Chromobacterium violaceum é uma bactéria de vida livre, móvel, que habita fontes de água e solos pobres de regiões tropicais e subtropicais. Nestes habitats, em que a concentração de fosfato é baixa, o regulon PHO encontra-se ativado. No laboratório, esta bactéria é capaz de crescer razoavelmente bem, apesar da limitação de fosfato, atingindo um rendimento celular similar ao observado na abundância deste nutriente. Mutações nos genes pstS, phoU, phoA1 e phoA2 foram construídas. As mutações pstS e phoU não causaram qualquer alteração no padrão de expressão da fosfatase alcalina, sugerindo que estes genes não participam da repressão dos genes de PHO. Porém, o mutante pstS mostrou-se deficiente na captação de Pi. Os mutantes phoA1 e phoA2 apresentaram, cada um, severa redução na atividade da fosfatase alcalina. Foi investigada a possibilidade de PhoA1 e PhoA2 formarem uma proteína heterodimérica. Fusões transcricionais entre phoU e phoB ao gene lacZ, revelaram que estes genes respondem à carência de Pi. Surpreendentemente, C. violaceum se mostrou pouco resistente a estresses ambientais. / Chromobacterium violaceum is a free-living, mobile bacterium that inhabits water sources and soils of tropical and subtropical regions, where the phosphate concentration is low. The PHO regulon is activated in response to low phosphate concentration in the environment. Surprisingly, the growth yield of C. violaceum under phosphate excess or under phosphate limitation is very similar. Mutations in pstS, phoU, phoA1 and phoA2 genes were constructed. The expression of alklaine phosphatase was not affected by the phoU and pstS mutations, suggesting that these genes do not participate in the repression of the PHO regulon. However, the pstS mutant was deficient in the uptake of Pi. The phoA1 and phoA2 mutants presented each one a severe reduction in alkaline phosphatase activity. The hypothesis that PhoA1 and PhoA2 form a heterodimeric protein was investigated. Transcriptional fusions between the promoters of phoB and phoU to lacZ showed that these genes respond to Pi starvation. Stress resistance assays showed that C. violaceum is generally sensitive to environmental stresses.

Chromobacterium violaceum é uma bactéria de vida livre, móvel, que habita fontes de água e solos pobres de regiões tropicais e subtropicais. Nestes habitats, em que a concentração de fosfato é baixa, o regulon PHO encontra-se ativado. No laboratório, esta bactéria é capaz de crescer razoavelmente bem, apesar da limitação de fosfato, atingindo um rendimento celular similar ao observado na abundância deste nutriente. Mutações nos genes pstS, phoU, phoA1 e phoA2 foram construídas. As mutações pstS e phoU não causaram qualquer alteração no padrão de expressão da fosfatase alcalina, sugerindo que estes genes não participam da repressão dos genes de PHO. Porém, o mutante pstS mostrou-se deficiente na captação de Pi. Os mutantes phoA1 e phoA2 apresentaram, cada um, severa redução na atividade da fosfatase alcalina. Foi investigada a possibilidade de PhoA1 e PhoA2 formarem uma proteína heterodimérica. Fusões transcricionais entre phoU e phoB ao gene lacZ, revelaram que estes genes respondem à carência de Pi. Surpreendentemente, C. violaceum se mostrou pouco resistente a estresses ambientais. / Chromobacterium violaceum is a free-living, mobile bacterium that inhabits water sources and soils of tropical and subtropical regions, where the phosphate concentration is low. The PHO regulon is activated in response to low phosphate concentration in the environment. Surprisingly, the growth yield of C. violaceum under phosphate excess or under phosphate limitation is very similar. Mutations in pstS, phoU, phoA1 and phoA2 genes were constructed. The expression of alklaine phosphatase was not affected by the phoU and pstS mutations, suggesting that these genes do not participate in the repression of the PHO regulon. However, the pstS mutant was deficient in the uptake of Pi. The phoA1 and phoA2 mutants presented each one a severe reduction in alkaline phosphatase activity. The hypothesis that PhoA1 and PhoA2 form a heterodimeric protein was investigated. Transcriptional fusions between the promoters of phoB and phoU to lacZ showed that these genes respond to Pi starvation. Stress resistance assays showed that C. violaceum is generally sensitive to environmental stresses.

