Yes / Computational models are perceived as an attractive alternative to mathematical models (e.g., ordinary differential equations). These models incorporate a set of methods for specifying, modeling, testing, and simulating biological systems. In addition, they can be analyzed using algorithmic techniques (e.g., formal verification). This paper shows how formal verification is utilized in systems and synthetic biology through qualitative vs quantitative analysis. Here, we choose two well-known case studies: quorum sensing in P. aeruginosas and pulse generator. The paper reports verification analysis of two systems carried out using some model checking tools, integrated to the Infobiotics Workbench platform, where system models are based on stochastic P systems. / EPSRC

There is an emerging threat of Neisseria gonorrhoeae strains that are resistant to all antibiotics. Because of this, the purpose of this research is to isolate, analyze, and partially characterize a new inhibitor(s) of N. gonorrhoeae. Since there is an unknown molecule secreted by Candida albicans that inhibits N. gonorrhoeae, this molecule can be partially characterized using 1H NMR Spectroscopy to assist in the development of a new antibiotic compound. It was hypothesized that quorum-sensing molecules, trans, trans- farnesol, tyrosol, phenylethyl alcohol, and tryptophol, could be possible candidates for the inhibitor. Because of this, 1H NMR spectra for these quorum-sensing molecules were obtained to serve as controls. Column chromatography and fractionation was used to isolate the inhibitor in large scale from C. albicans grown in salts-based media. Attempts to isolate the inhibitor in large scale, however, was unsuccessful since no inhibition of N. gonorrhoeae was observed. Because of this, analysis of growth media was conducted to test the media effect on producing the inhibitor. C. albicans was grown in liquid chocolate, liquid white chocolate, salts-based, and YPD media in aerobic and candle jar environments. Analysis of growth media in different environments suggests that liquid chocolate and salts-based media retain the inhibitory activity. 1H NMR spectra were obtained for the isolated molecule in liquid chocolate and salts-based media in both aerobic and candle jar environments. Analysis of this 1H NMR suggested that the inhibitor could be isolated from either the aerobic or candle jar environment for both liquid chocolate and salt-based media because a clear peak between 3.5 and 4.0 ppm was observed in all spectra. Comparison of 1H NMR spectra from quorum-sensing molecules with spectra from the isolated molecule suggests that the inhibitor is not a quorum-sensing molecule. The peaks represented by the inhibitor cannot be fully characterized and thus, either correspond to a single molecule or a complex molecular structure. It can be concluded that the inhibitor secreted by C. albicans to inhibit N. gonorrhoeae is a new unknown compound.

No description available.

Research on bacteria in their biofilm form is limited by the ability to artificially culture bacterial biofilms in a system that permits the visualization of individual cells. The experiments comprising this thesis research are on-going investigations of bacterial culture systems engineered to provide an environment that mimics biofilms while enabling real-time microscopy. Specifically, the microfluidic systems developed and assessed as part of this thesis permit the visualization of individual bacteria cells within consortia growing within a narrow space provided by a microfluidic device. This research demonstrates the versatility of these microfluidic systems across potentially high-throughput microbiological experiments utilizing genetically engineered Escherichia coli. Before demonstrating the efficacy of these systems, the development of the field of synthetic biology over the past half century is reviewed, focusing on synthetic genetic circuits and their applications (Chapter 2). The first and main microfluidic device explored in this research was developed to mimic the nutrient-deficient conditions within biofilms by forcing media to enter the culture area through a narrow, torturous channel. The microfluidic channel was thin enough (0.97 μm) to prevent the motility of 1-μm-wide E. coli cells, enabling visualization of individual cells. The bacteria cultured in the device contained either a simple Plux-driven quorum sensing receiver (Chapters 3 and 5) or a LacI- and TetR-driven genetic toggle switch (Chapter 4). Under the culture conditions, the quorum sensing reporter signal was detected even without addition of the signaling molecule (Chapter 3). The genetic toggle switch was stable when the system began in the high-LacI expression state, but after 5 days of culture, >5% of high-TetR expression cells began to consistently express the high-LacI state (Chapter 4). This system was also employed to track lineages of cells using real-time microscopy, which successfully characterized the inheritance of aberrant, enlarged cell phenotypes under stress (Chapter 5). Another microfluidic device, a droplet bioreactor, was also developed to culture small numbers of cells in an aqueous bubble suspended in oil (Chapter 6). Quorum sensing receiver cellswere cultured in this device, demonstrating that it is well suited for testing the effects of compounds on biofilms within water-in-oil droplets. / Ph. D.

