Abstract
Recombination and lateral transfer of genomic material between bacterial strains have long been recognised as important to the evolution of a species. Recent developments in sequencing technologies has resulted in a flood of sequencing data. Currently, there are ~2500 publicly available genomes of Pseudomonas aeruginosa, making it one of the most sequenced species, but still little is known about the role of recombination in the genome evolution within this species. One of the primary aims of this thesis was to detect recombination events in the genomes of P. aeruginosa isolates and understand the implications that these events may have had on the genomic evolution.
In 2014, Croucher et al., released an algorithm designed to recognise DNA that had been introduced by recombination into bacterial species. Within this study, the algorithm was tested against multiple datasets of P. aeruginosa genomes and used to assess horizontal DNA transfer in a well-annotated dataset. It was found that introduced blocks of DNA were not randomly distributed across the genomes. Instead, these recombination blocks occurred with high frequency at genomic locations often associated with virulence, including particular mobility elements, toxin/anti-toxin genes of the Type VI Secretion System, and an iron uptake system.
The frequency of recombination events detected in each isolate depended on the environmental source it was from. Genomes of clinical isolates had lower levels of recombination in comparison to other isolate types, despite similar levels of genetic diversity between isolates. Genes that were identified as having high levels of recombination were not associated with promoting long-term infections, such as those found in cystic fibrosis patients, but were rather associated with the establishment of infection. Low levels of recombination in cystic fibrosis isolates are likely due to longer term infections being fairly mono-cultural with less competition from other strains. Therefore, horizontal gene transfer between isolates was hypothesised to be important in facilitating colonisation in immunocompromised hosts and creating a selective advantage within nutrient-poor environments.
The iron uptake locus that was found to be a “hotspot” of recombination encodes genes for the production and transport of the iron chelating molecule called pyoverdine. The pyoverdine iron uptake system is crucial for isolates of P. aeruginosa to establish infection and survive in low iron conditions. Evolution of the pyoverdine locus is dependent upon horizontal gene transfer with different genes in the locus being transferred in different events.
Every isolate of P. aeruginosa produces one of three types of pyoverdine (type I/II/ III) whose synthesis, transporter, and regulator genes are transferred in multiple recombination events. Phylogenetic analysis of each gene within the iron uptake locus revealed that a recombination block containing the pyoverdine transporter and receptor genes were associated with the type of pyoverdine produced by each strain.
Genes belonging to a secondary recombination block detected in the pyoverdine locus were strongly associated with either pyoverdine type I/III or type II. The genes identified in this recombination block encoded a sigma factor FpvI, that promotes the production of the pyoverdine receptor FpvA, and an anti-sigma factor FpvR that binds FpvI preventing promotion of the receptor. Analysis of ~1400 genomes revealed that ~99% of genomes had FpvR and FpvI genes associated with pyoverdine type I/III or type II.
It was hypothesised that strains producing FpvI and FpvR associated with an alternative pyoverdine type would not be as efficient at regulating expression of the pyoverdine receptor compared to other strains. To test this hypothesis, alternate types of FpvI and FpvR were expressed in the reference strain PAO1 and expression from a FpvI induced promoter was measured. No discernible differences in gene expression could be detected in engineered strains expressing alternate FpvR and FpvI proteins. This suggested that selective pressures for obtaining the secondary recombination block encoding FpvR and FpvI may not be due to loss of efficiency in regulated gene expression in the pyoverdine locus.
Regulation of the anti-sigma factor FpvR is mediated by a regulated proteolytic cascade. With the exception of the site-2 protease RseP, the proteases involved in the proteolytic regulation of FpvR are unknown. A study of the cellular envelope proteases of P. aeruginosa revealed 18 candidate proteases. Phenotypic tests could not identify a single protease from these candidates that when interrupted prevented proteolytic cleavage of FpvR. However, multi-protease knockouts did prevent proteolysis of FpvR. Therefore, it is hypothesised that the initial proteolysis of FpvR is a 2-step event.
Overall this research represents the first thorough analysis of recombination in the species P. aeruginosa and the implications this has on the evolution and function of a virulence locus.