Bacteria, while traditionally studied in liquid suspensions, are often found in nature as biofilms, aggregates of cells enclosed in self-produced matrices. Cells in biofilms have a fitness advantage over those that are free-living, as the biofilm lifestyle is correlated with increased resistance to various assaults, including antimicrobials, UV exposure, and dehydration. These biofilm-associated characteristics have important clinical implications, as biofilm-based bacterial infections are a major cause of morbidity in immunocompromised individuals. With this increased resiliency, however, comes a major challenge that arises during biofilm growth: the formation of resource gradients. My thesis work focused on one particular gradient, that of oxygen, which is established in biofilms formed by Pseudomonas aeruginosa. This bacterium has multiple mechanisms for coping with limited access to oxygen, including a highly-branched respiratory system for optimal oxygen scavenging and production and utilization of redox-active molecules called phenazines. The purpose of this thesis has been to investigate the different strategies used by P. aeruginosa to deal with the oxygen limitation precipitated by the biofilm lifestyle. In Chapter 1, I will provide the necessary background for understanding the principles of redox balancing, metabolism, respiration, biofilm physiology, and phenazine utilization in P. aeruginosa. The work described in Chapter 2 provides evidence for the formation of a novel terminal oxidase complex that plays a biofilm-specific role in P. aeruginosa growth. The results in this chapter also suggest that specific terminal oxidase complexes differ in the timing of their contributions to biofilm growth and implicate the novel complex in mediating reduction of phenazines in biofilms. Chapter 3 expands upon the principle of metabolic versatility exemplified by the results discussed in Chapter 2. The research presented in this chapter looks at how varying the source of electrons that feed into the respiratory chain influences downstream electron transfer steps, including terminal oxidase activities and phenazine production and utilization. The data presented in Chapters 2 and 3 add to the growing body of evidence that bacterial growth in liquid culture is distinct from that in biofilms and underscores the need for more biofilm-based research that can inform treatment strategies for P. aeruginosa infections. The results described in Chapter 4 take an even broader look at the strategies used by P. aeruginosa to sustain efficient metabolism under conditions of potential stress. An important node of central metabolism is pyruvate, which can be transformed in a number of ways. In this chapter, I will consider two pathways of pyruvate metabolism: fermentation to lactate and carboxylation to oxaloacetate. I will present data indicating that a previously-uncharacterized lactate dehydrogenase contributes to P. aeruginosa growth under specific growth conditions and that pyruvate carboxylation contributes to optimal progress through central metabolic pathways. I will also describe experiments that characterize the contributions of another carboxylase, previously thought to function as the pyruvate carboxylase, to P. aeruginosa’s ability to grow on selected nutrient sources. Finally, I will discuss how redox state informs biofilm formation in a phylogenetically distinct bacterium, Bacillus subtilis, highlighting the universality of redox reactions in driving metabolic processes. In sum, the research presented in this thesis broadens our understanding of the immense respiratory and metabolic flexibility of P. aeruginosa and serves as an important reminder of the discrete factors that govern liquid culture and biofilm growth.

