The ability to synthesize pyrimidine nucleotides is essential for most organisms. Pyrimidines are required for RNA and DNA synthesis, as well as cell wall synthesis and the metabolism of certain carbohydrates. Recent findings, however, indicate that the pyrimidine biosynthetic pathway and its intermediates maybe more important for bacterial metabolism than originally thought. Maksimova et al., 1994, reported that a P. putida M, pyrimidine auxotroph in the third step of the pathway, dihydroorotase (DHOase), failed to produce the siderophore pyoverdin. We created a PAO1 DHOase pyrimidine auxotroph to determine if this was also true for P. aeruginosa. Creation of this mutant was a two-step process, as P. aeruginosa has two pyrC genes (pyrC and pyrC2), both of which encode active DHOase enzymes. The pyrC gene was inactivated by gene replacement with a truncated form of the gene. Next, the pyrC2 gene was insertionally inactivated with the aacC1 gentamicin resistance gene, isolated from pCGMW. The resulting pyrimidine auxotroph produced significantly less pyoverdin than did the wild type. In addition, the mutant produced 40% less of the phenazine antibiotic, pyocyanin, than did the wild type. As both of these compounds have been reported to be vital to the virulence response of P. aeruginosa, we decided to test the ability of the DHOase mutant strain to produce other virulence factors as well. Here we report that a block in the conversion of carbamoyl aspartate (CAA) to dihydroorotate significantly impairs the ability of P. aeruginosa to affect virulence. We believe that the accumulation of CAA in the cell is the root cause of this observed defect. This research demonstrates a potential role for pyrimidine intermediates in the virulence response of P. aeruginosa and may lead to novel targets for chemotherapy against P. aeruginosa infections.

Nucleotide pools were extracted and quantified from Pyr^+ and Pyr^- strains of P. aerucjinosa. Strains were grown in succinate minimal medium with and without pyrimidines, and nucleotides were extracted using trichloracetic acid (TCA; 6% w/v). The pyrimidine requirement was satisfied by uracil, uridine, cytosine or cytidine. Pyr^- mutants were starved for pyrimidines for two hours before nucleotide levels were measured. This starvation depleted the nucleotide pools which were restored to wild type levels by the addition of pyrimidines to the medium. When the pyrimidine analogue, 6-azauracil, known to inhibit OMP decarboxylase, was added to cultures of the wild type strain, the uridine and cytidine nucleotides were depleted to near zero. Thus, the nucleotide pool levels of Pseudomonas strains can be manipulated.

Previous research in our laboratory established that pyrB, pyrC or pyrD knock-out mutants in Pseudomonas aeruginosa required pyrimidines for growth. Each mutant was also discovered to be defective in the production of virulence factors. Moreover, the addition of exogenous uracil did not restore the mutant to wild type virulence levels. In an earlier study using non-pathogenic P. putida, mutants blocked in one of the first three enzymes of the pyrimidine pathway produced no pyoverdine pigment while mutants blocked in the fourth, fifth or sixth steps produced copious quantities of pigment, just like wild type P. putida. The present study explored the correlation between pyrimidine auxotrophy and pigment production in P. aeruginosa. Since the pigment pyoverdine is a siderophore it may also be considered a virulence factor. Other virulence factors tested included casein protease, elastase, hemolysin, swimming, swarming and twitching motilities, and iron binding capacity. In all cases, these virulence factors were significantly decreased in the pyrB, pyrC or pyrD mutants and even in the presence of uracil did not attain wild type levels. In order to complete this comprehensive study, pyrimidine mutants blocked in the fifth (pyrE) and sixth (pyrF) steps of the biosynthetic pathway were examined in P. aeruginosa. A third mutant, crc, was also studied because of its location within 80 base pairs of the pyrE gene on the P. aeruginosa chromosome and because of its importance for carbon source utilization. Production of the virulence factors listed above showed a significant decrease in the three mutant strains used in this study when compared with the wild type. This finding may be exploited for novel chemotherapy strategies for ameliorating P. aeruginosa infections in cystic fibrosis patients.

