Journal: Journal of Bacteriology

Article Title: Mycobacterium tuberculosis MtrB Sensor Kinase Interactions with FtsI and Wag31 Proteins Reveal a Role for MtrB Distinct from That Regulating MtrA Activities

doi: 10.1128/JB.01795-14

Figure Lengend Snippet: MtrB interactions with FtsI and Wag31. (A) BACTH analysis of MtrB interactions with FtsI and Wag31. E. coli BTH101 cotransformants with plasmids expressing Wag31/Wag31, Wag31/MtrB, Wag31/MtrBH305Y, MtrB/FtsI, and MtrBH305Y/FtsI were spotted on indicator agar plates as previously described (18), wherein blue and white spots indicate positive and negative interactions, respectively. Cotransformants with GCN4/GCN4 and empty vector/MtrB, representing positive and negative controls, respectively, were also spotted. (B) Recombinant colonies expressing the indicated combinations of proteins were propagated in LB broth, and β-galactosidase activity was measured as described in the text. Data shown are means ± standard deviations from three independent experiments. (C) Pulldown assays. Wag31 interacts with MtrB and not with FtsZ. E. coli lysates with MBP-Wag31 were incubated with His10-MtrBsol (i) or His10-FtsZ (iii) bound to Ni-NTA resin with rocking at 4°C. The Ni-NTA resin was washed five times, and the bound proteins were eluted with buffer containing imidazole (see below and Materials and Methods) (17). MBP-Wag31 was incubated with Ni-NTA (ii) and processed as described for panels i and iii. Various fractions were separated in SDS-polyacrylamide gels and transferred to PVDF membranes, and the proteins were visualized by immunoblotting using anti-MtrB, anti-Wag31, anti-FtsZ, or anti-MBP antibody (see Fig. S2 in the supplemental material). L, load; W, wash; E, elution fractions; W4 and W5, washes 4 and 5, respectively; E1, E2, and E3 are elutions with buffers containing 100 mM, 300 mM, and 1 M imidazole, respectively. For co-IP assays (iv) 1 ml of M. smegmatis lysate mixed with 60 μg of purified MBP-MtrB was incubated overnight with anti-Wag31 antibodies coupled to magnetic beads (BioMag Plus amine particles). The beads were washed five times with IP wash buffer, and the MtrB immunoprecipitates were eluted with 100 mM citrate (pH 3.1). The eluted proteins were neutralized with 1 N NaOH, separated by SDS-PAGE, and visualized following immunoblotting with anti-Wag31 or anti-MtrB antibody. P, pure protein markers for MBP-MtrB and His-Wag31; L, load; W, wash 5; E1 and E2, elutions 1 and 2; EC, elution from buffer control where beads were incubated with buffer instead of lysate. White arrowhead, MBP-MtrB, MtrB, or Wag31; black arrowhead, IgG band. M, molecular mass marker. α, anti.

Article Snippet: In vitro phosphorylation experiments revealed that MtrB phosphorylates MtrA ( 4 ) and that phosphorylated MtrA (MtrA∼P) binds to the origin of replication ( oriC ) and the promoters of fbpB , ripA , and dnaA ( 5 , – 7 ).

Techniques: Expressing, Plasmid Preparation, Recombinant, Activity Assay, Incubation, Western Blot, Co-Immunoprecipitation Assay, Purification, Magnetic Beads, SDS Page, Control, Marker

Journal: Journal of Bacteriology

Article Title: Mycobacterium tuberculosis MtrB Sensor Kinase Interactions with FtsI and Wag31 Proteins Reveal a Role for MtrB Distinct from That Regulating MtrA Activities

