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The accessory gene regulator (agr) quorum-sensing system is one of the major regulators of virulence factor production in the pathogen Staphylococcus aureus. Activation of the system depends on the production and sensing of a cyclic peptide signal called the autoinducing peptide (AIP). The biosynthesis of AIP depends on the coordinated action of the AgrB integral membrane endopeptidase and SpsB signal peptidase to process the peptide precursor AgrD into the final signal structure. The primary goal of this dissertation was to gain further insight on the role of AgrD and AgrB in the AIP biosynthesis mechanism. Studies in Chapter II were undertaken to better understand the role of AgrD domains in AgrB-mediated processing. A series of truncation and site-directed mutagenesis studies identified key residues in the AgrD C-terminus that were essential for AgrB processing and AIP production. In parallel, genetic manipulation of the N-terminal leader and AIP-encoding sequence revealed a role for these segments in AIP processing. For the first time, a complex of AgrD covalently linked to AgrB was identified, supporting proposals that this intermediate is an important precursor to AIP production. In Chapter III structure-function studies were performed on AgrB to gain further insight into the AIP biosynthetic mechanism. Initially, the agrBD genes were subjected to random mutagenesis and screened for deficiencies in AIP production. Single-site mutations at 20 different residues within AgrB and another 14 in AgrD were isolated. Interestingly, new mutations in the AgrD N-terminal leader were identified that affect AIP biosynthesis at different steps. In AgrB, most of the mutations blocked peptidase activity, but charge alterations to the K129-K131 region were defective in a later pathway step, separating the peptidase function from AIP ring formation and transport. To localize the AgrB mutations, we reevaluated the membrane topology using the substituted cysteine accessibility method. Our new model predicts four transmembrane helices and a reentrant loop, with both termini located outside of the cell. Finally, co-immunoprecipitation studies indicate that AgrB forms oligomeric structures within the membrane. Taken together, these findings provide a better understanding of the functional role of specific AgrD and AgrB regions in AIP biosynthesis.

O tratamento de infecções por Staphylococcus aureus tem sido problemático devido ao surgimento de cepas resistentes a múltiplios antibióticos. O antibiótico de escolha para o tratamento de infecções por S. aureus resistente a oxacilina é o glicopeptídeo vancomicina. Desde o primeiro isolamento de cepas com sensibilidade reduzida a vancomicina (VISA) em 1997, tem havido crescente preocupação com a disseminação da resistência a este antibiótico. Os mecanismos moleculares que levam à resistência de baixo nível a vancomicina ainda não foram elucidados. A detecção deste fenótipo na rotina de laboratório clínico é laboriosa, pois as técnicas disponíveis são de difícil execução e interpretação. Até agora, não há relato de transmissão horizontal de infecção por VISA, e todas as cepas com este fenótipo foram isoladas de pacientes que faziam o uso prolongado de vancomicina. Uma deficiência no locus regulador de genes acessórios (agr) foi postulado como fator de risco para a aquisição do fenótipo VISA por uma cepa sensível a este antibiótico. Para este estudo, foram selecionadas 47 cepas de S. aureus, com sensibilidades variadas a vancomicina, inclusive 5 cepas VISA isoladas no Brasil. Determinou-se nas cepas as concentrações inibitórias mínimas de vancomicina e oxacilina, a atividade hemolítica em ágar sangue de carneiro e de coelho, a capacidade de aderir ao poliestireno e o polimorfismo do locus agr. Determinou-se a integridade do locus agr por PCR-RFLP e sequenciamento de bases em 13 cepas representativas das 47 estudadas. A integridade do locus regulador acessório sarA também foi avaliada por sequenciamento de bases nestas 13 cepas. Foram escolhidas 18 cepas sensíveis a vancomicina com variadas características fenotípicas e estas foram submetidas à indução de resistência a vancomicina através da passagem seriada em concentrações crescentes deste antibiótico. A taxa de mutação que leva à capacidade de crescimento em 6 µg/mL de vancomicina foi avaliada em 8 cepas através de ensaios de flutuação. Não observou-se correlação entre a aquisição de resistência a vancomicina com as atividades hemolíticas ou capacidade de adesão das cepas. A maioria das cepas (82,9%) apresentou-se como pertencente ao grupo polimórfico agr I, inclusive as cepas VISA. Duas