Figure 1. Efficient Overexpression of PknD and Concomitant Induction of Rv0516c in Logarithmically Growing Mtb .

(A) Western blots showing kinase levels (anti-PknD) and kinase activity (anti-phosphothreonine) at 0, 2, 4, 8 and 24 h after induction of WT and Asp138Asn PknD (D138N). As a loading control, the amount of the KatG protein present at a constant level was monitored using anti-KatG antibodies. Addition of the inducer (acetamide) to WT Mtb (lacking an inducible PknD gene) caused no discernable change in the pattern of phosphoproteins (unpublished data). The presence of a band at the molecular weight of phosphorylated WT PknD at time zero may indicate that the kinase was already expressed (and autophosphorylated) at low levels in the absence of inducer. In contrast, phosphorylated Asn138Asn PknD accumulated only after the inducer was added.

(B) Cluster diagram showing the eight genes with the largest time-dependent changes in transcript levels upon PknD induction. Expression levels are shown relative to a reference pool in which equal amounts of RNA from each sample were mixed and the value at time zero was normalized to 1.0. Microarray analysis revealed >2-fold induction or repression of 137 genes (Table S1). Among the eight most altered transcripts shown here, we observed robust induction (blue) of the putative anti-anti–sigma factor Rv0516c. Rv0516c expression increased much more upon expression of WT PknD than the attenuated mutant (Asp138Asn). Genes labeled in red are among those most reduced in expression during logarithmic phase growth of an Mtb sigF knockout mutant [21]. Color unit is fold change of gene expression.