Les bactéries sont capables de communiquer entre elles afin de coordonner l’expression de certains gènes en fonction de leur densité de population. Ce système de communication utilise de petites molécules appelées autoinducteurs (AIs) comme messagers chimiques et est connu sous le nom de Quorum Sensing (QS). Chez les bactéries pathogènes, les gènes régulés sont, en particulier, ceux codant pour la production des facteurs de virulence et la structuration des biofilms. Ainsi des inhibiteurs du QS chez ces bactéries pourraient être de nouveaux agents anti-bactériens alternatifs aux antibiotiques actuels. Chez les bactéries à Gram-négatif, les AIs sont très majoritairement des acyl-homosérine lactones (AHLs). Utilisant deux approches, la conception rationnelle et le criblage virtuel, nous avons découvert cinq nouvelles familles d’antagonistes des AHLs, ainsi qu’un certain nombre d’agonistes. Nous avons également préparé des analogues d’antagonistes naturels de la famille des bromo-furanones, afin d’établir une étude structure-activité de ce type de composés. / The bacteria are able to communicate with each other for coordinating gene expression depending on their population density. This communication system use small molecules called autoinducers (AIs) as chemical messengers and is referred to as quorum sensing (QS). In pathogenic bacteria, the regulated genes are, in particular, those coding for the production of virulence factors and biofilms formation. Thus, inhibitors of bacterial QS could be used as new anti-bacterial agents providing an alternative to current antibiotics. In Gram-negative bacteria, the main AIs are acyl-homoserine lactones (AHLs). Using two approaches, rational design and virtual screening, we have discovered five new families of AHLs antagonists, and some agonists. We have also prepared analogues of natural bromo-furanones antagonists, in order to establish a structure-activity study of this type of compounds.