Horizontal gene transfer (HGT) and gene loss are important processes in the evolution of prokaryotic lineages. HGT involves the movement of genetic material between distantly related species, and can facilitate adaptation when gained genes confer advantageous phenotypes to recipient lineages. However, high levels of gene gain and loss are predicted to obfuscate patterns of vertical descent and homogenise nucleotide diversity across ecological and phylogenetic boundaries. Thus, a holistic understanding of the role of genome fluctuation in the emergence and maintenance of genetically and ecologically cohesive bacterial groups remains to be fully elucidated. In this thesis, I use the plant-associated bacterium Pseudomonas syringae as a model system to investigate the impact of HGT and gene loss on evolutionary processes such as adaptation, diversification and speciation. The Gram-negative Gammaproteobacterium P. syringae is an opportunistic plant pathogen, and has been used for decades as a model system with which to study the interaction between plants and their microbial pathogens. Recently, the diversification of lineages within this species has involved a number of host jumps onto a range of woody host plant species, resulting in the emergence of diseases such as bacterial canker of kiwi and bleeding canker of the European horse chestnut. Using whole-genome sequence data and a range of comparative genomics and phylogenetics methods, I quantitatively reconstruct the history of gene gain and loss in P. syringae and show HGT to be the predominant evolutionary force in this species. Genomes of this species are under constant permutation, are subject to a highly diverse HGT genepool and show marked differences in patterns of codon usage between imported and core genes. I then generate additional genome data for 26 strains of P. syringae that are pathogenic on a range of different woody plants, and investigate the contribution from HGT to the adaptation of these strains into the woody niche. Using a method that accounts for the underlying phylogenetic relationships among P. syringae strains, I look for the correlated evolution between gained genes and the woody niche, and find that a substantial proportion of the genome is associated with this ecological niche. I then investigate the recent adapitation of P. syringae pv. aesculi onto the European horse chestnut, and show that a number of genomic events that include both homologous and non-homologous recombination are likely to have led to the evolution of this bacterium onto its host, where it has become the causal agent of the bleeding canker disease that is currently epidemic across much of northern and central Europe. Overall, this thesis is an investigation into how HGT contributes to niche adaptation in P. syringae, and aims to further our understanding of the mechanisms that underlie bacterial evolution.

Broccoli head rot is a destructive disease found in most broccoli production areas. The main pathogen is the bacterium Pseudomonas fluorescens. P. fluorescens 5064, which was first isolated from an infected broccoli head in SE Scotland, produces biosurfactants that are important for bacterial establishment on the plant surface prior to causing disease in broccoli. Preliminary experiments performed in this study showed that biosurfactant production in P. fluorescens 5064 was cell density dependent, which is a typical characteristic of the quorum sensing mechanism. Quorum sensing is a bacterial communication mechanism, which controls a number of key processes in growth, reproduction and virulence via signalling molecules (quorum sensing signal) in many gram-negative bacteria. One aim of this study was to determine if biosurfactant production in P. fluorescens 5064 is controlled via quorum sensing. To do this, 35 surfactant-minus Tn5 mutants of P. fluorescens 5064 were screened for their abilities to produce a quorum sensing signal. Six of these biosurfactant-deficient mutants showed a large reduction in quorum sensing signal production. In one mutant 6423, which contains a single Tn5 insertion, the production of the quorum sensing signal was almost eliminated. Addition of quorum sensing signal, either synthetic or extracted from wild type P. fluorescens 5064, was able to restore biosurfactant production in mutant 6423. This strongly suggests that quorum sensing regulates biosurfactant production in P. fluorescens 5064. Attempts were made to clone and sequence the Tn5 disrupted gene in mutant 6423, but the identity of the gene remains inconclusive. The quorum sensing signal in wild type P. fluorescens 5064 was identified in this study by High Pressure Liquid Chromatography and Mass Spectrometry as N-3-hydroxyoctanoyl-homoserine lactone, which has been shown by other researchers to be present in P. fluorescens strain 2-79, but not in the strains F113, 7-14 and NCIMB 10586. The discovery that biosurfactant production in P. fluorescens 5064 is regulated by quorum sensing opens up a possibility for novel control of broccoli head rot. Although only the control of biosurfactant production by quorum sensing was examined in this study, it is possible that other virulence factors, such as pectic enzyme production, are also controlled by quorum sensing as in other pathogenic bacteria. By blocking the quorum sensing system, the pathogenic P. fluorescens that use this mechanism to control virulence could potentially be rendered avirulent. In greenhouse pathogenicity tests, a quorum sensing signal-degrading bacterium, Bacillus sp. A24, was evaluated for biocontrol of head rot disease caused by P. fluorescens 5064 on broccoli. However, the Bacillus sp. A24 showed only limited control effects, despite its strong quorum sensing signal-degrading ability towards the pathogen in vitro. A subsequent test proved that Bacillus sp. A24 is a surfactant producer itself and this could explain its ineffectiveness in disease control. When screening the quorum sensing signals of the 35 biosurfactant mutants, mutant 6418 was found to produce a potent antibiotic-like compound. This was identified by thin-layer chromatography as pyrrolnitrin. Unlike wild-type P. fluorescens 5064, mutant 6418 has lost its ability to produce virulence factors and is thus non-pathogenic. It was therefore of interest to determine if mutant 6418 could be used as a biocontrol agent to control broccoli head rot disease. In greenhouse pathogenicity tests, mutant 6418 significantly reduced disease by 41 %. The practical application of this research to bacterial disease control – via the manipulation of quorum sensing to inhibit virulence gene expression – is discussed.