The Pho regulon is controlled by the PstSCAB transporter, PhoU, and the two-component proteins, PhoB and PhoR. PhoU is a negative regulator of the Pho regulon under phosphate-replete conditions. How PhoU functions is unknown. Many PhoU homologues are found widely throughout prokaryotic domains. There are several conserved amino acid residues in the PhoU protein. It is hypothesized that these residues play an important role in the function of PhoU. To test this hypothesis, several site directed mutations in the phoU gene have been produced with single amino acid changes in conserved residues. After testing these mutants, it was found that some of the mutants abolished repression of the Pho regulon while other mutants had little or no effect. Further study of these mutants and their phenotypes will reveal more about how PhoU functions and help to better understand bacterial signaling in general.

Bordetellen sind Gram-negative Kokkobazillen, die phylogenetisch zu den β-Proteobakterien zählen und in der Familie der Alcaligenaceae eingeordnet sind. Der bedeutendste Vertreter der Gattung, die nach heutigem Kenntnisstand neun Arten umfasst, ist Bordetella pertussis, der Erreger des Keuchhustens. Der Keim ist obligat humanpathogen und besitzt zahlreiche Virulenzfaktoren, um die Epithelzellen des Respirationstraktes zu besiedeln und zu zerstören, wodurch es zu dem charakteristischen Krankheitsverlauf kommt. Neben B. pertussis werden noch B. bronchiseptica und B. parapertussis dem sogenannten B. bronchiseptica-Cluster zugeteilt. Alle Vertreter des B. bronchiseptica-Clusters sind in der Lage, bei verschiedenen Wirtsspezies respiratorische Erkrankungen mit unterschiedlichem Schweregrad auszulösen. Dabei weist B. bronchiseptica ein breiteres Wirtsspektrum auf und kann Atemwegserkrankungen in einer Vielzahl von Säugetieren auslösen, wohingegen B. parapertussis vornehmlich Schafe und Menschen infiziert und bei letzteren eine schwächere Form des Keuchhustens bewirkt. Das Hfq-Protein wurde ursprünglich als Wirtsfaktor identifiziert, welcher für die Replikation des RNA-Phagen Qβ in Escherichia coli benötigt wird (host factor for Qβ oder HF-1). Es ist in Struktur und Funktion homolog zu den Sm-Proteinen aus Eukaryoten, die am Splicing von mRNAs involviert sind. Die Beteiligung des Hfq-Proteins an regulatorischen Vorgängen, die durch kleine nicht-kodierende RNAs (sRNAs) vermittelt werden, wurde erstmals in einer Studie zum Mechanismus der rpoS-Regulation durch die kleine regulatorische RNA OxyS ersichtlich. Seitdem konnte für eine Vielzahl an sRNAs gezeigt werden, dass sie an Hfq gebunden vorliegen und die Hilfe des Proteins bei der post-transkriptionellen Kontrolle ihrer Ziel-mRNAs benötigen. In dieser Hinsicht übernimmt Hfq die Rolle eines RNA-Chaperons, indem es trans-kodierte sRNAs stabilisiert und die Basenpaarung mit ihren Ziel-mRNAs fördert. Dabei beeinflusst die Bindung der sRNA-Regulatoren an ihre Ziel-mRNAs deren Translation, sowohl aktivierend als auch inhibierend. Bislang wurden Hfq-Homologe in der Hälfte aller sequenzierten Gram-positiven und Gram-negativen Bakterienarten gefunden. Eine BLAST-Analyse ergab, dass B. pertussis und B. bronchiseptica Homologe zum Hfq-Protein aufweisen und diese in der veröffentlichten Genomsequenz bereits als Hfq-Protein annotiert sind. Fokus dieser Arbeit war weitestgehend, die Funktion des Hfq-Proteins in B. pertussis und vergleichend in B. bronchiseptica zu charakterisieren. Mittels Primer Extension-Analyse konnte zunächst der Startpunkt des hfq-Transkripts in B. pertussis und B. bronchiseptica unter logarithmischen Wachstumsbedingungen bestimmt werden. Dieser Startpunkt war zudem unter stationären Wachstumsbedingungen und nach Hitzestress aktiv, was in Diskrepanz zur Beobachtung in E. coli steht. Ferner konnte festgestellt werden, dass die hfq-Transkription nach Induktion verschiedener Stressformen in beiden Organismen erhöht war. Nach Generierung der jeweiligen Δhfq-Mutanten in beiden Organismen wurden diese charakterisiert. Die B. pertussis Δhfq-Mutante zeigte ein deutliches Wachstumsdefizit gegenüber dem Wildtyp, im Gegensatz zu B. bronchiseptica Δhfq, die sich im Wachstum wie der Wildtyp verhielt. Beide Mutanten zeigten sich sensitiver gegenüber H2O2-Stress als der Wildtyp, nicht jedoch gegenüber weiteren