Figure 1.

Overexpression of Rv0805 elicits changes in gene expression. A. Confirmation of Rv0805 overexpression in M. tuberculosis by immunoblotting using Rv0805-specific antibodies. Equal amounts of protein from cell lysates of strains containing empty vector (Control) or Rv0805 overexpression constructs (Rv0805) were used for western blotting. Robust overexpression of wild type Rv0805 was observed in three independent transformants (colonies A, B, C). B. Measurement of intracellular cAMP levels in M. tuberculosis overexpressing Rv0805 (Rv0805) compared to the empty vector (control). Bacterial cAMP was measured in the exponential phase of growth (Light transmittance at 600 nm ∼ 0.6). Expression of wild type Rv0805 led to decreased intracellular cAMP levels. Mean ± SEM of measurements done in triplicates from three independent biological replicates is plotted. C. Validation of microarray results by qRT-PCR was performed for three up-regulated (Rv1623c, Rv0287 and Rv2057c) and three down-regulated genes (Rv3197A, Rv1318c and Rv0467). rrs, coding for 16S rRNA, was used as the normalizing gene. Bars represent mean ± SEM of expression level of genes in M. tuberculosis-Rv0805 relative to M. tuberculosis-VC as measured from triplicate cultures. Numbers in parentheses represent fold regulation from microarray results. D. Analysis of cellular pathways affected by Rv0805 overexpression. Genes with altered expression levels (>±2 fold) on expression of Rv0805 were assigned functional categories according to Tuberculist (www.tuberculist.epfl.ch) annotation. Numbers represent percentage of total altered genes.