No description available.

Pseudomonas aeruginosa is found in numerous environments and is an opportunistic pathogen affecting those who are immunocompromised. Its large genome encodes tremendous metabolic and regulatory diversity that enables P. aeruginosa to adapt to various environments. We are interested in how P. aeruginosa senses and responds to the host-derived compounds, carnitine and acylcarnitines. Acylcarnitines can be hydrolyzed to carnitine, where the liberated carnitine and its catabolic product glycine betaine can be used as osmoprotectants, for induction of the virulence factor phospholipase C, and as sole carbon, nitrogen, and energy sources. P. aeruginosa is incapable of de novo synthesis of carnitine and acylcarnitines and therefore imports these compounds from exogenous source. Short-chain acylcarnitines are imported by the ABC transporter CaiX-CbcWV. Medium- and long-chain acylcarnitines are hydrolyzed extracytoplasmically and the liberated carnitine is transported through CaiX-CbcWV. Once in the cytoplasm, short-chain acylcarnitines are hydrolyzed by the L-enantiomer specific hydrolase, HocS. The transcriptional regulator CdhR is divergently transcribed from the carnitine catabolism operon and we have identified the upstream activating region, the binding site sequence, and essential residues required for CdhR binding and induction of the carnitine operon. Carnitine catabolism is repressed by glucose and glycine betaine at the transcriptional level. Furthermore, using two different cdhR translational fusions we show that CdhR enhances its own expression and that GbdR, a related transcription factor, contributes to cdhR expression by enhancing the level of basal expression. These studies are the first to determine the mechanism of O-acylcarnitine transport, metabolism, and the regulation of these processes, which contribute to utilization of these compounds for P. aeruginosa survival in diverse environments.

Bacterial communities engage in social activities, exhibiting behaviors such as communicating with small signaling molecules (quorum sensing [QS]) and building antibiotic-resistant biofilms. The opportunistic human pathogen Pseudomonas aeruginosa produces both freely diffusible QS molecules, as well as a QS molecule that is packaged or transported across cell membranes via the production of outer membrane vesicles. Despite the ubiquity of vesicle production in bacteria, the mechanism of outer membrane vesicle production has not been fully elucidated. In addition, most of our understanding of QS and biofilm formation arises from in vitro studies of bacterial communities containing large numbers of cells, often with greater than 10⁸ bacteria. However, many bacterial communities are comprised of small, densely packed aggregates of cells (≤10⁵ bacteria), and it is unclear how group behaviors and chemical interactions take place in densely packed, small populations. This dissertation has two main goals: i) to provide insights into the mechanism of bacterial membrane vesicle production, and ii) to understand how population size and the spatial distribution of cells affect cell-cell interactions and the nutritional microenvironment within a small (≤10⁵ bacteria) prokaryotic community. / text

Thesis (Ph. D.)--University of Wisconsin--Madison, 1952. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 74-78).

