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1.
FIGURE 7.

FIGURE 7. From: Septal Localization of the Mycobacterium tuberculosis MtrB Sensor Kinase Promotes MtrA Regulon Expression.

Characterization of the effects of mutant MtrA proteins on mtrB phenotype. Upper panel, shown is an autoradiogram evaluating the phosphorylation potential of MtrA proteins. MtrAWT, MtrAD13A, and MtrAY102C were incubated with EnvZ and [γ-32P]ATP, and products were resolved by SDS-PAGE and visualized by phosphorimaging. Lower panel, shown is morphology of ΔmtrB-producing mutant MtrA proteins. ΔmtrB transformed with plasmids producing MtrAWTmtrB + mtrAWT), phosphorylation-defective MtrA (ΔmtrB + mtrAD13A, ΔmtrB + mtrAD56N), or phosphorylation-proficient MtrA (ΔmtrB + mtrAY102C) were grown with acetamide for 16 h, and cells were visualized by brightfield microscopy. Note near restoration of WT phenotype with plasmids producing either mtrAY102C or mtrAD13A.

Renata Plocinska, et al. J Biol Chem. 2012 July 6;287(28):23887-23899.
2.
FIGURE 4.

FIGURE 4. From: Septal Localization of the Mycobacterium tuberculosis MtrB Sensor Kinase Promotes MtrA Regulon Expression.

Phenotypic characterization of mtrB mutant. Visualization of M. smegmatis (panel i) WT and (panel ii) ΔmtrB strains by brightfield microscopy is shown. Actively growing cultures in liquid were examined. Note: the black arrow in panel ii indicates cell lysis. Black arrowheads, buds, branches, and swollen cell morphology. Complementation of ΔmtrB phenotype: panel iii, Pami::mtrBTB (pRD102); panels iv-v, Ptet::mtrB-gfp (pKS4). Panel iv represents a brightfield image, whereas v represents a fluorescence image. White arrowhead, midcell MtrB-GFP band.

Renata Plocinska, et al. J Biol Chem. 2012 July 6;287(28):23887-23899.
3.
FIGURE 2.

FIGURE 2. From: Septal Localization of the Mycobacterium tuberculosis MtrB Sensor Kinase Promotes MtrA Regulon Expression.

MtrB localization under FtsZ depletion conditions. FZ3, M. smegmatis ΔftsZ, Pami::ftsZ strain (23) expressing Ptet::mtrB-gfp was grown with 10 ng/ml anhydrotetracycline but in the presence (i and ii) or in the absence (iii to vi) of acetamide for 6 h (iii and iv) and 9 h (v and vi) and examined by brightfield (panels i, iii, and v) and fluorescence (panels ii, iv, and vi) microscopy. Bars denote the length in microns. Note distinct Z-rings (marked with arrows) present in the panel ii are absent in panels iv and vi. Arrowhead, membrane localization.

Renata Plocinska, et al. J Biol Chem. 2012 July 6;287(28):23887-23899.
4.
FIGURE 6.

FIGURE 6. From: Septal Localization of the Mycobacterium tuberculosis MtrB Sensor Kinase Promotes MtrA Regulon Expression.

Cell wall hydrolysis activity of MtrB proteins. Cell wall preparations from M. smegmatis WT and ΔmtrB strains were prepared, labeled with fluorescein, and incubated with 1 mg/ml lysozyme in 50 mm Tris-HCl 8.0 and 1 mm EDTA for 4 h. Reactions were arrested by adding 4 m LiCl, and released labeled products in the supernatant relative to the total labeled input cell wall were measured in a Jasco FP 6500 fluorimeter. Average percent hydrolysis from three independent experiments is shown.

Renata Plocinska, et al. J Biol Chem. 2012 July 6;287(28):23887-23899.
5.
FIGURE 1.

FIGURE 1. From: Septal Localization of the Mycobacterium tuberculosis MtrB Sensor Kinase Promotes MtrA Regulon Expression.

Visualization of MtrB-GFP structures in M. tuberculosis and M. smegmatis. Mtb-Ptet::mtrB-gfp (i and ii), Msmeg-Ptet::mtrB-gfp (iii and iv), or Msmeg-Ptet::mtrBsol-gfp (v and vi) strains were examined by brightfield and fluorescence microscopy. Inducer anhydrotetracycline was added at a final concentration of 10 ng/ml. As controls, M. smegmatis dosS-gfp expressed from Ptet was tested (panels vii and viii). In panels ii, iv, and vi fluorescent structures are marked: arrow, midcell localization; arrowhead, polar localization. Note that Msmeg-Ptet::mtrBsol-gfp cells show diffuse fluorescence (panel vi), whereas Ptet::dosS-gfp cells do not show any distinct septal localization (panel viii).