doi: 10.1128/JB.01795-14

Figure Lengend Snippet: Loss of MtrB or depletion of FtsI increases phosphorylation of Wag31. (A) M. smegmatis WT, the ΔmtrB strain, FtsI-depleted cultures (FtsI depletion for 12 h), and the ΔmtrB Pami::mtrB, ΔmtrB Pami::mtrAY102C (where mtrAY102C is the Y-to-C change at position 102 encoded by mtrA) and ΔmtrB Pami::mtrBH305Y strains were grown as described in the legends to Fig. 3 and ​and4.4. The complemented ΔmtrB strains were grown with 0.2% acetamide. Wag31∼P/Wag31 ratios were determined by immunoblotting with anti-phospho-Ser/Thr and anti-Wag31 antibodies. Whole-cell lysates (5 μg protein) from the above strains were resolved by SDS-PAGE in 12% gels, and immunoblotting was performed with anti-phospho-Ser/Thr antibodies. The blots were then stripped and reprobed with anti-Wag31 antibodies. Wag31∼P/Wag31 (anti-phospho-Ser/Thr and anti-Wag31) ratios for various strains were normalized to those in the WT and plotted (B). The data shown are represented as the averages ± standard errors from three independent experiments. *, P < 0.05. (C) The Wag31 levels in the indicated strains were measured by immunoblotting, as previously described (36), and normalized to SigA levels. The data are represented as the means ± standard errors from three independent experiments.

Article Snippet: In vitro phosphorylation experiments revealed that MtrB phosphorylates MtrA ( 4 ) and that phosphorylated MtrA (MtrA∼P) binds to the origin of replication ( oriC ) and the promoters of fbpB , ripA , and dnaA ( 5 , – 7 ).

Techniques: Phospho-proteomics, Western Blot, SDS Page

Journal: Journal of Bacteriology

Article Title: Mycobacterium tuberculosis MtrB Sensor Kinase Interactions with FtsI and Wag31 Proteins Reveal a Role for MtrB Distinct from That Regulating MtrA Activities

doi: 10.1128/JB.01795-14

Figure Lengend Snippet: MtrB activity and localization are compromised in the absence of FtsI. (A and B) qRT-PCR analysis of MtrA targets. Total RNA was extracted from ΔftsI::Pami-ftsI (A) and Δwag31::Pami-wag31 (B) strains grown without and with 0.2% acetamide (see details in text) and reverse transcribed, and qRT-PCR was performed as described in Materials and Methods. The expression levels of select genes, that is, dnaA, ripA, fbpB, wag31, mtrA, mtrB, and ftsI, relative to the housekeeping gene sigA were compared, and the values are presented as fold difference. In panel A fold expression levels upon FtsI depletion (growth in the absence of acetamide for 12 h) were normalized to those in the presence of acetamide (FtsI+). In panel B fold expression levels upon Wag31 depletion (growth in the absence of acetamide for 15 h) were normalized to those in the presence of acetamide (Wag31+). Wag31 depletion beyond 15 h led to extreme cell distortion and eventually cell lysis; hence, expression studies were not carried out beyond 15 h. (C and D) MtrB-GFP localization. M. smegmatis ΔftsI::Pami-ftsI (C) and Δwag31::Pami-wag31 (D) strains expressing Ptet::mtrB-gfp were grown without and with 0.2% acetamide, as for panels A and B. For the visualization of MtrB-GFP, anhydrotetracycline was added at 10 ng/ml for 1 h. Bright-field (i and iii) and fluorescence (ii and iv) microscopy and imaging were carried out as described in the text. The ΔftsI::Pami-ftsI or Δwag31::Pami-wag31 strain was grown with (i and ii) or without (iii and iv) acetamide. For panel C, percent septal MtrB-GFP localizations from two independent experiments were scored, and averages ± standard errors are given in the respective fluorescent panels. Each experiment included >100 cells from each condition. White arrows, MtrB-GFP septal localization; black arrows, distorted cell shape upon Wag31 depletion.

Article Snippet: In vitro phosphorylation experiments revealed that MtrB phosphorylates MtrA ( 4 ) and that phosphorylated MtrA (MtrA∼P) binds to the origin of replication ( oriC ) and the promoters of fbpB , ripA , and dnaA ( 5 , – 7 ).

Techniques: Activity Assay, Quantitative RT-PCR, Reverse Transcription, Expressing, Lysis, Fluorescence, Microscopy, Imaging