La lysine décarboxylase (LDC) appartient à une famille d'enzymes décamériques dépendantes du cofacteur PLP qui sont connus pour catalyser la réaction transformant la L-Lysine en cadavérine tout en consommant un proton. Dans les entérobactéries comme Escherichia coli, nous trouvons deux paralogues, LdcI et LdcC. LdcI permet de faire face à la bactérie au conditions hostile de pH acide lors du passage à travers du tract gastro-intestinale. LdcC est produite pendant la phase stationnaire et aussi quand les bactéries font face aux traitements antibiotiques. La cadavérine produite par les LDCs est connue pour protéger les bactéries du stress oxydant. Cela s’explique par le fait que la cadavérine bloque les porines de la membrane externe, réduisant ainsi la perméabilité des molécules responsables du stress acides et oxydant. L'activité des LDCs chez E. coli est coordonnée avec la réponse stringente qui est mise en place lorsque les microorganismes sont dans des conditions pauvres en nutriments, afin d’éviter l’épuisement intracellulaire de la L-Lysine nécessaire pour la synthèse des protéines. Cependant, cette inhibition peut être levée par la formation d'un complexe en forme de cage avec son partenaire RavA, permettant ainsi aux bactéries de faire face aux stress multiples. Etant donné que la réponse au stress est importante pour que les bactéries puissent exhiber leur pathogénicité, nous nous sommes demandés si la bactérie opportuniste Pseudomonas aeruginosa pourrait employer LdcA pour contrer des conditions de stress qui ont déjà été décrites pour LdcI chez les entérobactéries. Au cours de ma thèse, nous avons abordé cette question en utilisant différentes approches complémentaires. Tout d'abord, nous avons utilisé des fusions promoteur-gène et de l'analyse par Western-blot pour déterminer les conditions dans lesquelles le gène ldcA a été exprimé et sa protéine synthétisée. Nous avons pu observer que ldcA est exprimé sur la phase stationnaire de croissance dans des conditions aérobies en milieux riches et également pendant des conditions anaérobies de respiration avec nitrate. Nous avons également confirmé que l'expression de ldcA est régulée par ArgR et elle est induite complètement lorsque l’acide aminé L-arginine est présente dans le milieu de croissance. Même si nous avons trouvé que les conditions de stress n'induisent pas l'expression de ldcA, nous avons obtenu de nouvelles données suggérant que d'autres mécanismes de régulation tels que le système de quorum sensing dépendant des quinolones (PQS) pourraient être impliqués dans l'expression de ldcA. En utilisant des souches mutantes de ldcA et son complémentée, nous avons évalué si LdcA était impliqué dans la réponse au stress acide et oxydatif. Bien que les données obtenues à l'aide des expériences dans notre laboratoire et des technologies à haut débit (Biolog) aient révélé que LdcA ne présente pas les mêmes fonctions que LdcI, nous avons découvert que la cadavérine produite par LdcA est nécessaire pour la croissance en milieu minimal avec L- Glutamate comme source de carbone. Nous avons également examiné si la présence de LdcA modifie la résistance aux antibiotiques et nous montré que les rends moins persistants face aux carbenicillines. Enfin, en combinant l'analyse phylogénétique et structurelle, nous avons découvert que LdcA appartient à un sous-groupe différent de LDCs bactériennes. Les alignements de séquences montrent que les résidus clés nécessaires pour lier le ppGpp ne sont pas présents dans le site de liaison prédit ce qui a été confirmer par l'analyse biochimique. Notre travail montre que, malgré le fait que LdcA catalyse la même réaction enzymatique et partage les mêmes caractéristiques structurelles que LdcI et LdcC, elle ne joue pas le même rôle que ses homologues. Son rôle est lié aux effets physiologiques de la cadavérine et à la relation entre la L-lysine et le catabolisme de la L-arginine. / The lysine decarboxylase (LDC) belongs to a family of decameric PLP-dependent enzymes that catalyse the reaction transforming L-Lysine into cadaverine while consuming a proton. They are known to be involved in polyamine metabolism and during acid and oxidative stress responses.In enterobacteria like Escherichia coli, two paralogs are present, LdcI and LdcC. LdcI takes part in acid stress response by buffering bacterial cytoplasm. LdcC is produced during stationary phase and also when bacteria face fluoroquinolone treatment. The cadaverine produced by LDCs is known to scavenge reactive oxygen species (ROS) and is capable of blocking outer membrane proteins, thus reducing the permeability of molecules responsible for acid and oxidative stresses. The activity of the LDCs from E. coli is coordinated with the stringent response (nutrient starvation) in order to prevent intracellular L-Lysine depletion. The stringent response signal molecule ppGpp is able to bind directly to LDCs and inhibit their enzymatic activity. However, the inhibition of the LdcI can be prevented by the formation of a cage-like complex with its partner RavA allowing bacteria to face the challenge of both acid and nutrient stresses.Since mechanisms allowing bacteria to counter stress challenges are important for displaying full virulence, we wondered if the opportunistic bacterium Pseudomonas aeruginosa could be using LdcA to counter stress conditions that have already been described for LdcI in enterobacteria. During my PhD, we addressed this question by using different but complementary approaches.First of all, we used promoter-gene fusions and western-blot analysis to determine the conditions in which ldcA was expressed and its product synthesized. We could observe that ldcA is expressed on stationary phase under aerobic conditions in rich media and also during nitrate-respiring anaerobic conditions. As previously described in literature, we also confirmed that ldcA expression is regulated by ArgR and fully induced when L-Arginine is present in the growth medium. Even though we found out that acid and oxidative stress conditions do not induce the expression of ldcA, we obtained new data suggesting that other regulation mechanisms such as the quinolone signal system (PQS) could be involved in ldcA expression.In paralell, we constructed an ldcA mutant and its complemented strain to understand whether LdcA was involved in acid and oxidative stress response. Although the data obtained by using manual screenings and high-throughput technologies (Biolog) revealed that LdcA is not displaying the same functions as LdcI, we discovered that the cadaverine produced by LdcA is needed for full growth fitness when growing in minimal medium using L-glutamate as carbon source. Since slow growing phenotypes are linked to heightened bacterial persistence and because cadaverine has been shown to reduce the persisters population, we also examined if the presence of LdcA is modifying the amount of persisters during carbenicillin treatment. Our data has confirmed that this is indeed the case.Finally, by combining phylogenetic and structural analysis, we discovered that LdcA belongs to a different subgroup of bacterial LDCs. Sequence alignments show that key residues needed for binding ppGpp are not present in the predicted binding site which also suggests that the enzymatic activity is not inhibited by this molecule. And biochemical analysis has confirmed that this is indeed the case as it is the case for Arginine decarboxylases.Our work shows that, in spite of the fact that LdcA catalyses the same enzymatic reaction and shares the same structural fold than LdcI and LdcC, it is not implicated in acid stress or oxidative stress responses. Its role is linked to physiological effects of cadaverine and to the relationship between L-lysine and L-Arginine catabolism.