A presença de plasmídeos conjugativos como IncP, IncU e IncFII carreando genes de resistência em Pseudomonas aeruginosa é de grande importância, pois podem ser trocados entre diferentes bactérias gram-negativas, disseminando a resistência aos antibióticos. Conhecer estes genes de resistência bem como os elementos genéticos que os carreiam é importante para entender os fatores que contribuem para a disseminação da resistência, auxiliando no controle da disseminação da resistência aos antibióticos. Ainda hoje não existe esquema para a tipagem de plasmídeos de P. aeruginosa, e são encontrados poucos trabalhos sobre estes plasmídeos. O objetivo deste estudo foi identificar os genes de resistência a antibióticos, o ambiente genético em que estes genes estão inseridos e a clonalidade dos isolados produtores de genes bla. No período do estudo, foram estudados 293 isolados de P. aeruginosa resistentes às cefalosporinas de 3ª e/ou 4ª gerações e/ou aos carbapenêmicos isoladas de pacientes de hospitais de Ribeirão Preto-SP, Belo Horizonte-MG, Franca-SP, Cuiabá-MT, de Barretos-SP e de Rio Branco-AC. Genes de resistência foram pesquisados por PCR. O perfil clonal dos isolados produtores de genes bla foi determinado por PFGE e MLST. A tipagem de plasmídeos foi feita por PFGE-S1 nuclease, hibridações com sondas específicas e tipagem de replicons (PBRT). Foram identificados 12 isolados carreando o gene blaSPM-1, 16 isolados carreando o gene blaCTX-M-2 e 3 isolados carreando o gene blaKPC-2. Em todos os 12 isolados produtores de SPM-1 foram identificadas duas cópias do elemento de inserção ISCR4, sendo uma cópia upstream e uma cópia downstream ao gene blaSPM-1 inseridos no cromossomo bacteriano. Em 13 dos 16 isolados produtores de CTX-M-2 o gene blaCTX-M-2 foi encontrado associado ao elemento de inserção ISCR1 e em 3 ao elemento de inserção ISEcp1 também inseridos no cromossomo bacteriano. Em 2 isolados o gene blaKPC-2 é carreado por um plasmídeo de ~3kb não tipável por PBRT e um em está inserido no cromossomo bacteriano. O ambiente genético do gene blaKPC-2 nos isolados estudados é diferente daqueles encontrados na literatura. Os isolados produtores de genes bla citados apresentaram diversidade clonal, tanto por PFGE quanto MLST demonstrando que vários clones estão envolvidos na disseminação desses genes. Este trabalho identificou e caracterizou 31 isolados produtores de ?-lactamases, o ambiente genético destes genes e a clonalidade de isolados de várias cidades do Brasil e em períodos diferenciados, demonstrando a disseminação desses genes em diferentes hospitais brasileiros. Esses dados auxiliam no conhecimento dos fatores que estão envolvidos na disseminação da resistência aos antibióticos e podem auxiliar as CCIHs dos hospitais a definirem estratégias para controlar a disseminação desses microrganismos prevenindo surtos de bactérias multirresistentes. / The presence of conjugative plasmids as IncP, IncU and Inc FII carrying resistance genes in Pseudomonas aeruginosa is very important because t these plasmids can be shared among different bacteria, spreading antibiotic resistance. Knowledge of these genes as well as genetic elements carrying these genes it is important to understend the factors that contribute to the spread of resistance, helping to control the spread of antibiotic resistance. Today there is no plasmid typing scheme to P. aeruginosa and few papers are found about this subject. The purpose of this study was to investigate resistance genes, genetic environment of these genes and clonal relationship of the isolates carrying these resistance genes. In the period of this study was studied 293 P. aeruginosa resistant to third and fourth generations of cephalosporins and/or carbapenens isolated of patients from hosptital from Ribeirão Preto, Belo Horizonte-MG, Franca-SP, Cuiabá-MT, Barretos-SP and Rio Branco-AC. Resistance genes were investigated by PCR. Twelve isolates were identified carrying blaSPM-1 gene, 16 isolates carrying blaCTX-M-2 gene and 3 isolates carrying blaKPC-2 gene. Clonal profiles of isolates producing resistance genes were investigated by PFGE and MLST. Plasmid typing was performed by PFGE-S1 nuclease, specific hybridizations and PCR replicon typing (PBRT). Two isolates presented a 3kb plasmid non-typeable by PBRT carrying blaKPC-2 gene. In all isolates SPM-1-producers were identified two copies of insertion sequence ISCR4, a copy upstream and a copy downstream to blaSPM-1 gene inserted in chromosomal DNA. In 13 of 16 isolates CTX-M-2-producers the blaCTX-M-2 gene was found associated to insertion sequence ISCR1 and in 3 isolates was associated to insertion sequence ISEcp1 also inserted in chromosomal DNA. Genetic environment of blaKPC-2 gene in the isolates studied it is different from those found in the literature. Isolates producing bla genes are clonally diversified using both PFGE and MLST showing that various clones are responsible to spread these resistance genes. This work identified and characterized 31 P. aeruginosa-?-lactamase-producing, the genetic environment of these genes and the clonal relationship of isolates collected from different periods from different cities of Brazil. These data can help us to understand the factors that are involved in the spread of antibiotics resistance and to help the Hospital Infection Control Committee to define strategies to control the spread of these microorganisms preventing outbreaks of resistant.

