Journal: Molecular microbiology

Article Title: A complex regulatory network controlling intrinsic multidrug resistance in Mycobacterium smegmatis

doi: 10.1111/mmi.12448

Figure Lengend Snippet: (a) Ten fold dilutions of gene replacement mutants in MSMEG_2538, MSMEG_5437 and MSMEG_6921 were spotted on Middlebrook 7H10 containing 30ng/ml of MMC with ΔMSMEG_5437 being the most sensitive of the mutants. (b) Ten fold serial dilutions of wild type and mc2155:Δ5437 were spotted on Middlebrook 7H10 containing indicated concentrations of isoniazid, chloramphenicol and tetracycline as well as a control lacking antibiotic. The mutant is hypersensitive to all three antibiotics tested. (c) Multiple sequence alignment of MSMEG_5437 with the eukaryotic like STPKs in M. tuberculosis showing the conserved signature motifs of bacterial ser/thr kinases and the conserved kinase domain in MSMEG_5437 shaded in grey. Deviations in the sequence of MSMEG_5437 are observed in the glycine rich motif and the catalytic loop and are marked. (d) Total cell lysate was prepared from wild type mc2155 and mc2155: Δ5437 and separated by 2-D gel electrophoresis. The gels were fixed and stained with a fluorescent phosphoprotein that stains all phosphorylated proteins. Differentially phosphorylated proteins were located using a combination of ImageQuant and DeCyder softwares. The white box and x and y_axis on each slide are for orientation purposes. Fifty one differentially phosphorylated proteins were identified and are circled and numbered. Twenty one protein spots were completely absent in mc2155: Δ5437 and were excised from the gel and identified by MALDI TOF/TOF (tandem mass spectrometry MS/MS). Identity of these spots is presented in Table 1.

Article Snippet: The phosphorylated residues were identified using MALDI tandem MS/MS (Applied Biomics, Inc.).

Techniques: Mutagenesis, Sequencing, Nucleic Acid Electrophoresis, Staining, Mass Spectrometry, Tandem Mass Spectroscopy

Journal: Molecular microbiology

Article Title: A complex regulatory network controlling intrinsic multidrug resistance in Mycobacterium smegmatis

doi: 10.1111/mmi.12448

Figure Lengend Snippet: Identification of phosphoproteins absent in mc 2 155: Δ5437 using MALDI-TOF Total cell lysate was prepared from wild type mc 2 155 and mc 2 155: Δ5437 were separated by 2-D gel electrophoresis and the phosphorylated proteins identified by fluorescent staining. Differentially phosphorylated proteins were located using a combination of ImageQuant and DeCyder softwares. Of the 51 differentially phosphorylated proteins 21 protein spots were completely absent in mc 2 155: Δ5437 and were excised from the gel and identified by MALDITOF/TOF (tandem mass spectrometry MS/MS).

Article Snippet: The phosphorylated residues were identified using MALDI tandem MS/MS (Applied Biomics, Inc.).

Techniques: Nucleic Acid Electrophoresis, Staining, Mass Spectrometry

Journal: Molecular microbiology

Article Title: A complex regulatory network controlling intrinsic multidrug resistance in Mycobacterium smegmatis

doi: 10.1111/mmi.12448

Figure Lengend Snippet: (a) MSMEG_6127 and (b) M. tuberculosis RsfB purified from E. coli were incubated with purified MSMEG_6129 in kinase buffer containing 1μCi of γ-P32 ATP at 25°C for 30 mins and separated on a 15% SDS-PAGE followed by autoradiography. The positions of MSMEG_6127 and RsfB are marked as well as the 15 kDa and 25 kDa standards. (c) The position of residues phosphorylated in MSMEG_6127 by MSMEG_6129 identified by MALDI TOF/TOF are indicated and include the consensus anti-sigma factor phosphorylation site at position S63.

Article Snippet: The phosphorylated residues were identified using MALDI tandem MS/MS (Applied Biomics, Inc.).

Techniques: Purification, Incubation, SDS Page, Autoradiography