Los biofilms o biopelículas, comunidades complejas de microorganismos, se encuentran colonizando diferentes ambientes bióticos como abióticos; los problemas generados por estos son diversos en diferentes actividades humanas. Ya que las biopelículas son de difícil erradicación, se han planteado diversas formas de control en cada una de las etapas del ciclo de una biopelícula. Una de ellas es la interrupción de las moléculas de señalización del quorum sensing, sistema que regula el proceso de la formación de biopelículas, para tal efecto se están buscando actualmente tanto antagonistas sintéticos como de origen natural. La presente investigación utiliza el Senecio calvus, una planta de uso medicinal, nativa del Perú. Se han expuesto extractos etanólico, butanólico, acuoso, metanólico y de diclorometano además de fracciones cromatográficas de Senecio calvus contra Pseudomonas aeruginosa, una bacteria oportunista muy utilizada debido a que es una bacteria que forma rápidamente biopelículas. Se mezclaron los cultivos de P. aeruginosa en fase de crecimiento junto a los extractos y fracciones de S. calvus para determinar el grado de inhibición de las bioopelículas; para tal efecto se usaron concentraciones subinhibitorias, previa prueba de concentración mínima inhibitoria (CMI). Los resultados arrojaron una inhibición de 92.9 y 76.4% en dos de los extractos y de hasta 88% en las fracciones cromatográficas, lo cual indica que Senecio calvus es un buen candidato para el aislamiento de una molécula inhibidora de biopelículas con potencial patentable. / Tesis

Cystic fibrosis (CF) is a hereditary multi-organ disorder characterized by formation of thick, viscous mucus in the lungs, leading to decreased fluid clearance and significant bacterial colonization. The bacteria form colonies, called biofilms, that are attached to the mucosal surface and produce a protective polymeric matrix. The matrix helps the biofilms form stable structures in the lungs while also protecting the embedded bacterial colonies from the host defense system and antimicrobials. Pseudomonas aeruginosa are opportunistic bacteria that commonly infect CF airways in the biofilm form. Current antibiotic treatment regimens fail to completely eradicate these biofilms, leading to chronic, persistent infections that over time lead to patient death. Therefore, there is a need to investigate antibacterial strategies that would completely eradicate these infections at reasonable doses and improve quality of patients’ lives. In this thesis, two strategies are investigated to better eradicate bacterial colonies – (1) the use of nutrient dispersion compounds for increasing the susceptibility of biofilm bacteria to the co-administered antibiotics, and (2) PEGylation of antimicrobial peptides to increase peptide retention in the lung airways. Clinical strains of P. aeruginosa isolated from lungs of CF patients were used in this research to better mimic the greater robustness of clinical biofilms compared to biofilms of laboratory bacterial strains. Growth curve studies were carried out to characterize the growth patterns of the bacterial strains. Antibiotic susceptibility of the planktonic (free-flowing) bacteria was studied using the minimum inhibitory concentration (MIC) assay. A method to grow and characterize 1-day and 4-day old biofilms in the minimum biofilm eradication concentration (MBEC) assay apparatus was developed and characterized. The MBECs of combination formulations consisting of an antibiotic and a nutrient dispersion compound for different treatment durations were measured against biofilms of the clinical isolates using four commonly used antibiotics, and sodium citrate as the nutrient dispersion compound. The growth curve studies allowed for better understanding of the clinical isolates’ growth rates in vitro, which could play an important role on their susceptibility to antibiotics. All bacterial strains displayed susceptibility to tobramycin sulfate and ciprofloxacin hydrochloride. Uniform bacterial growth was observed for 1-day old biofilms of both