O objetivo deste trabalho foi a seleção de bactérias capazes de biodegradar os pesticidas organoclorados dieldrin, DDD e PCP. Inicialmente, foram realizados os ensaios de tolerância visando à seleção das bactérias degradadoras; posteriormente foram realizados os ensaios de biodegradação em meio liquido utilizando a bactéria selecionada. Dentre as 14 linhagens bacterianas isoladas testadas, selecionou-se a linhagem Pseudomonas aeruginosa L2-1 por apresentar maior tolerância a todos os pesticidas. Os ensaios de biodegradação foram realizados em diferentes meios de cultura, variando-se a concentração de glicose, a fonte de nitrogênio e a presença de ramnolipídeo. Os ensaios de biodegradação foram realizados determinando-se a concentração de pesticida, a concentração de glicose, o número de células viáveis, e o pH. O meio de cultura que mais favoreceu a biodegradação dos três pesticidas foi o meio com nitrato de sódio e 0,5% de glicose, obtendo-se biodegradação de aproximadamente 50% para cada pesticida após três dias de incubação. Na ausência de glicose, o meio com nitrato de amônio e 0,1% de ramnolipídeo, favoreceu a biodegradação, obtendo-se após 14 dias de incubação 36,8% de biodegradação de dieldrin; 33,7% de DDD e 42,8% de PCP. De uma forma geral, as taxas de biodegradação pela P. aeruginosa L2-1 foram maiores em menores concentrações de glicose e na presença de ramnolipídeo. Ao alterar a fonte de nitrogênio foram observados resultados diversos sobre a taxa de biodegradação: na ausência de glicose, o nitrato de sódio favoreceu a biodegradação de PCP, enquanto o nitrato de amônio favoreceu a biodegradação de dieldrin, na presença de glicose observou-se o inverso. A taxa de biodegradação do DDD não foi significativamente alterada ao variar a fonte de nitrogênio. A bactéria selecionada P. aeruginosa L2-1 apresentou potencial para biodegradação de pesticidas organoclorados, sendo que as condições nutricionais do meio influenciaram diretamente a biodegradação. / The objective of this work was the selection of bacteria capable of biodegrading the organochloride pesticides dieldrin, DDD and PCP. Initially tolerance tests were conducted in order to select degrading bacteria subsequently, biodegradation tests were carried out in liquid medium using the selected bacteria. Among the 14 bacterial isolated strains, Pseudomonas aeruginosa L2-1 was selected due to its greater tolerance to all pesticides. The biodegradation tests were conducted on different culture media, varying the concentrations of glucose, nitrogen source and presence of rhamnolipid. Biodegradation studies were performed by measuring the concentration of pesticide, the concentration of glucose, the number of viable cell and pH during time. The best medium for the biodegradation of all three pesticides was composed of sodium nitrate and 0.5% glucose, giving approximately 50% yield after three days of incubation. In the absence of glucose, the medium containing ammonium nitrate and 0.1% rhamnolipid improved biodegradation yielding, after 14 days of incubation, 36.8% biodegradation of dieldrin; 33.7% DDD and 42.8% of PCP. In general, the biodegradation rates of pesticides by P. aeruginosa L2-1 were greater at lower concentrations of glucose and in the presence of rhamnolipid. Nitrogen source had different effects on the rate of biodegradation: in the absence of glucose, sodium nitrate favored the biodegradation of PCP, whereas ammonium nitrate favored the biodegradation of dieldrin; and in the presence of glucose, it was observed the opposite. The biodegradation rate of the DDD was not significantly altered by the nitrogen source tested. The selected bacteria, P. aeruginosa L2-1, showed potential for the biodegradation of organochloride pesticides demonstrating that nutritional conditions has a direct effect on degradation yields.