Renata Plocinska, et al. J Biol Chem. 2012 July 6;287(28):23887-23899.
6.
FIGURE 9.

FIGURE 9. From: Septal Localization of the Mycobacterium tuberculosis MtrB Sensor Kinase Promotes MtrA Regulon Expression.

qRT-PCR analysis of MtrA targets. Total RNA from WT and ΔmtrB cells was extracted and reverse-transcribed, and qRT-PCR was performed as described under “Materials and Methods.” Expression levels of select genes relative to housekeeping gene sigA were compared, and the values are presented as -fold difference in the expression of mutant relative to WT cells. A, shown are qRT-PCR expression levels of select targets in ΔmtrB background. Panel i includes the -fold expression levels of wag31, dnaA, ripA, ftsI, and fbpB, whereas panel ii includes the expression levels of chiZ, ftsZ, and pfkB. B, qRT-PCR expression levels of select targets in ΔmtrB background-overproducing mutant MtrAY102C. Uninduced refers to cultures grown in the absence of acetamide, whereas induced indicates cultures grown in the presence of 0.2% acetamide for 12 h. Panel i shows the expression levels of mtrA, wag31, and ftsI; panel ii shows the data for dnaA, ripA, and fbpB, and panel iii shows the qRT-PCR data for chiZ, ftsZ, and pfkB. Note the y axis scale in panel iii is different from panels i and ii. C, shown are qRT-PCR expression levels of select targets in ΔmtrB background overproducing mutant MtrAD13A. Other details are as described for panels i–iii under B.

Renata Plocinska, et al. J Biol Chem. 2012 July 6;287(28):23887-23899.
7.
FIGURE 3.

FIGURE 3. From: Septal Localization of the Mycobacterium tuberculosis MtrB Sensor Kinase Promotes MtrA Regulon Expression.

Construction of M. smegmatis mtrB mutant. A, shown is a schematic depicting the 9.3-kb mtrB region of M. smegmatis (top panel) and the mutant mtrB region (bottom panel). The region is not drawn to scale. The locations of NcoI and BglII sites are shown. B, shown is a Southern blot confirming the deletion of mtrB. Genomic DNA was isolated from WT M. smegmatis, two SCO (A and B under SCO), and four DCO strains, digested with BglII or NcoI enzymes, transferred to nitrocellulose membrane, and probed with 32P-labeled mtrA fragment (black bar shown in the top panel). Using this probe, we detected bands corresponding to 7.8 kb for mutant and 3.9 kb for wild type with BglII digestion but 1.98 kb for mutant and 1.38 kb for WT with NcoI. Positions of SCO, DCO and chromosomal copy mtrB bands are marked. C, shown is immunoblotting of M. smegmatis mtrB strains. For confirmation of the loss of MtrB protein, protein lysates from select DCOs along with WT strain were prepared, 5 μg of protein was resolved by SDS-PAGE in 12% gels, and immunoblotting was performed with affinity purified α-MtrB antibodies. Parallel blots were processed and probed with α-SigA antibodies.

Renata Plocinska, et al. J Biol Chem. 2012 July 6;287(28):23887-23899.
8.
FIGURE 5.

FIGURE 5. From: Septal Localization of the Mycobacterium tuberculosis MtrB Sensor Kinase Promotes MtrA Regulon Expression.

In vitro phosphorylation and in vivo localization of mutant MtrB-GFP structures. A, recombinant MtrB, MtrBH305Y, and MtrBH305D proteins were tested for autophosphorylation activity in buffer containing Ca2+ and Mg2+ and [γ-32P]ATP as described in the Materials and Methods section. Samples were incubated for 30 min and resolved by SDS-PAGE, and the radioactivity in the protein bands was visualized in a Bio-Rad Molecular Imager Fx (top panel). A parallel gel was stained with Coomassie Blue and visualized (bottom panel). M, protein size markers. Note that distinct phosphorylation was seen with wild-type MtrB but not with mutant proteins. B, visualization of MtrBH305Y-GFP structures in M. smegmatis ΔmtrB strain is shown. Panels i shows the bright-field image, whereas panel ii shows the fluorescent image. Note: these cells remained filamentous but contained distinct bands at septa. Black arrow, bulged morphology; white arrow, membrane localization; white arrowhead, septal localization.

Renata Plocinska, et al. J Biol Chem. 2012 July 6;287(28):23887-23899.
9.
FIGURE 10.

FIGURE 10. From: Septal Localization of the Mycobacterium tuberculosis MtrB Sensor Kinase Promotes MtrA Regulon Expression.

