Inoculation with plant pathogens induces a diverse range of plant responses which potentially contribute to disease resistance or susceptibility. Plant responses occuring in consequence of pathogen infection include activation of classical defence pathways and changes in metabolic activity. The main defence route against hemibiotrophic bacterial pathogens such as Pseudomonas syringae is based on the phytohormone salicylic acid (SA). SA-mediated responses are strictly regulated and have also been shown to depend on external factors, e.g. the presence of light. A major goal of this work was to provide a better understanding of the light dependency of plant defence responses mediated through SA. The second part of the project focussed on the influence of plant sterols on plant resistance. I analyzed leaf lipid composition and found that accumulation of the phytosterol stigmasterol in leaves and in isolated (plasma) membranes is a significant plant metabolic process occurring upon pathogen infection. / Eine Infektion mit Pathogenen veranlasst Pflanzen zur Aktivierung zahlreicher Abwehrreaktionen, welche entscheidend dazu beitragen können, ob die Pflanze anfällig ist und erkrankt oder eine erfolgreiche Resistenz ausbilden kann. Die Abwehr gegen hemibiotrophe bakterielle Pathogene basiert vor allem auf der verstärkten Bildung des Pflanzenhormons Salicylsäure (SA) und der Aktivierung SA-vermittelter Abwehrreaktionen. Beides ist nicht nur intern genau reguliert, sondern auch von externen Faktoren beeinflusst. So trägt zum Beispiel die Verfügbarkeit von Licht wesentlich zum Ausmaß und zum Erfolg dieser Abwehrreaktionen bei. Ein Ziel dieser Arbeit ist es, zu einem besseren Verständnis des Einflusses von Licht auf die Pathogenabwehr beizutragen. Das zweite Projekt dieser Arbeit stellt die Lipidzusammensetzung pathogen-infizierter Blätter in den Mittelpunkt. Bei der Analyse pflanzlicher Sterole zeigte sich unter anderem, dass das C22-ungesättigte Sterol Stigmasterol in Blättern und in daraus isolierten Plasmamembranen nach Pathogenbehandlungen akkumuliert.

Sphingolipide (SPL) sind wichtige und ubiquitar verbreitete Bestandteile von Biomembranen. Aufgrund der enormen Vielfalt, der komplexen Struktur und diverser physiko-chemischer Eigenschaften der Sphingolipide gestaltet sich die qualitative und quantitative Untersuchung der Sphingolipide allerdings schwierig. In dieser Arbeit konnten, basierend auf publizierten Methoden, analytische Verfahren entwickelt werden, mit deren Hilfe sich die Gehalte spezifischer Sphingolipide in A. thaliana quantitativ nachweisen lassen. Unter Einsatz eines targeted metabolite profiling-Ansatzes wurde die Rolle spezifischer Sphingolipide in der Pflanzen-Pathogen Interaktion charakterisiert. Infiltration von avirulenten P. syringae pv. tomato (Pst) in Blätter von A. thaliana führte zu schnell und transient erhöhten Gehalten der freien Sphingobase Phytosphingosin (t18:0). Im Gegensatz zu avirulenten Pst kam es nach Infiltration von virulenten Pst zu einer schnellen Rückkehr auf Basalniveau und nicht zu einer hypersensitiven Antwort (HR), was auf eine positiv regulatorische Rolle von t18:0 in Abwehrreaktionen von Pflanzen hinwies, z.B. bei der HR. Damit konnte in der vorliegenden Arbeit zum ersten Mal gezeigt werden, dass die Spiegel freier Sphingobasen der Pflanze, insbesondere von t18:0, in Antwort auf bakterielle Pathogene reguliert werden. Diese spezifische Regulation korreliert, in Abhängigkeit von der Pathogeninfektion, mit dem Verlauf der HR. Im Unterschied zu avirulenten Stämmen sind virulente Pst in der Lage, Abwehrreaktionen des Wirtsorganismus zu unterdrücken. Daher tritt keine HR auf, welche die Ausbreitung des Pathogens stoppen könnte. Die unterschiedliche Beeinflussung der t18:0 Gehalte virulenter und avirulenter Stämme zeigte sich auch in Experimenten mit einem anderen P. syringae Stamm. Freie Sphingobasen zeigten in dieser Arbeit typische Merkmale von Signalmolekulen: geringe basale Spiegel, schnelle und transiente Gehaltsanderungen, präzise Regulation sowie spezifische Wirkeffekte. Sphingolipide stellen somit, neben den etwa durch PAMPs ausgelösten und durch Phytohormone vermittelten, weitere Signalwege in der Pflanzen Pathogen Interaktion dar. Die Infiltration von Pst in Blätter der A. thaliana Mutante sbh1-1 führte zu transient erhöhten d18:0 Spiegeln. In dieser Mutante ist die Funktion von einer der zwei Sphingobasen-Hydroxylasen gestört. Wie sich nach Totalhydrolyse zeigte, sind die