Abstract
Pseudomonas aeruginosa is a significant opportunistic pathogen that like other organisms requires iron for survival. One way that P. aeruginosa acquires iron is via an iron-scavenging siderophore called pyoverdine. Pyoverdine is regulated by two sigma factors, FpvI and PvdS. FpvI induces the expression of the outer membrane receptor, fpvA, for uptake of iron-loaded pyoverdine (ferri-pyoverdine). PvdS induces the expression of the pyoverdine synthesis genes. Anti-sigma factor FpvR inhibits the activities of FpvI and PvdS in the absence of pyoverdine. A unique feature of this regulatory mechanism is that two sigma factors are inhibited by one anti-sigma factor. The work described in this thesis investigated the interactions between FpvR, FpvI and PvdS for regulation of the pyoverdine signalling pathway.
A protocol was developed for the purification of two forms of the anti-sigma domain (ASD) of FpvR (FpvR1-67 or FpvR1-89). Analysis of the pure protein showed FpvR1-67 and FpvR1-89 to have mostly α-helix structure. This was similar to the crystallised ASD for other anti-sigma factors. The protein was sufficiently pure for crystallisation trials but crystals were not obtained. FpvR1-67 and FpvR1-89 were also co-expressed with FpvI and PvdS. Complexes of FpvR/FpvI and FpvR/PvdS were purified, confirming that these proteins interact.
The expression of fpvR, fpvI and pvdS in P. aeruginosa was measured by RT-qPCR in order to demonstrate the effect of iron on gene expression. Expression of these genes was regulated by the amount of iron in the growth medium via the iron-sensing Fur repressor. A strong increase in pvdS expression relative to fpvI during iron starvation conditions indicated that more PvdS than FpvI is present in iron-starved cells, which could mean greater expression of pyoverdine synthesis genes compared to fpvA expression. The amount of FpvR protein in a pyoverdine mutant was also measured and shown to be much lower than previously measured amounts of PvdS. This indicated that in the absence of pyoverdine PvdS activity may be regulated by other mechanisms, in addition to FpvR, to limit induction of pyoverdine synthesis genes due to the excess PvdS.
The affinity that FpvR has for FpvI and PvdS was measured. FpvR was able to repress the activities of FpvI and PvdS to a similar level, when the sigma factors were competing for binding to FpvR, indicating that FpvR has a similar affinity for both sigma factors. This would mean that the relative amounts of FpvI and PvdS would dictate the amount of each sigma factor bound to FpvR in P. aeruginosa rather than the relative affinities. The similar affinity also suggested that FpvR binds to FpvI and PvdS through a similar mechanism.
FpvI and PvdS share conserved functional regions with house-keeping sigma factors, called region two and four. Single mutations were introduced into region four of FpvI and PvdS, which binds to the -35 site of promoters and core RNA polymerase, to investigate the effect on interactions with FpvR1 89. The FpvI single mutations disrupted interactions with FpvR providing evidence that FpvR binds to this region of FpvI and inhibits activity through steric hindrance of core RNA polymerase binding. Some evidence was also provided for PvdS region four in binding to FpvR.
The research presented in this thesis showed strong similarities between FpvI and PvdS in interactions with FpvR, indicating that FpvR inhibits both sigma factors through a similar mechanism. It is likely that differences in gene expression and the predicted amounts of FpvI and PvdS determine the amount of fpvA and pyoverdine synthesis gene transcription.