cepas não conseguiram ser induzidas à resistência a vancomicina. O tempo levado para a aquisição de resistência não se correlacionou com nenhuma característica fenotípica ou genotípica de um grupo de cepas. A taxa de mutação que leva à capacidade de crescimento em 6µg/mL de vancomicina apresentou-se maior para uma cepa pertencente ao clone endêmico brasileiro (CEB) cujo locus agr pertence ao grupo I e não apresentou variação de acordo com funcionalidade ou tipo do locus agr. Apenas uma das cepas VISA apresentou uma mutação no locus agr que o torna disfuncional. Os loci agr das outras cepas estudadas apresentaram-se íntegros. O locus sarA das cepas estudadas apresentou-se íntegro e com polimorfismos funcionais agrupados de acordo com a linhagem clonal das cepas. Pôde-se concluir que a integridade funcional do locus agr não é uma condição sine qua non para a aquisição de resistência de baixo nível a vancomicina por parte de uma cepa sensível a este antibiótico. O grupo polimórfico agr II não tem maior predisposição à aquisição de resistência de baixo nível a vancomicina, como havia sido sugerido por alguns trabalhos disponíveis na literatura. / The treatment of staphylococcal infections has lately been a strenuous undertaking due to the resistance of Staphylococcus aureus to multiple antibiotics. The antimicrobial drug of choice for the treatment of methicillin resistant S. aureus (MRSA) is the glycopeptide vancomycin. Since the first isolation of S. aureus with reduced susceptibility to vancomycin (VISA) in 1997, there has been growing concern as to the dissemination of this resistance phenotype among isolates of this species. The molecular mechanisms that result in low level resistance to vancomycin have not yet been completely elucidated. The correct detection of this phenotype in the clinical laboratory is tricky, for the techniques available for this purpose are hard to execute and interpret. Until now, lateral transmission (dissemination) of VISA has not been reported and all strains bearing this phenotype have been isolated from patients who had been making prolonged use of vancomycin. A deficiency in the accessory gene regulator (agr) has been proposed as a risk factor for the acquisition of a VISA phenotype by a susceptible strain. For this study, 47 nosocomial VISA strains, that had been isolated in another study, were used. These strains were isolated from multiple geographical regions of Brazil, and included 5 VISA strains. The minimal inhibitory concentrations (MIC) of vancomycin and oxacillin, as well as haemolysis in sheep and rabbit agar, adhesion to polystyrene and agr polymorphism were determined in all of these strains. The integrity of the agr locus was determined by PCR-RFLP and by nucleotide sequencing in a sample of 13 strains chosen to be representative of the 47 strains studied. The integrity of the Staphylococcal accessory regulator sarA was also determined by nucleotide sequencing in these 13 strains. Another representative sample of 18 strains that were susceptible to vancomycin were submitted to induction of resistance to vancomycin by serial passage in increasing concentrations of this drug. The mutation rate of a mutation that leads to the ability of growing in a concentration of 6 µg/mL of vancomycin was determined for 8 strains by fluctuation assays. There was no correlation between the acquisition of resistance to vancomycin with either haemolysis or adhesion to polystyrene. Most strains (82.9%) bore a group I agr polymorphism, including all of the VISA strains. Two strains could not be induced to resistance. The time taken for each strain to acquire resistance to vancomycin did not correlate with any phenotypic or genotypic characteristic pertaining to a group of strains. The rate of mutation that leads to the ability of growing in 6µg/mL of vancomycin proved to be higher for a strain belonging to the Brazilian Endemic Clone (BEC) bearing an agr group I polymorphism, and did not vary according to presence or type of agr locus. Only one of the VISA strains presented a mutation in the agr locus that renders it disfunctional. The agr loci of the other strains studied presented themselves to be intact. The sarA loci of the strains evaluated were intact however presented functional polymorphisms that were groups according to the clonal lineage of the strains. It can thus be concluded that the functional integrity of the agr locus is not a sine qua non condition for the acquisition of low level resistance to vancomycin by a susceptible strain. Bearing of an agr group II polymorphism does not predispose a strain to acquire resistance to vancomycin, as has been previously suggested in literature.