Gesamtgehalte von t18:0 in der Mutante allerdings nicht reduziert. Dies spricht dafür, dass der pathogenabhängige transiente Anstieg von t18:0 durch de novo Synthese aus d18:0 entsteht und nicht durch Freisetzung aus komplexen Sphingolipiden mittels spezifischer Lipasen. Somit ist die Hydroxylase SBH1 für den schnellen signalvermittelten Anstieg von t18:0 verantwortlich. Neben t18:0 lösen auch strukturell ähnliche freie Sphingobasen, z.B. d18:1 und d18:0, Abwehrreaktionen und Zelltod aus, während andere Sphingobasen (d20:0 und d20:1) sowie Ceramide keine Reaktionen auslösten. Dies weist auch direkt auf die Spezifität der beteiligten Mechanismen hin. / Sphingolipids (SPL) are important and ubiquitously distributed constituents of biological membranes. Due to the tremendous variety, complex structure and diverse physicochemical properties of sphingolipids, qualitative and quantitative analysis has only recently been possible due to newly developed methods in mass spectrometry and chromatography. In this work, analytical methods to quantitatively detect the SPL content in A. thaliana leaves were established based on published literature. Using a targeted metabolic profiling approach, the role of specific SPL in the plant‐pathogen interaction was characterized. In line with the production of reactive oxygen species (ROS), a hallmark of biotic stress, infiltration of the avirulent form of the phytopathogen P. syringae pv. tomato (Pst) led to a fast and transient increase of the free long chain base Phytosphingosine (t18:0). Virulent Pst showed also a fast and transient, but clearly less prolonged elevation of t18:0 levels. Also, no HR was elicited in response to the infiltration, pointing to a positive regulatory role of t18:0 in this plant defense response. This work shows, for the first time, that SPL, namely t18:0, were regulated in response to bacterial pathogens. The t18:0 kinetics showed a strong correlation with the course of the pathogen‐elicited HR. There was also evidence, that virulent Pst influences the plants own biosynthetic and regulatory mechanisms to inhibit the SPL mediated defense response. This was also the case with another tested Pseudomonas syringae strain. In this work, free long chain bases showed characteristics typical for signaling molecules: low basal levels, a fast and transient increase in response to pathogens and a tight regulation. Hence, SPL may represent members of signaling pathways in plant‐pathogen interactions in addition to or besides PAMP‐triggered and hormonal mediated signaling pathways. Infiltration of Pst into leaves of the A. thaliana hydroxylase mutant sbh1-1 led to transiently increased d18:0 levels in leaves. In this mutant, one of the two functional sphingobase hydroxylases of A. thaliana is impaired. As the total pool of t18:0 was not significantly reduced in the mutant after total hydrolysis, we argue that the pathogen‐dependent transient increase of t18:0 was due to de novo synthesis from d18:0 and not to the action of specific lipases. Furthermore SBH1 was responsible for the fast increase of t18:0 levels. In addition to t18:0, also other free long chain bases, e.g. d18:0, elicited plant reactions and cell death, whereas other long chain bases (d20:0 and d20:1) or ceramides elicited no response. Apparently, the specific lipid structure plays a major role for the efficiency in different signaling pathways.

A unique undosciribed gramnegative rod is extensively characterized in this study. The cells of this unusual water isolate measure 1.2 X 6.5 microns, The most distinguishing characteristic of the bacterium is a polar tuft of 35-40 flagella that aggregate to function as a single organelle which is visible under phase contrast. Aging cells deposit poly- -hydroxybutyric acid granules which are bound by an inclusion membrane made up of four distinct layers. It also possesses an unusual exterior membrane outside the cell wall which contains large fibrils of protein running at a slight angle to the longitudinal axis of the cell. The guanosine-cytosine ratio was found to be 62.2$. The organism's taxonomic position was further investigated by immunological, morphological, and biochemical methods. It was found to be most closely akin to members of the genus Pseudo onas, although somewhat divergent from other species classified in this genus. After careful evaluation of the findings obtained during this study, the new bacterium was subsequently named Pseudomonas multiflagella.

