Abstract
In cell-surface signalling pathways, the binding of an iron chelate to a specific outer membrane receptor induces gene expression in the cytoplasm via extra-cytoplasmic function sigma proteins. In these pathways gene expression is controlled by an antisigma protein that spans the inner membrane and interacts with both receptor and sigma proteins. In the pathogenic bacterium Pseudomonas aeruginosa, the binding of the siderophore pyoverdine to the ferri-pyoverdine receptor induces the activity of sigmas PvdS and FpvI by relieving the activity of the anti-sigma FpvR. The aim of the research described in this thesis was to determine and characterise the molecular mechanisms underlying signal transduction in the pyoverdine signalling pathway, and to determine the generality of these mechanisms.
Tools were developed for the detection of FpvR under different signalling conditions. Truncated FpvR sub-fragments were detected when sigma-dependent gene expression was inhibited. In contrast no FpvR was detected when pyoverdine was available and gene expression occurred. This indicated a role for proteolysis in the regulation of FpvR activity. The role of the FpvR cytoplasmic and extra-cytoplasmic domains in anti-sigma and signal transduction functions of FpvR was investigated using prematurely truncated and tagged chromosomal fpvR alleles. The FpvR cytoplasmic domain could inhibit gene expression, but was not to produce the same level of anti-sigma activity as the wild-type protein. The involvement of candidate protease genes algW, mucD, rseP, and prc on the proteolysis of FpvR and on sigma-dependent gene expression was investigated. Strains lacking rseP accumulated novel FpvR degradation fragments, implicating RseP in the proteolysis of FpvR. Sigma-dependent gene expression was reduced in strains lacking rseP indicating this gene is a positive regulator of sigma activity. The generality of these findings was investigated in the ferrioxamine (Fox) and ferrichrome (Fiu) cellsurface signalling pathways. Anti-sigmas FoxR and FiuR were subjected to proteolysis in the absence of cognate siderophore and rseP was critical for siderophore-mediated gene expression in these systems.
Overall this research identified regulated intramembrane proteolysis as a key regulatory mechanism in the pyoverdine, ferrioxamine, and ferrichrome cell-surface signalling systems. Furthermore it is likely that these findings extend to all cell-surface signalling pathways in P. aeruginosa and perhaps many more Gram-negative bacteria.