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.

The pathogenic bacterium Pseudomonas aeruginosa uses a variety of highly efficient chelating compounds (siderophores) to acquire sufficient iron for growth and virulence. These siderophores can either be endogenous or acquired from exogenous sources such as other bacteria or fungi. The transport of the endogenous siderophore pyoverdine activates a signal-transduction pathway that increases the synthesis of both the ferripyoverdine receptor protein (FpvA) and pyoverdine itself. Signal-transduction systems similar to this have three specific proteins involved: a receptor protein specific for one siderophore in the outer membrane, an anti-sigma factor in the cytoplasmic membrane and a sigma factor that activates gene expression in the cytoplasm. The aim of the research presented in this thesis was to study the roles of the proteins in three different iron uptake and signalling pathways of P. aeruginosa. The substrates for each receptor protein were confirmed and the roles of each protein in the pathways were compared to the P. aeruginosa pyoverdine signalling pathway. The pyoverdine, desferrioxamine and ferrichrome transport pathways were studied to find whether interactions occur between them and if so, the mechanism(s) for that interaction. Furthermore, a technique for analysing gene expression of P. aeruginosa in sputum from the cystic fibrosis (CF) lung was developed. This technique was subsequently used to study the levels of iron responsive gene expression. The receptor, sigma factor and anti-sigma factors were all found to have a role in the siderophore-induced expression of their own signalling pathway. The experimental data provide evidence of similarities in the roles of the sigma and receptor proteins within each pathway but different roles for the anti-sigma factors. In the absence of the cognate sigma factor or anti-sigma factor the expression of the desferrioxamine and ferrichrome receptors could not be upregulated. Without its cognate sigma factor fpvA could no longer be upregulated in the presence of pyoverdine. However, unlike the other systems, in the absence of the cognate anti-sigma factor, expression of fpvA was always observed. This is consistent with the anti-sigma factors being required for the activity of the cognate sigma factor in the ferrichrome and desferrioxamine signalling pathways but not the pyoverdine signalling pathway. The siderophore signalling pathways were found to be upregulated in the presence of multiple siderophores, but generally to a lesser extent than if only one siderophore was available. This suggests that in the presence of multiple siderophores, P. aeruginosa uses all available iron chelators. The study of the role of the receptor, sigma factor and anti-sigma factor into these effects indicate sigma factor competition for RNA polymerase has a major role in the effects of multiple siderophores on pathways upregulation. The gene expression studies of P. aeruginosa in sputum from the lungs of CF patients provided support for the hypothesis that the bacteria were growing in an environment where iron levels were sufficient for bacterial growth, but not storage of iron. The expression of the sigma factor gene pvdS that is required for pyoverdine synthesis was studied because expression of this gene is a sensitive reporter of intracellular iron levels. It was found to be downregulated in bacteria in sputum compared to laboratory grown bacteria. This result suggests the bacteria are inhabiting a more iron-replete environment within the lung. This finding advances our understanding of the CF lung environment and the impact it has on P. aeruginosa infection. This knowledge has medical implications for the development of novel therapies to combat P. aeruginosa infection.