O tratamento de infecções por Staphylococcus aureus tem sido problemático devido ao surgimento de cepas resistentes a múltiplios antibióticos. O antibiótico de escolha para o tratamento de infecções por S. aureus resistente a oxacilina é o glicopeptídeo vancomicina. Desde o primeiro isolamento de cepas com sensibilidade reduzida a vancomicina (VISA) em 1997, tem havido crescente preocupação com a disseminação da resistência a este antibiótico. Os mecanismos moleculares que levam à resistência de baixo nível a vancomicina ainda não foram elucidados. A detecção deste fenótipo na rotina de laboratório clínico é laboriosa, pois as técnicas disponíveis são de difícil execução e interpretação. Até agora, não há relato de transmissão horizontal de infecção por VISA, e todas as cepas com este fenótipo foram isoladas de pacientes que faziam o uso prolongado de vancomicina. Uma deficiência no locus regulador de genes acessórios (agr) foi postulado como fator de risco para a aquisição do fenótipo VISA por uma cepa sensível a este antibiótico. Para este estudo, foram selecionadas 47 cepas de S. aureus, com sensibilidades variadas a vancomicina, inclusive 5 cepas VISA isoladas no Brasil. Determinou-se nas cepas as concentrações inibitórias mínimas de vancomicina e oxacilina, a atividade hemolítica em ágar sangue de carneiro e de coelho, a capacidade de aderir ao poliestireno e o polimorfismo do locus agr. Determinou-se a integridade do locus agr por PCR-RFLP e sequenciamento de bases em 13 cepas representativas das 47 estudadas. A integridade do locus regulador acessório sarA também foi avaliada por sequenciamento de bases nestas 13 cepas. Foram escolhidas 18 cepas sensíveis a vancomicina com variadas características fenotípicas e estas foram submetidas à indução de resistência a vancomicina através da passagem seriada em concentrações crescentes deste antibiótico. A taxa de mutação que leva à capacidade de crescimento em 6 µg/mL de vancomicina foi avaliada em 8 cepas através de ensaios de flutuação. Não observou-se correlação entre a aquisição de resistência a vancomicina com as atividades hemolíticas ou capacidade de adesão das cepas. A maioria das cepas (82,9%) apresentou-se como pertencente ao grupo polimórfico agr I, inclusive as cepas VISA. Duas cepas não conseguiram ser induzidas à resistência a vancomicina. O tempo levado para a aquisição de resistência não se correlacionou com nenhuma característica fenotípica ou genotípica de um grupo de cepas. A taxa de mutação que leva à capacidade de crescimento em 6µg/mL de vancomicina apresentou-se maior para uma cepa pertencente ao clone endêmico brasileiro (CEB) cujo locus agr pertence ao grupo I e não apresentou variação de acordo com funcionalidade ou tipo do locus agr. Apenas uma das cepas VISA apresentou uma mutação no locus agr que o torna disfuncional. Os loci agr das outras cepas estudadas apresentaram-se íntegros. O locus sarA das cepas estudadas apresentou-se íntegro e com polimorfismos funcionais agrupados de acordo com a linhagem clonal das cepas. Pôde-se concluir que a integridade funcional do locus agr não é uma condição sine qua non para a aquisição de resistência de baixo nível a vancomicina por parte de uma cepa sensível a este antibiótico. O grupo polimórfico agr II não tem maior predisposição à aquisição de resistência de baixo nível a vancomicina, como havia sido sugerido por alguns trabalhos disponíveis na literatura. / The treatment of staphylococcal infections has lately been a strenuous undertaking due to the resistance of Staphylococcus aureus to multiple antibiotics. The antimicrobial drug of choice for the treatment of methicillin resistant S. aureus (MRSA) is the glycopeptide vancomycin. Since the first isolation of S. aureus with reduced susceptibility to vancomycin (VISA) in 1997, there has been growing concern as to the dissemination of this resistance phenotype among isolates of this species. The molecular mechanisms that result in low level resistance to vancomycin have not yet been completely elucidated. The correct detection of this phenotype in the clinical laboratory is tricky, for the techniques available for this purpose are hard to execute and interpret. Until now, lateral transmission (dissemination) of VISA has not been reported and all strains bearing this phenotype have been isolated from patients who had been making prolonged use of vancomycin. A deficiency in the accessory gene regulator (agr) has been proposed as a risk factor for the acquisition of a VISA phenotype by a susceptible strain. For this study, 47 nosocomial VISA strains, that had been isolated in another study, were used. These strains were isolated from multiple geographical regions of Brazil, and included 