SOS Racisme est une association antiraciste née en 1984 dont le but, selon ses statuts, est de « faire entreprendre toute action humanitaire susceptible de résoudre les problèmes nés du racisme ». L’association a mobilisé un grand nombre de jeunes, à la fois sur des actions très médiatisées et au travers d’un militantisme de base. Ses responsables eux-mêmes étaient de jeunes militants, certains cependant ayant déjà une expérience politique ou syndicale.Cette association qui peut être qualifiée de mouvement de jeunesse a donc eu, par la pratique surtout, à mettre en action les jeunes qui y militaient ou qui l’animaient. D’où une fonction d’école parallèle qui leur a permis d’acquérir compétences, savoir-faire et aussi réseaux de connaissance. Toutes ré-investissables dans une vie professionnelle. Cette recherche vérifie que les anciens responsables de SOS Racisme, occupent maintenant des fonctions dans les domaines politique et social et qu’ils attribuent en grande partie leur évolution professionnelle aux compétences acquises et mises en œuvre lors de leur prise de responsabilités à SOS Racisme. Ils continuent à intervenir dans le champ social et font le lien entre leurs engagements passés et les actuels. L’étude empirique, appuyée sur la réalisation de quinze entretiens avec d’anciens responsables de SOS-racisme et deux grands témoins de ses activités, entretiens enregistrés, transcrits et analysés, est à la fois portée et enrichie par l’utilisation de notions très présentes dans l’analyse sociologique, en particulier, celles d’habitus, de capital -social, culturel ou militant, de réseau et de compétences. D’une façon générale donc, notre travail est conçu comme une contribution à la connaissance des effets formateurs du militantisme et de leur efficacité ultérieure. / SOS Racisme is an anti-racist association founded in 1984 whose purpose is to "undertake any humanitarian action likely to solve the problems arising from racism". The association has mobilized a large number of young people, both on high-profile actions and through grassroots activism. Its leaders themselves were young activists, some of them however having political or trade union experience.This association, which can be described as a youth movement, has had, by way of practice above all, put into action the young people who militated or animated it. Hence a parallel school function that enabled them to acquire skills, know-how and also networks of knowledge. All re-investable in a professional life.This research verifies that the former SOS Racisme officials now occupy political and social functions and that they largely attribute their professional development to the skills acquired and implemented when taking up their responsibilities at SOS Racisme. They continue to intervene in the social field and make the link between their past and present commitments.The empirical study, based on fifteen interviews with former officials and major witnesses of its activities.

Ras proteins are mutated in 30% of all human tumours contributing to several malignant phenotypes including abnormal cell growth, proliferation and apoptosis. The activity of Ras is controlled by the inter-conversion between GTP- and GDP- bound forms. This conversion is partly regulated by the binding of protein Son of Sevenless (Sos), a guanine nucleotide exchange factor. The mechanism of Ras activation via its interactions with Sos remains unclear making it challenging as an effective drug target. The aim of this work is to use Nuclear Magnetic Resonance (NMR) spectroscopy and other biophysical methods to understand the molecular activation of Ras via its interactions with Sos. In this thesis, the backbone and Cβ, as well as the partial side-chain NMR assignment for human K-Ras•GDP were completed at pH 7.4. We also revealed significant chemical shift differences between apo, GDP and GTPϒS-bound H-Ras states from the TROSY spectra. In addition, the monitoring of shift perturbations for H-Ras reveals several residues that appear to be central in Sos binding and may provide a starting point in the search for possible inhibition sites for future drug design. To gain a further understanding into the binding events of the Ras:Sos complex, we have expressed and purified the Sos construct containing the REM and Cdc25 domains (SosCat) for titration studies. Here, we have implemented a relatively novel approach to study large complexes (Stoffregen et al. 2012), by selectively labelling the [13C-] Met and Ile methyl groups of SosCat. This approach has provided an assignment for eight reporter signals. In addition, monitoring the shift perturbations of Met [13C-] methyls in the NMR spectra allowed us to examine individual residues at the two Ras binding sites (allosteric and catalytic sites) of SosCat. Disruption of H-Ras•GTPγS binding at the allosteric site (via SosCat W729E mutant) significantly weakens the interactions of Ras at the catalytic site. The data suggests a positive co-operative binding mechanism between the allosteric and catalytic sites, which is consistent with the allosteric feedback model. We have also measured the binding affinities of SosCat (by NMR spectroscopy and fluorescence) with wild type and Ras mutants using different GTP analogues. Our 15N-relaxation data of the H-Ras•GTPϒS:SosCat complex reveal dynamical changes in several regions of Ras other than the P-loop, switch I and II regions. In addition, the backbone NMR relaxation studies revealed that a complex between H-Ras•GTPϒS and SosCat proteins is dynamic and transiently formed. The reported work could be a significant step towards understanding the activation of Ras via its interactions with Sos; and in time the data may influence new anti-cancer treatments.

