Antibiotics are the guns and swords of bacterial warfare. In the early 1900s, the antibiotic penicillin was used to control bacterial infections in humans. Since then, the use and misuse of antibiotics has led to various resistant strains of bacteria. One way in which bacteria can resist being killed by antibiotics but which does not require the uptake of a resistance gene is through the stringent response. This stress response is regulated by the small molecule (p)ppGpp. High levels of (p)ppGpp lead to downregulation of transcription, translation, DNA replication, in addition to upregulation of motility, biofilm production, and virulence. In Gram positive organisms, including both pathogens and non-pathogens, (p)ppGpp levels are regulated by a bifunctional synthetase/hydrolase with a C-terminal regulatory domain, RelA. In the model system Bacillus subtilis, two minor synthetases, YjbM and YwaC also affect cellular (p)ppGpp levels. These minor synthetases share homology to the synthesis domain of RelA, however, the regulatory domain of RelA has been poorly characterized. Furthermore, while it is clear that RelA is regulated by metabolic signals in response to starvation conditions, the exact mechanism by which this occurs is not yet known. Therefore, two aims of the present work are to characterize the domains of B. subtilis RelA through both a global domain deletion analysis and random mutagenesis. B. subtilis will also be used as a heterologous system for the characterization of the RelA proteins from pathogenic organisms from phylogenetically diverse species. The structure-function knowledge gathered in this study should pave the way for development of small molecules that are capable of being used in junction with antibiotics to prevent the survival of these bacteria.
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