Uncoupling flagellar gene expression from flagellar assembly

Bacterial locomotion plays a key role during the life cycle of over seventy percent of bacterial species. The most common way bacteria achieve movement is by using a cell surface structure known as the bacterial flagellum. The structural organisation of the flagellum is often paralleled to the propeller of a motorboat, consisting of a motor anchored into the cell surface and a long external filament that acts as the propeller. Two key issues in assembly of external structures such as flagella are: how does a cell monitor assembly? And how is the gathered information processed? Assembly of an individual flagellum is a step-wise process that requires individual flagellar building blocks to be transported out of the cell by a dedicated transport system. As with every construction site this process requires site managers. Without site managers the efficiency of construction is reduced leading to a waste of energy, as the production and delivery of the required building materials is not regulated. In the case of flagellar assembly the site managers are a group of proteins known as secretion chaperones. In the pathogen Salmonella enterica the secretion chaperones of flagellar assembly must 1) coordinate delivery of specific building blocks and 2) oversee the timely synthesis of a subset of the flagellar building blocks in response to the construction status of the growing flagellum. During this proposal we will investigate how two flagellar secretion chaperones regulate the production of flagellar building blocks in response to intermediate stages of flagellar assembly. To achieve our goal we will characterise mutations isolated in the secretion chaperones unable to process signals obtained from the growing flagellar. We will also use these mutations to study the overall effect loss of flagellar gene regulation by secretion chaperones has upon flagellar assembly. Our study will advance our understanding of how cells perceive and regulate fundamental aspects of their life cycles.

How do cells sense the assembly status of 'extracellular' structures and react to changes in assembly status? Flagellar assembly is a temporal process, requiring secretion of flagellar subunits via a dedicated secretion apparatus through the base of the growing structure and incorporated at its tip. The secretion apparatus associated with the flagellum belongs to the type III secretion (T3S) family. Other T3S systems are associated with an injectisome, which injects virulence determinants into host cells to facilitate pathogenesis. The bacterial cell is posed with a major problem in sensing the assembly status of a flagellum due to its external nature and length. How bacteria overcome this problem has lead to flagellar assembly, becoming a leading model system used to study the coupling of gene expression to macromolecular structure assembly. Substrate recognition by the T3S secretion apparatus is facilitated by the T3S chaperones. Two flagellar T3S chaperones FlgN and FliT in Salmonella enterica are bi-functional also sensing the progression of flagellar assembly, feeding the perceived signals back into the system at the level of flagellar gene expression. Our current objectives are to identify how FlgN and FliT regulate flagellar gene expression and to determine the effect of removing the regulation of flagellar gene expression by the T3S chaperones with respect to flagellar gene expression. We aim to achieve our goals by isolating and characterizing mutants in the regulatory functions of FlgN and FliT. Regulatory mutants will then be used in combination with other flagellar mutations to study flagellar gene expression with respect to time in bioluminescence based real time growth assays.