El sistema SOS de Escherichia coli ha sido durante mucho tiempo el modelo de referencia para su estudio en otras especies. Este sistema se encuentra en otros microorganismos incluyendo bacterias gramnegativas, grampositivas y otras. Recientemente se han encontrado diferencias entre las cajas SOS y los genes que integran el regulón SOS de E. coli con respecto a otras especies bacterianas.El propósito de este trabajo ha sido determinar y caracterizar las cajas SOS de dos especies bacterianas, Ralstonia metallidurans y Deinococcus radiodurans, pertenecientes a los grupos b-Proteobacterias y Deinococcus-Thermus, respectivamente.En primer lugar se realizó la clonación y secuenciación de los genes recA y lexA de R. metallidurans, el primero mediante hibridación con una sonda del gen recA de Agrobacterium tumefaciens y el segundo utilizando programas informáticos en los que se usó la secuencia del gen lexA de E. coli para identificar dicho gen en la secuencia parcial del genoma de R. metallidurans. Tras un análisis de sus regiones promotoras, se determinó que ambas contenían un motivo regulador, CTGT-N8-ACAG, idéntico al de E. coli.Se comprobó que esta caja reguladora era funcional tanto en R. metallidurans como en E. coli, evaluando su capacidad de inducir el gen recA frente a lesiones en el DNA. Adicionalmente se determinó que la caja SOS de este microorganismo se encontraba en varios genes que, en E. coli forman parte del regulón SOS, como recA, lexA y un hipotético gen de la familia impB/samB/mucB. En cambio, no se identificó dicha caja en los hipotéticos genes uvrA, ruvAB y dinG, los cuales, en E. coli, también integran el regulón SOS. Mediante el análisis cuantitativo por RT-PCR on-line de los tránscritos se demostró que la expresión de todos estos genes era inducible al lesionar el DNA. Asimismo, mediante ensayos de movilidad electroforética, utilizando extractos proteicos de LexA de E. coli purificada y extractos crudos de R. metallidurans y R. metallidurans LexA(Def), se determinó que la proteína LexA era la responsable de la regulación de los genes recA, lexA y el hipotético gen de la familia impB/samB/mucB. Por el contrario, los hipotéticos genes uvrA, ruvAB y dinG no están bajo el control de la proteína LexA. Por lo tanto, si bien R. metallidurans posee una caja SOS idéntica a la de E. coli, solo algunos de los genes integrados en el regulón SOS de E. coli forman parte de este regulón en R. metallidurans. Además, el hecho de que los hipotéticos genes uvrA, ruvAB y dinG sean inducibles por lesiones en el DNA indica que deben estar sometidos a algún control independiente de LexA.Para determinar la caja SOS de D. radiodurans, se procedió a clonar el gen lexA obteniéndose su secuencia mediante programas informáticos que permiten localizar secuencias de un genoma con un determinado grado de similitud a otras secuencias conocidas. Una vez clonado dicho gen, se sobreexpresó la proteína LexA de este microorganismo y el extracto proteico obtenido se utilizó para realizar ensayos de movilidad electroforética frente al promotor del gen lexA de D. radiodurans, demostrándose que el gen lexA se autorregula. Seguidamente y mediante ensayos de movilidad electroforética se acotó al máximo la región promotora del gen lexA hasta identificar un posible motivo regulador. Mediante mutagénesis dirigida de las diferentes bases de dicho motivo se determinó que la proteína LexA de D. radiodurans reconoce específicamente el palíndromo CTTG-N8-CAAG como motivo de unión, siendo las bases señaladas en negrita las más importantes para la unión proteína-DNA. Finalmente se demostró que otros genes como recA o el hipotético lexA2 no tienen la misma caja SOS ni son regulados por LexA. Se concluye que la proteína LexA de D. radiodurans tiene un motivo regulador diferente a los anteriormente descritos para otros grupos