oxidativen Stressbedingungen oder Membranstress induzierenden Substanzen. Die Δhfq-Mutante in B. pertussis war zudem in ihrer Fähigkeit zur Biofilmbildung beeinträchtigt, was jedoch nicht für B. bronchiseptica Δhfq galt. Da Hfq an sRNA-mRNA-Interaktionen, welche die Translation der mRNAs beeinflussen, beteiligt ist, sollte über 2D-Gelelektrophorese das Hfq-regulierte Proteom in B. pertussis und B. bronchiseptica bestimmt werden. Auffällig war, dass viele periplasmatische Transport-bindeproteine von der Δhfq-Mutation betroffen waren. Es zeigten sich aber auch Stoffwechselenzyme und wichtige Housekeeping-Faktoren, wie z. B. der Elongationsfaktor EF-Tu und das Chaperon GroEL, in der Δhfq-Mutante dereguliert. Generell scheint das Hfq-regulierte Proteom in B. pertussis und B. bronchiseptica nur einen kleinen Teil des gesamten Proteoms auszumachen. Zudem ist das Hfq-regulierte Proteom variabel zwischen verschiedenen Wachstumsbedingungen, aber auch zwischen den beiden Organismen trotz der engen Verwandtschaft. Die Expression ausgewählter Virulenzfaktoren zeigte keinen Unterschied zwischen Δhfq-Mutante und B. pertussis-Wildtyp. / Bordetellae are Gram-negative coccobacilli phylogenetically belonging to the β-group of proteobacteria and therein to the family of Alcaligenaceae. The most prominent member of the genus comprising nine species so far is Bordetella pertussis, the etiological agent of whooping cough. This organism is an obligatory human pathogen and expresses a variety of virulence factors in order to colonize and destroy the epithelial cells of the respiratory tract causing the characteristic symptoms of the disease. In addition to B. pertussis, B. bronchiseptica and B. parapertussis are assigned to the so-called B. bronchiseptica-cluster. All members of the B. bronchiseptica-cluster have the ability to cause respiratory symptoms with varying severity. B. bronchiseptica exhibits a broad host range causing respiratory symptoms in a variety of mammals, whereas B. parapertussis infects sheep and humans causing a milder form of whooping cough in the latter. The Hfq protein was originally identified as a host factor necessary for the replication of the RNA-phage Qβ in Escherichia coli (host factor for Qβ or HF-1). It is functionally and structurally homologous to Sm-proteins involved in splicing of mRNAs in eukaryotes. The involvement of Hfq in regulatory processes caused by small non-coding RNAs (sRNAs) was first recognized in a study on the mechanism of rpoS-regulation by the small regulatory RNA OxyS. Since then a variety of sRNAs were shown to be bound to Hfq and require its help for post-transcriptional control of their target-mRNAs. In this regard, Hfq functions as an RNA-chaperone by stabilizing trans-encoded sRNAs and their basepairing to target-mRNAs. Binding of the sRNA-regulators to their target-mRNAs thereby either activates or inhibits their translation. To date Hfq homologues were identified in half of all sequenced Gram-positive and Gram-negative bacterial species. BLAST analysis revealed that B. pertussis and B. bronchiseptica possess an Hfq homologue which has already been annotated as such in the published genome sequence. The main focus of this work was to characterize the function of the Hfq protein in B. pertussis as well as in B. bronchiseptica. By primer extension analysis we could identify the start of the hfq-transcript in B. pertussis and B. bronchiseptica under logarithmic growth conditions. This transcriptional start site was also active under stationary growth conditions and after heat shock which is discrepant from the observations in E. coli. Furthermore, it could be shown that the hfq-transcription was elevated in both B. pertussis and B. bronchiseptica under various stress conditions. Δhfq-mutants were established and characterized in both organisms. The Δhfq-mutant of B. pertussis exhibited a pronounced growth deficit in comparison to the wildtype whereas the Δhfq-mutant of B. bronchiseptica showed the same growth properties as the wildtype. Both Δhfq-mutants expressed a higher sensitivity to stress caused by H2O2 compared to the wildtype. However, there was no increased sensitivity of the Δhfq-mutants to other oxidative stress agents or membrane stress inducing agents. Furthermore, the Δhfq-mutant of B. pertussis