Journal: Journal of Bacteriology

Article Title: Mycobacterium tuberculosis MtrB Sensor Kinase Interactions with FtsI and Wag31 Proteins Reveal a Role for MtrB Distinct from That Regulating MtrA Activities

doi: 10.1128/JB.01795-14

Figure Lengend Snippet: FtsI localization and activity are altered in the absence of MtrB. (A) GFP-FtsI localization was examined in M. smegmatis WT (i and ii), the ΔmtrB strain (iii and iv), and merodiploid strains overproducing (↑) MtrB (v and vi) or MtrBH305Y (vii and viii). In all of the strains, the GFP-FtsI fusion protein was produced from Pami::gfp-ftsI following induction with 0.2% acetamide for 3 h, visualized by bright-field (left panels) and fluorescent (right panels) microscopy, and imaged as described in the text. (B) The exponential cultures of the M. smegmatis WT strain (i and ii) and the ΔmtrB strain (iii and iv) were grown in the presence of Van-FL for 2 h and were imaged by bright-field (i and iii) and fluorescence (ii and iv) microscopy. (C) Loss of MtrB increases sensitivity to vancomycin. M. smegmatis WT and ΔmtrB strains were grown for 6 h, and 1 × 105 cells were spread on 7H10 agar plates. Etest antibiotic strips (ampicillin or vancomycin) were placed on the agar plates, plates were incubated for 4 days at 37°C, and MICs were measured as per the supplier's protocol.

Article Snippet: In vitro phosphorylation experiments revealed that MtrB phosphorylates MtrA ( 4 ) and that phosphorylated MtrA (MtrA∼P) binds to the origin of replication ( oriC ) and the promoters of fbpB , ripA , and dnaA ( 5 , – 7 ).

Techniques: Activity Assay, Produced, Microscopy, Fluorescence, Incubation

Journal: Journal of Bacteriology

Article Title: Mycobacterium tuberculosis MtrB Sensor Kinase Interactions with FtsI and Wag31 Proteins Reveal a Role for MtrB Distinct from That Regulating MtrA Activities

doi: 10.1128/JB.01795-14

Figure Lengend Snippet: Wag31 localization is altered in the absence of MtrB. (A) Pami::wag31-mCherry expressing M. smegmatis WT (i and ii), the ΔmtrB strain (iii and iv), and M. smegmatis overproducing MtrB (v and vi) or MtrBH305Y (vii and viii) were grown with 0.2% acetamide for 3 h and visualized as described in the legend to Fig. 3A. Top panels are bright-field images; bottom panels are fluorescence images. Arrow, septal localization; arrowhead, polar localization. Percent septal Wag31-mCherry localizations from two independent experiments were scored, and averages ± standard errors are given in the respective fluorescent panels. Each experiment included >132 cells from each strain. Black arrows, septa in multiseptate ΔmtrB strain; white arrows and arrowheads, septal and polar localizations, respectively. (B) FtsZ-GFP localization in WT (i and ii) and the ΔmtrB strain (iii and iv). Arrow, FtsZ-GFP rings.

Article Snippet: In vitro phosphorylation experiments revealed that MtrB phosphorylates MtrA ( 4 ) and that phosphorylated MtrA (MtrA∼P) binds to the origin of replication ( oriC ) and the promoters of fbpB , ripA , and dnaA ( 5 , – 7 ).

Techniques: Expressing, Fluorescence

Journal: Journal of Bacteriology

Article Title: Mycobacterium tuberculosis MtrB Sensor Kinase Interactions with FtsI and Wag31 Proteins Reveal a Role for MtrB Distinct from That Regulating MtrA Activities

doi: 10.1128/JB.01795-14

Figure Lengend Snippet: MtrB interacts with PknA and PknB. Interactions of MtrB and MtrA with PknA or PknB were examined using BACTH assays, as previously described (17). MtrA, MtrB, FtsI, PknA, and PknB fusions to the T18 and T25 fragments of adenylate cyclase in BACTH vectors (see Table S1 in the supplemental material) were used to transform E. coli BTH101, and the recombinants were plated on LB agar supplemented with X-Gal and isopropyl-β-d-thiogalactopyranoside. Green-blue colonies, indicative of positive interactions, were subsequently propagated in LB broth, and β-galactosidase activity was measured as described in the text. GCN4/GCN4 and MtrB/MtrA are shown as positive controls, and MtrB/empty vector (vector control) is shown as the negative control. The data shown are the means ± standard deviations from three independent experiments.

Article Snippet: In vitro phosphorylation experiments revealed that MtrB phosphorylates MtrA ( 4 ) and that phosphorylated MtrA (MtrA∼P) binds to the origin of replication ( oriC ) and the promoters of fbpB , ripA , and dnaA ( 5 , – 7 ).

