Visualising a bacterial stress response: gene product localisations movements and gene regulation at single molecule level in live bacterial cells

Bacteria comprise one of the most abundant life forms on the planet, and contribute hugely to, for example, the major carbon, nitrogen, sulphur and phosporus cycles and transformations that characterise a functional and healthy global living environment. Despite huge advances in understanding how the components of the bacterial cell work, little is known about how they are organised within the living cell, nor how such organisations impact upon the functionality of the cell. Using a method that enables us to visualise single molecules within living cells, we will look in a quantitative way at the dynamics of the cellular components that let the cell survive a stress that damages its membrane. In effect we will be studying the cell in action, and will be able to describe the cell in its pre-stressed state as well as in its adapted state. Gaining an appreciation and understanding of how the cells components are organised for function is important in establishing principles of signaling and the networks of interactions that allow cells to grow and adapt to new environments.

To date, much of our in depth understanding of cell functionality has been at the level of the detailed biochemical/biophysical characterisations of key cellular components. Advances in the field of single molecule, single cell imaging now make it possible to obtain quantitative information on the cellular localisations, interactions, dynamics and gene expressions of key effector and control proteins in important microbial cells. We plan to examine at the single molecule and single cell level how the E.coli cell adapts to stress that causes its inner membrane to lose integrity. This stress is important in a range of significant contexts, including protein export scenarios that support virulence in bacteria. One protein we will study, PspA, has a homologue in VIPP1, needed for thylakoid biogenesis. We will study in vivo (i) the states of self association of PspA and its localisations, to understand how it switches from negative regulator to effector, (ii) the localisations and dynamics of the effector protein PspG, the localisations and self associations of the transcriptional control protein PspF, and finally (iii) the expression dynamics of the pspG promoter. The latter will for the first time allow us to determine if expression of an activated and complexly controlled promoter does or does not show the geometric distribution of expression bursts seen with simple repression control as in lac. At present it is even unknown whether expression bursts are generally seen or not. The gene expression studies are important in understanding how variations in expression levels between cells might be caused, and for deducing whether different types of gene regulation strategies give rise to rather different types of outputs. Different types of outputs may well be better suited to some purposes than others, dependepending upon the type of gene product being produced.