M. tuberculosis was grown in 7H9-OADC-TW broth at 37°C, and lysates prepared using a bead beater. About 500 g protein was separated in 10-40% sucrose gradient. A. The ODs of the separated fractions were measured (manually) at 260 nm. B. The proteins in the fractions were then

precipitated with ethanol and separated on SDS-PAGE, transferred to nitrocellulose membranes, and probed with anti-Obg antiserum (1:500 dilution), followed by peroxidase-labeled anti-rabbit IgG (1:10,000 dilution, Sigma). The blots were developed with an ECL kit (Amersham) and autoradiographed. Lane C is a whole-cell extract from M. tuberculosis. Lanes 1-15 represent fractions from the top (10% sucrose) to the bottom (40% sucrose) of the sucrose gradient. Fraction 16 was not analyzed in immunoblot. M. FK228 manufacturer tuberculosis Obg interacts with UsfX Scott et al [41] were the first to observe I-BET151 that B. subtilis Obg interacts with upstream regulators of the stress sigma factor SigB. In this respect,

this bacterium’s Obg resembles B. subtilis RsbT and RsbW, both of which also interact with SigB in this species [41]. More recently, the Obg proteins of E. coli [20] and V. harveyi [21] have been shown to interact with SpoT, a stringent response regulator. Since SigB, RsbW and SpoT-related genes are present in M. tuberculosis, we asked whether M. tuberculosis Obg interacts with any or all of these proteins, in the yeast two-hybrid system. The M. tuberculosis genes coding for Obg (Rv2240c), UsfX (homologue of RsbW, Rv3287c), SigF (homologue of SigB of B. subtilis, Rv3286c) and RelA (a stringent response regulator related to SpoT, Rv2853c) were cloned in yeast vectors, and transformed into the yeast strain AH109. Table 1 shows that M. tuberculosis Obg strongly interacts with UsfX, but not with the SpoT-related RelA protein. The strength of this interaction is comparable to the interaction of M. tuberculosis UsfX with its cognate

sigma factor SigF. In the same experiment, we looked for interaction of M. tuberculosis Obg with various other putative anti-anti sigma factors that we have described earlier for this bacterium [42], including RsbU (Rv1364c), RsfA (Rv1365c), RsfB (Rv3687c), Rv0516c, Rv1904 and Rv2638. However, we observed no significant interaction of Obg with any of the Cediranib (AZD2171) above anti-anti sigma factors (data not shown), indicating that the interaction of M. tuberculosis Obg is limited to UsfX. In light of the known stress response role of UsfX [43], its specific interaction with Obg suggests that Obg plays a role in the M. tuberculosis stress response. Table 1 Interaction of Obg with stress related proteins in the yeast two-hybrid system.   *Plasmids SD Minimal Medium Mel-l (α-gal) in SD plates Mel-1 (α-gal) in SD broth**     -Leu/ -Trp -His/ -Leu/-Trp -Ade/-His/ -Leu/-Trp     1. pGADT7-T + + + +++ 3.512 ± 0.709   pGBKT7-53           2. pGADT7-T + – - – -   pGBKT7-Lam           3. pGA3287c + + + ++ 2.367 ± 0.354   pGB3286c           4. pGA3287c + + + ++ 2.