Quorum sensing (QS) cell-cell communication systems are utilized by bacteria to coordinate their behaviour according to cell density. Several different types of QS signal molecules have been identified, among which acyl-homoserine lactones (AHLs) produced by Proteobacteria have been studied to the greatest extent. QS has been shown to be involved in many aspects of bacterial life, including virulence, bioluminescence, symbiosis, antibiotic production, swarming and swimming motility, biofilm formation, conjugation and growth inhibition. Although QS has been studied extensively in cultured microorganisms, little is known about the QS systems of uncultured microorganisms and the roles of these systems in microbial communities. To extend our knowledge of QS systems and to better understand the signalling that takes place in the natural environment, in the first part of this thesis, isolation and characterization of new QS systems from metagenomic libraries constructed using DNA from activated sludge and soil were described. Using an Agrobacterium biosensor strain, three cosmids (QS6-1, QS10-1 and QS10-2) that encode the production of QS signals were identified and DNA sequence analysis revealed that all three clones encode a novel luxI family AHL synthase and a luxR family transcriptional regulator. Thin layer chromatography revealed that these LuxI homolog proteins are able to synthesize multiple AHL signals. Tandem mass spectrometry analysis revealed that LuxIQS6-1 directs the synthesis of at least three AHLs, 3-O-C14:1 HSL, 3-O-C16:1 HSL and 3-O-C14 HSL; LuxIQS10-1 directs the synthesis of at least 3-O-C12 HSL and 3-O-C14 HSL; while LuxIQS10-2 directs the synthesis of at least C8 HSL and C10 HSL. Two possible new AHLs, C14:3 HSL and (?)-hydroxymethyl-3-O-C14 HSL, were also found to be synthesized by LuxIQS6-1. Agrobacterium tumefaciens is a plant pathogen that causes crown gall disease. Its ability to transfer and integrate foreign DNA into plant genome also makes it a useful tool for plant genetic engineering. Ethylene, the gaseous plant hormone, has been reported to be important for both crown gall development and A. tumefaciens mediated transformation efficiency to plants. ACC deaminase, an enzyme that can break down ACC, the direct precursor of ethylene biosynthesis in plants, is a mechanism used by some plant growth promoting bacteria (PGPB) to promote plant growth by reducing stress ethylene levels. In the second part of this thesis, the effect of ACC deaminase on A. tumefaciens induced crown gall development and on A. tumefaciens mediated transformation efficiency was studied. By either introduction of an ACC deaminase encoding gene into the virulent strain A. tumefaciens C58 or co-inoculation of A. tumefaciens C58 with an ACC deaminase containing PGPB P. putida UW4, using different plant systems including tomato plants and castor bean plants, it was found that the presence of an ACC deaminase significantly inhibited crown gall development. It was also found that introduction of an acdS gene into the disarmed A. tumefaciens strain GV3101::pMP90 reduced the ethylene levels evolved by plants during infection and cocultivation process and increased the transformation efficiency of commercialized canola cultivars. The A. tumefaciens D3 strain was reported to contain an ACC deaminase encoding gene (acdS). In this study it was determined that this strain is an avirulent strain and shows plant growth promoting activity. When co-inoculated with A. tumefaciens C58 on castor bean stems, both the wild type and the acdS knockout mutant showed biocontrol activity and were able to significantly inhibit crown gall formation, with the wild type strain showing slightly better tumor inhibition effects. The mutation of acdS and its regulatory gene lrpL in A. tumefaciens D3 was also found to affect QS signal production of this strain, which indicates a cross talk between the two sets of genes.

