Characterisation and Function of Campylobacter Jejuni Flagellar Proteins FlhF and FlhG

Campylobacter jejuni is the dominant foodborne bacterial pathogen in the UK. Its virulence depends crucially on the expression of flagella. Recent research showing that flagella form a secretary channel for host invasion proteins underlines this dependence. However the genetics and biogenesis of flagella in C. jejuni and other polar flagellate bacteria remain poorly understood. In model peritrichous bacteria like Escherichia coli, flagellar genes have been investigated in detail and are well-characterised. In contrast flagellar genes flhF and flhG (aka fleN) are absent from E. coli and remain neglected. The proteins encoded by these genes are key flagellar components in C. jejuni, and also occur in polar flagellate bacteria that are important as animal and plant pathogens as well as environmentally and industrially. The functions of these proteins are unclear but they appear to influence flagellar number and polar positioning. FlhF is a distant orthologue of FtsY, a signal recognition particle-associated protein involved in targeting of proteins for export and membrane insertion. FlhG is an orthologue of MinD, a septum site- determining protein found in many bacteria. The sparse data on FlhF and FlhG are contradictory but suggest that they are involved in regulation of number and position of polar flagella. This proposal seeks to explore their function in the important foodborne pathogen, and tractable small genome model organism, C. jejuni. It builds on the applicants extensive experience investigating a variety of flagellar genes and proteins. Prior work explores the function of essential flagellar regulatory genes encoding alternative sigma factors s54 and s28, and s54-activator protein FlgR, the anti-s28 factor FlgM and the hook length sensor and export pathway specificity protein FliK. It includes construction and characterisation of knock-out mutants in flhF and flhG in C. jejuni, revealing that flhF is essential for flagellar expression while flhG is involved in regulation of flagellar number and position. <P>
The project will investigate the cellular locations of FlhF and FlhG, and their interaction with other flagellar proteins, the transcriptional regulation of the genes and effects on expression of other flagellar genes. Findings will shed light on their functions, with implications also for other important bacteria in which they occur. Methods used will include the following. Translational fusions of the proteins to green fluorescent protein will enable their detection and locations by fluorescence microscopy. Flagellar subcellular fractions will be analysed by proteomics methods, to determine whether or not these proteins are associated with the flagellar basal body. Two-hybrid experiments will be done to screen FlhF and FlhG for protein-protein interactions. Microarray experiments, real-time PCR and promoter mapping will be used to analyse transcription in flhF and flhG and other flagellar gene knockout mutants.<P> In total, these experiments will yield data that will significantly advance knowledge of the cellular location and function of these proteins.