Diseases caused by mycobacteria are among the most serious of the world's public health problems. However, very little is currently known about how these bacteria control the expression of genes that are active in the free living state as well as those that are uniquely expressed in the infected host. Free-living microorganisms must possess mechanisms that enable them to adapt to and survive environmental challenges such as temperature extremes, radiation and nutrient deprivation. The most commonly described examples of response to environmental stress are the SOS and heat shock responses in prokaryotes and sporulation in bacteria (the genera Bacillus, Myxobacteria and Streptomyces) and fungi (Saccharomyces and Schizosaccharomyces). Similarly, pathogenic microorganisms are faced with environmental stresses when they invade a host, especially those that grow intracellularly, e.g. Mycobacterium, Shigella, Salmonella, Listeria, Legionella, etc. When a member of these latter genera infects a vertebrate, it is immediately confronted and engulfed by host macrophages. The bacterium is subjected to heat shock, oxidative and nitric oxide stress in phagolysosomes. New proteins must be synthesized to allow the parasite to survive and multiply in the host. Formerly quiescent genes must be activated to program the synthesis of these new proteins, some of which are virulence factors. Our laboratory, for the last 10 years of support under grant GM 19693, has studied the regulatory mechanisms of sporulation, a global response to environmental stress, in Bacillus subtilus. With the theoretical knowledge gained and experimental approaches developed in these studies, we have changed the focus of our work to study related questions in mycobacteria. We plan to investigate how the mycobacterial cell responds to environmental conditions that it can expect to encounter in the host. However, very little is presently known about the basic transcriptional machinery in the genus Mycobacterium and even less about environmental stress responses. We have therefore begun to study the cellular machinery involved in mycobacterial gene expression. We have purified highly active RNA polymerase holoenzyme and have cloned genes that code for proteins of the transcriptional apparatus. In the next grant period, we will extend these studies, including in vitro and in vivo approaches, to further characterize the cellular machinery involved in basic transcription and in the sensing of environmental conditions and the resulting effector responses. Our experiments should provide information on the repertoire of alternative modes of gene expression available in the genus Mycobacterium, which will be immediately applied to similar investigations of mycobacterial virulence.
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