Fusobacterium nucleatum is a saccharolytic Gram-negative anaerobic organism belonging to the so-called ‘orange complex’ which is believed to play an important role in the microbial succession associated with the pathogenesis of periodontal disease. Its genome contains niche-specific genes shared with the other inhabitants of dental plaque, which may help to explain its ability to survive and grow in the changing environmental conditions experienced in the gingival sulcus during the progression from health to disease. The pH of the gingival sulcus increases during the development of periodontitis and is thought to occur by the metabolism of nutrients supplied by gingival crevicular fluid. Studies have shown that F. nucleatum is partly responsible for the rise in pH and have concluded that in comparison to other plaque inhabitants, F. nucleatum has the greatest ability to neutralise acidic environments. In common with a number of other oral bacteria, F. nucleatum has also been shown to produce intracellular polyglucose (IP) from simple sugars such as glucose, galactose and fructose. Its response and adaptation to stressful environmental conditions such as pH is unknown. The overall aim of this study was, therefore, to determine how F. nucleatum copes with environmental stresses induced by pH changes. F. nucleatum was grown by continuous culture in a chemically defined medium at a growth rate corresponding to those measured in vivo. The effect on protein expression, and IP synthesis was examined during steady-state growth at high (>7.2<7.8) or low pH (pH 6.4). The present study also investigated the response of F. nucleatum to growth at pH 8.2. It was found that the organism grew as a biofilm and this corresponded with an increase in cellular hydrophobicity and decreased IP levels. Optimal growth pH’s differed between the different sub-species used in this study. In response to pH stress, F. nucleatum changed its amino acid and glucose utilisation and increased IP synthesis at the expense of cell numbers. Pulsing the chemostat with glutamic acid or serine produced an increase in IP synthesis and the pattern of end-products observed was dependent upon the amino acid being fermented. The effect on IP synthesis in response to increased levels of exogenous fermentable amino acids was also compared during concomitant fructose or glucose fermentation. Growth media containing fermentable amino acids and supplemented with fructose produced higher cell numbers and non-detectable levels of IP compared to media containing glucose. The differential expression of cytoplasmic- and cell envelope-proteins induced by changes in pH were identified by two-dimensional gel electrophoresis. The results represent the first proteomic investigation of F. nucleatum. Twenty-two cytoplasmic proteins were found to have altered expression in response to external pH. At low (sub-optimal) pH, proteins associated with the generation of ATP and ammonia were up-regulated, the latter contributing to the alkalinisation of the gingival sulcus. Conversely, neutral to alkaline pH conditions led to the upregulation of enzymes involved in energy storage. The study also identified several proteins associated with iron limitation and fatty acid synthesis which might not otherwise have been identified as part of the pH-dependent response. In response to growth at pH 7.8, 14 cell envelope proteins were identified as having significantly altered expression. Down-regulated proteins included those associated with uptake of C4 di-carboxylates and phosphorus, a potential membrane protease and an enzyme associated with amino acid fermentation. The up-regulation of a transcriptional regulator linked to the repression of sugar metabolism was also reported along with proteins linked to the transport of iron. The periplasmic chaperone, peptidyl prolyl cis trans isomerase, which is responsible for the folding of outer membrane proteins, was also found to be up-regulated. In conclusion, the proteomic investigation of protein expression by F. nucleatum identified gene products which form part of the organism’s coordinated stress response to changes in environmental pH. In addition to these, the physiological based studies also presented help to explain the organism’s persistence during the transition from health to disease in vivo. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1339503 / Thesis (Ph.D.) - University of Adelaide, Dental School, 2008

No description available.