Biodegradation of poorly water soluble hydrocarbons, such as n-alkanes and polycyclic aromatic hydrocarbons (PAHs) is often limited by the low availability of the pollutant to microbes. Adhesion of microorganisms to the oil-water interface can influence this availability. Our approach was to study a range of compounds and mechanisms to promote the adhesion of a hydrophilic PAH degrading bacterium, Pseudomonas fluorescens LP6a, to an oil-water interface and examine the effect on biodegradation of phenanthrene by the bacteria. The cationic surfactants cetylpyridinium chloride (CPC), poly-L-lysine and chlorhexidine gluconate (CHX) and the long chain alcohols 1-dodecanol, 2-dodecanol and farnesol increased the adhesion of P. fluorescens LP6a to n-hexadecane from ca. 30% to ca. 90% of suspended cells adhering. The alcohols also caused a dramatic change in the oil-water contact angle of the cell surface, increasing it from 24° to 104°, whereas the cationic compounds had little effect. In contrast, cationic compounds changed the electrophoretic mobility of the bacteria, reducing the mean zeta potential from –23 to –7 mV in 0.01M potassium phosphate buffer, but the alcohols had no effect on zeta potential. This results illustrate that alcohols acted through altering the cell surface hydrophobicity, whereas cationic surfactants changed the surface charge density. Phenanthrene was dissolved in heptamethylnonane and introduced to the aqueous growth medium, hence forming a two phase system. Introducing 1-dodecanol at concentrations of 217, 820 or 4100 mg/L resulted in comparable increases in phenanthrene biodegradation of about 30% after 120 h incubation with non-induced cultures. After 100 h of incubation with LP6a cultures induced with 2-aminobenzoate, 4.5% of the phenanthrene was mineralized by cultures versus more than 10% by the cultures containing initial 1-dodecanol or 2-dodecanol concentrations of 120 or 160 mg/L. The production and accumulation of metabolites in the aqueous phase responded similarly to the addition of 1-dodecanol. Further experiments showed that the positive influence of the alcohols could not be attributed to the changes in surface and interfacial tension or increase in biomass concentration. The results suggest that enhanced adhesion of bacterial cells to the oil-water interface was the main factor responsible for the observed increase in phenanthrene biodegradation by P. fluorescens LP6a. / Chemical Engineering

Butane-grown Pseudomonas butanovora oxidized butane by a soluble butane monooxygenase through the terminal pathway yielding 1 -butanol as the predominant product. Alcohol dehydrogenases (ADHs) involved in butane oxidation in P. butanovora were purified and characterized at the biochemical, genetic and physiological levels. Butane-grown P. butanovora expressed a type I soluble quinoprotein 1 -butanol dehydrogenase (BOH), a soluble type II quinohemoprotein 1 -butanol dehydrogenase (BDH) and an NAD���-dependent secondary ADH. Two additional NAD���-dependent secondary ADHs were also detected in cells grown on 2-butanol and lactate. BDH was purified to near homogeneity and characterized. BDH is a monomer of 66 kDa consisting of one mole of pyrroloquinoline quinone (PQQ) and 0.25 mole of heme c as the prosthetic groups. BOH was partially purified and its deduced amino acid sequence suggests a 67-kDa ADH containing a PQQ as a cofactor. BOH and BDH exhibited high activities and preference towards I -butanol and fair preference towards butyraldehyde. While BDH could not oxidize 2-butanol, BOH is capable of 2-butanol oxidation and has a broader substrate range than that of BDH. Genes encoding BOH and BDH and their deduced amino acid sequences were identified. BOH and BDH mRNAs and 1-butanol oxidation activity were induced when cells were exposed to butane. Primary C��� and C��� alcohols were the most effective inducers for boh and bdh. Some secondary alcohols, such as 2-butanol, were also inducers for BOH mRNA, but not for BDH mRNA. Insertional inactivation of boh or bdh affected unfavorably, but did not eliminate, butane utilization in P. butanovora. The P. butanovora mutant strain with both boh and bdh genes disrupted was unable to grow on butane and 1-butanol. This result confirmed the involvement of BOH and BDH in butane and 1-butanol metabolism in P. butanovora. Roles of B011 and BDH in butane and 1-butanol metabolism were further studied at the physiological level. There are no substantial differences between BOH and BDH in the mRNA expressions in response to three different 1- butanol levels tested and in their abilities to respond to 1-butanol toxicity. Different bioenergetic roles of BOH and BDH in butane and 1-butanol metabolism were suggested. A model of 1 -butanol- dependent respiratory systems was proposed where the electrons from 1 -butanol oxidation follow a branched electron transport chain. The role of BOH was suggested to function primarily in energy generation because B011 may couple to ubiquinone with the electrons being transported to a cyanide-sensitive terminal oxidase. BDH may be more important in the detoxification of 1 -butanol because the electrons from BDH may be transferred to a terminal oxidase system that is less sensitive to cyanide, which is not capable of energy generation. / Graduation date: 2003

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. / February 2006

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.

