Experimental Characterization of the <italic>Mycobacterium tuberculosis</italic> Gene Regulatory Network

Abstract

Tuberculosis is a massive public health problem on a global scale and the success of <italic>Mycobacterium tuberculosis</italic> is linked to its ability to persist within humans for long periods without causing overt disease symptoms. Hypoxia is predicted to be a key host-induced stress limiting growth of the pathogen in vivo. However, studies indicate that <italic>M. tuberculosis</italic> coordinates a complex transcriptional program in response to long-term changes in oxygen tension in vitro, with the differential regulation of >20% of all transcriptional regulatory proteins. The results presented in this dissertation describe our efforts to create an experimental framework to define the control logic underpinning transcript regulation in <italic>M. tuberculosis</italic>. Creating a multi-platform experimental foundation for gene regulatory network construction is a critical step in generating predictive models of complex responses in prokaryotes. We describe a workflow that couples a defined perturbation to a regulatory response using chromatin immunoprecipitation followed by high throughput sequencing and transcriptional profiling by tiling microarray. We implement this experimental platform to reconstruct the transcriptional regulatory network of <italic>M. tuberculosis</italic> with particular attention to oxygen-responsive DNA binding proteins. This network allows us to generate predictive models of gene expression during an in vitro time course of hypoxia and reaeration. In this context, we describe the physiological consequences of aerobic induction of the early hypoxia-responsive regulator DosR. We find that <italic>M. tuberculosis</italic> growth is unaffected upon the upregulation of dosR and the DosR regulon - in support of the hypothesis that growth inhibition as a result of oxygen limitation is mediated by a complex regulatory response. We also report studies that define the degradation rate of the mRNA pool in <italic>M. tuberculosis</italic> under different experimental conditions. We find that <italic>M. tuberculosis</italic> contains an unusually stable pool of mRNA and that this pool can be further stabilized by physiologically relevant alterations to the bacterial environment. There are obvious and pressing needs for greater understanding of the basic biology of <italic>M. tuberculosis</italic>, and this dissertation describes advancements we have made toward achieving this goal.