Pseudomonas aeruginosa is a metabolically versatile environmental bacterium that grows in extremely diverse habitats—from sea water to jet fuel—and is able to infect a large variety of organisms. It is a significant cause of human disease and is one of the most frequent healthcare-associated infections. P. aeruginosa uses a sophisticated gene regulatory network to adapt its growth strategy to these diverse environmental niches and the fluctuating conditions it encounters therein. The las and rhl “quorum sensing” (QS) intercellular communication systems play integral roles in this regulatory network and control the expression of factors important to the bacterium’s ecological fitness, including many secreted factors involved in nutrient acquisition, microbial competition, and virulence. These QS systems use diffusible acyl-homoserine lactone (AHL) signalling molecules to infer environmental parameters, including bacterial population density, and to coordinate behaviour across bacterial communities. This dissertation describes an investigation into the relationship between QS and small molecule primary metabolism, using metabolomic methods based on nuclear magnetic resonance spectroscopy and mass spectrometry. Analysis of extracellular metabolic profiles (the bacteria’s “metabolic footprint”) established that QS can modulate the uptake and excretion of primary metabolites and that this effect was strongest during the transition from exponential to stationary phase cell growth. Analysis of the cellular metabolome and proteome demonstrated that QS affected most major branches of primary metabolism, notably central carbon metabolism, amino acid metabolism and fatty acid metabolism. These data indicate that QS repressed acetogenesis and the oxidative C02-evolving portion of the TCA cycle, while inducing the glyoxylate bypass and arginine fermentation. QS also induced changes to fatty acid pools associated with lower membrane fluidity and higher chemical stability. Elevated levels of stress-associated polyamines were detected in QS mutants, which may be a consequence of a lack of QS-dependent adaptations. These findings suggest that wild-type QS directs metabolic adaptations to stationary phase stressors, including oxidative stress. Previous work, including several transcriptomic studies, has suggested that QS can play a role in primary metabolism. However, there has been no previous study of the global impact of AHL QS on the metabolome of P. aeruginosa. Research presented here demonstrates that QS induces a global readjustment in the primary metabolism and provides insight into QS- dependent metabolic changes during stationary-phase adaptation.

The alarming rise of antibacterial drug resistance and the dwindling supply of novel antibiotics highlight the need for innovative approaches in combating bacterial infections. Traditionally, antibacterial drug discovery campaigns have largely been conducted in rich media. Such growth conditions are not representative of the host environment and render many metabolic pathways, otherwise needed for survival and infection, dispensable. Such pathways have been overlooked in conventional drug discovery campaigns despite their validity as potential antibacterial targets. The work presented in this thesis focuses on the development and validation of a screening strategy for the identification and mechanism of action determination of novel inhibitors of metabolic pathways in bacteria under nutrient-limited conditions. This screen led to the identification of MAC168425, MAC173979 and MAC13772 as inhibitors that target glycine metabolism, p-aminobenzoic acid biosynthesis and biotin biosynthesis, respectively. Moreover, it established this approach as a general platform that can be applied for different organisms with synthetic or natural product libraries. Additional mechanistic studies of the biotin biosynthesis inhibitor, MAC13772, resulted in solving the crystal structure of BioA in complex with MAC13772. Analysis of the co-structure confirmed our proposed mode of inhibition and provided information for strategies for rational drug design. Investigation of the antibacterial activity of MAC13772 revealed its potency against a number of pathogens. Furthermore, we show how MAC13772 acts synergistically with rifampicin in clearing growing mycobacterial cultures. The potential of this inhibitor as a lead for preclinical pharmacokinetic studies and for antibacterial drug development is discussed. We also discuss our current efforts to develop a metabolomic platform for the characterization of novel antibacterials that can be used in concert with our current approach to chart the metabolic response of bacteria to chemical perturbants and to generate testable hypotheses regarding the mode of action of novel inhibitors of bacterial metabolism. / Thesis / Doctor of Philosophy (PhD)

