To understand how Mycobacterium tuberculosis survives and grows in an infected host, we are studying the mycobacterial transcriptional machinery and its response to stresses encountered in vitro and in vivo. Much has been learned about sigma factors and other transcriptional regulators concerning their roles in controlling mycobacterial gene expression. It has recently been shown that sigma A is the essential housekeeping sigma factor and the alternative sigma factor sigma B, not essential for growth in a laboratory setting, is required for a robust protective response to various environmental stresses. We are also studying the mechanism by which the R522H mutation in sigma A prevents the transcription of certain genes, including some that are believed necessary for virulence. Also under investigation is the mycobacterial iron acquisition apparatus and its regulation, as metabolism of this essential element plays a key role in microbial pathogenesis. We have identified and characterized the major mycobacterial iron regulator IdeR that blocks the synthesis of the iron uptake machinery and have identified target genes in M. smegmatis and M. tuberculosis that are directly repressed by IdeR. Recent studies have examined the control of M. tuberculosis gene expression in vivo. Among these new approaches are an in vivo expression technology system to identify M. tuberculosis genes that are induced in macrophages and mice and a novel RT-PCR method that allows an accurate comparison between the levels of specific mRNAs in M. tuberculosis grown in vitro with those found in bacteria growing in human macrophages.