: The experiments outlined in this proposal are designed to elucidate the sigE regulon of M. tuberculosis and the role it plays in the infection process. Recent data demonstrate the induction of sigE when M. tuberculosis is phagocytized by the macrophage. The in vivo signal and the genes regulated by SigE are not known. The objectives of this proposal are to: (1) determine if SigE is required for intracellular survival in macrophage, (2) identify genes regulated by SigE, and (3) characterize the regulation of sigE at the transcriptional and post-translational level. A delta sigE strain of M. tuberculosis will be constructed, and its ability to survive and replicate in the macrophage will be assessed. This strain will also be used to identify genes that are regulated by SigE. The expression of sigE will be controlled in the strain by cloning this gene downstream of an acetamide inducible promoter located in a shuttle vector. The protein expression profiles of strains harboring this plasmid in the presence and absence of acetamide will be compared to identify genes regulated by SigE. To assess the regulation of sigE, a delta orf1 strain of M. tuberculosis will be constructed, and the levels of SigE in wild type and this strain will be compared by western blot. The transcript levels of sigE in both strains also will be examined.
| Posey, James E; Shinnick, Thomas M; Quinn, Frederick D (2006) Characterization of the twin-arginine translocase secretion system of Mycobacterium smegmatis. J Bacteriol 188:1332-40 |