A pouca solubilidade do ferro impõe desafios para sua captação por bactérias e outros organismos. Uma solução eficaz para este problema é a utilização de sideróforos próprios ou exógenos para solubilizar o ferro do ambiente ou de proteínas do hospedeiro e transportá-lo para o interior da célula bacteriana. Neste trabalho, identificamos vias de produção e captação de ferro por sideróforos e definimos o papel destas moléculas na virulência da bactéria Chromobacterium violaceum, um abundante componente da microbiota de solo e água que ocasionalmente causa graves infecções em humanos. Por meio de análises in silico, vários genes relacionados com a síntese e captação de sideróforos foram encontrados no genoma de C. violaceum ATCC 12472 em dois clusters de síntese de metabólitos secundários. Obtenção de linhagens mutantes de vários destes genes e caracterização destas linhagens por ensaios de CAS, curvas de crescimento em carência de ferro e ensaios de estimulação de crescimento revelaram que C. violaceum produz sideróforos endógenos. Essa produção mostrou-se dependente do percursor comum 2,3-DHBA produzido pelas enzimas codificadas pelos genes entCEBA (CV_1485-84-83-82) e de duas enzimas sintetases de peptídeo não ribossomais (NRPSs CV_1486 e CV_2233), as quais provavelmente montam dois sideróforos distintos do tipo catecolato. Cada sideróforo foi captado por um receptor dependente de TonB (RDTB) específico, com o sideróforo produzido via NRPS CV_1486 sendo captado pelo RDTB CV_1491, e o sideróforo produzido via NRPS CV_2233 sendo captado pelo RDTB CV_2230, uma vez que mutantes sem esses RDTBs acumularam sideróforos no meio externo no ensaio de CAS. Além de seus sideróforos endógenos, C. violaceum foi capaz de utilizar xenosideróforos do tipo catecolato de outras bactérias via o RDTB CV_1491. Ensaios de infecção em camundongos revelaram que tanto a síntese quanto a captação de seus sideróforos endógenos são importantes para a virulência de C. violaceum, pois as linhagens mutantes que não produzem sideróforos (?CV_1485-84- 83-82, ?CV_1485-84-83-82/1486::pNPT e ?CV_1486/2233::pNPT) ou são incapazes8 de captá-los (?CV_2230/1491) tiveram sua virulência diminuída em relação a linhagem selvagem. Os dados mostrando que o mutante que não capta ambos sideróforos de C. violaceum teve atenuação mais acentuada da virulência e induziu menor produção de NET em ensaios com neutrófilos in vitro sugerem que o acúmulo de sideróforos na infecção pode ser benéfico para o hospedeiro. Por fim, demonstramos a possibilidade de gerar mutantes de transposon em C. violaceum e ao realizarmos varredura de uma coleção destes mutantes identificamos ao menos um potencial novo fator de transcrição envolvido na regulação da síntese de sideróforo nesta bactéria. Portanto, os dados obtidos neste trabalho revelaram que C. violaceum utiliza-se de diferentes sideróforos endógenos para captação de ferro e que estas moléculas são importantes para seu estabelecimento no hospedeiro. / The low solubility of iron imposes challenges for its uptake by bacteria and other organisms. An effective solution to this problem is the use of own or exogenous siderophores to solubilize the iron from environmental or host sources and transport it into the bacterial cell. In this work, we identified pathways for production and uptake of siderophores, and we defined the role of these molecules in virulence of the bacterium Chromobacterium violaceum, an abundant component of the microbiota of soil and water, which occasionally causes serious infections in humans. By performing an in silico analysis, we found several genes related with synthesis and uptake of siderophores in the genome of C. violaceum ATCC 12472 within two secondary metabolite biosynthesis gene clusters. Obtaining mutant strains from several of these genes and characterizing these strains by CAS assays, growth curves under iron deficiency and growth stimulation assays revealed that C. violaceum produces endogenous siderophores. This production was shown to be dependent on the common precursor 2,3-DHBA produced by the enzymes encoded by the genes entCEBA (CV_1485-84-83-82) and on two non-ribosomal peptide synthetase enzymes (NRPSs CV_1486 and CV_2233), which probably build two distinct catecholate siderophores. Each siderophore was picked up by a specific TonB-dependent receptor (RDTB), with the siderophore produced via NRPS CV_1486 being picked up by RDTB CV_1491, and the siderophore produced via NRPS CV_2233 being picked up by RDTB CV_2230, since mutants without those RDTBs accumulated siderophores in the external environment in the CAS assays. In addition to its endogenous siderophores, C. violaceum was able to use catecholate-type xenosiderophores from other bacteria via the RDTB CV_1491. Infection assays in mice revealed that both the synthesis and the uptake of its endogenous siderophores are important for the virulence of C. violaceum, since mutant strains that do not produce siderophores (?CV_1485-84-83- 82, ?CV_1485-84-83- 82/1486 :: pNPT and ?CV_1486/2233 :: pNPT) or are unable to uptake them (?CV_2230/1491) had their virulence decreased relative to the wild type strain. The data showing that the mutant strain unable to uptake both siderophores of10 C. violaceum had more pronounced attenuation of virulence and induced lower NET production in in vitro neutrophil assays suggest that the accumulation of siderophores in the infection may be beneficial to the host. Finally, we demonstrated the possibility of generating transposon mutants in C. violaceum, and by screening a collection of these mutants we identified at least one potential novel transcription factor involved in the regulation of siderophore synthesis in this bacterium. Therefore, the data obtained in this work revealed that C. violaceum uses different endogenous siderophores for iron uptake and that these molecules are important for its establishment in the host.