clinical isolates across all pegs. Growing 4-day old biofilms using 100% MHB without refreshing the bacterial suspension over 4 days gave uniform biofilm bacterial growth across the pegs. Four-day old biofilms displayed greater biomass than 1-day old biofilms for 2 out of 3 bacterial strains. Combination formulations eradicated 1-day and 4-day old biofilms at lower antibiotic concentrations than the antibiotic alone, with further improvement in eradication after increasing the duration of treatment. Sodium citrate did not enhance the metabolic activity of the biofilm bacteria. The antimicrobial peptide CaLL was conjugated with different MW polyethylene glycol (PEG) molecules using disulfide and maleimide linkages, and the effect of PEGylation on its antibacterial activity against P. aeruginosa laboratory strain PAO1 was evaluated. PEGylation was observed to reduce bacterial growth inhibition by CaLL, with the disulfide-linked CaLL-PEG less efficacious than the maleimide-linked CaLL-PEG. Time-kill assays demonstrated the longer duration of action of PEGylated peptides compared to non-PEGylated peptides, probably due to prevention of enzymatic degradation of the peptide by the PEG molecule. This research will shed light on antibacterial strategies for complete and rapid eradication of bacterial biofilms, thereby reducing development of antibiotic resistance and prevent recurrence of infection, reducing progressive lung damage caused in people with CF, and improve their quality of life.

Pulmonary surfactant is a critical surface-active substance consisting of dipalmitoylphosphatidylcholine (DPPtdCho) and key apoproteins that are produced and secreted into the airspace from alveolar type II epithelial cells. Surfactant deficiency leads to severe lung atelectasis, ventilatory impairment, and gas-exchange abnormalities. These are features of the acute lung injury syndrome, characterized by a strong pro-inflammatory component where cytokines or bacteria infections greatly impair surfactant DPPtdCho biosynthesis. The key enzyme needed to produce surfactant DPPtdCho is a rate-limiting enzyme CTP: phosphocholine cytidylyltransferase (CCTalpha). Calmodulin (CaM), rather than disruption of an NH2-terminal PEST sequence, stabilizes CCTalpha from actions of the proteinase, calpain. Mapping and site-directed mutagenesis of CCTalpha uncovered a motif (LQERVDKVK) harboring a vital recognition site, Q243, whereby CaM directly binds to the enzyme. Mutagenesis of CCTalpha Q243 not only resulted in loss of CaM binding, but also led to complete calpain resistance in vitro and in vivo. These data suggest that CaM, by antagonizing calpain, serves as a novel binding partner for CCTalpha that stabilizes the enzyme under pro-inflammatory stress. We further show that CCTalpha does not undergo polyubiquitination and proteasomal degradation. Rather, the enzyme is monoubiquitinated at a molecular site (K57) juxtaposed near its NLS resulting in disruption of its interaction with importin, nuclear exclusion, and subsequent degradation within the lysosome. Importantly, by using CCTalpha-ubiquitin hybrid constructs that vary in the intermolecular distance between ubiquitin and the NLS, we show that CCTalpha monoubiquitination masks its NLS resulting in cytoplasmic retention. These results unravel a unique molecular mechanism whereby monoubiquitination governs the trafficking of a critical regulatory enzyme in vivo. Last, we identify FBXL2 as a novel F-box E3 ubiquitin ligase that targets CCTalpha for degradation. Interestingly, FBXL2 also interacts with CaM, and CaM directly disrupts CCTalpha and FBXL2 interaction. This study demonstrates in the first time that adenoviral gene transfer of CaM attenuates the deleterious effects of P. aeruginosa infection by improving several parameters of pulmonary mechanics in animal models of sepsis-induced acute pulmonary injury. Collectively, these studies reveal a novel regulatory mechanism for phosphatidylcholine synthesis that may provide important clues to understanding the pathobiology of acute lung injury.

