sci. USA, 91, 7573–7577. [PubMed]
  • Matthews, B. W. (1968). J. Mol. Biol. 33, 491–497. [PubMed]
  • Park, S. T., Kang, C.-M. & Husson, R. N. (2008). Proc. Natl Acad. Sci. USA, 105, 13105–13110. [PubMed]
  • Rodrigue, S., Provvedi, R., Jacques, P.-É, Gaudreau, L. & Manganelli, R. (2006). FEMS Microbiol. Rev. 30, 926–941. [PubMed]
  • Sachdeva, P., Misra, R., Tyagi, A. K. & Singh, Y. (2010). FEBS J. 277, 605–626. [PubMed]
  • Saïd-Salim, B., Mostowy, S., Kristof, A. S. & Behr, M. A. (2006). Mol. Microbiol. 62, 1251–1263. [PubMed]
  • Sanger, F., Nicklen, S. & Coulson, A. R. (1977). Proc. Natl Acad. Sci. USA, 74, 5463–5467. [PubMed]
  • Sklar, J. G., Makinoshima, H., Schneider, J. S. & Glickman, M. S. (2010). Mol. Microbiol. 77, 605–617. [PMC free article] [PubMed]
  • Song, T., Dove, S. L., Lee, K. H. & Husson, R. N. (2003). Mol. Microbiol. 50, 949–959. [PubMed]
  • Winn, M. D. et al. (2011). Acta Cryst. D67, 235–242. [PMC free article] [PubMed]

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