but not the Δhfq-mutant of B. bronchiseptica was impaired in its ability to form biofilms. Since Hfq is involved in sRNA-mRNA-interactions affecting the efficient translation of mRNAs, the Hfq-regulated proteome of B. pertussis and B. bronchiseptica was determined by 2D-gelelectrophoresis. Strikingly, a variety of periplasmic binding proteins involved in transport were affected by the Δhfq-mutation. In addition, enzymes of various metabolic pathways and important housekeeping factors, such as elongation factor EF-Tu and the protein chaperone GroEL, were deregulated in the Δhfq-mutant. The Hfq-regulated proteome comprises generally only a small part of the complete proteome in B. pertussis and B. bronchiseptica. Furthermore, this Hfq-regulated proteome differs between certain growth conditions as well as between the two closely related organisms. No difference could be observed in the expression of selected virulence factors between B. pertussis Δhfq and wildtype.

Le Quorum sensing (QS) est un système de communication bactérienne capable de réguler divers processus cellulaires qui dépendent de la densité de la population microbienne. Chez les bactéries à Gram négatif, cela se produit par production de molécules de signalisation auto-inductrices (AI), les acyl homosérine lactones (AHL). La libération d´AHL à l'extérieur de la cellule est détectée par la population bactérienne provoquant en réponse la régulation de l'expression de certains gènes (régulon QS).Notre laboratoire a étudié et identifié un système QS fonctionnel dans la souche Acidithiobacillus ferrooxidans ATCC 23270T. En outre, nous avons montré que des analogues synthétiques d´AHL modulent l´adhésion d´At. ferrooxidansT sur un substrat minéral, tels des coupons de soufre.Dans ce projet de recherche, nous nous proposons d'identifier les gènes qui sont régulés par le QS chez At. ferrooxidansT, en particulier ceux impliqués dans la biogénèse du biofilm. Notre hypothèse est que des analogues synthétiques d’AHL induisent le système QS. Ainsi, nous nous proposons de moduler l´adhésion sur substrat minéral grâce à l'utilisation de ces molécules. L'utilisation de ces AHL permettra de caractériser le régulon QS dans cette souche bactérienne.L'identification d'analogues synthétiques d´AHL qui favorisent l'adhésion à des coupons de soufre nous a permis d'étudier le transcriptome d´At. ferrooxidansT dans des conditions où le régulon QS est stimulé. Puces à ADN d´At. ferrooxidansT avec/sans ces analogues synthétique d´AHLs nous a permis de caractériser le régulon QS et les gènes impliqués dans la biogénèse du biofilm dans les conditions utilisées. / Quorum sensing (QS) is a bacterial communication system capable of controlling several cellular processes dependent on the density of the microbial population. In Gram-negative bacteria, it occurs mainly through the production by bacteria of small diffusible signaling molecules, termed autoinducers (AI), of the acyl homoserine lactones type (AHLs). The release of AHLs outside the cell is detected by the bacterial population generating the regulation of the expression of several genes (regulon QS).Our laboratory has studied and identified a functional QS system in the Acidithiobacillus ferrooxidans ATCC 23270T type strain. Besides, by using synthetic analogs of AHLs, we have shown that AHL-type QS molecule analogs modulate adhesion of At. ferrooxidansT to minerals, such as sulfur coupons. In this research, we propose to identify the genes that are regulated by QS in At. ferrooxidansT, particularly those that are associated with biofilm formation. For this, we propose to modulate the adhesion of At. ferrooxidansT to mineral substrate through the use of a synthetic AHL analog. Our working hypothesis postulates that AHLs molecules induce the QS system, and that their use will allow the characterization of the QS regulon of this bacterial strain by transcriptomic analysis.The identification of synthetic AHLs improving adherence of At. ferrooxidansT on sulfur coupons allowed us to study the transcriptome of this organism in conditions in which QS regulon is stimulated. DNA microarrays of At. ferrooxidansT with/without one of these AHLs synthetic analogues allowed us to identify the QS regulon and to determine genes involved in biofilm formation.