The historical notion of a microbial population has been of a clonal population of identical swimming planktonic cells in a laboratory flask. As the field has advanced, we have grown to appreciate the immense diversity in microbial behaviors, from their propensity to grow in dense surface-attached communities as a biofilm, to the consequences of social dilemmas between cells, to their ability to form spores able to survive nearly any environmental insult. However, the historically biased view of the clonal microbial population still persists – even when a rare phenotype is investigated, the focus simply shifts to that narrower focal population - and this bias can lead to some of the broader questions relating to the consequences of phenotypic diversity within populations to be overlooked. This work seeks to address this gap by investigating the evolutionary causes and consequences of phenotypic heterogeneity, with a focus on clinically relevant phenotypes. We first develop and experimentally validate a theoretical model describing the evolution of a microbial population faced with a trade-off between survival and fecundity phenotypes (e.g. biofilm and planktonic cells), which suggests that simultaneous investment in both types maximizes lineage fitness in heterogeneous environments. This model helps to inform the experimental studies in the following chapters. We find that biofilm-mediated phenotypic resistance to antibiotics is evolutionarily labile, and responsive to antibiotic dose and whether biofilm or planktonic cells are passaged. We also show that persistence in E. coli is age-independent, supporting the current hypothesis of stochastic metabolic fluctuations as the cause of this rare phenotype. Finally, we explore phenotypic variation across a library of natural isolates of P. aeruginosa, and find few organizing principles among key phenotypes related to virulence. Together these results suggest that phenotypic heterogeneity is a crucial component in the ecology and evolution of microbial populations, and directly affects pressing applied concerns such as the antibiotic resistance crisis.

Pseudomonas aeruginosa é um importante patógeno humano oportunista capaz de causar severas infecções em pacientes imunodeprimidos e em pacientes que apresentam fibrose cística. Para desencadear uma resposta efetiva contra a infecção pela P. aeruginosa é necessário uma primeira linha de reconhecimento desta bactéria pelo sistema imunológico inato. Apesar do desencadeamento da resposta imune inata ser benéfica no controle da infecção pela P. aeruginosa, esta resposta deve ser controlada. Estudos recentes têm começado a esclarecer como os miRNAs desempenham papéis fundamentais na regulação de processos como infecção, resposta imune e inflamação participando da modulação da resposta imune. Dada a relevância da bactéria P. aeruginosa nos processos infecciosos em humanos e estimulados pela necessidade de desvendar os mecanismos de regulação da reposta imune contra esta bactéria, o objetivo deste trabalho foi avaliar o papel do mmu-miR-155-5p e mmu-miR-146b-5p expressos em camundongos infectados pelas cepas ATCC 27853 e PA14 da bactéria P. aeruginosa. Para avaliar o nível de expressão dos mmu-miR-155-5p e mmu-miR-146b-5p e das citocinas IL-1β, IL-6 e IL-12 in vitro, o RNA total foi extraído de células Raw 264.7, macrófagos derivados da medula óssea (BMDM) e de células dendríticas derivadas da medula óssea (BMDC) para análise de PCR em tempo real. Após a infecção das células Raw 264.7, BMDMs e BMDCs foi possível observar que a expressão dos miRNAs e das citocinas ocorreu de forma dependente em cada tipo celular infectado. Para as análises in vivo camundongos C57BL/6 foram infectados via intratraqueal com as cepas ATCC 27853 e PA14 de P. aeruginosa para análises de UFCs, RT-qPCR, histopatologia e estereologia. Apenas a cepa PA14 foi recuperada no pulmão e baço dos animais, onde não foi observado variação na expressão do mmu-miR-155-5p nem aumento significativo de uma série de citocinas efetoras. As análises histopatológicas demostraram intenso processo inflamatório difuso apresentando número maior de células inflamatórias, diminuição no número de alvéolos e do volume da estrela quando comparados aos animais não infectados e animais infectados pela cepa avirulenta. No pulmão dos animais infectados com a cepa ATCC 27853, observou-se aumento de expressão de mmu-miR-155-5p e mmu-miR-146b-5p, das citocinas inflamatórias. A identificação das redes de regulação dos miRNAs em estudo mostrou importantes alvos diretos e indiretos associados à resposta imune inata que podem estar comprometidas durante a expressão diferencial de mmu-miR-155-5p e mmu-miR-146b-5p em favorecimento ou em detrimento da resolução da infecção pela P. aeruginosa. Os resultados obtidos até o momento permitem sugerir que a infecção pela P. aeruginosa exerce uma