A análise de fluxos metabólicos para a produção de PHAMCL por Pseudomonas sp. LFM046 revelou que a baixa eficiência de conversão de carboidratos em PHA (60-70% do valor máximo teórico) deve estar associada à utilização principal da via das pentoses (VP) para o metabolismo de carboidratos. Eficiências significativamente maiores poderiam ser obtidas se uma parcela maior da glicose fosse metabolizada pela via Entner-Doudoroff (ED). Assim, neste trabalho foi realizada a análise de fluxos metabólicos utilizando glicose marcada (C13) e o melhoramento genético de Pseudomonas sp. LFM046 utilizando estratégia de engenharia metabólica. Experimentos com glicose marcada confirmaram um maior fluxo de carboidratos pela VP em relação a ED. A superexpressão de genes específicos de ED em Pseudomonas sp. LFM046 demonstrou apenas pequenos aumentos na eficiência de conversão de carboidratos em PHA pelas linhagens recombinantes. / The metabolic flux analysis for PHAMCL production by Pseudomonas sp. LFM046 revealed that the low efficiency of carbohydrates conversion into PHA (60-70% of the maximum theoretical value) should be associated with the main use of pentose phosphate pathway (PP) for the carbohydrate metabolism. Significantly higher efficiencies could be obtained if a larger portion of the glucose was metabolized via the Entner-Doudoroff (ED). Therefore, the metabolic fluxes analysis was performed in this work using labeled glucose (13C) and the genetic improvement of Pseudomonas sp. LFM046 by using metabolic engineering approach. Experiments with labeled glucose confirmed an increased flow of carbohydrates by PP compared to ED. The overexpression of specific genes from ED in Pseudomonas sp. LFM046 showed only small increases in the efficiency of carbohydrates conversion into PHA by recombinant strains.

by Woo Yiu Ho, Anthony. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 162-171). / Abstract also in Chinese. / Abstract --- p.i / Acknowledgements --- p.iii / Table of Contents --- p.iv / List of Figures --- p.ix / List of Tables --- p.xi / List of Abbreviations --- p.xii / Chapter 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Bacterial Adhesion and Aggregation --- p.1 / Chapter 1.1.1 --- Significance of Bacterial Adhesion Studies --- p.1 / Chapter 1.1.2 --- Definitions --- p.4 / Chapter 1.1.3 --- Colonization Process --- p.7 / Chapter 1.1.4 --- Specific and Nonspecific Interactions --- p.8 / Chapter 1.1.5 --- Models of Bacterial Adhesion and Aggregation Processes --- p.14 / Chapter 1.1.6 --- Experimental Systems in Adhesion Research --- p.16 / Chapter 1.1.7 --- Experimental Systems in Aggregation Research --- p.19 / Chapter 1.2 --- Pseudomonas aeruginosa --- p.21 / Chapter 1.2.1 --- General Description and Clinical Significance --- p.21 / Chapter 1.2.2 --- Adhesins of Pseudomonas aeruginosa --- p.22 / Chapter 1.2.3 --- "Alginate, Mucoidity, Biofilm Formation and Cystic Fibrosis" --- p.23 / Chapter 1.2.4 --- Lipopolysaccharides --- p.26 / Chapter 1.2.5 --- Pili --- p.29 / Chapter 1.2.6 --- Flagella --- p.30 / Chapter 1.2.7 --- Lectins --- p.31 / Chapter 1.2.8 --- Other Adhesins --- p.31 / Chapter 1.2.9 --- Rhamnolipids --- p.32 / Chapter 1.3 --- Current Study --- p.33 / Chapter 2 --- MATERIALS AND EQUIPMENT --- p.35 / Chapter 2.1 --- Bacterial Strain --- p.35 / Chapter 2.2 --- Solid Surfaces --- p.35 / Chapter 2.3 --- Chemicals --- p.36 / Chapter 2.4 --- Recipes --- p.38 / Chapter 2.5 --- Equipment --- p.38 / Chapter 3 --- METHODS --- p.40 / Chapter 3.1 --- Maintenance and Culturation --- p.40 / Chapter 3.1.1 --- Maintenance of Bacterial Strains --- p.40 / Chapter 3.1.2 --- Seed Culture Preparation --- p.40 / Chapter 3.1.3 --- Culturation in Defined Growth Media --- p.40 / Chapter 3.2 --- Bacterial Adhesion and Aggregation Assay Methods --- p.41 / Chapter 3.2.1 --- Bacterial Adhesion on Glass Assay --- p.41 / Chapter 3.2.2 --- Bacterial Adhesion on Plastic Assay --- p.44 / Chapter 3.2.3 --- Bacterial Adhesion under Shear Assay --- p.44 / Chapter 3.2.4 --- Bacterial Aggregation Examination by Adhesion on Glass Assay --- p.45 / Chapter 3.2.5 --- Bacterial Aggregation Examination by Top-agar Assay --- p.45 / Chapter 3.2.6 --- Bacterial Aggregation Examination by Epi-fluorescence Microscopy --- p.46 / Chapter 3.2.7 --- Bacterial Aggregation Screening Test --- p.46 / Chapter 3.3 --- Determination of the Effects of Various Factors on Adhesion and Aggregation --- p.47 / Chapter 3.3.1 --- Culturation Period --- p.47 / Chapter 3.3.2 --- Osmotic Shock during the Washing Procedure --- p.47 / Chapter 3.3.3 --- Growth Media --- p.48 / Chapter 3.3.4 --- Assay Conditions --- p.48 / Chapter 3.3.5 --- Cell Pretreatments --- p.48 / Chapter 3.4 --- Isolation of Aggregation-deficient Mutants --- p.49 / Chapter 3.5 --- Outer