Traditional bacterial disease therapies utilize compounds that ultimately kill the target bacteria but it exerts a strong selective pressure on the bacteria to develop multi-drug resistance mutants. The increasing occurrence of resistance in common pathogens has highlighted the need to identify new anti-microbials that target the control of bacterial pathogenicity in a non-extermination manner to reduce the incidence of bacteria resistance. One new strategy exploits the discrete signaling molecules that regulate the various bacterial signaling pathways, which are responsible for the expression of pathogenicity traits. Halogenated furanones (fimbrolides) from the marine red alga, Delisea pulchra have been shown to interfere with the key signaling pathway present in Gram-negative bacteria by competitively displacing the cognate signaling molecule from the transcription protein. This project focused on the design and synthesis of 1,5-dihydropyrrol-2-ones, a new class of fimbrolide derivatives capable of displaying strong antagonistic properties of the fimbrolides. Primary synthetic methodologies examined include the halolactamization of allenamides and the direct lactone-lactam transformation. No doubt, both methodologies yielded the lactam ring, the former failed to introduce the crucial C-5 bromomethylene group essential for bioactivity. A facile high yielding two-step lactone-lactam transformation method was developed and using this method, a wide range of substituted 5-bromomethyl- and 5-dibromomethylene-1,5-dihydropyrrol-2-ones were synthesized. Furthermore, a new class of tricyclic crown-ether type compounds with no literature precedent were discovered. To vary the diversity of the compounds, a related class of compounds, 5,6-dihydroindol-2-ones, were examined. A general versatile method for the synthesis of 7-substituted 5,6-dihydroindol-2-ones was developed. The synthetic strategy proceeds via the established Suzuki-Miyaura cross-coupling reaction of halogenated dihydroindol-2-ones with arylboronic acids/esters. The Suzuki methodology was found to be reliable in furnishing a wide range of 7-substituted products in high yields. A preliminary molecular modeling approach was used to assist in the design of new anti-microbials via the ligand-docking analyses of the TraR and LasR protein. A positive correlation was observed between the docking scores and biological activity and the methodology was further developed into an initial screening tool to filter potential active and non-active compounds. The newly synthesized compounds were analysed for their efficacy in reducing the expression of the Green Fluorescent Protein (GFP) in the presence of natural AHL signaling molecules in an AHL-monitor strain, indicative of the inhibition of bacterial phenotype expression. The dihydropyrrol-2-one class of compounds showed significant biological activity and this highlighted their potential for further development.

Thesis (Ph. D)--UCLA, 2008. / Includes bibliographical references (leaves 324-350).

Quorum sensing (QS) is a population-dependent signaling process bacteria use to control multiple processes including virulence, critical for establishing infection. There are two major pathways of QS systems. Type 1 is species specific or intra-species communication in which N-acylhomoserine lactones (Gram-negative bacteria) or oligopeptides (Gram-positive bacteria) are employed as signaling molecules (autoinducer one). Type 2 is inter-species communication in which S-4,5-dihydroxy-2,3-pentanedione (DPD) or its borate esters are used as signaling molecules. The DPD is biosynthesized by LuxS enzyme from S-ribosylhomocysteine (SRH). Recent increase in prevalence of bacterial strains resistant to antibiotics emphasizes the need for the development of new generation of antibacterial agents. Interruption of QS by small molecules is one of the viable options as it does not affect bacterial growth but only virulence, leading to less incidence of microbial resistance. Thus, in this work, inhibitors of both N-acylhomoserine lactone (AHL) mediated intra-species and LuxS enzyme, involved in inter-species QS are targeted. The γ-lactam and their reduced cyclic azahemiacetal analogs, bearing the additional alkylthiomethyl substituent, were designed and synthesized targeting AHL mediated QS systems in P. aeruginosa and Vibrio harveyi. The γ-lactams with nonylthio or dodecylthio chains acted as inhibitors of las signaling in P. aeruginosa with moderate potency. The cyclic azahemiacetal with shorter propylthio or hexylthio substituent were found to strongly inhibit both las and rhl signaling in P. aeruginosa at higher concentrations. However, lactam and their azahemiacetal analogs were found to be inactive in V. harveyi QS systems. The 4-aza-S-ribosyl-L-homocysteine (4-aza-SRH) analogs and 2-deoxy-2-substituted-S-ribosyl-L-homocysteine analogs were designed and synthesized targeting Bacillus subtilis LuxS enzyme. The 4-aza-SRH analogs in which oxygen in ribose ring is replaced by nitrogen were further modified at anomeric position to produce pyrrolidine, lactam, nitrone, imine and hemiaminal analogs. Pyrrolidine and lactam analogs which lack anomeric hydroxyl, acted as competitive inhibitors of LuxS enzyme with KI value of 49 and 37 µM respectively. The 2,3-dideoxy lactam analogs were devoid of activity. Such findings attested the significance of hydroxyl groups for LuxS binding and activity. Hemiaminal analog of SRH was found to be a time-dependent inhibitor with IC50 value of 60 µM.