de microorganismos y que debe haber un tipo de regulación diferente a los anteriormente descritos para los genes involucrados en procesos reparativos. / The SOS system in Escherichia coli has been the reference model for its study of other species. This system is present in other microorganisms including gramnegative, grampositive and other bacteria. Lately, some differences have been found between E. coli and other bacterial species in the SOS boxes and genes that compose the SOS regulon.The purpose of this work is to determine and characterize SOS boxes from two bacterial species, Ralstonia metallidurans and Deinococcus radiodurans, belonging to the b-Proteobacteria and Deinococci group, respectively.First, recA and lexA genes from R. metallidurans were cloned and sequenced, the former one by a hybridisation using an Agrobacterium tumefaciens recA gene's probe. The latter one was isolated with the help of informatics' programs using E. coli 's lexA gene' s sequence to find this gene in the partial sequenced genome from R. metallidurans. The promoter regions of both genes were analysed and it was discovered that they have the regulating motif, CTG-N8-ACAG, identical with the one of E. coli.The functionality of this regulating box in R. metallidurans and E. coli was demonstrated by determining the recA gene expression due to damage induction in both species. It was also approved that this SOS box controls the expression of some genes like recA, lexA, and the hypothetical gene of impB/samB/mucB family, which are included in E. coli SOS regulon. In contrast, this SOS box was not identified in other hypothetical genes like uvrA, ruvAB and dinG, which normally belong to E. coli 's SOS regulon. It was demonstrated that all of these genes' expression was induced by DNA damage, using a RT-PCR on-line technique to quantitatively analyse the transcripts. Furthermore, EMSAs (Electrophoretic Mobility Shift Assay), using purified E. coli 's LexA protein and R. metallidurans and R. metallidurans LexA(Def) crude protein extracts, confirmed that the LexA protein was responsible for the regulation of recA, lexA, and the hypothetical gene of impB/samB/mucB family genes. In contrast, hypothetical genes uvrA, ruvAB and dinG are not under the LexA protein control. Therefore, only some genes from E. coli's regulon conform R. metallidurans SOS regulon, even though R. metallidurans has a SOS box identical to E. coli's. Moreover, the fact that hypothetical genes uvrA, ruvAB and dinG are induced by DNA damage indicates that they most likely are under a LexA independent control.To determine the SOS box from D. radiodurans, the lexA gene was cloned. It's sequence was obtained using informatics programs that allow to locate regions from a genome similar to other known sequences. After cloning the lexA gene, the D. radiodurans 's LexA protein was overexpressed, and the protein extract obtained was used to perform EMSAs with the promoter region from the lexA gene of this microorganism. It was proved that this gene regulates itself. Using the same technique, serial deletions of the lexA promoter region were done, identifying a possible regulating motif. Each of the bases conforming the motif were directly mutagenized and it was determined that the D. radiodurans LexA protein recognizes specifically the palindrome CTTG-N8-CAAG as union motif; the bold printed bases are the most important ones for DNA-protein union. Finally, it was shown that other genes like recA or hypothetical gene lexA2 do not have the same box, and that they are not regulated by the LexA protein. In conclusion, D. radiodurans LexA protein has a regulating motif different from the ones formerly known for other bacterial groups. Most likely, it must be a regulation system which is different from those already defined for genes involved in reparation.