Although arsenic is ubiquitous in the environment, certain forms of arsenic are added deliberately in ever increasing quantities. The addition of large quantities of the trivalent inorganic form (arsenite) could pose a serious hazard because of the toxicity of this form explicable in terms of its action of sulfhydryl containing enzymes and/or coenzymes. In the environment B.rsenite is rapidly converted to arsenate (the pentavalent inorganic form). Since a biological explanation for the oxidation of arsenite to arsenate was available only for cattle dips (a unique environment), this study was undertaken to examine sewage for the presence of arsenite oxidizing bacteria. The isolation of bacteria capable of arsenite oxidation would allow a study of cultural and physiological properties and thus provide information useful in making rational decisions regarding the continued use of inorganic arsenicals as pesticides; as well as elucidating one segment of the arsenic cycle. Thirty-four different strains of arsenite oxidizing pseudomonads were isolated from sewage and classified into two major groups, Pseudomonas fluorescens-arsenoxydans and Pseudomonas acidovorans-arsenoxydans, with three and four ecotypes in each group. One particularily rapid arsenite oxidizing strain (designated Pseudomonas acidovorans-arsenoxydans YE56) was extensively studied. A minimal, defined medium was used to study the relationship of temperature, pH, aeration and nutrition to growth of this organism versus the ability of the organism to convert arsenite to arsenate under the same conditions. The conversion of arsenite to arsenate in YE56 was shown to be due to the appearance at stationary phase of an enzyme and/or component of the electron transport system when the organism is grown in chemically defined medium. In contrast, induction of the arsenite oxidizing system occurs in late exponential growth phase when the organism is grown in a rich medium. Thus, even though cell populations may never reach stationary phase in sewage, the ability of organisms to oxidize arsenite is probably constant. No energy could be detected from the oxidation of arsenite as measured by increase in growth rate or total cell yield. The adaptability of this organism to the changing environmental conditions of a sewage plant helps explain the findings that pseudomonads are the predominant species in sewage. A simple, unambiguous method for the separation of arsenite, arsenate and phosphate, using a combination of thin layer chromatography and paper chromatography, is described which makes it possible for investigators to define arsenic determinations to include the form (organic or inorganic), oxidation state (trivalent or pentavalent) and the quantity present in the samples.

Utilization of toluene by Pseudomonas putida as its sole carbon and energy source affects morphology, outer membrane protein composition, and glycolipid production. Two strains of P. putida were found to utilize toluene and to coexist in continuous and batch culture. The two strains were designated translucent and opaque, based upon their readily identifiable coloration when grown on Luria agar. The translucent strain was the dominant strain in continuous culture conditions. The outer membrane proteins of P. putida were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis. When toluene is the carbon and energy source, the trend in protein composition was towards a general increase in concentration of lower molecular weight proteins (wt). A similar decrease occurred in the concentration of higher molecular weight proteins in the range of 70X104-9X104 mol wt. P. putida produces glycolipids when grown on toluene as a sole carbon and energy source. Three glycolipids have been isolated from chemostat and batch culture spent media, using thin layer chromatography on silica gel GF254· The glycolipids are believed to be previously reported mono- and di-rhamnolipids that function as biosurfactants. The release of glycolipid into the media is believed to function to emulsify toluene, aiding in toluene uptake.