modulação na expressão de miRNAs e consequentemente na resposta imune contra esta bactéria e que a virulência de diferentes cepas de P. aeruginosa influenciam na expressão dos miRNAs mmu-miR-155-5p e mmu-miR-146b-5p, de citocinas pró-inflamatórias e na patologia. Mais estudos são necessários para desvendar os mecanismos pelos quais cepas virulentas da P. aeruginosa conseguem subverter a resposta imune e garantir a sua replicação no hospedeiro. / Pseudomonas aeruginosa is an important opportunistic human pathogen capable of causing severe infections in immunocompromised patients and in patients with cystic fibrosis. To trigger an effective response against infection by P. aeruginosa is required a first line of recognition of this bacterium by the innate immune system. Although the innate immune response is beneficial in the control of P. aeruginosa infection, this response should be controlled. Recent studies have begun to clarify how miRNAs play key roles in regulating processes such as infection, immune response and inflammation by participating in the immune response modulation. Due to the importance of the P. aeruginosa bacterium in infectious processes in humans and stimulated by the need to uncover the mechanisms of regulation of the immune response against this bacterium, the objective of this study was to evaluate the role of mmu-miR-155-5p and mmu-miR-146b -5p expressed in mice infected with strains ATCC 27853 and PA14 of the bacterium P. aeruginosa. To assess the level of expression of mmu-miR-155-5p and mmu-miR-146b-5p and the cytokines IL-1β, IL-6 and IL-12 in vitro, the total RNA was extracted from Raw 264.7 cells, bone marrow derived macrophages (BMDM) and bone marrow-derived dendritic cells (BMDC) for real-time PCR analysis. After infection of the Raw 264.7, BMDMs and BMDCs cells, it was possible to observe that the expression of miRNAs and cytokines occurred in a dependent manner in each infected cell type. For the in vivo analyzes C57BL / 6 mice were infected intratracheally with the strains ATCC 27853 and PA14 of P. aeruginosa for analysis of CFU, RT-qPCR, histopathology and stereology. Only the PA14 strain was recovered in the lungs and spleens of the animals, where no variation in the expression of mmu-miR-155-5p or a significant increase in a series of effector cytokines was observed. Histopathological analyzes demonstrated an intense diffuse inflammatory process, presenting a larger number of inflammatory cells, a decrease in the number of alveoli and the volume of the star when compared to uninfected animals and animals infected by the avirulent strain. In the lung of animals infected with ATCC 27853 strain, increased expression of mmu-miR-155-5p and mmu-miR-146b-5p, of inflammatory cytokines was observed. The identification of regulatory networks of the miRNAs under study showed important direct and indirect targets associated with the innate immune response that may be compromised during the differential expression of mmu-miR-155-5p and mmu-miR-146b-5p in favor or detriment of P. aeruginosa infection. The results obtained to date suggest that the infection by P. aeruginosa exerts a modulation in the expression of miRNAs and consequently in the immune response against this bacterium and that the virulence of different strains of P. aeruginosa influence the expression of miRNAs mmu-miR- 155-5p and mmu-miR-146b-5p, of proinflammatory cytokines and in pathology. Further studies are needed to uncover the mechanisms by which virulent P. aeruginosa strains can subvert the immune response and ensure replication in its host. / Fundação de Amparo à Pesquisa do Estado de Minas Gerais - FAPEMIG

by Li Sze Kwan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1990. / Bibliography: leaves 234-259. / ACKNOWLEDGEMENT --- p.i / ABSTRACT --- p.ii / CONTENTS : / Chapter CHAPTER 1: --- GENERAL INTRODUCTION --- p.1 / Chapter 1.1 --- Our Environment Is Polluted --- p.1 / Chapter 1.2 --- Heavy Metal Contamination --- p.3 / Chapter 1.3 --- The Effect of Cadmium and Some Related Metals on Environment --- p.5 / Chapter 1.4 --- The Uses of Microorganisms in Cleaning Up Environment --- p.9 / Chapter 1.5 --- Mechanisms of Cadmium Uptake in Cadmium Accumulating Strains --- p.10 / Chapter 1.6 --- Techniques for Cell Immobilization --- p.13 / Chapter 1.7 --- Prospect --- p.20 / Chapter CHAPTER 2: --- ISOLATION OF CADMUIM ACCUMULATNIG MICROORGANISMS --- p.22 / Chapter 2.1 --- Introduction --- p.22 / Chapter 2.2 --- Materials and Methods --- p.25 / Chapter 2.2.1 --- Recipes Used for Growing Various Organisms --- p.25 / Chapter 2.2.2 --- Methods Used for Collecting Organisms to be Tested --- p.27 / Chapter 2.2.3 --- Observation of Samples by Microscope --- p.28 / Chapter 2.2.4 --- Enrichment of Cadmium Resistant Microorganisms --- p.28 / Chapter 2.2.5 --- Selection and Isolation of Cadmium Resistant Microorganisms --- p.29 / Chapter 2.2.6 --- Purification of Microbial Colonies --- p.30 / Chapter 2.2.7 --- Preliminary Classification of Selected Microorganisms --- p.30 / Chapter 2.2.8 --- Screening of Cadmium Accumulating Strains --- p.30 / Chapter 2.2.9 --- Cadmium Analysis --- p.31 / Chapter 2.3 --- Result --- p.32 / Chapter 2.3.1 --- Selection of Cadmium Resistant --- p.32 / Chapter 2.3.2 --- Cadmium Resistance of Isolates --- p.36 / Chapter 2.3.3 --- Screening of Cadmium Accumulating Microorganisms --- p.38 / Chapter 2.4 --- Discussion --- p.39 / Chapter CHAPTER 3: --- GENERAL CHARACTERIZATION OF STRAIN 1000A --- p.43 / Chapter 3.1 --- Introduction --- p.43 / Chapter 3.1.1 --- Various Factors Affecting the Accumulation of Cadmium of Strain 1000A --- p.43 / Chapter 3.1.2 --- Identification --- p.44 / Chapter 3.2 --- Materials and Methods --- p.45 / Chapter 3.2.1 --- "Preparation of Solutions, Antibiotics and Reagents" --- p.45 / Chapter 3.2.2 --- Culture Media Used --- p.47 / Chapter 3.2.3 --- Growth Kenetics Determination --- p.48 / Chapter 3.2.4 --- Determination of the Effect of Cadmium Concentration on Cd-uptake in Free Cells --- p.49 / Chapter 3.2.5 --- Determination of the Effect of Phosphate Concentration on Cd-uptake in Free Cell --- p.49 / Chapter 3.2.6 --- Determination of the Cd-uptake in Free Cells in Continuous Cultures --- p.50 / Chapter 3.2.7 --- Determination of Antibiotic Resistance of Strain 1000A --- p.51 / Chapter 3.2.8 --- Dstermination of Relationship between Chloramphenicol Resistance and Cd-uptake --- p.52 / Chapter 3.2.9 --- Cadmium Analysis --- p.52 / Chapter 3.2.10 --- Determination of Inorganic Precipitation of Cadmium --- p.53 / Chapter 3.2.11 --- Assimilation Tests --- p.54 / Chapter 3.2.12 --- Identification of Strain 1000A --- p.55 / Chapter 3.3 --- Result --- p.55 / Chapter 3.3.1 --- Growth Kinetics of Strain 1000A in Cadmium Supplemented Peptone Medium --- p.55 / Chapter 3.3.2 --- Cd-uptake of Strain 1000A at Various Cadiuin Concentration --- p.65 / Chapter 3.3.3 --- Effect of Phosphate concentration on Cd-uptake of Strain 1000A --- p.65 / Chapter 3.3.4 --- Cd-uptake of Strain 1000A in Continuous Cultures --- p.70 / Chapter 3.3.5 --- Inorganic Precipitation of Cadmium Phosphate --- p.75 / Chapter 3.3.6 --- Determination of Antibiotic-Resistance of Strain 1000A --- p.78 / Chapter 3.3.7 --- Effect of Chloramphenicol on Cd-uptake and Cadmium Resistance of Strain 1000A --- p.82 / Chapter 3.3.8 --- Determination of the Effect of Tetracyclin --- p.85 / Chapter 3.3.9 --- Assimilation Tests --- p.94 / Chapter 3.3.10 --- Identification of Strain 1000A --- p.94 / Chapter 3.4 --- Discussion --- p.97 / Chapter CHAPTER 4: --- DETERMINATION OF CADMIUM UPTAKE MECHANISM IN P. PICKETTI 1000A --- p.102 / Chapter 4.1 --- Introduction --- p.102 / Chapter 4.2 --- Materials and Methods --- p.105 / Chapter 4.2.1 --- Preparation of Solutions and Reagents --- p.105 / Chapter 4.2.2 --- Preparation of Reagents for SDS-PAGE --- p.105 / Chapter 4.2.3 --- Recipes for Growing Cells --- p.107 / Chapter 4.2.4 --- Protein Determination --- p.108 / Chapter 4.2.5 --- Examination of Cadmium Accommodation in P. picketti 1000A by Transmission Electron Microscope --- p.108 / Chapter 4.2.6 --- SDS-polyacrylamide Gel Electrophoretic Determination of Protein Profiles --- p.109 / Chapter 4.2.7 --- Phosphate Assay --- p.111 / Chapter 4.2.8 --- Orthophosphate Estimation --- p.112 / Chapter 4.2.9 --- Sulphide Analysis --- p.112 / Chapter 4.2.10 --- Cadmium Analysis --- p.113 / Chapter 4.2.11 --- Cd-binding Determination through Column Separation --- p.113 / Chapter 4.2.12 --- Cd-binding Determinate ion through SDS Electrophoresis --- p.114 / Chapter 4.2.13 --- Determination of Cadmium Distribution of Cells --- p.115 / Chapter 4.3 --- Result --- p.116 / Chapter 4.3.1 --- SDS-PAGE Determination of Protein Profiles of P. picketti 1000A --- p.116 / Chapter 4.3.2 --- Determination of Cd-binding Protein of P. picketti 1000A --- p.121 / Chapter 4.3.3 --- "Determination of the Relationship of Cellular Cadmium, Sulphide and Phosphate" --- p.131 / Chapter 4.3.4 --- Examination of Cadmium Accumulation of P. picketti 1000A by Transmission Electron Microscope --- p.142 / Chapter 4.3.5 --- Cadmium Distribution of Cadmium-Accommodated Cells --- p.148 / Chapter 4.4 --- Discussion --- p.152 / Chapter CHAPTER 5: --- CORRELATION AMONG METALS IN HEAVY METAL UPTAKE --- p.158 / Chapter 5.1 --- Introduction --- p.158 / Chapter 5.2 --- Materials and Methods --- p.158 / Chapter 5.2.1 --- Preparation of Solutions --- p.159 / Chapter 5.2.2 --- "Determination of Effect of Zn+2 ," --- p.160 / Chapter 5.2.3 --- Determination of Effect of Cu+2 . --- p.161 / Chapter 5.2.4 --- "Correlation among Cd+2, Cu+2 and Zn+2" --- p.161 / Chapter 5.2.5 --- Growth Kenetics Determination --- p.162 / Chapter 5.2.6 --- Cell Sample Preparation --- p.162 / Chapter 5.2.7 --- Orthophosphate