O gênero Methylobacterium é composto por bactérias de coloração rósea, metilotróficas facultativas (PPFM - pink-pigmented facultative methylotrophic), que podem fixar nitrogênio, nodular a planta hospedeira, produzir o fitohormônio citocinina e as enzimas pectinase e celulase, podendo dessa forma promover o crescimento vegetal devido à disponibilidade de nitrogênio e à indução de resistência sistêmica. Methylobacterium spp. têm sido descritas como endófitos ou epífitas em diferentes plantas hospedeiras, onde a sua colonização e distribuição no hospedeiro podem ser influenciadas pelo genótipo da planta ou por interações com outros microrganismos associados ao hospedeiro. Neste contexto, poucos trabalhos têm sido desenvolvidos visando um melhor entendimento da interação Methylobacterium-planta e da diversidade deste gênero bacteriano que tem sido isolado de diferentes plantas hospedeiras, exercendo diferentes funções ainda pouco conhecidas. Portanto, este trabalho tem como objetivo estudar a diversidade genética de Methylobacterium spp., por meio do seqüenciamento parcial dos genes 16S rRNA e mxaF; analisar os genes de responsáveis pela interação da Methylobacterium com a planta hospedeira e analisar os genes envolvidos na interação Methylobacterium (endófito)- Xylella fastidiosa (patógeno). Os resultados mostraram que existe uma resposta adaptativa de Methylobacterium spp. específica para cada planta hospedeira. Da mesma forma, foi observado que esta adaptação específica da bactéria à planta, também pode levar à seleção de genótipos específicos para cada planta hospedeira, embora eventos aleatórios também possam ser responsáveis pela diversidade de Methylobacterium na planta hospedeira. Na análise de expressão gênica da interação Methylobacterium-planta, foi observado que o gene relacionado ao metabolismo do metanol (mxaF) não apresentou mudança no padrão de expressão. Genes relacionados a estresse crtI (estresse sentido pela bactéria) e acdS (estresse sentido pela planta), tiveram suas expressões reduzidas na presença da planta, mostrando que a presença de exsudados das plantas não representou um estresse ao desenvolvimento bacteriano. Os genes relacionados à patogenicidade patatin e phoU não foram alterados, confirmando que Methylobacterium é um endófito, e possuem expressão induzida de tais genes quando interagindo com a planta hospedeira. Os resultados permitem concluir que nas condições avaliadas os exsudados das plantas não causam estresse à bactéria (SR1.6/6). Por meio da análise de expressão gênica in vitro de X. fastidiosa em co-cultivo com M. mesophilicum, foi observado que este fitopatógeno vascular apresentou diminuição do crescimento e da formação de biofilme. Os resultados aqui apresentados mostram que a diversidade deste grupo de endófitos é parcialmente determinada pela planta hospedeira, onde tais bactérias interagem tanto com a planta como com outros grupos, como fitopatógenos presentes neste nicho. / The genus Methylobacterium, constituted by PPFMs - pink-pigmented facultative methylotrophic, are able to fix nitrogen, nodule the host plant, produce cytokines and enzymes involved in induction of systemic resistance such as pectinase and cellulase, inducing plant growth. Methylobacterium sp. has been described as endophyte or epiphyte in different host plants, where the colonization and distribution on the host can be influenced by plant genotype or by interaction with other microorganisms associated to the host. In this context, few studies aims the better understanding of the diversity of this genus in different host, the interaction Methylobacterium-plant, and the interaction Methylobacterium-other bacteria. Therefore, this study aims to study the genetic diversity of Methylobacterium spp., by sequencing the 16S rRNA and mxaF gene; to analyze the genes responsible for the Methylobacterium-plant host interaction and to analyze the genes involved in Methylobacterium (endophyte) - Xylella fastidiosa (pathogen) interaction. Results show differential adaptive responses of Methylobacterium spp. in distinct plant species. However, the clustering according to the host plant was observed for a subset of isolates, suggesting that this diversity could be driven by stochastic events, although plant genotype may contribute to this diversity. Analyzing the Methylobacterium-plant interaction gene expression it was observed that genes related to metabolism of methanol (mxaF) was not amended. The genes related to stress such as crtI (stress sensed by the bacteria) and acdS (stress sensed by the plant) had its expression reduced with the plant showing that the plant exudates did not represent a stress to the bacteria development. The genes related to pathogenicity like patatin and phoU were not amended, confirming that Methylobacterium is an endophyte that do not induce when the bacteria interacts with the plant host. Using a genetic expression analyses of X. fastidiosa in vitro in co-cultive with M. mesophilicum, it was seen that this phytopathogen presented the growth and biofilm formation reduction. These results show that the diversity of this endophyte group is partially determinate by the plant host, where this bacterium interacts with the plant and with other groups, such as phytopathogen present in this niche.

