Analysis of ATc-regulated <i>vapC</i> expression in mycobacteria.
2011-06-29T00:20:14Z (GMT) by
<p>Expression was analyzed by RT-PCR (<b>A</b> and <b>B</b>) and detection of expressed proteins by epitope tagging (<b>C</b>). RT-PCR was carried out using mRNA samples obtained from (<b>A</b>) induced cultures of wild type Mtb (6 h treatment with ATc at 25 ng/ml); and (<b>B</b>) ATc-induced and uninduced cultures of <i>M. smegmatis</i> (1 h treatment with ATc at 50 ng/ml) over-expressing various <i>vapC</i> toxins under the control of the P<sub>myc1</sub><i>tetO</i> promoter. Samples obtained with (+) and without (-) reverse transcription (RT) were compared to distinguish cDNA from genomic DNA contamination. (<b>C</b>) Cellular fractions isolated from <i>M. smegmatis</i> were subjected to Western blot analysis using the anti-FLAG M2 antibody to detect the epitope-tagged VapC Rv2546 and Rv2549c proteins. Cultures were grown in either the presence (+) or absence (-) of 50 ng/ml ATc for 3 h. (<b>D</b>) The effect of epitope tagged Rv2549c expression on the growth of <i>M. smegmatis</i> on solid media. Ten-fold serial dilutions of cells were spotted on 7H10 agar without or with the ATc (1, 2, 3 and 50 ng/ml) and incubated for 24–48 h. (<b>E</b>) Comparison of the relative abundance of Rv2549c induced with varying concentrations of ATc. Cultures were grown in either the presence (+) or absence (−) of 50 ng/ml ATc for 3 h. Equal amounts (3 µg) were subjected to Western blot analysis using the anti-FLAG M2 antibody to detect the epitope-tagged Rv2549c protein.</p>