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Yes / N-acylhomoserine lactone (AHL)-based quorum sensing (QS) is important for the regulation of proteobacterial virulence determinants. Thus, the inhibition of AHL synthases offers non-antibiotics-based therapeutic potentials against QS-mediated bacterial infections. In this work, functional AHL synthases of Pseudomonas aeruginosa LasI and RhlI were heterologously expressed in an AHL-negative Escherichia coli followed by assessments on their AHLs production using AHL biosensors and high resolution liquid chromatography–mass spectrometry (LCMS). These AHL-producing E. coli served as tools for screening AHL synthase inhibitors. Based on a campaign of screening synthetic molecules and natural products using our approach, three strongest inhibitors namely are salicylic acid, tannic acid and trans-cinnamaldehyde have been identified. LCMS analysis further confirmed tannic acid and trans-cinnemaldehyde efficiently inhibited AHL production by RhlI. We further demonstrated the application of trans-cinnemaldehyde inhibiting Rhl QS system regulated pyocyanin production in P. aeruginosa up to 42.06%. Molecular docking analysis suggested that trans-cinnemaldehyde binds to the LasI and EsaI with known structures mainly interacting with their substrate binding sites. Our data suggested a new class of QS-inhibiting agents from natural products targeting AHL synthase and provided a potential approach for facilitating the discovery of anti-QS signal synthesis as basis of novel anti-infective approach. / University of Malaya High Impact Research (HIR) Grant (UM-MOHE HIR Grant UM.C/625/1/HIR/MOHE/CHAN/14/1, no. H-50001-A000027) given to K.G.C. and National Natural Science Foundation of China (no. 81260481) given to H.W.

Chez les Vibrio spp., le QS est impliqué dans de nombreuses fonctions comme la colonisation de niche écologiques, les stratégies de survie ou encore la virulence. Cependant, pour la majorité des espèces de Vibrio, la diversité des AHLs produites reste largement sous-estimée et l'étude du QS est encore limitée à quelques espèces modèles ou pathogènes. Toutefois, dans les environnements aquatiques, ces espèces sont minoritaires et les espèces les plus abondantes ne sont que très peu étudiées. Nos résultats ont révélé une importante diversité d'AHLs mais aussi, de façon surprenante, une hétérogénéité dans les phénotypes de production d'AHL au sein d'une même espèce de Vibrio. Pour la première fois, nous avons mis en évidence qu'une même souche de Vibrio pouvait présenter des phénotypes de production d'AHLs différent au cours du temps et une approche statistique a révélé l'implication de certains déterminants biotiques et abiotiques dans ces variations temporelles. Par ailleurs, une approche à micro-échelle a révélé une structuration des populations de Vibrio en unités fonctionnelles constituées de souches phylogénétiquement proches qui partagent des niches écologiques spécifiques et des comportements sociaux. Nos résultats ont mis en évidence que les modalités de communication pouvaient être hétérogènes suggérant l'absence d'un langage commun au sein de ces unités fonctionnelles. En conclusion, ce travail de thèse a permis d'apporter de nouvelles connaissances sur le QS chez les Vibrio dans l'environnement marin, de la souche à la population, et propose une vision intégrée des mécanismes de régulation de la production d'AHLs dans l'environnement. / Quorum sensing is an important mechanism among Vibrio species and is involved in many vital functions such as niche colonization, survival strategies or virulence. However, AHL diversity still largely underestimated for the majority of Vibrio species and the current knowledge on AHL-mediated QS is limited to a few pathogenic or bioluminescent species. Nonetheless, these species are weakly abundant in seawater while dominant species in the environment are poorly studied. Our results revealed a unexpected diversity of AHL molecules but also a quite surprising intra-species diversity of AHL production phenotypes. For the first time, we showed that different isolates of a single genotype switched between different AHL production phenotypes among time and we revealed the potential involvement of abiotic and biotic parameters in these variations. However, it appears that when studied at a microscale, Vibrio populations are showing a functional structuration in ecological units consisting of phylogenetically close strains sharing habitat and social traits. In this context, it was necessary to determine if these different AHL production phenotypes were associated to different micro-habitats in the water column. We did not demonstrate that a common language was spoken within ecological populations. This thesis work provide new insights on AHL-mediated QS among a broader range of species and among Vibrio populations and depicts the potential impact of multiple aspects of marine environments on AHL production.