Two key proteins, LexA and RecA, are involved in regulation of the SOS expression system in bacteria. LexA and RecA act as the transcriptional repressor and inducer of the SOS operon, respectively. LexA downregulates the expression of at least 43 unlinked genes and activated RecA interacts with the repressor LexA and therefore, LexA undergoes self-cleavage. The ability of the LexA protein to dimerize is critical for its ability to repress SOS-regulated genes in vivo, as the N-terminal domain (NTD) alone has a lower DNA-binding affinity without the C-terminal domain (CTD) and the components for the dimerization of LexA are located in the CTD. Two antiparallel β-strands (termed β-11) in the CTD at the dimer interface of LexA are involved in the dimerization. LexA interacts with the active form of RecA in vivo during the SOS response. It was determined experimentally that monomeric and non-cleavable LexA binds more tightly to RecA and is resistant to self-cleavage. Therefore, we reasoned that if we can produce such LexA mutants we would be able to stabilize the LexA and active RecA complex for crystallization. Therefore, in this experiment, we attempted to make a non-cleavable and predominantly monomeric LexA that interacts intimately with RecA. We produced four single mutations at the dimer interface of the non-cleavable and NTD-truncated mutant of LexA (∆68LexAK156A) in order to weaken the interactions at the interface. The predominant forms of LexA mutants and the affinities of interaction between the mutant LexA proteins and RecA were examined. ∆68LexAK156AR197P mutant was found as predominantly monomeric at a concentration of 33.3 μM both by gel filtration chromatography and dynamic light scattering (DLS) experiments. It also bound RecA more tightly than wild-type LexA. Another mutant, ∆68LexAK156AI196Y, was also found as predominantly monomeric at a concentration of 33.3 μM by DLS. Both these proteins were subjected to crystallization with wild-type RecA protein. We were able to produce some predominantly monomeric LexA with good binding affinity for RecA; however, we were unsuccessful in co-crystallization.

El sistema SOS és una xarxa multigénico induïble davant del dany al DNA. Les seves funcions estan relacionades amb la replicació, reparació del DNA, mutagènesi I control del cicle cel·lular. Aquesta xarxa ha estat caracteritzada per diferents bacteris grampositius i gramnegatius, trobant-se per a tots ells un motiu d'unió del seu repressor, la proteïna LexA.El present treball de Tesi es centra en la caracterització del motiu d'unió de la proteïna LexA a Xylella fastidiosa, Anabaena sp. i Fibrobacter succinogenes. Mitjançant recerques amb el programa TBLASTN, els gens lexA d'aquests microorganismes han estat identificats. Després de procedir a la seva clonació, els productes que codifiquen han estat expressats I purificats mitjançant sistemes d'afinitat a la cua d'histidines o a la GST. Assaigs de mobilitat electroforética i de footprinting utilitzant el promotor de lexA i proteïna purificada, ens han permès definir el motiu d'unió de la proteïna LexA a tots tres microorganismes: TTAGN6TACTA per a X. fastidiosa, RGTACNNNDGTWCB per a Anabaena i TGCNCN4GTGCA per a F. succinogenes.Aquests motius d'unió han estat utilitzats per determinar la composició del reguló LexA a l'ordre Xanthomonadals i als phyla Cianobacteris i Fibrobacter. Aquest estudi ens ha permès descriure una important variabilitat en la composició d'aquests regulons i la presència de gens induïbles davant del dany al DNA de manera independent de LexA a X.fastidiosa.Estudis de mutagènesis dirigida utilitzant les seqüències d'unió de la proteïna LexA a Anabaena i F. succinogenes i estudis filogenètics amb les proteïnes LexA i RecA ens han permès determinar la història evolutiva del motiu d'unió de LexA al Domini Bacteria, demostrant que a la subdivisió 'Alphaproteobacteria' s'ha perdut la còpia del gen lexA heretada verticalment essent la que presenten actualment aquests bacteris una adquisició per transferència horitzontal a partir d'un ancestre d'una cianobactèria o espècie relacionada.Els següents articles donen suport a les dades i conclusions del treball, que ha estat redactat com a compendi d'aquestes publicacions :Campoy, S., Mazón, G., Fernández de Henestrosa, A.R., Llagostera, M., Monteiro, P.B. i Barbé, J. 2002. A new regulatory DNA motif of the gamma subclass Proteobacteria: identification of the LexA protein binding site of the plant pathogen Xylella fastidiosa. Microbiology 148: 3583 - 3597.Mazón G., Lucena J.M., Campoy, S., Fernández de Henestrosa, A.R., Candau, P. i Barbé J. 2004. LexA-binding sequence in Gram-positive and cyanobacteria are closely related. Mol. Gen. Genomics 271: 40 - 49.Mazón, G., Erill, I., Campoy, S., Cortés, P., Forano, E. i Barbé, J. 2004. Reconstruction of the evolutionary history of the LexA binding sequence. Microbiology 150: 3783-3795. / The SOS network is a DNA-damage inducible multigenic network whose functions are involved in DNA replication, DNA repair, mutagenesis and control of