Techniques: Activity Assay, Plasmid Preparation, Control, Negative Control

Journal: Nucleic Acids Research

Article Title: Splice-shifting oligonucleotide (SSO) mediated blocking of an exonic splicing enhancer (ESE) created by the prevalent c.903+469T>C MTRR mutation corrects splicing and restores enzyme activity in patient cells

doi: 10.1093/nar/gkv275

Figure Lengend Snippet: RHC-Glo splicing reporter minigene assay. Upper panel: schematic representation of the RHC-Glo splicing reporter minigene and the sub-cloned sequences. Lower panel: Agarose gel electrophoresis of RT-PCR minigene splicing products expressed in HEK293 cells. Inclusion or exclusion of exon 2 is indicated on the right. The gel pictures are representative results from two experiments.

Article Snippet: Mutations in the MTRR minigene were introduced by GeneScript Inc. (GenScript, Piscataway, NJ, USA).

Techniques: Mini Gene Assay, Clone Assay, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction

Journal: Nucleic Acids Research

Article Title: Splice-shifting oligonucleotide (SSO) mediated blocking of an exonic splicing enhancer (ESE) created by the prevalent c.903+469T>C MTRR mutation corrects splicing and restores enzyme activity in patient cells

doi: 10.1093/nar/gkv275

Figure Lengend Snippet: The balanced interplay between an hnRNP A1 binding ESS and two SRSF1 binding ESEs dictate MTRR pseudoexon activation. ( A ) Schematic representation of the sequences from MTRR -minigenes used and the nucleotide changes introduced. A pictogram of hnRNP A1 position weight matrix is shown above the putative ESS and the invariant position 2 of this motif is underscored. A pictogram of the SRSF1 position weight matrix is shown above the previously described ESE1 and the ACADM like ESE. Indicated positions 361, 362 and 365 are relative to the ACADM coding sequence. Nucleotide positions that were changed are underscored. ( B ) Splicing minigene assay. MTRR -minigenes were transiently transfected into HEK293. After RNA isolation the splicing products were analyzed by RT-PCR. The upper panel shows the average of pseudoexon inclusion from two measurements of each duplicate. Error bars represent the range. Quantification of PCR products was performed using a Fragment Analyzer instrument. The lower panel shows a sample agarose gel electrophoresis displaying pseudoexon inclusion levels in the different cell lines. The lower bands represent correctly spliced exons, whereas the upper bands represent MTRR pseudoexon inserted between minigene exons. Ψ marks the pseudoexon. ( C ) Binding of hnRNPA1 and SRSF1 proteins. Biotinylated RNA oligonucleotides were used in a pull-down experiment with HeLa nuclear extract followed by SDS PAGE and western blot analysis using antibodies against SRSF1 and hnRNP A1. Blank indicates a control lane from pull down without RNA oligonucleotides. NE is nuclear extract. The displayed blots are representative result from at least three pull-down experiments.

Article Snippet: Mutations in the MTRR minigene were introduced by GeneScript Inc. (GenScript, Piscataway, NJ, USA).

Techniques: Binding Assay, Activation Assay, Sequencing, Mini Gene Assay, Transfection, Isolation, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, SDS Page, Western Blot, Control

Journal: Nucleic Acids Research

Article Title: Splice-shifting oligonucleotide (SSO) mediated blocking of an exonic splicing enhancer (ESE) created by the prevalent c.903+469T>C MTRR mutation corrects splicing and restores enzyme activity in patient cells

doi: 10.1093/nar/gkv275

Figure Lengend Snippet: SSO treatment in HEK293 cells transfected with wild-type, mutant and TCGGGA MTRR -minigenes. ( A ) Splicing minigene assay. MTRR -minigenes were transiently transfected into HEK293. 24 h after the cells were transfected with SSOs targeting either the 5′ss, 3′ss or both ESEs. After RNA isolation the splicing products were analyzed by RT-PCR. The upper panel shows a sample agarose gel electrophoresis displaying pseudoexon inclusion levels. The lower bands represent correctly spliced exons, whereas the upper bands represent MTRR pseudoexon inclusion. Ψ marks the pseudoexon. ( B ) Quantification of pseudoexon inclusion. Error bars represent the range. Quantification of PCR products was performed using a Fragment Analyzer instrument.

Article Snippet: Mutations in the MTRR minigene were introduced by GeneScript Inc. (GenScript, Piscataway, NJ, USA).