Evaluation of select mtrA targets under conditions affecting MtrB-septal assembly. A, qRT-PCR expression levels of select mtrA targets under conditions affecting FtsZ-septal assembly. Actively growing M. smegmatis FZ-3, ftsZ conditional expression strain, with acetamide (referred to as FtsZ+) was harvested, washed with acetamide-free medium, and grown without acetamide (FtsZ-depleted) or with acetamide (FtsZ+) for 9 h. Total RNA from FtsZ+ and FtsZ-depleted cultures was prepared, and qRT-PCR was performed as described under Fig. 9. Panel i shows expression profiles of mtrA, mtrB, wag31, and ftsI; panel ii shows data for dnaA, ripA, and fbpB; panel iii shows data for chiZ and pfkB. Note: expression levels of chiZ and pfkB in panel iii were modestly affected. B, shown is the effect of mitomycin C exposure on MtrB-GFP and FtsZ-GFP structures. M. smegmatis expressing ftsZ-gfp (panels i–iv) or mtrB-gfp (panels (v–viii) were exposed to 0.5 μg/ml mitomycin C (panels iii, iv, vii, and viii) or grown untreated (panels i, ii, v, and vi) for 6 h, and cells were visualized by microscopy. Panels i, iii, v, and vii show brightfield images, whereas panels ii, iv, vi, and viii show fluorescent images. Septal localizations of FtsZ-GFP (panel ii and iv) and MtrB-GFP (panel vi) are marked with arrowheads. Note that majority of mitomycin C-treated cultures did not contain predominant FtsZ-GFP (panel iv) or MtrB-GFP (panel viii) septal localizations. Aberrant FtsZ-GFP localizations are marked with arrows (panel iv). C, qRT-PCR expression profiles of select MtrA-targets upon exposure to mitomycin C are shown. Total RNA from untreated and 6-h mitomycin C-treated cultures of M. smegmatis was prepared, and qRT-PCR analysis of select targets was performed as described above. Panel i shows the expression profiles of mtrA, wag31, ftsI, and mtrB; panel ii shows data for dnaA, ripA, and fbpB, whereas panel iii shows data for chiZ, ftsZ, and pfkB.

Renata Plocinska, et al. J Biol Chem. 2012 July 6;287(28):23887-23899.
10.
FIGURE 8.

FIGURE 8. From: Septal Localization of the Mycobacterium tuberculosis MtrB Sensor Kinase Promotes MtrA Regulon Expression.

MtrA binds to ripA promoter. A, Logo analysis shows the MtrA motif in the 5′-upstream regions of the ripA gene. The 250-bp upstream sequences (also called the promoter region) of the ripA-coding region of M. smegmatis and M. tuberculosis were analyzed by MEME analysis (50). MtrA-motif like sequences were identified and used to generate MtrA-motif logo. Although not shown, the M. tuberculosis PripA region has two motifs starting at positions 90 and 142 in the + strand and at 214 in the − strand, whereas M. smegmatis has two motifs at positions 62 and 115 in the + strand and 214 in the − strand. Potential 9-base motif is underlined. B, ChIP experiments are shown. M. tuberculosis cell lysates prepared after formaldehyde cross-linking were used for immunoprecipitation with α-MtrA or mock antibodies. Protein-DNA complexes obtained were processed for removal of cross-links and subjected to PCR using primers specific to PftsZ, PripA, and PfbpB. PCR reactions were done in duplicate for different dilutions of template, products were resolved by agarose gel electrophoresis, gels were visualized by staining with SYBR Green dye in a Bio-Rad Molecular Imager Fx, and bands were quantified using Quantity One software (Bio-Rad). Results are presented as ratios of immunoprecipitates (IP) to mock and normalized against FtsZ values, and data shown are the average of at least three independent experiments. C, shown is polyacrylamide gel analysis of PripA- MtrAWT complexes. MtrAWT protein was phosphorylated by EnvZ protein in the presence of ATP. MtrAWT and MtrAWT∼P were incubated individually with 200 fmol of 6-carboxyfluorescein-labeled PripA for 15 min at 37 °C, and samples were resolved by polyacrylamide gel electrophoresis and visualized in a Bio-Rad Molecular Imager Fx. The positions of ripA probe and MtrAWT-PripA complexes are marked. Note MtrAWT∼P bound PripA better than MtrA. MtrAWT or MtrAWT∼P was used at 0.1, 0.25, 0.5, and 1.0 μm. D, shown is a polyacrylamide gel analysis of PripA-MtrA/MtrAD13A complexes. All details are as in panel C, except that MtrAD13A was used at 5, 10, 15, 20, and 25 μm.

Renata Plocinska, et al. J Biol Chem. 2012 July 6;287(28):23887-23899.

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