Pseudomonas aeruginosa is a versatile Gram-negative pathogen that can infect a diversity of immunocompromised patients. Interest in alternatives to traditional antibiotics has inspired our investigation of R- and F-type pyocins as novel therapeutics. These phage tail-like bacteriocins are produced by P. aeruginosa to kill competing strains via pore formation in target cells. We aimed to characterize the diversity of pyocins and bacteriophages generated by diverse P. aeruginosa strains so as to identify pyocins of therapeutic value. Strategies to delineate pyocin and phage activities included physical methods, the modulation of pyocin regulation, and antibody-based detection of tail-like pyocins. We have identified the dominance of R- and F-type pyocins in impacting P. aeruginosa populations and revealed a small number of strains producing particularly potent pyocins. In addition, the co-regulation of phages and pyocins, the dependence of pyocins on pili for activity, and the striking diversity of pyocin susceptibility have all been recognized.

We studied the host specificity, interactions with plant immune systems, and virulence factors of the phytopathogenic Type III secretion system-carrying bacterium Pseudomonas syringae. In studying host specificity, we ran growth and pod assays using seventeen pathovars of P. syringae on kidney bean hosts. We tracked bacterial growth numbers over six days and compared pathovar growth patterns. To study immune interactions with host plants, we performed effector-triggered immunity induction and suppression assays with individual effectors in Arabidopsis thaliana to determine whether effector evolutionary age was related to resultant plant immune responses. No correlations were observed. To generate candidate virulence effectors, we sequenced mRNA from seven P. syringae pathovars grown in inducing media and pulled out hits to virulence-related genes.

Pseudomonas aeruginosa is a versatile Gram-negative pathogen that can infect a diversity of immunocompromised patients. Interest in alternatives to traditional antibiotics has inspired our investigation of R- and F-type pyocins as novel therapeutics. These phage tail-like bacteriocins are produced by P. aeruginosa to kill competing strains via pore formation in target cells. We aimed to characterize the diversity of pyocins and bacteriophages generated by diverse P. aeruginosa strains so as to identify pyocins of therapeutic value. Strategies to delineate pyocin and phage activities included physical methods, the modulation of pyocin regulation, and antibody-based detection of tail-like pyocins. We have identified the dominance of R- and F-type pyocins in impacting P. aeruginosa populations and revealed a small number of strains producing particularly potent pyocins. In addition, the co-regulation of phages and pyocins, the dependence of pyocins on pili for activity, and the striking diversity of pyocin susceptibility have all been recognized.

I have investigated the evolutionary response of Pseudomonas aeruginosa to salt (NaCl) stress, and the physiological mechanisms responsible for this adaptation. Populations of P. aeruginosa founded from the same ancestral genotype were selected at three different concentrations of NaCl, low, moderate and high for about 660 generations with four independent replicates for each concentration. Adaptation was measured as the fitness of the evolved populations relative to the ancestor assessed in direct, head-to-head competition experiments conducted in the same environment in which they were selected (direct response) as well as in all alternative environments (correlated response). Results suggest that selection in each salt environment led to adaptation to that environment and a modest degree of specialization that evolved because correlated responses to selection were smaller than direct responses. In order to identify the physiological mechanisms contributing to the populations' adaptation in high NaCl concentration, I chose a sample of evolved lines that showed the strongest evidence for specialization to salt and competed them against the common ancestor in KCl and sucrose. Results suggested that increased Na+ /H+ antiporter activity is probably the primary mechanism behind adaptation to high NaCl concentration, however alternative mechanisms cannot be excluded. Tolerance curves, which measure the performance of a genotype across a gradient of salt concentrations, suggested no change in the high salt group’s ability to tolerate extreme concentrations of NaCl. We conclude that high salt evolved population showed improvements to its ionic/osmotic stress resistance strategies mainly to Na+ efflux strategies but with no changes to salt niche.