The PstSCAB2 complex serves the dual function of being a phosphate transporter as well as the primary sensor of phosphate for the Pho regulon. PhoU is an integral protein required for the signal from PstSCAB2 to be transmitted to PhoR. Our hypothesis is that conformational changes of PstSCAB2 during the phosphate transport process are the mechanism by which information about environmental phosphate levels are transduced to the cell. Additionally, we propose that direct protein-protein interactions between PhoU and the alternating conformations of PstSCAB2 mediate PhoU interactions with PhoR. By means of genetic and biochemical approaches, we have found substantial evidence supporting both these hypotheses.

Quorum sensing is a bacterial signaling system that controls gene expression in a population density-dependent manner. In Gram-negative proteobacteria, the cell density control of luminescence was first observed in the symbiotic marine bacterium Vibrio fischeri and this system is one of the best studied quorum sensing systems. Two-dimensional sodium dodecyl sulfate-polyacrylamide (2D-SDS) gel electrophoresis analysis previously identified several non-Lux proteins in V. fischeri MJ-100 whose expression was dependent on LuxR and 3-oxo-hexanoyl-L-homoserine lactone (3-oxo-C6-HSL). A lacZ reporter was used to show that the promoters for qsrP, acfA, and ribB were directly activated via LuxR-3-oxo-C6-HSL in recombinant Escherichia coli. The sites of transcription initiation were established via primer extension analysis. Based on the position of the lux box-binding site near position â 40, all three promoters appear to have a class II-type promoter structure. Real-time reverse transcription-PCR was used to study the temporal expression of qsrP, acfA, and ribB during the exponential and stationary phases of growth, and electrophoretic mobility shift assays were used to compare the binding affinities of LuxR to the promoters under investigation. In order to fully characterize the LuxR regulon in V. fischeri ES114, microarray analysis was performed in the Greenberg lab (University of Washington) and 18 LuxR-3-oxo-C6-HSL regulated promoters were found including 2 genes (qsrP and acfA) identified previously in MJ-100 in addition to the well-studied lux operon. In collaboration with them, full-length purified LuxR protein was used to show direct interaction between the LuxR protein and 7 genes/operons newly identified out of 13 genes/operons examined. The binding affinity between LuxR proteins and those genes was also measured. Based on the sequence of the lux boxes of the known genes regulated by LuxR and LitR, a position specific weight matrix (PSWM) was created and used to search through the intergenic regions of the V. fischeri ES114 genome. Some potential LuxR and LitR-regulated genes with high score were tested experimently to confirm direct activation. For the LuxR regulon, these possible LuxR-regulated promoters were cloned into a lacZ reporter and tested for their LuxR dependence. Beyond the genes found in microarray, the promoter of the intergenic region VFA0658-0659 was found to be activated by LuxR and 3-oxo-C6-HSL. For the LitR regulon, two LitR-regulated genes found in the microarray were also identified using PSWM and confirmed by real-time PCR to be dependent on LitR for expression. EMSA experiments showed that LitR can specifically bind to the litR boxes of LitR-regulated genes, litR and VF0170 which confirmed that the regulation is direct. / Ph. D.