cell cycle. This network has been characterized in different gram-positive and gram-negative bacterial species. A binding-motif for their repressor, the LexA protein, has been already determined.The present work focuses in the characterization of the LexA-binding motif of Xylella fastidiosa, Anabaena sp. and Fibrobacter succinogenes. Using TBLASTN searches, their respective lexA genes have been identified and cloned, and their products expressed and purified using histidine-tag and GST-tag systems. Electrophoretic mobility shift assays and foot-printing experiments performed using purified LexA proteins and their lexA promoter fragments revealed the presence of the specific LexA-binding motif for these microorganisms: TTAGN6TACTA for X. fastidiosa, RGTACNNNDGTWCB for Anabaena and TGCNCN4GTGCA for F. succinogenes.These three binding motifs have been used to elucidate the LexA regulon composition in the Order Xanthomonadales and the Cyanobacteria and Fibrobacter phyla, showing an important variability in their regulon composition and the presence of LexA-independent DNA-damage inducible genes in X. fastidiosa.Directed mutagenesis of the Anabaena and F. succinogenes LexA-binding sequences and phylogenetic analyses of LexA and RecA proteins have revealed the evolutionary history of the LexA-binding motif in the Bacteria Domain, with the loss of the vertically inherited lexA gene in 'Alphaproteobacteria' and the presence of a lateral gene transfer in these group resulting in a new lexA copy acquired from a cyanobacterium ancestor or related species.This work is supported on data published in the following papers:Campoy, S., Mazón, G., Fernández de Henestrosa, A.R., Llagostera, M., Monteiro, P.B. i Barbé, J. 2002. A new regulatory DNA motif of the gamma subclass Proteobacteria: identification of the LexA protein binding site of the plant pathogen Xylella fastidiosa. Microbiology 148: 3583 - 3597.Mazón G., Lucena J.M., Campoy, S., Fernández de Henestrosa, A.R., Candau, P. i Barbé J. 2004. LexA-binding sequence in Gram-positive and cyanobacteria are closely related. Mol. Gen. Genomics 271: 40 - 49.Mazón, G., Erill, I., Campoy, S., Cortés, P., Forano, E. i Barbé, J. 2004. Reconstruction of the evolutionary history of the LexA binding sequence. Microbiology 150: 3783-3795.

No description available.

El sistema SOS és una xarxa multigènica sota el control negatiu del repressor LexA, amb importància vital per a la supervivència cel·lular ja que la seva desrepressió es dóna quan la cèl·lula presenta danys en el material genètic que poden ser transmesos a la descendència. Aquest fet, junt amb la seva amplia distribució en el domini Bacteria ens presenten el coneixement del seu funcionament en els diferents grups com una eina filogenètica idònia.Per a tal fi, i com a fil conductor d'aquest treball, és va procedir a l'estudi del reguló LexA a Dehalococcoides ethenogenes, un bacteri verd no sulfurós; Magnetococcus sp. soca MC-1,un bacteri magnetotàctic; i Leptospira interrogans serovar Lai, la primera espiroqueta amb un lexA identificat. Per a tal es va procedir a la clonació i purificació del gen lexA de cada un d'ells i a la purificació del seu producte.En el primer d'ells es va definir la caixa d'unió de LexA (AGAACN4GTTCT), comprovant a partir d'ella els seus vincles amb els bacteris grampositius, així com la no regulació del gen recA, considerat com a canònic, pel mateix.A Magnetococcus sp. soca MC-1, la caixa SOS descrita fou GTTCN7GTTC. Fins el moment, aquest microorganisme es trobava ubicat dins la subclasse Alfa del Proteobacteris, però tant el coneixement del motiu d'unió del seu LexA com els gens que integraven el reguló, ens el mostren com una branca estretament relacionada amb aquesta classe, però clarament independent.A Leptospira interrogans serovar Lai, el resultat fou sorprenent, ja que la caixa SOS d'aquest microorganisme, la varem haver d'identificar a partir de la regió promotora de recA, ja que no era present en la de lexA, esdevenint així el primer exemple en el que LexA no autoregula la seva expressió. Aquesta dada així com el fet de no poder detectar cap altre gen sota el seu control, ens mostren Leptospira, com un pas entremig en la tendència evolutiva seguida en les espiroquetes de perdre el seu gen lexA.Els següents articles es presenten a la secció d'annexes, i en ells es descriuen i discuteixen els resultats sobre els quals està basada aquesta tesis.:I. Fernandez de Henestrosa, A.R., J. Cuñé, I. Erill, J.K. Magnuson, i J. Barbé. 2002. A green nonsulfur bacterium, Dehalococcoides ethenogenes, with the LexA binding sequence found in gram-positive organisms. J. Bacteriol. 184(21): 6073 - 6080.II. Fernandez de Henestrosa, A.R., J. Cuñé, G. Mazón, B.L. Dubbels, D.A. Bazylinski, i J. Barbé. 2003. Characterization of a new LexA binding motif in the marine magnetotactic bacterium strain MC-1. J. Bacteriol. 185: 4471 - 4482.III. Cuñé, J., P. Cullen, G. Mazon, S. Campoy, B. Adler, i J. Barbé. 2005. The Leptospira interrogans lexA gene is not autoregulated. J. Bacteriol. 187: 5841 - 5845.

