Mapping combinatorial stress responses in bacteria
Bacteria respond to changing environments by redirecting gene expression to cope with the applied stress, during infection and in the environment as nutrient and abiotic conditions vary. How the complex signal transduction pathways, the associated metabolic factors and protein factors inter-relate to achieve the necessary adaptive changes in the cell has not been well studied at an integrated level. In particular how combinatorial stresses cause adaptive change in the cell is unknown. This project used non-native bio-synthetic regulatory proteins to rewire signal transduction pathways to reprogram E. coli and M. tuberculosis to elicit specific gene expression changes, uncoupled from the cognate native cues of gene expression. The main strength of synthetic proteins to study complex biological systems is that they can be made to function independent from the native physiological context. Hence the complex control feedback mechanisms that regulatory systems usually employ to regulate genetic and metabolic flow can be separated out. We plan to produce and characterise bio-synthetic domain exchanged (chimeric) transcription activators of the bacterial RNA polymerase to control genetic flow under defined sets of stress conditions. These chimera are used to dissect genetic and metabolic control of nitrogen regulation and other stresses and to gauge the relative contributions of genetic and metabolic factors to cell adaptation. Methodologies include quantitative proteomics of key players, metabolic profiling and array technologies to measure protein-DNA interactions and transcript responses linked to advanced modelling approaches. Results of the project are anticipated to contribute significantly to biotechnology, infection research and emerging fields of synthetic and systems biology. Synthetic biology can be applied to uncover design principles of complex genetic networks through dissecting the functional performance of modular system components of the cell.
Sub-project 1: Nitrogen limitation of Mycobacteria
The overall aim of Study 1 was to determine systems changes due to nitrogen limitation stress conditions of Mycobacteriium smegmatis and Mycobacterium tuberculosis.