Brucella abortus and Agrobacterium tumefaciens are pathogenic bacteria that infect animals and plants, respectively. These bacteria are genetically similar and are found within the same Class, Alphaproteobacteria, and Order, Rhizobiales, of the domain Eubacteria; however, they survive and replicate in vastly different environmental niches. In Order to adapt to different environments, bacteria utilize several mechanisms of gene regulation to tightly control gene expression. Two of these mechanisms include transcriptional regulators and small regulatory RNAs (sRNAs), which can activate and repress gene expression through various interactions with DNA, mRNA, and proteins. A well-conserved transcriptional regulator among the Rhizobiales is VtlR, a virulence-associated transcriptional LysR regulator. The objectives of this dissertation were three fold: 1) characterize the known regulon of VtlR in B. abortus with regards to gene regulatory function and virulence, 2) determine the regulon of VtlR in A. tumefaciens and define the mechanism by which this regulation occurs, and 3) define the role of an ABC-type transport system indirectly regulated by VtlR in B. abortus that putatively imports the non-proteinogenic amino acid gamma-aminobutyric acid (GABA). VtlR was characterized in B. abortus as a virulence-associated transcriptional regulator that directly activates four genes: the sRNA AbcR2, and the three small hypothetical proteins BAB1_0914, BAB2_0512, and BAB2_0574; and deletion of vtlR led to a significant defect in the ability of B. abortus to cause infection in vitro and in vivo. Since dysregulation of abcR2 alone could not account for the defect in virulence, it was hypothesized that one or all three hypothetical proteins could be responsible for a virulence phenotype observed in ΔvtlR. This turned out to not be the case, as a deletion of the entire VtlR regulon displayed no difference in virulence compared to the parental strain. Further characterization of the small hypothetical proteins is outlined in Chapter 2 and the data revealed bona fide translation of each small protein, and the deletion strain of the VtlR regulon displayed a growth defect when grown in the presence of the sugar fucose. This phenotype was subsequently observed in ΔvtlR as well. This led to the identification of a putative fucose transport and metabolism locus in B. abortus that has yet to be studied. In A. tumefaciens, VtlR is necessary for proper attachment to plant cells and biofilm formation and regulates over 200 genes, significantly more than the four genes VtlR regulates in B. abortus. The mechanism by which this occurs was unknown, and the relationship between VtlR and AbcR1 or AbcR2 was uncharacterized. The data in Chapter 3 outline the VtlR network by showing that VtlR regulation of myriad genes in A. tumefaciens is primarily indirect via the direct regulation of a few sRNAs. This direct interaction was shown experimentally and a VtlR binding box was identified in the A. tumefaciens genome. This project outlines the divergence of a regulatory element between phylogenetically related organisms that occupy different environmental niches. The AbcR sRNAs are conserved throughout the Rhizobiales and regulate numerous ABC-type transport systems within these bacteria. In A. tumefaciens, one of these transport systems specifically transports the amino acds proline and GABA. B. abortus contains homologs of this system, which led to the hypothesis that the brucellae may also transport GABA but for a yet unknown purpose. The data in Chapter 4 revealed that B. abortus also transports GABA in vitro and this transport is under the regulation of AbcR1 and AbcR2. This transport was increased under extreme nutrient limitations and was uninhibited by the presence of other amino acids. Metabolic studies showed GABA is not utilized by B. abortus under aerobic conditions, and transcriptomic data revealed increased expression of several loci in the presence of GABA. Altogether, this study uncovers a putative signaling role for the amino acid GABA that has been understudied in bacterial pathogens that infect animal hosts. Overall, the work presented in this dissertation is focused on further elucidating the biological role of downstream regulatory targets of both VtlR and the sRNAs AbcR1 and AbcR2 in the related organisms Brucella abortus and Agrobacterium tumefaciens. Findings show that while there are similarities between the two systems, there are also many differences that may be attributed to the vastly different lifestyles of each organism. / Doctor of Philosophy / Brucella abortus and Agrobacterium tumefaciens are two highly related bacterial pathogens that infect mammals and plants, respectively. Although genetically related, both organisms survive and replicate in vastly different environmental niches with one living in the soil (i.e., A. tumefaciens) and the other living within immune cells of the infected host (i.e., B. abortus). In Order to quickly adapt to changing environmental conditions, the bacteria must rapidly control gene expression through multiple regulatory mechanisms. The works presented in this dissertation will focus on further characterizing one of these regulatory systems and comparing the homologous systems shared by B. abortus and A. tumefaciens. This includes uncovering a putative sugar transport and metabolism system, as well as discovering the potential for host-pathogen signaling via the well-studied neurotransmitter GABA.