El sistema SOS és una xarxa multigènica controlada negativament per la proteïna LexA, i està format per un conjunt de gens implicats en el manteniment de la viabilitat cel·lular davant de lesions en el DNA. Aquest sistema es troba en la majoria d'espècies bacterianes, malgrat que existeixen diferències tant en la seqüència d'unió de la proteïna LexA, com en el contingut genètic del reguló. En la present memòria es descriu el sistema SOS de Pseudomonas putida, un bacteri gramnegatiu pertanyent al grup Gamma. Primerament s'han clonat els dos gens lexA, anomenats lexA1 i lexA2, i s'han obtingut els seus productes gènics mitjançant sobreexpressió i purificació per columnes d'afinitat. Ambdues proteïnes s'han utilitzat en assaigs de mobilitat electroforètica (EMSA) amb els promotors de cada un dels gens lexA. Així s'ha pogut identificar la seqüència d'unió de la proteïna LexA1 (CTGTN8ACAG) i de la proteïna LexA2 (AGTACN4GTGCT). Posteriorment, utilitzant RT-PCR, s'ha vist com el gen lexA2 constitueix una única unitat transcripcional amb els gens que el segueixen, formant el casset lexA2-imuA-imuB-dnaE2. Aquest casset s'ha vist que és induïble per danys en el DNA, i que es troba àmpliament distribuït en el domini Bacteria. Seguidament, s'han obtingut dues soques mutants defectives pels gens lexA1 i lexA2, i s'ha analitzat l'expressió gènica de cada una d'elles, respecte la soca salvatge, utilitzant xips de DNA (microarrays). Els resultats obtinguts han demostrat que la proteïna LexA1 controla la majoria de gens del sistema SOS, que a més corresponen amb la resposta convencional del seu grup filogenètic; mentre que la proteïna LexA2 només regula l'expressió de la seva pròpia unitat transcripcional, i la d'un gen (PP3901) pertanyent a un profag resident de P. putida. A més, aquest gen també es troba controlat per la proteïna LexA1, essent l'únic que comparteix les dues regulacions. L'obtenció d'un mutant defectiu pel gen PP3901 ha demostrat que l'expressió d'aquest és necessària per a la transcripció dels gens del profag resident. L'expressió d'aquest profag, però, no origina cap efecte deleteri apreciable sobre el creixement de P. putida. / The SOS system is a multigenic network negatively controlled by the LexA protein, and is composed of a set of genes involved in maintaining cell viability against DNA lesions. This system is present in most bacterial species, despite the existence of differences in the binding sequence of the LexA protein, and in the genetic content of regulon. The present report describes the SOS system of Pseudomonas putida, a Gram negative bacteria belonging to the Gamma proteobacteria group. Firstly we have cloned the two lexA genes, called lexA1 and lexA2 and we have obtained their genetic products through gene overexpression and purification by affinity columns. Both proteins have been used in electroforetic mobility shift assays (EMSA) with the promoters of each of the lexA genes. By this way we could identify the recognition sequence of the LexA1 protein (CTGTN8ACAG) and the LexA2 protein (AGTACN4GTGCT). Subsequently, using RT-PCR, we could see that the lexA2 gene forms a single transcriptional unit with the genes that follow it, forming the cassette lexA2-imuA-imuB-dnaE2. This cassette has been seen that is inducible by DNA damage, and that it is present in many Proteobacteria families. Then, we constructed two defective mutant strains of lexA1 and lexA2 genes, and their gene expression has been analyzed using DNA chips (microarrays). The results have shown that the LexA1 protein controls most of the SOS genes, which also correspond with the conventional response of its phylogenetic group; while LexA2 protein only regulates its own transcriptional unit expression, and a gene (PP3901) belonging to a resident P. putida prophage. In addition, this gene is also controlled by the LexA1 protein, being the only one who shares the two regulations. The construction of a PP3901 defective strain, dempnstrated that the expression of this gene is required for the transcription of the resident prophage genes. However, the expression of the prophage genes, do not cause any significant deleterious effect on the growth of P. putida.