5 VISA strains. The minimal inhibitory concentrations (MIC) of vancomycin and oxacillin, as well as haemolysis in sheep and rabbit agar, adhesion to polystyrene and agr polymorphism were determined in all of these strains. The integrity of the agr locus was determined by PCR-RFLP and by nucleotide sequencing in a sample of 13 strains chosen to be representative of the 47 strains studied. The integrity of the Staphylococcal accessory regulator sarA was also determined by nucleotide sequencing in these 13 strains. Another representative sample of 18 strains that were susceptible to vancomycin were submitted to induction of resistance to vancomycin by serial passage in increasing concentrations of this drug. The mutation rate of a mutation that leads to the ability of growing in a concentration of 6 µg/mL of vancomycin was determined for 8 strains by fluctuation assays. There was no correlation between the acquisition of resistance to vancomycin with either haemolysis or adhesion to polystyrene. Most strains (82.9%) bore a group I agr polymorphism, including all of the VISA strains. Two strains could not be induced to resistance. The time taken for each strain to acquire resistance to vancomycin did not correlate with any phenotypic or genotypic characteristic pertaining to a group of strains. The rate of mutation that leads to the ability of growing in 6µg/mL of vancomycin proved to be higher for a strain belonging to the Brazilian Endemic Clone (BEC) bearing an agr group I polymorphism, and did not vary according to presence or type of agr locus. Only one of the VISA strains presented a mutation in the agr locus that renders it disfunctional. The agr loci of the other strains studied presented themselves to be intact. The sarA loci of the strains evaluated were intact however presented functional polymorphisms that were groups according to the clonal lineage of the strains. It can thus be concluded that the functional integrity of the agr locus is not a sine qua non condition for the acquisition of low level resistance to vancomycin by a susceptible strain. Bearing of an agr group II polymorphism does not predispose a strain to acquire resistance to vancomycin, as has been previously suggested in literature.

Made available in DSpace on 2014-08-20T13:42:05Z (GMT). No. of bitstreams: 1 Dissertacao_Milena_Tomasi_ Bassani.pdf: 628517 bytes, checksum: af4a756ae84e13ef34c451b2d486162e (MD5) Previous issue date: 2009-10-27 / he accessory gene regulator (agr) is a S. aureus global regulator of virulence factors, as the staphylococcal enterotoxins (SE), responsible for the staphylococcal food poisoning. There are four different agr groups due to the polymorphism in the amino acids sequence of the agrC and agrD. In the literature is described a relationship among agr groups and virulence factors, diseases, preferential host and antibiotic resistance. In this context, it was aimed to characterize the agr groups through biplex PCR, and relationship among agr groups, enterotoxigenic and antimicrobian profiles of S. aureus isolated from foods and bovine mastitis milk. A total of 115 strains were used to characterize the agr groups , being 30 isolated from f bovine mastitis milk and 85 isolated from several sources of foods. To assess the relationship between agr groups and enterotoxin production were used 14/85 strains previously characterized for the presence of some enterotoxins (sea, seb, sec, sed e cluster egc). To determine the profile of antibiotics resistance were used 71/115 strains. We observed a prevalence of agr group II with19.1% (22/115 strains), followed by the agr I with 8.6% (10/115 strains), agr III with 7.8% (9 / 115 strains), and agr IV with6.0% (7 / 115 strains). Among the strains isolated from bovine mastitis milk agr group I was prevailed with 20% (6/30 strains), whereas in the strains isolated from several food sources was observed prevalence of agr group II with 32.7% (18/85 strains), especially among those from chicken meat. Among the 14 strains (14/85) that contained enterotoxin genes, the majority of them contained the cluster egc (70%) belonged to agr II, whereas no relationship was found with those who had the genes for the classical SE (sea, seb, sec, sed). Considering the antibiotic resistance 100% of bovine mastitis milk strains and from various sources of food were resistant to penicillin, ampicillin, cefoxitin, and vancomycin. Relationship was observed between food strains, which were resistant to vancomycin and agr II, however, no relationship was found between antibiotic profile and agr groups among the strains isolated from bovine mastitis milk. These results demonstrated the prevalence of agr II among food strains and agr I