Estimation --- p.162 / Chapter 5.2.8 --- Metal Analysis --- p.163 / Chapter 5.3 --- Result --- p.163 / Chapter 5.3.1 --- Effect of Zn+2 --- p.163 / Chapter 5.3.2 --- Effect of Cu+2 --- p.173 / Chapter 5.3.3 --- "Correlation among Cd+2, Cu+2 and Zn+2" --- p.178 / Chapter 5.4 --- Discussion --- p.195 / Chapter CHAPTER 6: --- HEAVY METAL UPTAKE OF IMMOBILIZED CELL --- p.197 / Chapter 6.1 --- Introduction --- p.197 / Chapter 6.2 --- Materials and Methods --- p.199 / Chapter 6.2.1 --- Preparation of Solutions and Medium --- p.199 / Chapter 6.2.2 --- Harvesting of Cells --- p.199 / Chapter 6.2.3 --- Immobilization of Cells --- p.199 / Chapter 6.2.4 --- Determination of the Effect of Temperature --- p.200 / Chapter 6.2.5 --- Determination of Optimum Cell Concentration in Polyacrylamide Gel --- p.201 / Chapter 6.2.6 --- Determination of pH Effect on Cd-uptake --- p.201 / Chapter 6.2.7 --- Pretreatment with 70% Methanol --- p.202 / Chapter 6.2.8 --- Combined Pretreatment with Methanol and NaOH --- p.202 / Chapter 6.2.9 --- Effect of Phosphate on Cd-uptake of Immobilized Cell --- p.202 / Chapter 6.2.10 --- Comparison of Cadmium- and Copper-uptakes in Cells Immobilized in K-carrageenan and in Polyacrylamide --- p.203 / Chapter 6.3 --- Result --- p.204 / Chapter 6.3.1 --- Effect of Temperature on Cd-uptake --- p.204 / Chapter 6.3.2 --- Determination of Optimum Cell Concentration in Polyacrylamide Gel --- p.204 / Chapter 6.3.3 --- Effect of pH on Cd-uptake of Immobilized Cells --- p.207 / Chapter 6.3.4 --- Effect of Methanol on Cd-uptake --- p.210 / Chapter 6.3.5 --- Combined Effect of pH and Methanol on Cd-uptake --- p.213 / Chapter 6.3.6 --- Effect of Phosphate on Cd-uptake of Immobilized Cells --- p.213 / Chapter 6.3.7 --- Comparison between Cadmium- and Copper-uptake of Cells Immobilized in K-carrageenan and in Polyacrylamide --- p.220 / Chapter 6.4 --- Discussion --- p.228 / Chapter CHAPTER 7: --- CONCLUSION --- p.232 / REFERENCES --- p.234

The bronchiectatic lung is a diseased state in which the airways are chronically damaged and dilated. This state is found in the clinical entities of cystic fibrosis and non-cystic fibrosis bronchiectasis. These are two highly relevant chronic suppurative lung diseases in which an understanding of the microbiology of these patients is considered key to appropriate management. This has traditionally been via the use of traditional culture techniques. However, with the development of molecular methodologies, the previously perceived wisdom is being challenged. In both cystic fibrosis and non-cystic fibrosis bronchiectasis, Pseudomonas aeruginosa is considered the most significant pathogen. In CF there has been considerable concern surrounding the risk of transmission of Pseudomonas aeruginosa between patients on the basis of a significant quantity of research into this matter. In contrast, there has been very little research performed into the equivalent risk in non-cystic fibrosis bronchiectasis. In this thesis we describe an extensive single-centre epidemiological review of Pseudomonas aeruginosa spanning both these diseases. Via this we have shown evidence of cross-infection within a non-cystic fibrosis bronchiectasis cohort. This epidemiological review has included multiple genotyping methods including multilocus sequence typing and whole genome sequencing, As an extension of the epidemiological review, we have performed an in silico prediction of hypermutator status from the whole genome sequencing data to provide greater understanding of the likelihood of cross-infection, and have also demonstrated a culture-independent adaption of multilocus sequence typing for potential screening for cross-infection. In addition to Pseudomonas aeruginosa, we have also looked at the wider bacterial community in the lungs of patients with these two conditions via culture-independent techniques. We have shown that whilst Pseudomonas aeruginosa is often an important component, these are clearly complex communities. We have primarily investigated the cohort with non-cystic fibrosis bronchiectasis, but we have demonstrated associations between clinically-relevant markers and complexity of the bacterial communities within the lungs of both these cohorts of patients. Whilst we have used the gold-standard technique of 16S rRNA sequencing, we have also shown the validity of a simple and potentially more feasible profiling technique for standard clinical care. In summary, through the application of culture-dependent and independent molecular techniques, this research has shed light on the epidemiology of Pseudomonas aeruginosa within our respiratory cohorts, and the complexity and clinical relevance of the wider microbial communities within these patients. Such studies are essential if we are to advance our understanding of the bronchiectatic lung and optimise strategies for patient management.