ABSTRACT Sclerotinia sclerotiorum is a ubiquitous ascomycetous fungal pathogen that causes disease in over 400 crop species, specifically in soybean and canola plants, where stem rot is the most common disease symptom. Pseudomonas chlororaphis PA23 was previously isolated from the rhizosphere of soybean and has demonstrated excellent antifungal activity against S. sclerotiorum in vitro, greenhouse and field experiments. To elucidate the molecular mechanisms involved in PA23 biocontrol, random mutagenesis experiments were initiated. Several mutants were isolated that could be divided into three general classes. Biocontrol activity of various Pseudomonas spp. is highly regulated by a GacS/GacA two-component global regulatory system. Class I PA23 mutants harboured Tn5 insertions in the gacS-coding region, resulting in pleiotropic defects including deficiency in secondary metabolite production and biocontrol activity. Complementation with the wild type gacS allele in trans restored wild type phenotypes. These findings suggest that the ability of P. chlororaphis PA23 to suppress S. sclerotiorum causing stem rot in canola is dependent on a functional GacS/GacA global regulatory system. This is the first study assessing disease symptoms on canola (Brassica napus L.) plants inoculated with a gacS minus strain of P. chlororaphis. Phenazine compounds are considered to be a key secondary metabolite contributing to the antagonistic and antifungal activity of P. chlororaphis. In P. chlororaphis PA23, mutations in phenazine biosynthetic genes exhibited equal or more antifungal activity in vitro, compared to the wild type. To assess the effect of the deficiency in phenazine production, a Class II mutant , harbouring a Tn5 insertion in phzE was tested for a number of biocontrol traits including secondary metabolite production, motility, and suppression of Sclerotinia pathogenic traits. Since no other traits were markedly affected beyond phenazine production, it was concluded that phenazine is not the major product contributing to S. sclerotiorum biocontrol. A single Class III mutant was isolated harbouring a Tn5 insertion in a gene encoding a transcriptional regulator of the LysR family. This mutant exhibited no antifungal activity on plate assays and was unable to protect against S. sclerotiorum in green house assays. A number of secondary metabolites were no longer produced by this mutant, suggesting that this LysR-type transcriptional regulator is either directly or indirectly involved in controlling several genes in P. chlororaphis PA23.

Cystic Fibrosis is one of the most common genetic recessive diseases among Caucasians and is caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene on chromosome 7. There are different classes of CFTR mutation, leading to differences in disease severity among patients. In addition to the CFTR genotype, secondary genetic factors, modifier genes, also influence CF phenotypes. Due to the dysfunction of CFTR protein and production of thickened mucus, bacterial infection in the lungs is favored and can lead to further clinical complications in CF patients. Pseudomonas aeruginosa is one of the most common bacteria detected among patients. The aim of this project was to investigate four candidate modifier genes, Factor B, Complement Factor 3, Toll-like Receptor 4 and Heme oxygenase-1, which might affect the status of Pseudomonas aeruginosa infection. A total of 22 single nucleotide polymorphisms (SNPs) were selected in these four genes and they were tested against five phenotypic traits, including age of diagnosis, FEV1% predicted andstandard deviation value, age of first Pseudomonas aeruginosa infection and Pseudomonas aeruginosa infection status. Among the selected SNPs, both case-control studies and family-based analysis were performed in order to establish any correlation between the genotypes and the phenotypes. In addition, haplotype analysis was performed to determine whether there was interaction between SNPs or whether there were unidentified SNPs in the vicinity of the selected ones that might contribute to the observed phenotypic traits. Among the 22 chosen SNPs, 13 of them were found to be significantly linked to one or more of the tested phenotypes. The three most significant associations were BF_2557 with lung function, HMOX1_9531 with lung function and BF_7202 with age of diagnosis. Several haplotypes were significantly associated with one of the five phenotypes. There was no evidence for the presence of unidentified SNPs or interaction between SNPs. Most of haplotype associations were likely due to the presence of a single SNP which was found to be significantly linked to the phenotype. Conclusively, both SNPs and haplotype analyses suggest that the four candidate genes are modifiers of disease severity in CF.