La fibrose kystique (FK) est la maladie génétique mortelle la plus répandue chez les jeunes Canadiens. La colonisation des poumons par une bactérie opportuniste, Pseudomonas aeruginosa, est la principale cause de morbidité et de mortalité chez les patients FK. La maladie est causée par des mutations du gène codant pour la protéine CFTR (Cystic Fibrosis Transmembrane conductance Regulator) qui agit comme canal à ions chlorures. Ces modifications entraînent notamment une surexpression d'oligosaccharides fucosylés à la surface de l'épithélium pulmonaire. Le processus d'adhésion de la bactérie à la surface des cellules de l'épithélium pulmonaire est causé par la présence de deux lectines à la surface de la bactérie. Nous nous intéressons principalement à l'une d'entre elles, une lectine calcium dépendante qui reconnaît particulièrement le L-fucose (PA-IlL). Des études cristallographiques menées sur PA-IlL, en complexe avec divers ligands naturels tel que le Lewis a, ont permis d'identifier plusieurs éléments essentiels à l'obtention d'une forte interaction ligand-lectine. Basé sur ces études, le projet de recherche a consisté en la synthèse de différents analogues d'un disaccharide composé d'une unité fucose et glucosamine du type L-Fuc-a(1→4)-D-GIcNAcβ intimement impliqué dans le site de liaison de la protéine. Différents glycoclusters du disaccharide ont été synthétisés en utilisant la 'Click Chemistry'. De plus, quelques disaccharides modifiés en position C-2 et C-6 ont aussi été synthétisés. Les disaccharides ainsi que les glycoclusters ont été testés sur la PA-IlL en utilisant un test d'inhibition compétitive (ELLA). Les dérivés disaccharidiques ont montrés une constante de dissociation (Kd = 310 nM) dans le même ordre de grandeur que celle du meilleur ligand naturel Lewis a (Kd = 210 nM) connu jusqu'à maintenant pour la PA-IlL. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Pseudomonas aeruginosa, Fibrose kystique, Lectine PA-IlL, Lewis a, Glycosylation, «click chemistry», Glycoclusters.

Differential gene expression of P. aeruginosa in a rhizosphere biofilm on poplar tree roots was examined in order to identify new virulence factors from this human pathogen. Changes in gene expression for poplar trees contacted with P. aeruginosa was examined as well to identify the response of poplar roots to P. aeruginosa infection. This is the first study of the whole-transcriptome analysis of P. aeruginosa on a plant tree root. The 20 most highly-induced genes of P. aeruginosa were examined for their role in biofilm formation, rhizosphere colonization, barley germination, and poplar tree killing assays. Seven previously uncharacterized virulence genes (PA1385, PA2146, PA2462, PA2463, PA2663, PA4150, and PA4295) were identified. The role of PA2663, a hypothetical protein discovered in the microarrays of P. aeruginosa while killing poplar trees, was examined in further detail. Expression of PA2663 protein increases biofilm formation in P. aeruginosa PAO1 drastically. By complementing the PA2663 mutation in trans and by studying with DNA microarrays and RT-PCR the PA2663 mutant vs. the wild-type strain, PA2663 was confirmed to be related to biofilm formation and was found that it is the first protein to control the psl operon in P. aeruginosa PAO1. Furthermore, PA2663 protein increases pyoverdine synthesis and quorum sensing (QS)- regulated phenotypes. A biofilm formation-related hypothetical protein, PA0939, was identified in this study. The effects of indole and 7-hydroxyindole on P. aeruginosa virulence factors were also examined for the first time. Indole and 7HI repressed expression of mexGHI-opmD multidrug efflux pump genes and genes involved in synthesis of QS-regulated virulence factors (pyocyanin, rhamnolipid, PQS, and pyoverdine production). In addition, the effects of an anti-cancer uracil analog, 5-fluorouracil (5-FU) on P. aeruginosa virulence factors and E. coli K-12 biofilm formation were examined. 5-FU repressed biofilm formation, abolished quorum-sensing phenotypes, and reduced virulence in P. aeruginosa. DNA microarray and biofilm studies with 5-FU in E. coli revealed that 5-FU controls biofilm formation through the AriR protein in E. coli K-12 strain. The effects of lsrR and lsrK mutations on E. coli biofilm formation were also examined by flow cell experiments.