Pseudomonas aeruginosa is extremely harmful to immunocompromised individuals. An atomic force microscope was used to measure the surface forces of this bacteria’s exopolymers. These forces were characterized with the AdG force model, which is a function of brush length, probe radius, temperature, separation distance and an indefinite density variable, s. This last parameter could represent the root spacing or mesh spacing of the exopolymers. This study aims to clarify s by obtaining force values as a function of temperature. The data suggest that s represents the mesh spacing. If s is the root spacing it should remain constant regardless of the changing polymer lengths, on the other hand if it is the mesh spacing it will vary with changing temperature, as shown by the data presented in this research. This knowledge will aid in understanding and characterizing how bacteria cause infections.

Bactérias Gram-negativas não fermentadoras são frequentemente encontradas em águas superficiais, sendo muitas vezes carregadoras de múltipla resistência. O presente trabalho teve como objetivo principal analisar a participação de Pseudomonas sp. e Acinetobacter sp. na manutenção da resistência a antimicrobianos em quatro pontos na laguna de Tramandaí e se a presença deles poderia contribuir para a sua permanência. As amostras de água superficial foram coletadas em quatro pontos de coleta na laguna e submetidas a tratamento com os antimicrobianos: ácido nalidíxico, ceftazidima, imipenem e tetraciclina na concentração de 20mg/L. Em cada ponto de coleta uma das alíquotas não foi suplementada com os mesmos sendo utilizada como controle. Os isolados de Pseudomonas sp. foram identificados por provas bioquímicas e MALDI-TOF MS, enquanto que os isolados de Acinetobacter sp. somente por provas bioquímicas. O perfil de suscetibilidade de ambos foi avaliado pela técnica de disco-difusão e a produção de ESBL pela técnica de disco combinado. Os pontos 3 e 4 foram os que exibiram maior número de isolados resistentes. Os maiores percentuais de resistência estiveram associados às amostras submetidas ao tratamento com antimicrobianos. Em todos os pontos de coleta foram encontrados isolados de P. aeruginosa e Acinetobacter sp. multirresistentes. Ambas amostras (com e sem tratamento) exibiram diferentes padrões de resistência nos diferentes pontos de coleta e tanto isolados de P. aeruginosa como de Acinetobacter sp. exibiram isolados produtores de ESBL. A presença de P. aeruginosa e Acinetobacter sp. multirresistentes na Laguna de Tramandaí atenta para o risco de disseminação de resistência neste ambiente e que o mesmo pode estar atuando como reservatório de resistência. / Non-fermenting Gram-negative bacteria are often found in surface water, and are often carriers of multiple resistance to antimicrobials. The present work had as main objective to analyze the role of Pseudomonas sp. and Acinetobacter sp. in maintaining antimicrobial resistance at four points in the Tramandaí lagoon. The surface water samples were collected at four sampling points in the lagoon and treated with the antimicrobials: nalidixic acid, ceftazidime, imipenem and tetracycline at a concentration of 20mg/L. At each collection point, one of the aliquots was not supplemented with the antimicrobial and were used as the control for the treatment. The isolates of Pseudomonas sp. were identified by biochemical tests and MALDITOF MS, whereas the isolates of Acinetobacter sp. were identified only by biochemical tests. The susceptibility profile of both was evaluated by the disc diffusion method and the ESBL production by the combined disk method. Sampling points 3 and 4 showed the highest number of resistant isolates. The highest percentages of resistance were associated with the samples that were submitted to antimicrobial treatment. In all sampling points, multiresistant P. aeruginosa and Acinetobacter were isolated. Both samples (with and without treatment) showed different resistance patterns within the sampling points. P. aeruginosa and Acinetobacter sp. isolates exhibited ESBL producers. The presence of multiresistant P. aeruginosa and Acinetobacter sp. in the Tramandaí Lagoon attempted to the risk of spreading resistance in the aquatic environment, since it can act as a reservoir of resistance.

