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. 2024 May 13;13:RP92136. doi: 10.7554/eLife.92136

Figure 1. PhoP contributes to the maintenance of mycobacterial 3′,5-cyclic adenosine monophosphate (cAMP) level.

(A) Intra-mycobacterial cAMP levels were determined by a fluorescence-based assay as described in ‘Materials and methods’, and compared for indicated mycobacterial strains, grown under normal or specific stress conditions. For acid stress, mycobacterial strains were initially grown to the mid-log phase (OD600 0.4–0.6), and then transferred to acidic pH (7H9 media, pH 4.5) for further 2 hr of growth at 37°C. For NO stress, cells grown to the mid-log phase were exposed to 0.5 mM DetaNonoate for 40 min. The data represent average values from three biological repeats (*p≤0.05, **p≤0.01). (B) To compare the secretion of cAMP by WT and phoPR-KO, cAMP levels were also determined in the corresponding culture filtrates (CF) (ns., non- significant). (C) Immunoblotting analysis of 10 µg of cell lysates (CL) and 20 µg of CF of indicated M. tuberculosis strains. α-GroEL2 was used as a control to verify cytolysis of cells, CFP-10 detected as a secreted mycobacterial protein in the CFs, and RpoB used as the loading control.

Figure 1—source data 1. cAMP estimation in WT, phoPR-KO and the complemented mutant.
Figure 1—source data 2. Quantification of cAMP secretion of WT, phoPR-KO and the complemented mutant.
Figure 1—source data 3. GroEL2 levels in cell lysates of WT-H37Rv and phoPR-KO.
Figure 1—source data 4. GroEL2 levels in cell lysates of WT-H37Rv and phoPR-KO.
elife-92136-fig1-data4.pptx (333.6KB, pptx)
Figure 1—source data 5. Anti- RpoB blot as a loading control of comparable amount of cell lysates.
Figure 1—source data 6. Anti-RpoB blot as a loading control of comparable amount of cell lysates.
elife-92136-fig1-data6.pptx (321.8KB, pptx)
Figure 1—source data 7. CFP-10 in culture filtrates of H37Rv and phoPR-KO.
Figure 1—source data 8. CFP-10 in the culture filtrates of H37Rv and phoPR-KO.
elife-92136-fig1-data8.pptx (260.5KB, pptx)

Figure 1.

Figure 1—figure supplement 1. Defined and indicated stress conditions do not influence in vitro growth of mycobacterial strains.

Figure 1—figure supplement 1.

To determine the impact of in vitro stress on bacterial growth under conditions when intra-mycobacterial 3′,5-cyclic adenosine monophosphate (cAMP) levels were measured, WT bacilli, phoPR-KO, and the complemented mutant (Compl.) were exposed to carefully controlled stress conditions as detailed in ‘Materials and methods’, and CFUs enumerated. The results clearly suggest that bacterial viability under indicated conditions of stress does not impact intra-mycobacterial cAMP level.
Figure 1—figure supplement 1—source data 1. CFU of indicated mycobacterial strains under normal and stress conditions.