Protein-protein interactions (PPIs) across the cell envelope of Gram-negative bacteria are critical for mediating signal transduction pathways that underpin cellular homeostasis. The Ton and Tol Pal systems are two conserved, ancestrally related protein networks that are also required for bacterial pathogenesis. Both Ton and Tol-Pal traverse the periplasm to effect different functions at the outer membrane (OM). Tol-Pal is composed of a homologous complex of three inner membrane proteins, TolQ-TolR-TolA (linked to proton motive force) and two additional periplasmic proteins TolB and Pal. The physiological role of the Tol-Pal system is to stabilise the OM, however the mechanism involved is unknown. TolA is however known to form a crucial protein-protein interaction via its C-terminus with the disordered N-terminus of TolB. Prior to this thesis, determination of the molecular features underlying a protein-protein complex between TolA and an endogenous binding partner TolB had never been accomplished. In this work, I describe the first structure comprising the TolA-TolB complex from Gram negative bacteria. The structure of this complex was determined from Pseudomonas aeruginosa by solution NMR spectroscopy. I determined the interaction between P. aeruginosa TolA and a TolB N terminal peptide to be relatively weak using fluorescence anisotropy. I found that TolB interacts with TolA through an analogous mechanism to that seen in TonB-dependent transporters. Based on these studies and bioinformatics analyses, I hypothesize that the evolutionary resilience of the Tol-Pal system to external pressures is contingent on the preservation of the TolA-TolB interface. Structure-based mutations within the TolA-TolB complex were also evaluated for their effect on in vivo function of the Tol-Pal complex and impact on complex formation in vitro. Taken together, the results demonstrate that protein networks which transduce energy to the OM through PMF-dependent systems in bacterial cells appear to follow a common β-strand augmentation mechanism.

Orientadores: Anete Pereira de Souza, Ricardo Aparício / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T00:47:17Z (GMT). No. of bitstreams: 1 Santos_CleltonAparecidodos_D.pdf: 17722787 bytes, checksum: bfe46a8c0582a66be16a3f8b35fc9ada (MD5) Previous issue date: 2013 / Resumo: Xylella fastidiosa é uma bactéria responsável por inúmeras doenças de plantas em culturas economicamente importantes ao redor do mundo, incluindo a clorose variegada dos citros. Após a infecção de seu hospedeiro, as células de X. fastidiosa é apta a formarem uma estrutura de biofilme que bloqueia os vasos xilemáticos, levando a uma condição de estresse hídrico na planta hospedeira e desencadeando o desenvolvimento da doença. Tendo como estímulo a relevância econômica da citricultura para o Brasil e, visando reduzir os prejuízos provocados pelos problemas fitossanitários que acometem esta cultura, foi realizado um consórcio de pesquisa com o intuito de se conhecer completamente o genoma da linhagem 9a5c de X. fastidiosa. Inúmeras proteínas associadas com patogenicidade, adaptação e sobrevivência bacteriana foram identificadas, incluindo XfDsbC (proteína disulfeto isomerase), Xf5'-Nt (5'-nucelotidase), XfTolB (proteína de translocação B) e XfPal (lipoproteína associada ao peptidoglicano) que foram caracterizadas neste estudo. Empregando ferramentas de caracterização de proteínas, aspectos funcionais e estruturais destas quatro proteínas alvos foram avaliados. Dentre os resultados destaca-se a imunodetecção de XfDsbC, Xf5'-Nt, XfTolB e XfPal durante as diferentes fases de formação e desenvolvimento do biofilme de X. fastidiosa, que é tido como o principal mecanismo de patogenicidade deste fitopatógeno, confirmando a predição inicial de tais proteínas como associadas à patogenicidade bacteriana. Adicionalmente, resultados funcionais e estruturais revelaram detalhes finos do papel biológico desempenhado por cada uma das proteínas estudadas. Juntos, os resultados apresentados neste trabalho contribuem para o melhor entendimento de patogenicidade bacteriana, especialmente com respeito ao fitopatógeno X. fastidiosa / Abstract: Xylella fastidiosa is a plant pathogen bacterium responsible for numerous economically important crops diseases around the world, including the citrus variegated chlorosis. Following the host infection, the X. fastidiosa cells are able to form a biofilm structure which block the xylem vessels, leading to a hydric stress condition in the host plant and triggers the disease development. Given the economic relevance of citriculture for Brazil and in order to reduce the damage caused by phytosanitary problems that affect the citrus production, a research consortium was established with the aim to elucidate the complete genome sequence of the X. fastidiosa 9a5c strain. Numerous proteins associated with bacterial pathogenicity, adaptation and survival have been identified, including XfDsbC (protein disulfide isomerase), Xf5'-Nt (5'-nucleotidase), XfTolB (protein translocation B) and XfPal (peptidoglycan-associated lipoprotein) which were characterized in this study. Using tools for protein characterization, structural and functional aspects of these four protein targets were evaluated. Among the results, we highlight the immunodetection of XfDsbC, Xf5'-Nt, XfTolB and XfPal during the different stages of X. fastidiosa biofilm formation and development which is considered the primary mechanism of pathogenicity of this pathogen. These findings, confirming the initial prediction that relates such proteins as associated with bacterial pathogenicity. Additionally, structural and functional results revealed accurate details of the biological role played by each protein studied. Taken together, the findings presented in this study contribute to a better understanding of bacterial pathogenesis, especially with regard to the plant pathogen X. fastidiosa / Doutorado / Genetica de Microorganismos / Doutor em Genetica e Biologia Molecular