Membrane Protein Profiles --- p.50 / Chapter 3.5.1 --- Isolation of Outer Membrane Fraction --- p.50 / Chapter 3.5.2 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis --- p.51 / Chapter 3.6 --- Determination of the Mobility of the Bacteria on Surfaces --- p.52 / Chapter 3.6.1 --- Subsurface Twitching Assay --- p.52 / Chapter 3.6.2 --- Soft-agar Swarm Assay --- p.53 / Chapter 3.7 --- Detection of Alginate Production --- p.53 / Chapter 3.7.1 --- Extraction of Alginate from Spent Growth Medium --- p.53 / Chapter 3.7.2 --- Releasing Cell Surface-associated Alginate --- p.54 / Chapter 3.8 --- Other Assay Methods --- p.55 / Chapter 3.8.1 --- Protein Assay --- p.55 / Chapter 3.8.2 --- Carbohydrate Determination --- p.55 / Chapter 3.8.3 --- Alginate Determination --- p.55 / Chapter 4 --- RESULTS --- p.57 / Chapter 4.1 --- Standardization of the Assays for Bacterial Adhesion and Aggregation --- p.57 / Chapter 4.1.1 --- Effects of Cell Density and Exposure Time on the Number of Adhered Bacteria Detected in Bacterial Adhesion on Glass Assay --- p.57 / Chapter 4.1.2 --- Characterization of Bacterial Aggregation by Different Examination Methods --- p.62 / Chapter 4.1.3 --- Effects of Culturation Period on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.67 / Chapter 4.1.4 --- Effects of Osmotic Shock during Washing on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.70 / Chapter 4.1.5 --- Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 as a Function of Time under the Standard Assay Condition --- p.71 / Chapter 4.1.6 --- Consistency of Bacterial Adhesion on Glass Assay --- p.74 / Chapter 4.2 --- Effects of Growth Media on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.77 / Chapter 4.3 --- Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 in Different Assay Media --- p.77 / Chapter 4.3.1 --- Effects of Various Buffers on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.77 / Chapter 4.3.2 --- Effects of pH on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.78 / Chapter 4.3.3 --- Effects of Various Electrolytes on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.81 / Chapter 4.3.4 --- Concentration Effects of Monovalent and Divalent Cations on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.88 / Chapter 4.3.5 --- Concentration Effects of Phosphate Buffers on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.93 / Chapter 4.3.6 --- Concentration Effects of Ammonium Sulfate and Cyclohexylammonium Sulfate on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.96 / Chapter 4.3.7 --- Effects of Cation Chelation on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.99 / Chapter 4.3.8 --- Effects of Sugars on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.100 / Chapter 4.3.9 --- Effects of Amino Acids on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.101 / Chapter 4.4 --- Adhesion and Aggregation after Pretreatments of the Cells --- p.103 / Chapter 4.4.1 --- Effects of Protease Treatments on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.103 / Chapter 4.4.2 --- Effects of Externally Added Proteins on Adhesion and Aggregation of Pronase-treated Cells --- p.107 / Chapter 4.4.3 --- Effects of Acid or Base Treatments on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.108 / Chapter 4.4.4 --- Effects of Heat Treatment on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.108 / Chapter 4.4.5 --- Effects of Extensive Washing on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.110 / Chapter 4.5 --- Isolation and Growth Characteristics of Aggregation-deficient Mutants --- p.111 / Chapter 4.6 --- Comparisons of the Adhesion and Aggregation Characters of Pseudomonas aeruginosa ATCC 10145 and Mutant 9 --- p.115 / Chapter 4.6.1 --- Under Standard Condition --- p.115 / Chapter 4.6.2 --- On Different Surfaces and in Different Electrolytes --- p.115 / Chapter 4.6.3 --- Under Shear --- p.118 / Chapter 4.6.4 --- Adhesion and Aggregation of Combined Suspensions of Pseudomonas aeruginosa ATCC 10145 and Mutant 9 --- p.122 / Chapter 4.7 --- Characterization of the Cell Surface Properties of Pseudomonas aeruginosa ATCC 10145 and Mutant 9 --- p.125 / Chapter 4.7.1 --- Outer Membrane Protein Profiles --- p.125 / Chapter 4.7.2 --- Pili-elicited Twitching Mobility --- p.125 / Chapter 4.7.3 --- Mobility Due to Flagella --- p.128 / Chapter 4.7.4 --- Production of Alginate --- p.128 / Chapter 5 --- DISCUSSIONS --- p.130 / Chapter 5.1 --- Choice of the Materials --- p.130 / Chapter 5.2 --- Development of the Assay Methods --- p.130 / Chapter 5.2.1 --- Development of the Procedures for Bacterial Adhesion Assays --- p.130 / Chapter 5.2.2 --- Development of the Assay Methods for Bacterial Aggregation --- p.132 / Chapter 5.2.3 --- Standardization of the Assays --- p.133 / Chapter 5.2.4 --- Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 as a Function of Time under the Standard Assay Condition --- p.134 / Chapter 5.2.5 --- Consistency of Bacterial Adhesion on Glass Assay --- p.135 / Chapter 5.2.6 --- Limits of Bacterial Adhesion on Glass Assay --- p.135 / Chapter 5.3 --- Effects of Growth Media on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.135 / Chapter 5.4 --- Effects of Various Chemicals in the Assay Media on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.136 / Chapter 5.4.1 --- Effects of Electrolytes on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.137 / Chapter 5.4.2 --- Effects of Aggregation on Adhesion --- p.140 / Chapter 5.4.3 --- Effects of Cyclohexylammonium Sulfate and Ammonium Sulfate on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.141 / Chapter 5.4.4 --- Effects of Sugars and Amino Acids on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.143 / Chapter 5.5 --- Effects of Various Cell-surface Modifications on Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 --- p.144 / Chapter 5.6 --- Isolation and Growth Characteristics of Aggregation-deficient Mutants --- p.146 / Chapter 5.7 --- Comparisons of the Adhesion and Aggregation Characters of Pseudonomas aeruginosa ATCC 10145 and Mutant 9 --- p.147 / Chapter 5.7.1 --- Adhesion and Aggregation of Pseudonomas aeruginosa ATCC 10145 and Mutant 9 on Different Surfaces In Different Electrolytes --- p.147 / Chapter 5.7.2 --- Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 and Mutant 9 Under Shear --- p.147 / Chapter 5.7.3 --- Adhesion and Aggregation of Combined Suspensions of Pseudomonas aeruginosa ATCC 10145 and Mutant 9 --- p.148 / Chapter 5.8 --- Characterization of the Cell Surface Properties of Pseudomonas aeruginosa ATCC 10145 and Mutant 9 --- p.148 / Chapter 5.9 --- General Discussions --- p.151 / Chapter 6 --- APPENDIX --- p.154 / Chapter 6.1 --- Visual Examination of Adhesion and Aggregation of Pseudomonas aeruginosa ATCC 10145 on Glass --- p.154 / Chapter 6.2 --- Fractal Analysis of Bacterial Aggregates --- p.154 / Chapter 7 --- REFERENCES --- p.162

Le Pseudomonas aeruginosa est un pathogène nosocomial majeur. Le clone ST235 est le plus prévalent des clones internationaux dits à hautris que. Ce clone est très fréquemment multi résistant aux antibiotiques, ce qui complique la prise en charge des infections dont il est à l’origine.Malgré son importance clinique, la base moléculaire Du succès du clone ST235 n’est pas comprise.Dans ce travail, nous avons cherché à comprendre l’origine spacio temporelle de ce clone et les bases moléculaires de son succès. A l’aide d’outils bio informatiques existants ,nous avons trouvé que le clone ST235 a émergé en Europe en 1984 et que tous les isolates ST235 produisent l’exotoxine ExoU. Nous avons également identifié 22 gènes Contigus spécifiques de ce clone et impliqués dans l’efflux transmembranaire, dans le traitement de l’ADN et dans la transformation bactérienne. Cette combinaison unique de gènes a pu contribuer à la gravité des infections dues à ce clone et à sa capacité à acquérir des gènes de résistance aux antibiotiques. Ainsi, la diffusion mondiale de ce clone a probablement été favorisée par l’utilisation extensive des fluoroquinolones, puis il est de venu localement résistant aux amino glycosides, aux β-lactamines, et aux carbapénèmes par mutation et acquisition d’éléments de résistance. Nous avons majoritairement utilisé des outils existants,mais avons découvert que les programmes de détection des séquences d’insertions (IS, ayant un rôle important dans l’évolution des génomes bactériens) ne sont pas adaptés aux données dont nous disposions. Nous avons ainsi mis au point un outil (appelé panISa) qui détecte de façon précise et sensible les IS à partir de données brutes de séquençage de génomes bactériens. / Pseudomonas aeruginosa is a major nosocomial pathogen with ST235 being the most prevalent of the so-called ‘international’ or ‘high-risk’ clones. This clone is associated with poor clinical outcomes in part due to multi- and high-level antibiotic resistance. Despite its clinical importance, the molecular basis for the success of the ST235 clone is poorly understood. Thus this thesis aimed to understand the origin of ST235 and the molecular basis for its success, including the design of bioinformatics tools for finding insertion sequences (IS) of bacterial genomes.To fulfill these objectives, this thesis was divided into 2 parts.First, the genomes of 79 P. aeruginosa ST235 isolates collected worldwide over a 27-year period were examined. A phylogenetic network was built using Hamming distance-based method, namely the NeighborNet. Then we have found the Time to the Most Recent Common Ancestor (TMRCA) by applying a Bayesian