In natural habitats bacteria predominantly grow and survive as biofilms, which are densely populated assemblages of cells encased in self-produced matrices. Biofilms face the challenge of resource limitation due to poor substrate diffusion and consumption by cells closer to the periphery. When terminal electron acceptors for metabolism, such as oxygen, are limiting, reducing equivalents accumulate in the cell, leading to an imbalanced redox state and disruption of metabolic processes. The opportunistic pathogen Pseudomonas aeruginosa possesses various redox-balancing strategies that facilitate disposal of excess reducing power, including (i) production of phenazines, redox-active compounds that mediate extracellular electron shuttling; (ii) use of nitrate as an electron acceptor via the denitrification pathway, and (iii) fermentation of pyruvate. However, if the biofilm grows to a point where these metabolic strategies become insufficient, the community adopts a “structural” strategy: the cells collectively produce extracellular matrix to form wrinkle features, which increase surface area and oxygen availability, ultimately oxidizing (i.e., rebalancing) the cellular redox state. Though the broad physiological effects of these metabolic and structural strategies are known, details of their regulation and coordination in biofilm communities have remained elusive. The work presented in this thesis was aimed at elucidating the (cross-)regulation and coordination of different redox-balancing strategies in biofilms of P. aeruginosa strain PA14. Studies described in Chapter 2 demonstrate novel regulatory links between phenazines and microaerobic denitrification, including a redox-mediated mechanism for control of the global transcription factor Anr, which is traditionally thought to be regulated solely by oxygen. This chapter also presents observations of the spatial segregation of denitrification enzymes in a colony biofilm, which is suggestive of metabolic specialization and substrate crossfeeding between different groups of cells. Chapters 3 and 4 describe work examining the physiological functions and regulation of pyruvate and lactate metabolism in P. aeruginosa. These studies were motivated by pyruvate’s role as a “hub” for central metabolism, the unique structural biochemistry of the P. aeruginosa pyruvate carboxylase, and the intriguing complement of “lactate dehydrogenase” genes in P. aeruginosa. These genes include two that encode canonical and non-canonical respiration-linked L-lactate dehydrogenases. My results in Chapter 3 show that the non-canonical L-lactate dehydrogenase gene can substitute for the canonical one to support aerobic L-lactate utilization and that it is induced specifically by the L- enantiomer of lactate. This enzymatic redundancy for L-lactate utilization could be an adaptation that enhances virulence, given that host organisms (e.g. humans and plants) produce L-lactate but not D-lactate. In addition, Chapter 3 includes studies of pyruvate-lactate metabolism in the context of biofilm communities, where aerobic and anaerobic zones coexist in proximity. Evidence is provided that cells in biofilms have the potential to engage in crossfeeding of anaerobically generated D-lactate, which would constitute a new instance of bacterial multicellular metabolism. Finally, Chapter 4 shows that mutants of pyruvate carboxylase, which converts pyruvate to oxaloacetate, have a matrix-overproducing, hyperwrinkling biofilm phenotype indicative of an imbalanced cellular redox state. This result suggests that disruption of pyruvate carboxylase shunts metabolic flow through pyruvate dehydrogenase, converting pyruvate to acetyl-CoA and generating an excess of reducing power. Together, the findings presented in Chapter 3 and 4 underscore the importance of pyruvate metabolism in the contexts of redox homeostasis and community behavior. When metabolic strategies are insufficient to balance the redox state, biofilms can ameliorate the problem of electron acceptor limitation by forming wrinkle structures, which increase the community’s surface area-to-volume ratio. Wrinkle formation depends on the production of extracellular matrix. Matrix production is also required for the formation of pellicles, biofilms that reside at air-liquid interfaces. Experiments described in Chapter 5 investigate properties of the P. aeruginosa matrix from a socio-evolutionary perspective. My results show that matrix production confers a competitive advantage in pellicle biofilms but not in colony biofilms. The evolutionary landscape of matrix production in biofilms is complex and context-specific; i.e., each microenvironment selects for a subset of phenotypes that confers fitness only in that specific microenvironment. Chapter 6 describes the dynamic processes of pellicle formation in the gram-positive bacterium Bacillus subtilis as well as the gram-negative P. aeruginosa in a time-resolved manner. In these two distantly related species, we observed a conserved mechanism for pellicle formation that involves motility, chemotaxis and aerotaxis. These findings indicate that motility is more than just a unicellular behavior: cells collectively migrate to a microniche and initiate biofilm formation. Finally, Appendix A describes efforts to characterize proteinaceous components of the matrix isolated from P. aeruginosa PA14. In conclusion, this work has elucidated mechanistic details of various redox-balancing strategies in P. aeruginosa, particularly from the perspective of multicellular community development.