Le système Tol-Pal est un complexe de l’enveloppe d’Escherichia coli composé de CINQ protéines. Les protéines ToIQ, ToIR, TolA forment un complexe dans la membrane interne; la lipoprotéine Pal interagit avec le peptidoglycane avec la protéine périplasmique TolB. Ce système est conservé chez la plupart des bactéries à Gram négatif. Il joue un rôle important dans le maintien de la stabilité de l’enveloppe et dans l’étape tardive de la division cellulaire. Une interaction entre TolA et Pal relie les membranes interne et externe, et dépend des protéines ToIQ, TolR et de la force proton motrice (PMF). Les protéines ToIQ-R-A formeraient un moteur moléculaire utilisant la PMF afin de relier membranes interne, externe et le peptidoglycane. Mon travail a consisté à étudier l’organisation des segments transmembranaires (STs) de TolQ et TolR au sein de la membrane interne d’E. coli en utilisant l’approche expérimentale du « cysteine scanning ». Ainsi, nous avons pu identifier les résidus impliqués dans les interactions entre les STs et améliorer la connaissance de l’organisation moléculaire de ce système. Nous avons aussi démontré la dimérisation du ST de TolRet l’importance de la dynamique dans le fonctionnement de ce moteur. Le système Tol est parasité par certaines toxines (comme les colicines) et par des phages filamenteux. Les colicines sont produites par des souches d’Escherichia coli et active contre les entérobactéries. Je me suis aussi intéressé à l’organisation structurale de la protéine d’immunité de la colicine A. La colicine A forme un canal ionique dans la membrane interne pour tuer la bactérie cible. Les cellules produisant la Colicine A synthétisent également une protéine d’immunité (Cai) qui les protègent de l’action de la colicine A. Par une approche combinant « cysteine scanning » classique et un « anti-cysteinescanning », nous avons pu apporter des informations nouvelles sur l’organisation des quatre STs deCai. Nous avons montré que Cai forme un dimère dans la membrane et que ce dimère se dissocie au contact de sa cible, la colicine A. / The Tol-Pal system is a protein complex of the Escherichia coli cell envelope. It consists offive proteins. The ToIQ, TolR, TolA proteins form a complex in the inner membrane, the lipoproteinPal interacts with the peptidoglycan and with the periplasmic protein TolB. This system is conservedin most Gram-negative bacteria. It plays an important role in maintaining the integrity of the outermembrane and in the late stage of cell division. The interaction between Pal and TolA connects innerand outer membranes and depends on ToIQ, TolR and the proton motive force (pmf). The ToIQ-R-Aproteins are suspected to form a molecular motor using pmf to connect the inner and outermembranes and the peptidoglycan. The first aim of my work was to study the organization of thetransmembrane helices (TMHs) of E. coli TolQ and TolR using the cysteine scanning approaches. Weidentified residues involved in the interactions between the TMHs and improved the knowledge ofthe molecular organization of this system. We have also demonstrated the dimerization of the TMHof TolR and the importance of its dynamic movement in the system. The second aim of my work wasto analyze the structural organization of the immunity protein to colicin A. The colicins are producedby certain strains of E. coli and are active against other Enterobacteriaceae. The colicin A forms anion channel in the bacterial inner membrane which kill the bacteria. It hijacks the Tol system to enterin the cell. Cells producing colicin A also synthesize the colicin A immunity protein (Cai) whichprotects the producing cells against the action of colicin A. The approaches combining "cysteinescanning" and "anti-cysteine-scanning”, we found that Cai form a dimer in the membrane whichdissociates upon contact with its target, the colicin A

Rhizobium leguminosarum is a Gram negative nitrogen-fixing soil bacterium. Due to the limited bioavailability of iron, bacteria utilize siderophores that scavenge and bind available iron. The transport of iron-siderophore complexes is achieved by the TonB-ExbB-ExbD complex. We have previously shown that a functional TonB protein is necessary for iron transport by creating ΔtonB mutants and assessing their growth and 55Fe-siderophore transport ability. We attempted to identify and characterize the roles of putative exbB and exbD genes using a similar approach. Growth curves and sequence analyses suggest putative exbB and exbD may be the tolpal-associated genes tolQ and tolR. Phenotypic and sensitivity assays showed mutants do not exhibit the characteristic tol phenotype and are not sensitive to detergents or changes in ionic strength of the growth medium. We also expressed and purified the 120 amino acid fragment of the TonB C-terminus for further physical and chemical characterization.