A habilidade de sensoriar o ambiente extracelular e responder às suas mudanças é inerente para a maioria das bactérias. As concentrações de nutrientes direcionam os processos metabólicos relacionados à sobrevivência e proliferação. O fosfato inorgânico (Pi) é um dos nutrientes cuja regulação, sensoriamento e sinalização são bastante conservados em bactéria. Um dos mecanismos de captação do íon fosfato com alta afinidade é o sistema Pst, um transportador do tipo ABC (ATP-Binding Cassette) , localizado na membrana interna das células. Este transportador, juntamente com as proteínas PhoR/PhoB que formam um sistema de dois componentes (Two-Component Regulatory System) , são capazes de sensoriar e monitorar os níveis deste íon nas células. Ambos os sistemas pertencem ao chamado regulon Pho, conjunto de genes envolvidos no transporte, captação e metabolização do fosfato. Estudos tem mostrado que a interação entre os sistema Pst e o sistema doiscomponentes PhoR/PhoB é mediada pela proteína PhoU, um regulador negativo cujo gene encontra-se no mesmo operon do transportador. Apesar de muito estudados em Escherichia coli , poucas informações existem sobre as características destes sistemas em Xanthomonas citri subsp. citri , bactéria responsável pelo cancro cítrico e de grande importância econômica para o país. Estudos realizados pelo nosso grupo mostraram que X. citri conserva a maioria dos genes descritos como pertencentes ao regulon Pho, incluindo o sistema Pst, as proteínas PhoR/PhoB e PhoU. Este trabalho, portanto, tem como objetivos, a caracterização funcional e estrutural da proteína PhoU de X. citri e a análise da possível interação de PhoU com a proteína PhoR, a histidina quinase do sistema dois componentes. Para tal, as proteínas foram expressas em linhagens de E. coli Tuner e purificadas por cromatografia de afinidade a metal, seguida de exclusão molecular. Visando a caracterização biofísica e estrutural da proteína PhoU, foram realizados ensaios de dicroísmo circular, cristalização, análises de bioinformática e modelagem molecular. Os resultados de bioinformática mostraram que PhoU conserva características estruturais e funcionais quando comparada com ortólogos. Após sua purificação, a proteína foi produzida na sua forma enovelada e mostrou interação com ligantes, conforme descrito na literatura para ortólogos. A expressão da proteína PhoR também foi obtida e ensaios de pull-down foram realizados para a caracterização da interação entre PhoU-PhoR. Adicionalmente, foram realizados estudos de expressão das proteínas em diferentes condições de cultivo, utilizando-se anticorpos policlonais anti-PhoU e anti-PhoR. Os resultados apresentados neste projeto são de grande importância uma vez que se obteve a padronização dos processos de produção de ambas as proteínas e ensaios biofísicos e estruturais para a futura caracterização do complexo, o que será de grande relevância para a compreensão do papel destes sistemas na fisiologia da bactéria. / The ability to sensor the extracellular environment and respond to its changes is inherent to most bacteria. Nutrient concentrations direct metabolic processes related to survival and proliferation. Inorganic phosphate (Pi) is one of the nutrients whose regulation, sensing and signalling are quite preserved in bacteria. One of the mechanisms for phosphate ion uptake with high affinity is the Pst system, composed by an ABC transporter (ATP-Binding Cassette), located on the inner membrane of the cells. This transporter, along with the PhoR/PhoB proteins, which form a Two Component Regulatory System, are capable of sensing and monitoring the levels of phosphate in cells. Both systems belong to the called regulon Pho, set of genes involved in phosphate transport, uptake and metabolism. Studies have shown that the interaction between the Pst system and the two component PhoR/PhoB system is mediated by the PhoU protein, a negative regulator, whose gene is located in the same operon of Pst system. Although much studied in Escherichia coli , there are few information about of these systems in Xanthomonas citri subsp. citri , the major causative of citric canker. Studies conducted by our group showed that X. citri conserves most of the genes described as belonging to regulon Pho, including the Pst system, the proteins PhoR/PhoB and PhoU. This work, therefore, aimed at performing functional and structural characterization of the X. citri PhoU protein and analyzing the possible interaction of PhoU with the PhoR protein, the histidine kinase of the Two Component System. For this, the proteins were expressed in E. coli Tuner strains and purified by metal affinity chromatography, followed by size exclusion chromatography. Aiming at the biophysical and structural characterization of the PhoU protein, we performed circular dichroism, crystallization, bioinformatics and molecular modeling. The results of bioinformatics showed that PhoU retains structural and functional characteristics when compared with orthologs. After purification, the protein was produced in its folded form and showed interaction with ligands, as described in the literature for orthologs. Expression of the PhoR protein was also obtained and Pull Down assays were performed for the characterization of the interaction between PhoUPhoR. In addition, protein expression studies were carried out under different culture conditions using polyclonal anti-PhoU and anti-PhoR antibodies. The results presented in this project are of great importance, once the standardization of the production processes of both proteins has been obtained, as well as biophysical and structural information. These information will be important for future characterization of the complex, which will be of great relevance for the understanding of the role of these systems in the physiology of bacteria.