Doctor of Philosophy / Cystic fibrosis (CF) is the most common inherited lethal disorder among Caucasian populations. Chronic pulmonary infections, particularly from Pseudomonas aeruginosa, are the major determinant of the morbidity and mortality of people with CF. It is generally accepted that people with CF acquire this pathogen independently from their surrounding environment, and that individual CF patients carry unique strains different from others. The spread of this pathogen from patient to patient is thought to be rare and occurs particularly among closely contacted cases such as CF siblings. However, over the past decade, there have been several reports of an emergence of clonal P. aeruginosa strains commonly found infecting a number of CF patients. One such report is from the CF paediatric clinic at the Royal Children’s Hospital in Melbourne in which more than half of the patients were infected with a single strain or clone, subsequently called Australian epidemic strain 1 or AES-1. A preliminary survey showed that AES-1 had spread extensively along the Australian eastern seaboard among CF patients attending other CF centres in Melbourne, Sydney and Brisbane, including adult patients at the Royal Prince Alfred Hospital (RPAH), Sydney. Another clonal strain, subsequently called AES-2, was identified in both CF adults and children at the Prince Charles Hospital and the Royal Children’s Hospital, in Brisbane. The total extent of prevalence of the AES-1 and AES-2 strains at the RPAH as well as the clinical status of patients who carried these strains was unknown. Moreover, the pathogenicity of these two clonal strains had not been investigated. The studies presented in this thesis investigated the prevalence of these clonal strains among CF patients attending the adult CF clinic at RPAH, Sydney by using pulsed-field gel electrophoresis. Overall, 50% of 112 patients with P. aeruginosa were found to be infected with clonal strains. The AES-1 and AES-2 strains were identified in 38% and 5% of the patients respectively. Two new clonal strains, called Sydney-1 and Sydney-2, were also identified. Patients with clonal strains had a significant increase in their number of exacerbations and hospitalisation days, and tended to have lower pulmonary functions when compared to patients infected with non-clonal strains. By using a variety of bioassays to examine the pathogenicity of the clonal and non-clonal strains, it was found that both AES-1 and AES-2 produced more virulence factors and were more resistant to antibiotics when compared to the non-clonal strains. AES-1 and AES-2 were associated with increased production of proteases, including elastase, alkaline protease and protease IV. Overall the results presented in this thesis suggest that there may be a link between virulence and transmissibility of this pathogen. The studies presented in this thesis also compared the biofilm forming capacities of the AES-1 and non-clonal isolates. AES-1 was shown to have greater biofilm-forming capacity than the non-clonal strains, when they were grown on a glass surface, suggesting a possible association between clonality and biofilm formation. A model for the study of bacteria grown in conditions similar to CF sputum was also developed. P. aeruginosa grown in this model was found to develop into clumps which may be comparable to the biofilm structure in the CF lung. This model was shown to be beneficial for transcriptomic and proteomic studies which are underway within the research group. AES-1 was also found to have phenotypic variations between isolates. By applying the amplified fragment length polymorphism technique, more subtypes of this clone were revealed. However, these detected subtypes did not correlate with the different phenotypes, suggesting minor mutations such as single point polymorphisms may be responsible for the phenotypic diversity within the clone. The final part of this thesis was devoted to examining the safety of a novel CF treatment: hypertonic saline (HS) inhalation. HS was shown to increase airway mucociliary clearance, while increased osmolarity associated with the use of HS was also shown to have an inhibitory effect on the formation of biofilms. Findings in this study proved that there was no evidence of strain selection in patients who received the long-term treatment with HS. The study also demonstrated that AES-1 was significantly more persistent in the CF lung than the non-clonal strains. The present thesis not only defines the clonal strains of P. aeruginosa and their implications for infected patients, but also provides a general understanding into the pathogenesis of both clonal and non-clonal strains infecting CF lungs.

Professional paper (MS)--Montana State University--Bozeman, 2008. / Typescript. Chairperson, Graduate Committee: Michael Franklin. Includes bibliographical references (leaves 37-46).

Thesis (Ph.D.)--Ohio State University, 1977. / Includes vita. Includes bibliographical references (leaves 127-140). Available online via OhioLINK's ETD Center.

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [67]-74).