approach. Additionally, we have identified antibiotic resistance determinants, CRISPR-Cas systems, and ST235-specific genes profiles. The results suggested that the ST235 sublineage emerged in Europe around 1984, coinciding with the introduction of fluoroquinolones as an antipseudomonal treatment. The ST235 sublineage seemingly spreads from Europe via two independent clones. ST235 isolates then appeared to acquire resistance determinants to aminoglycosides, β-lactams, and carbapenems locally. Additionally, all the ST235 genomes contained the exoU-encoded exotoxin and identified 22 ST235-specific genes clustering in blocks and implicated in transmembrane efflux, DNA processing and bacterial transformation. These unique genes may have contributed to the poor outcome associated with P. aeruginosa ST235 infections and increased the ability of this international clone to acquire mobile resistance elements.The second part was to design a new Insertion Sequence (IS) searching tool on next-generation sequencing data, named panISa. This tool identifies the IS position, direct target repeats (DR) and inverted repeats (IR) from short read data (.bam/.sam) by investigating only the reference genome (without any IS database). To validate our proposal, we used simulated reads from 5 species: Escherichia coli, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Staphylococcus aureus, and Vibrio cholerae with 30 random ISs. The experiment set is constituted by reads of various lengths (100, 150, and 300 nucleotides) and coverage of simulated reads at 20x, 40x, 60x, 80x, and 100x. We performed sensitivity and precision analyses to evaluate panISa and found that the sensitivity of IS position is not significantly different when the read length is changed, while the modifications become significant depending on species and read coverage. When focusing on the different read coverage, we found a significant difference only at 20x. For the other situations (40x-100x) we obtained a very good mean of sensitivity equal to 98% (95%CI: 97.9%-98.2%). Similarly, the mean of DR sensitivity of DR identification is high: 99.98% (95%CI: 99.957%-99.998%), but the mean of IR sensitivity is 73.99% (95%CI: 71.162%-76.826%), which should be improved. Focusing on precision instead of sensibility, the precision of IS position is significantly different when changing the species, read coverage, or read length. However, the mean of each precision value is larger than 95%, which is very good.In conclusion, P. aeruginosa ST235 (i) has become prevalent across the globe potentially due to the selective pressure of fluoroquinolones and (ii) readily became resistant to aminoglycosides, β-lactams, and carbapenems through mutation and acquisition of resistance elements among local populations. Concerning the second point, our panISa proposal is a sensitive and highly precise tool for identifying insertion sequences from short reads of bacterial data, which will be useful to study the epidemiology or bacterial evolution.

Bactérias aclimatadas a cobre foram isoladas a partir de amostras de solo e água coletadas na região da Mina de Sossego (Vale, Carajás, PA). Pseudomonas putida sp. foi escolhida, pois, apresentou a maior capacidade sortiva de Cu2+, Q = 40 mg/g. O cultivo em regime de batelada, meio mineral, com glicerol como fonte de carbono, resultou fator de conversão de glicerol a células, YX/S, de 0,49 g/g e velocidade específica máxima de crescimento, mmax, de 0,11 h-1. Alta concentração celular, 90 g/L, foi alcançada em cultivos em regime de batelada alimentada. Promoveu-se sorção de cobre pelas células, por meio de adição contínua ou em pulsos, de solução de CuSO4. A maior sorção específica de cobre, Q, de 30 (mg de Cu2+/g de células), foi verificada na adição por pulsos. Fotos de MET da bactéria na ausência e presença de Cu2+ mostram acúmulo de cobre na membrana e internamente, caracterizando biossorção e bioacumulação. / Bacteria acclimated to copper were isolated from soil and water samples collected in Mina de Sossego (Vale, Carajás, PA). Pseudomonas putida sp. was chosen as it had the highest sorptive capacity for Cu2+, Q = 40 mg/g. The fed-batch culture in mineral medium with glycerol as the carbon source resulted in a glycerol-to-cell conversion factor, YX/S of 0.49 g/g and maximum specific growth rate, mmax of 0.11 h-1. High cell concentration, 90 g/L, was achieved in cultures in fed-batch regimen. Cooper sorption by cells was promoted, by continuous or pulse addition of CuSO4 solution. The highest specific copper sorption, Q, 30 (mg Cu+2/g of cells) was seen with the addition by pulses. TEM photos of the bacteria in the absence and presence of Cu+2 show copper accumulation in the membrane and internally, featuring biosorption and bioaccumulation.