Ce travail vise à comprendre les mécanismes dinduction du développement chez les Streptomyces. Chez ce genre bactérien, le facteur de transcription DasR a été identifié comme le premier régulateur global à la charnière entre le métabolisme primaire et le métabolisme secondaire, capable de percevoir l'état nutritionnel de l'environnement et d'adapter en conséquence la réponse des différents processus associés au développement (Rigali et al., 2006 et Rigali et al., 2008). La première ambition de cette thèse de doctorat a été d'évaluer l'ampleur du régulon DasR chez S. coelicolor afin d'obtenir une liste - exhaustive et appuyée par des critère de fiabilité - des gènes ciblés par ce régulateur global. L'hypothèse à la base de ce travail de prédiction était qu'au sein du régulon DasR se trouvaient peut-être des gènes de fonction encore inconnue mais essentiels au développement. Comprendre le rôle de ces protéines pouvait déboucher sur l'élucidation de nouvelles voies d'induction du développement et donc peut-être de nouvelles voies à exploiter pour induire le réveil des gènes cryptiques. La première démarche visant à répondre à ce premier objectif nous a amené à constater les lacunes des programmes web de prédiction des régulons existant, notamment le manque de flexibilité au niveau des critères de recherche et au niveau de l'exploitation des résultats obtenus, ainsi que l'absence de moyen permettant d'estimer la fiabilité de la prédiction. C'est pourquoi, dans un premier temps, nous nous sommes lancés le défi de créer un nouvel outil de prédiction des régulons procaryotiques qui répondrait davantage aux attentes des biologistes. Ce programme, nommé PREDetector (Prokaryotic Regulatory Elements Detector), a été réalisé en collaboration avec le Professeur Louis Wehenkel (Department of Electrical Engineering and Computer Science, Université de Liège) (Hiard et al., 2007). Les caractéristiques et les possibilités offertes par PREDetector sont expliquées et illustrées dans le premier chapitre des résultats. Le second chapitre des résultats - ainsi que l'annexe de cette thèse - sont quant à eux dédiés à l'exploitation de ce programme et la caractérisation de l'ensemble des cibles de DasR prédites chez S. coelicolor. Ensuite, et toujours motivé par un souci de présenter un travail fiable pour d'ultérieures investigations, une comparaison des régulons DasR prédits chez différents streptomycètes et autres actinomycètes (prédictions et démarches détaillées dans l'annexe de thèse), vient appuyer notre définition du noyau dur du régulon DasR - conservon DasR - déduit pour l'organisme modèle S. coelicolor. Enfin, nous passons à l'étape d'utilisation de ces données bioinformatiques avec l'étude de gènes/protéines dont l'expression est dépendante de DasR dans le but de peut-être établir de nouvelles connexions entre le métabolisme primaire et le développement chez la souche modèle S. coelicolor. De manière intéressante, le régulon DasR de S. coelicolor comprend plusieurs gènes codant pour des systèmes de transport ABC de sucre. A l'instar du PTSGlcNAc, ces transporteurs situés à l'interface cellule/environnement peuvent aussi être les senseurs de molécules cruciales pour l'induction du développement. Après le PTSGlcNAc, nous nous sommes donc concentrés sur l'étude du premier transporteur ABC de sucre régulé par DasR - en termes de fiabilité des prédictions et de conservation inter-espèces/-genres -, c'est-à-dire, le système DasABC. Dans le troisième chapitre des résultats nous démontrerons ainsi que DasA est une protéine impliquée dans le processus de différenciation morphologique de S. coelicolor (Colson et al., 2008).