A Clorose Variegada dos Citros (CVC), doença que causa graves prejuízos à citricultura no estado de São Paulo, é causada pela bactéria Xylella fastidiosa que é transmitida pelas cigarrinhas Bucephalogonia xanthophis (Berg), Dilobopterus costalimai (Young), Acrogonia citrina (Marucci & Cavichioli) e Oncometopia facialis (Signoret). Durante a alimentação em plantas afetadas, esses insetos adquirem a bactéria, que coloniza o pré-cibário e o cibário, e depois são capazes de transmitir a doença para plantas sadias. Colonizando o xilema das plantas de citros encontram-se também bactérias endofíticas, que são microrganismos capazes de colonizar internamente tecidos de plantas sem causar dano aparente, e que podem interagir com patógenos no interior do hospedeiro. Este trabalho teve como objetivo avaliar a comunidade bacteriana associada as cigarrinhas vetoras de CVC, e observar as possíveis interações que ocorrem entre insetos vetores de X. fastidiosa e bactérias endofíticas de citros. Primeiramente foi feito um isolamento das bactérias da cabeça de cigarrinhas coletadas em pomares de citros afetados com CVC. Foram isoladas um total de 17230 bactérias de três espécies de cigarrinhas (O. facialis, D. costalimai e A. citrina) em três datas diferentes (22/março, 05/maio e 14/junho de 2002), que foram primeiramente classificadas em 9 grupos morfológicos. Do total, 120 bactérias representantes foram avaliadas por ARDRA e classificadas em 16 haplótipos, dos quais alguns foram identificados, por seqüenciamento da região 16S do rDNA, como Methylobacterium sp. e Curtobacterium sp., que são bactérias endofíticas de citros já descritas e que interagem com X. fastidiosa em citros. Primers específicos para estas bactérias foram utilizados para PCRs com o DNA total da cabeça de cigarrinhas e variou de 39,1% a 89,6% nas diferentes espécies. A comunidade bacteriana associada às cigarrinhas foi também avaliada por DGGE e apresentou variações quanto ao inseto hospedeiro e quanto à época das avaliações. Um isolado de M. mesophilicum expressando o gene da proteína verde fluorescente (GFP ? Green Fluorescent Protein) foi utilizado para experimento de transmissão deste endófito por B. xanthophis. Os insetos se alimentaram em membrana contendo a solução da bactéria e depois foram colocados em plantas de Catharanthus roseus. Das plantas, 13% apresentaram a bactéria fluorescente colonizando tecidos endofiticamente, o que indica que a cigarrinha é capaz de transmitir o endófito. Testes para a avaliação da produção de moléculas de quorum sensing (AHLs - Acil Homocerina Lactonas) foram feitos para M. mesophilicum e M. extorquens. Foram ainda desenvolvidos mutantes de M. extorquens, defectivos para a produção de biofilme e AHLs. / The Citrus Variegated Chlorosis (CVC), a very important disease which attacks citrus trees in the state of São Paulo, is caused by the xylem-limited bacteria, Xylella fastidiosa, which is transmitted by four species of sharpshooters (Cicadellidae) Bucephalogonia xanthophis (Berg), Dilobopterus costalimai (Young), Acrogonia citrina (Marucci & Cavichioli) and Oncometopia facialis (Signoret) which are capable of acquiring X. fastidiosa from the xylem while feeding. In plants, endophytic bacteria, which inhabit the interior of aerial plant parts developing an asymptomatic infection and show potential benefits as biocontrol agents of pests and diseases, may colonize the same niche of pathogens, such as X. fastidiosa, what could allow endophytes to interact with the pathogen. Bacteria were isolated from head of insect vectors, collected on affected citrus plants. From 17230 bacteria isolated from the heads of three insect species (O. facialis, D. costalimai and A. citrina) in three different days (22/march, 05/may and 14/june of 2002), 120 bacteria were tested with ARDRA and classified into 16 different haplotypes. The most frequent strains had their 16S rDNA fragment sequenced and they were identified as Methylobacterium sp. and Curtobacterium sp., bacteria which have been described as citrus endophytes which interact with X. fastidiosa. Total DNA of insect heads was used in Nested PCRs for detection of these citrus endophytes in insects. The frequencies of detection ranged from 39,1% to 89,6%. The bacterial community associated to sharpshooters was also evaluated by DGGE and presented variations due to insect host and time of the evaluations. One M. mesophilicum isolate, expressing the GFP (Green Fluorescent Protein), was used to test the transmission of endophytes by B. xanthophis. Insects fed on membranes containing a bacterial solution and after were trapped on Catharanthus roseus. From the evaluated plants, 13% presented the fluorescent protein colonizing endophytically, indicating that the insect is able to transmit the endophytic bacteria. Biofilm and quorum sensing molecules (AHLs - Acyl Homocerine Lactones) production by Methylobacterium sp. were tested, and M. extorquens mutants for both features were produced.

Serratia sp. ATCC 39006 (S39006) is known for producing carbapenem and prodiginine antibiotics; 1-carbapen-2-em-3-carboxylic acid (car) and prodigiosin. It displays different motility mechanisms, such as swimming and swarming aided by flagellar rotation and biosurfactant production. In addition, S39006 produces gas vesicles to float in aqueous environments and enable colonization of air-liquid interfaces. Gas vesicles are thought to be constructed solely from proteins expressed from a gene cluster composed of two contiguous operons, gvpA1-gvpY and gvrA-gvrC. Prior to this study, three cognate regulators, GvrA, GvrB, and GvrC, encoded by the right hand operon were known to be essential for gas vesicle synthesis. Post-transcriptional regulators such as RsmA-rsmB were also known to be involved in the inverse regulation of gas vesicles and flagella based motility. Furthermore, gas vesicle formation, antibiotic production, and motility in S39006 were affected by cell population densities and de-repressed at high cellular densities through a quorum sensing (QS) system. The aim of this research study was to identify novel regulatory inputs to gas vesicle production. Mutants were generated by random transposon mutagenesis followed by extensive screening, then sequencing and bioinformatic identification of the corresponding mutant genes. After screening, 31 mutants and seven novel regulatory genes impacting on cell buoyancy were identified. Phenotypic and genetic analysis revealed that the mutations were pleiotropic and involved in cell morphology, ion transport and central metabolism. Two new pleiotropic regulators were characterized in detail. Mutations in an ion transporter gene (trkH) and a putative transcriptional regulator gene (floR) showed opposite phenotypic impacts on flotation, flagella-based motility and prodigiosin, whereas production of the carbapenem antibiotic was affected in the transcription regulator mutant. Gene expression assays with reporter fusions, phenotypic assays in single and double mutants, and proteomics suggested that these regulatory genes couple different physiological inputs to QS and RsmA-dependent and RsmA-independent pathways.

