Abstract
Pseudomonas aeruginosa requires iron for growth and metabolism, but is able to grow in environments where bioavailable iron is scarce. As such, the bacteria must scavenge iron from their environment using a variety of iron acquisition systems, including the secretion and uptake of siderophores. Siderophores are compounds secreted by microbes, which bind iron in the environment. Once iron is bound to the siderophore, bacteria are able to uptake the siderophore-bound iron. P. aeruginosa secretes and produces a siderophore, pyoverdine, and its uptake is controlled by the well-studied fpv cell surface signalling (CSS) system. P. aeruginosa can also uptake iron from exogenous siderophores secreted by other microbes – desferrioxamine and ferrichrome. The uptake of these iron bound siderophores is controlled by the fox and fiu CSS systems, respectively.
The general components of the CSS systems are as follows: membrane receptor, FpvA/FoxA/FiuA; sigma regulator, FpvR20 /FoxR/FiuR; and extracytoplasmic function (ECF) sigma factor, FpvI/FoxI/FiuI. In the presence of cognate siderophore, the sigma factor becomes active.
The downstream targets of active sigma factor protein are the outer membrane receptor gene promoters. The FpvR20 protein also regulates the activity of a second sigma factor, PvdS, which targets the promoters of genes for pyoverdine biosynthesis and virulence factor production. In the well-studied fpv system, deletion of the fpvR gene causes constitutive activity of FpvI, indicating that FpvR20 acts as an anti-sigma of FpvI; however in the fox and fiu systems, deletion of the foxR/fiuR gene causes FoxI/FiuI to be inactive, regardless of the presence of cognate siderophore.
This observation forms the basis of the overarching question addressed in this research: why is the sigma regulator required for activity of the sigma factor in the fiu and fox systems but not in the fpv system? This research question was addressed in three ways by this project: (a) are there differences in the cellular amount of sigma factor? (Chapter 3); (b) are differences in regulation due to differences in genome organisation (operons)? (Chapter 4); and (c) do the signalling systems respond to iron or siderophore differently? (Chapter 5).
There were differences in the amounts of sigma factor proteins per cell, but these differences did not offer insight as to why FpvR20 has anti-sigma activity whereas FoxR/FiuR have are required for activity. The addition of an epitope tag to the N-terminus of the sigma factor proteins caused changes in activity for all three sigma factors tested, but with different effects in each case
The operonic expression of foxIR and fiuIR operons were found to be important for activity of FoxI, and proper regulation of FiuI. The activities of the three sigma factors in response to extracellular stimuli, such as changing iron or siderophore concentrations were different, indicating that there is likely a hierarchy of promoters inhibited by Fur, the master regulator of iron uptake genes.
Taken together, the results of this research show that superficially similar systems which carry out analogous functions are regulated differently and may be more specific than appearances indicate. This thesis sheds light on differences in mechanisms of the CSS systems studied and revealed surprising differences between analogous proteins in bacteria.