Characterisation of the stringent response and polyphosphate biosynthesis in the intracellular pathogens Burkholderia pseudomallei, Francisella tularensis and Yersinia pestis

The paucity of novel antibiotics to treat intracellular pathogens has become a matter of intense concern for the scientific community. Similarly, innate, emerging and even engineered antibiotic resistance is of disquiet for pathogens of concern including those of interest in biodefence. Therefore there is a significant need to identify novel classes of antibiotics.Under conditions of nutrient limitation, bacteria initiate the stringent response, co-ordinated by the signalling nucleotides guanosine tetra- and penta-phosphate, collectively termed (p)ppGpp. During starvation, (p)ppGpp accumulates and coordinates diverse transcriptional alterations. (p)ppGpp levels are controlled by two enzymes, RelA and SpoT, which are a monofunctional (p)ppGpp synthetase and a bifunctional (p)ppGpp synthetase/hydrolase, respectively.#Inorganic polyphosphate, a global regulatory molecule, has also been linked to the stringent response. Levels of polyphosphate are controlled by a polyphosphate kinase (PPK) and an exopolyphosphatase (PPX). Mutation of the genes relA and spoT results not only in abrogation of (p)ppGpp production, but also results in lower levels of polyphosphate accumulation. However, the interaction of the stringent response with the polyphosphate regulon is not yet clearly understood.The aim of this project is to inactivate the key genes involved in (p)ppGpp and polyphosphate metabolism in intracellular pathogens of interest to defence. The characterisation of these mutants in vitro and in vivo and the analysis of the global stringent response regulon are discussed in this thesis