El sistema SOS és una xarxa multigènica controlada negativament per la proteïna LexA, i està format per un conjunt de gens implicats en el manteniment de la viabilitat cel·lular davant de lesions en el DNA. Aquest sistema es troba en la majoria d'espècies bacterianes, malgrat que existeixen diferències tant en la seqüència d'unió de la proteïna LexA, com en el contingut genètic del reguló. En la present memòria es descriu el sistema SOS de Pseudomonas putida, un bacteri gramnegatiu pertanyent al grup Gamma. Primerament s'han clonat els dos gens lexA, anomenats lexA1 i lexA2, i s'han obtingut els seus productes gènics mitjançant sobreexpressió i purificació per columnes d'afinitat. Ambdues proteïnes s'han utilitzat en assaigs de mobilitat electroforètica (EMSA) amb els promotors de cada un dels gens lexA. Així s'ha pogut identificar la seqüència d'unió de la proteïna LexA1 (CTGTN8ACAG) i de la proteïna LexA2 (AGTACN4GTGCT). Posteriorment, utilitzant RT-PCR, s'ha vist com el gen lexA2 constitueix una única unitat transcripcional amb els gens que el segueixen, formant el casset lexA2-imuA-imuB-dnaE2. Aquest casset s'ha vist que és induïble per danys en el DNA, i que es troba àmpliament distribuït en el domini Bacteria. Seguidament, s'han obtingut dues soques mutants defectives pels gens lexA1 i lexA2, i s'ha analitzat l'expressió gènica de cada una d'elles, respecte la soca salvatge, utilitzant xips de DNA (microarrays). Els resultats obtinguts han demostrat que la proteïna LexA1 controla la majoria de gens del sistema SOS, que a més corresponen amb la resposta convencional del seu grup filogenètic; mentre que la proteïna LexA2 només regula l'expressió de la seva pròpia unitat transcripcional, i la d'un gen (PP3901) pertanyent a un profag resident de P. putida. A més, aquest gen també es troba controlat per la proteïna LexA1, essent l'únic que comparteix les dues regulacions. L'obtenció d'un mutant defectiu pel gen PP3901 ha demostrat que l'expressió d'aquest és necessària per a la transcripció dels gens del profag resident. L'expressió d'aquest profag, però, no origina cap efecte deleteri apreciable sobre el creixement de P. putida. / The SOS system is a multigenic network negatively controlled by the LexA protein, and is composed of a set of genes involved in maintaining cell viability against DNA lesions. This system is present in most bacterial species, despite the existence of differences in the binding sequence of the LexA protein, and in the genetic content of regulon. The present report describes the SOS system of Pseudomonas putida, a Gram negative bacteria belonging to the Gamma proteobacteria group. Firstly we have cloned the two lexA genes, called lexA1 and lexA2 and we have obtained their genetic products through gene overexpression and purification by affinity columns. Both proteins have been used in electroforetic mobility shift assays (EMSA) with the promoters of each of the lexA genes. By this way we could identify the recognition sequence of the LexA1 protein (CTGTN8ACAG) and the LexA2 protein (AGTACN4GTGCT). Subsequently, using RT-PCR, we could see that the lexA2 gene forms a single transcriptional unit with the genes that follow it, forming the cassette lexA2-imuA-imuB-dnaE2. This cassette has been seen that is inducible by DNA damage, and that it is present in many Proteobacteria families. Then, we constructed two defective mutant strains of lexA1 and lexA2 genes, and their gene expression has been analyzed using DNA chips (microarrays). The results have shown that the LexA1 protein controls most of the SOS genes, which also correspond with the conventional response of its phylogenetic group; while LexA2 protein only regulates its own transcriptional unit expression, and a gene (PP3901) belonging to a resident P. putida prophage. In addition, this gene is also controlled by the LexA1 protein, being the only one who shares the two regulations. The construction of a PP3901 defective strain, dempnstrated that the expression of this gene is required for the transcription of the resident prophage genes. However, the expression of the prophage genes, do not cause any significant deleterious effect on the growth of P. putida.