Techniques: Transfection, Mutagenesis, Mini Gene Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis

Journal: Nucleic Acids Research

Article Title: Splice-shifting oligonucleotide (SSO) mediated blocking of an exonic splicing enhancer (ESE) created by the prevalent c.903+469T>C MTRR mutation corrects splicing and restores enzyme activity in patient cells

doi: 10.1093/nar/gkv275

Figure Lengend Snippet: SSO treatment restores MTRR splicing in patient cells. ( A ) Fibroblasts from a patient harboring the c.903+469T>C mutation were transfected with SSOs that target either the 5′ss, the 3′ss, both ESEs (ESE-SSO), or a non-targeting sequence (NT). Control fibroblasts were treated in parallel. RNA was extracted after 48 h. A representative agarose gel electrophoresis of the RT-PCR products is shown. The lower bands represent correctly spliced exons, whereas the upper bands represent MTRR pseudoexon inclusion between exon 6 and exon 7. Ψ marks the pseudoexon. ( B ) Quantification of pseudoexon inclusion in patient fibroblasts following SSO treatment. Error bars represent the range. Quantification of PCR products was performed using a Fragment Analyzer instrument.

Article Snippet: Mutations in the MTRR minigene were introduced by GeneScript Inc. (GenScript, Piscataway, NJ, USA).

Techniques: Mutagenesis, Transfection, Sequencing, Control, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction

Journal: Nucleic Acids Research

Article Title: Splice-shifting oligonucleotide (SSO) mediated blocking of an exonic splicing enhancer (ESE) created by the prevalent c.903+469T>C MTRR mutation corrects splicing and restores enzyme activity in patient cells

doi: 10.1093/nar/gkv275

Figure Lengend Snippet: SSO treatment partially restores enzymatic activity of methionine synthase reductase (MTRR). Fibroblasts from a patient homozygous for the c.903+469T>C mutation were transfected with an SSO that target both ESEs (ESE-SSO) or a non-targeting sequence (Ctr). Fibroblasts were then labeled with [ 57 Co]cyanocabalamine and MTRR activity was determined indirectly by measuring methylcobalamine (MeCbl) synthesis (expressed as% of total labeled cobalamin derivatives). Adenosylcobalamine (AdoCbl) synthesis was measured as a control. The vertical lines represent the range of duplicate determinations in a representative experiment.

Article Snippet: Mutations in the MTRR minigene were introduced by GeneScript Inc. (GenScript, Piscataway, NJ, USA).

Techniques: Activity Assay, Mutagenesis, Transfection, Sequencing, Labeling, Control

Journal: Nucleic Acids Research

Article Title: Splice-shifting oligonucleotide (SSO) mediated blocking of an exonic splicing enhancer (ESE) created by the prevalent c.903+469T>C MTRR mutation corrects splicing and restores enzyme activity in patient cells

doi: 10.1093/nar/gkv275

Figure Lengend Snippet: Splicing model of the MTRR pseudoexon activation induced by the c.903+469T>C mutation. The wild-type sequence harbors a very weak SRSF1 binding ESE1, a flanking ACADM like SRSF1 binding ESE and a hnRNP A1 binding ESS. In the wild-type MTRR gene binding of hnRNP A1 to the TAGGGA high affinity ESS inhibits splicing because this initiates cooperative spreading of hnRNPA1 to weaker sites in a 3′-to-5′ direction thereby directly blocking access to the 5′ splice site and other splicing regulatory elements. Simultaneously binding of hnRNP A1 to the ESS inhibits binding of SRSF1 both to the very weak SRSF1 motif in ESE1 and to the ACADM -like ESE motif. This also affects recognition of the weak 5′ss by U1 snRNP, since this may be dependent on SRSF1 binding to the ESEs. When the c.903+469T>C mutation is present it changes the weak SRSF1 motif in ESE1 to a very strong site, which may act synergistically with the ACADM -like SRSF1 ESE to recruit U1snRNP to the weak 5′ss despite binding of hnRNP A1 to the flanking ESS does not seem to be affected. SRSF1 binding to the ESEs also antagonizes the cooperative spreading of hnRNPA1 binding from the TAGGGA high affinity ESS. Together this leads to pseudoexon activation and the abnormal splicing of the MTRR transcript observed in patients.

Article Snippet: Mutations in the MTRR minigene were introduced by GeneScript Inc. (GenScript, Piscataway, NJ, USA).

Techniques: Activation Assay, Mutagenesis, Sequencing, Binding Assay, Blocking Assay