<p>By their very nature, attempts by civil society organizations to promote democracy abroad involve cooperation and contact across the borders of nation states. The dissemination of the ideas and practices of the promoters is often essential; in the case of Swedish democracy promotion, popular education or folkbildning has been important.</p><p>This thesis investigates the dissemination of ideas and practices by civil society organizations in Sweden and Estonia in the field of popular education. More specifically, a number of projects run by member organizations of the Swedish study association ABF (Workers’ Educational Association) and member organizations of the Estonian AHL (Open Education Association) are studied. These projects are also part of democracy-promoting activities whose funding comes mainly from Swedish donor agencies. The thesis aims to understand not only what has been spread but also how ideas and practices have been translated to fit the Estonian context. The primary theoretical concept used is therefore translation, even though one aim of the thesis is to systematize the field of study concerning processes of dissemination and to enrich the concept of translation with the aid of previous work on diffusion and socialization.</p><p>The main contribution of the thesis consists of the in-depth study of cooperation between Swedish and Estonian popular education organizations. It also contributes to the theoretical development of the concept of translation by identifying different phases in this kind of process and important elements of these phases. Popular education is an important part of Swedish democracy promotion and this study also contributes to this rather undertheorized subject, and also to the discussions of transnational civil society cooperation, by demonstrating an analytical framework that can be of use in future research into these issues.</p>

By their very nature, attempts by civil society organizations to promote democracy abroad involve cooperation and contact across the borders of nation states. The dissemination of the ideas and practices of the promoters is often essential; in the case of Swedish democracy promotion, popular education or folkbildning has been important. This thesis investigates the dissemination of ideas and practices by civil society organizations in Sweden and Estonia in the field of popular education. More specifically, a number of projects run by member organizations of the Swedish study association ABF (Workers’ Educational Association) and member organizations of the Estonian AHL (Open Education Association) are studied. These projects are also part of democracy-promoting activities whose funding comes mainly from Swedish donor agencies. The thesis aims to understand not only what has been spread but also how ideas and practices have been translated to fit the Estonian context. The primary theoretical concept used is therefore translation, even though one aim of the thesis is to systematize the field of study concerning processes of dissemination and to enrich the concept of translation with the aid of previous work on diffusion and socialization. The main contribution of the thesis consists of the in-depth study of cooperation between Swedish and Estonian popular education organizations. It also contributes to the theoretical development of the concept of translation by identifying different phases in this kind of process and important elements of these phases. Popular education is an important part of Swedish democracy promotion and this study also contributes to this rather undertheorized subject, and also to the discussions of transnational civil society cooperation, by demonstrating an analytical framework that can be of use in future research into these issues.