Pseudomonas aeruginosa et Candida albicans sont deux pathogènes fréquemment isolés dans les communautés microbiennes des unités de soins intensif. Nous avons auparavant montré que la colonisation des voies aériennes par C. albicans protégeait contre les lésions pulmonaires induites par P. aeruginosa. Le but de cette étude était de caractériser les mécanismes cellulaires et moléculaires responsables de la protection des voies aériennes induite par C. albicans. La colonisation des voies aériennes par C. albicans permet le recrutement et l'activation de cellules NK, de cellules lymphoïdes innées, de macrophages et de cellules dendritiques. Ce recrutement est associé à la sécrétion d'IL-22. L'inhibition de l'IL-22 est responsable d'une perte de la protection conférée par C. albicans. Nous avons identifié par cytométrie en flux que les cellules lymphoïdes innées étaient la seule source cellulaire d'IL-22. La déplétion spécifique des cellules lymphoïdes innées par des anticorps anti-CD90.2 était responsable d'une diminution de la sécrétion d'IL-22 et d'une diminution de la survie lors de la pneumonie à P. aeruginosa. Nos résultats démontrent que la production d'IL-22, principalement par les cellules lymphoïdes innées, est une étape inductible majeure dans la protection contre les lésions pulmonaires induites par P. aeruginosa. Cette cytokine pourrait ainsi représenter une cible thérapeutique pour prévenir les lésions pulmonaires induites par P. aeruginosa. / Pseudomonas aeruginosa and Candida albicans are two pathogens frequently encountered in the intensive care unit microbial community. We have demonstrated that C. albicans airway exposure protected against P. aeruginosa-induced lung injury. The goal of this study was to characterize the cellular and molecular mechanisms associated to C. albicans-induced protection. Airway exposure by C. albicans led to the recruitment and activation of NK cells, innate lymphoid cells (ILC), macrophages and dendritic cells. This recruitment was associated with the secretion of IL-22 whose neutralization abolished C. albicans-induced protection. We identified, by flow cytometry, ILCs as the only cellular source of IL-22. Depletion of ILCs by anti-CD90.2 antibodies was associated with a decreased IL-22 secretion and an impaired survival after P. aeruginosa challenge. Our results demonstrate that production of IL-22, mainly by ILCs, is a major and inducible step in protection against P. aeruginosa-induced lung injury. This cytokine may represent a clinical target in Pseudomonas aeruginosa induced lung injury.

Psychotrophic bacteria produce heat-stable lipase, protease, and phospholipase. Pervious studies indicate the production of multiple enzymes in several strains of Pseudomonas fluorescens, but conclusive evidence is lacking. The influence of culture conditions on the production and thermostability of phospholipase, protease, and lipase was investigated in 17 raw milk and environmental isolates. Production and thermostability of the enzymes were influenced by strain, stage of growth, and the culture medium. Cross-reactivity of antibodies raised to a purified protease and a commercial lipase indicated the immunological diversity of the enzymes. Protease purification was undertaken to investigate the production of multiple proteases within a single strain. A single monomeric protease with a molecular weight of 52 kDa was purified from P. fluorescens RO98. Biochemical characterization of the enzyme revealed that it was a zinc-metallo acidprotease with pH and temperature optima of 5.0 35°C, respectively. The enzyme was thermostable with a D55 of 41 min and a D62.5 of 18 h. Although sensitive assays exist for proteases, they are not suitable for detection of protease activity in milk in the presence of milk proteins. Existing immunoassays approach the required sensitivity but take about 6 h and cannot distinguish between active and inactive enzyme. An immunoassay that can be completed within 2 h and that can detect and distinguish both total and active enzyme was explored. The ratio of these two forms gives insight into the history of the milk. The ability of the purified protease to hydrolyze hydrophobic peptides associated with bitterness in Cheddar cheese was also investigated. Results demonstrated that the protease had the potential to debitter Cheddar cheese because it was able to hydrolyze the bitter peptides commonly found during aging. Two lipolytic enzymes with molecular weights of 50 (Pf-lip1) and 12 kDa (Pf-lip2) were purified from P. fluorescens RO98. Differences were observed in their biochemical properties. D62.5-values of 12.7 and 29.9 h were determined for Pf-lip1 and Pf-lip2, respectively. Pf-lip1 preferred longer chain length fatty acids, and Pf-lip2 preferred shorter chain length substrates. Pf-lip1 hydrolyzed milk fat and emulsified triolein, but Pf-lip2 did not, indicating that the latter was an esterase. This information is of significance to the dairy industry because activity tests that assay both the lipolytic enzymes need to be used in order to direct raw milk to short shelf-life products during processing and ensure quality of long shelf-life products