Les Escherichia coli entérohémorragiques (EHEC) sont responsables de toxi–infections alimentaires conduisant à des colites hémorragiques pouvant se compliquer d'un syndrome hémolytique et urémique. Le facteur majeur de pathogénicité est la production de Shiga-toxines (Stx), dont la toxine Stx2. Nous avons étudié la production de toxine Stx2 in vitro par des souches STEC provenant de diverses origines (bovine ou clinique), appartenant à divers séropathotypes, et codant pour différents variants Stx2. Nous avons montré que les souches O157:H7 les plus pathogènes possèdent le variant stx2 et produisent de fortes quantité de Stx2 en conditions basales comme en présence d'un inducteur du système SOS. Les souches non-O157 présentant ces caractéristiques pourraient représenter un risque pour la santé humaine. Nous avons ensuite étudié l'effet de molécules présentes dans le tube digestif sur la synthèse de Stx2 par E. coli O157:H7. Les auto-inducteurs AI-2 et AI-3 du quorum sensing, produits par le microbiote intestinal, n'influencent pas la synthèse de Stx2, non plus que l'hormone intestinale norépinéphrine. Cependant, la protéine régulatrice QseA impliquée dans une voie de signalisation par le quorum sensing serait un activateur transcriptionnel de stx2. Enfin, nous avons étudié la production de Stx2 par la souche EHEC O157:H7 EDL 933 dans un milieu se rapprochant le plus possible de celui rencontré in vivo par les EHEC, en particulier grâce à un modèle de rats associés au microbiote intestinal humain. Nous avons ainsi montré que le microbiote humain inhibe la transcription de stx2 par l'inhibition de la transcription de recA même lors de l'induction du système SOS, et que cette inhibition peut être en partie attribuée à l'espèce Bacteroides thetaiotaomicron.

Depuis son isolement en 1977, les épidémies de pneumopathies à Legionella pneumophila (Lp) sont en constante progression. La contamination se fait essentiellement par inhalation d'aérosols contaminés, générés à partir de l'eau des réseaux de distribution. L'écologie de Lp est complexe, faisant intervenir des protozoaires dans lesquels elle se multiplie et des biofilms dans lesquels elle persiste à l'état viable mais non cultivable.<br />L'objectif de ce travail était de mieux comprendre cette écologie. Des incubateurs permettant le prélèvement de biofilm ont été installés in situ dans un réseau, la colonisation en eau froide et en eau chaude a été suivie pendant 1 an. Par ailleurs, un pilote reproduisant un réseau intérieur permis de comparer l'efficacité de différents traitements de désinfection. Enfin, la recherche de molécules auto-inductrices qui pourraient expliquer l'exacerbation de la virulence lors de la multiplication intra-cellulaire a été menée dans différentes conditions.<br />Les incubateurs ont permis l'amplification de la quantité de légionelles, rendant possible la détection par culture en eau froide et entraînant une stabilisation de la population en eau chaude. L'utilisation de sondes spécifiques n'a pas permis d'améliorer la détection dans les biofilms.<br />Lors des essais sur pilote, les populations des biofilms ont été analysées grâce à des méthodes moléculaires (FISH, marquage de vitalité) et par culture. Le classement des traitements par ordre d'efficacité décroissante est le suivant : dioxyde de chlore > chlore > ozone > monochloramine > cuivre-argent. Seul le dioxyde de chlore montre une activité résiduelle dans les bras morts rincés quotidiennement avec de l'eau traitée. Les amibes résistent aux traitements, et Lp retrouve son niveau de population initial quelques jours seulement après leur interruption.<br /> Enfin, aucune sécrétion d'auto-inducteur n'a été mise en évidence, et l'incubation de Lp avec ces molécules ne semble pas modifier l'invasion des amibes.