Pseudomonas aeruginosa é uma gamma-proteobacteria ubíqua, sendo a principal causa de infecção hospitalar dentre todos os patógenos relacionados com pneumonia em UTI. A defesa do hospedeiro se dá por vários mecanismos como a liberação, por fagócitos, de espécies reativas de oxigênio, como o ânion superóxido (O2?-), peróxido de hidrogênio (H2O2) e o radical hidroxila (OH?) para combater o patógeno. LsfA pertence à família das peroxirredoxinas (Prx) e ao sub-grupo de Prxs que contém somente uma cisteína catalítica (denominadas 1-Cys Prx). Prxs são enzimas capazes de remover peróxidos (incluindo peroxinitrito) em velocidades muito elevadas. Além disso, LsfA está relacionada a patogenicidade de P. aeruginosa. Dentro desse contexto, o objetivo do presente trabalho é a caracterização bioquímica e estrutural de LsfA; que pode possibilitar a identificação de inibidores dessa enzima antioxidante. Por outro lado, caracterizando cineticamente reações de oxido-redução de LsfA e caracterizando seus mecanismos de ação, podemos identificar seus substratos biológicos. Dessa maneira, utilizando diferentes técnicas, determinamos as constantes de segunda ordem de LsfA com H2O2 (na ordem de 107 M -1.s -1); para terc-butilhidroperóxido (na ordem de 106 M-1.s-1) e peroxinitrito (na ordem de 107 M-1.s-1). A redução de LsfA por ascorbato foi descrita previamente por nosso grupo (na ordem de 103 M-1.s-1); e aqui apresentamos dados preliminares sobre a redução dessa 1-Cys Prx por GSH. Além disso, fomos capazes de determinar a estrutura cristalográfica de LsfA em sua forma oxidada e superoxidada, com resolução de 2.6 e 2.0 ? respectivamente; que, como esperado, se apresentou no estado dimérico, em ambos os casos. Descrevemos aqui características sobre a estrutura do sítio ativo de LsfA, que apresenta mais eletronegativo, com a cisteína peroxidásica desprotonada, e mais hidrofóbico. Na estrutura de LsfA superoxidada, observamos a co-cristalização dessa enzima com uma molécula de polietileno glicol que pode estar mimetizando um substrato. Portanto, esses estudos levantaram importantes informações estruturais e bioquímicas de uma enzima antioxidante envolvida com a virulência de P. aeruginosa / Pseudomonas aeruginosa is a ubiquous gamma-proteobacteria that is the main cause of hospitalar infections among all pathogens related with pneumonia. Host defenses against pathogens are mainly by phagocytes, which releases reactive oxygen species, such as superoxide (O2?-), hydrogen peroxide (H2O2) and hydroxyl radical (OH?) to fight against pathogen. LsfA belongs to peroxiredoxins (Prx) family; and to the 1-Cys Prx sub-group (Prx6 sub-family) that possess only one catalytic cysteine. Prx are enzymes can remove peroxides with extremely high efficiency. LsfA was already related with P. aeruginosa virulence. So, the aim of the present work is the structural and biochemical characterization of LsfA, which may enable the discovery of inhibitors. Furthermore, the investigation of the kinetics and the mechanism of catalysis of LsfA may give insights on the chemical nature of its biological substrates. Therefore, using different techniques; the second order rate constants of LsfA with H2O2 (107 M -1.s -1), tert-butylhydroperoxide (106 M -1.s -1) and peroxynitrite (107 M -1.s -1) were determined. Our group has already determined the rate constant between ascorbate and LsfA (103 M -1.s -1) and preliminary data on the reduction of this 1-Cys Prx by glutathione is described. Furthermore, two crystallographic structures of LsfA were elucidated in distinct oxidative states (sulfenic and sulfonic acid in the CP), both in the dimeric state; at 2.6 and 2.0 ? resolution respectively. Features in the LsfA active site are also described here, such as poor exposure to the solvent. In the LsfA crystal structure where Cp is hyperoxidized to sulfinic acid, we observed the presence of an electronic density compatible with a PEG molecule that might be mimicking one of the possible substrates. Therefore, relevant structural and biochemical information were gained with our studies about an antioxidant enzyme involved with P aeruginosa virulence