The rapidly increasing emergence of antibiotic resistance amongst pathogenic bacteria is a major clinical and public health problem. The increase in resistant pathogens, accompanied with the small number of new antibiotics introduced in recent years, has limited the number of effective antimicrobials. The classical paradigm suggests that antibiotic resistance emerges by selection for pre-existing mutants in the bacterial population exposed to antibiotics. In contrast, recent data suggested that mutations evolve after cells encounter antibiotic therapy. This kind of mutation is known as adaptive mutation, which is activated by the SOS DNA repair and mutagenesis pathways. Accumulation of single-stranded DNA (ss-DNA) is the signal that induces the SOS response by promoting the formation of the RecA filament, which in turn activates the auto-cleavage activity of LexA and allows expression of SOS genes, including the SOS error-prone polymerases. In this project, phthalocyanine tetrasulfonic acid (PcTs)-based RecA inhibitors were characterized. PcTs molecules were found to potentiate the activity of bactericidal antibiotics and reduce the ability of bacteria to acquire antibiotic resistance mutations. This study highlights the ability of RecA inhibitors to potentiate the activity of antibiotics and provides a strategy for prolonging the life span of existing and newly developed antibiotics. We predicate that RecA inhibitors will be part of an antibiotic “cocktail” that enhances the activity of antibiotics and blocks resistance, which will ultimately prolong antibiotic lifespan.

Two key proteins, LexA and RecA, are involved in regulation of the SOS expression system in bacteria. LexA and RecA act as the transcriptional repressor and inducer of the SOS operon, respectively. LexA downregulates the expression of at least 43 unlinked genes and activated RecA interacts with the repressor LexA and therefore, LexA undergoes self-cleavage. The ability of the LexA protein to dimerize is critical for its ability to repress SOS-regulated genes in vivo, as the N-terminal domain (NTD) alone has a lower DNA-binding affinity without the C-terminal domain (CTD) and the components for the dimerization of LexA are located in the CTD. Two antiparallel β-strands (termed β-11) in the CTD at the dimer interface of LexA are involved in the dimerization. LexA interacts with the active form of RecA in vivo during the SOS response. It was determined experimentally that monomeric and non-cleavable LexA binds more tightly to RecA and is resistant to self-cleavage. Therefore, we reasoned that if we can produce such LexA mutants we would be able to stabilize the LexA and active RecA complex for crystallization. Therefore, in this experiment, we attempted to make a non-cleavable and predominantly monomeric LexA that interacts intimately with RecA. We produced four single mutations at the dimer interface of the non-cleavable and NTD-truncated mutant of LexA (∆68LexAK156A) in order to weaken the interactions at the interface. The predominant forms of LexA mutants and the affinities of interaction between the mutant LexA proteins and RecA were examined. ∆68LexAK156AR197P mutant was found as predominantly monomeric at a concentration of 33.3 μM both by gel filtration chromatography and dynamic light scattering (DLS) experiments. It also bound RecA more tightly than wild-type LexA. Another mutant, ∆68LexAK156AI196Y, was also found as predominantly monomeric at a concentration of 33.3 μM by DLS. Both these proteins were subjected to crystallization with wild-type RecA protein. We were able to produce some predominantly monomeric LexA with good binding affinity for RecA; however, we were unsuccessful in co-crystallization.