among bovine mastitis milk strains. Moreover, the strains that carried the cluster egc were predominant agr II, which could indicate the occurrence of a clonal group among those. Another important result obtained in this study was the high rate of S. aureus multiresistant strains isolated from food, which emphasizes the importance of dissemination of these strains among foods. / O accessory gene regulator (agr) é um regulador global de fatores de virulência em S. aureus, como as enterotoxinas estafilocócicas (EE), responsáveis pela intoxicação alimentar estafilocócica. São conhecidos quatro distintos grupos agr devido ao polimorfismo na seqüência dos aminoácidos de agrC e agrD. Na literatura descreve-se relação entre fatores de virulência, patogenias, hospedeiro preferencial e perfil de resistência a antibióticos com grupos agr. Neste contexto, objetivou-se caracterizar grupos agr através de biplex PCR, e relacioná-los com os perfis enterotoxigênico e antimicrobiano de cepas de S. aureus isoladas em alimentos e leite de vacas mastíticas. Para caracterização dos grupos agr foram utilizadas 115 cepas, sendo 30 isoladas em leite de vacas mastíticas e 85 em diversas fontes de alimentos. Para a relação entre grupos agr e produção de enterotoxina foram utilizadas 14/85 cepas previamente caracterizadas quanto à presença de alguma enterotoxina (eea, eeb, eec, eed e cluster egc), já para determinar o perfil de resistência a antibióticos utilizaram-se 71/115 cepas. Observou-se uma prevalência do grupo agr II, com 19,1% (22/115 cepas), seguido do agr I, com 8,6% (10/115 cepas), agr III 7,8% (9/115 cepas), e agr IV, 6,0% (7/115 cepas). Entre as cepas isoladas em leite de vacas com mastite houve predomínio do grupo agr I, com 20% (6/30 cepas); já nas cepas isoladas de diversas fontes de alimentos observou-se prevalência do grupo agr II, com 32,7% (18/85 cepas), especialmente entre as provenientes de carne de frango. Entre as 14/85 cepas que carreavam genes de enterotoxinas, a maioria que albergava o cluster egc (70%), pertencia ao grupo agr II, enquanto nenhuma relação foi observada com aquelas que possuíam os genes para as EE clássicas (eea, eeb, eec, eed). Com relação ao perfil de resistência antimicrobiana, 100% das cepas isoladas de leite de vacas com mastite e das diversas fontes de alimentos apresentaram resistência à penicilina, ampicilina, cefoxitina e vancomicina. Observou-se relação entre cepas isoladas de alimentos, que eram resistentes à vancomicina e grupo agr II, entretanto, nenhuma relação foi observada entre perfil antimicrobiano e grupos agr entre as cepas isoladas em leite de vacas com mastite. Através destes resultados demonstra-se a prevalência do grupo agr II entre as cepas isoladas de alimentos e do grupo agr I em cepas isoladas de leite de vacas mastíticas. Além disso, nas cepas que carreiam o cluster egc houve predominância do grupo agr II, podendo indicar um grupo clonal entre essas. Outro resultado relevante obtido neste estudo foi à elevada taxa de cepas de S. aureus multiresistentes isoladas em alimentos, o que ressalta a importância da disseminação de cepas multiresistentes entre os alimentos.

Master of Science / Division of Biology / Revathi Govind / Toxigenic Clostridium sordellii causes uncommon but highly lethal infections in humans and animals. Recently, an increased incidence of C. sordellii infections has been reported in women undergoing obstetric interventions. Pathogenic strains of C. sordellii produce numerous virulence factors, including sordellilysin, phospholipase, neuraminidase, and two large clostridial glucosylating toxins, TcsL and TcsH. Recent studies have demonstrated that TcsL toxin is an essential virulence factor for the pathogenicity of C. sordellii. In this study, we identified and characterized TcsR as the toxin gene (tcsL) regulator in C. sordellii. High-throughput sequencing of two C. sordellii strains revealed that tcsR lies within a genomic region that encodes TcsL, TcsH, and TcsE, a putative holin. By using ClosTron technology, we inactivated the tcsR gene in strain ATCC 9714. Toxin production and tcsL transcription were decreased in the tcsR mutant strain. However, the complemented tcsR mutant produced large amounts of toxins, similar to the parental strain. Expression of the Clostridium difficile toxin gene regulator tcdR also restored toxin production to the C. sordellii tcsR mutant, showing that these sigma factors are functionally interchangeable.

Bacillus subtilis is the model organism for low GC Gram-positive bacteria and is of great biotechnological interest. Protein phosphorylation is an important regulatory mechanism in bacteria and it has not been extensively studied yet. Recent site-specific phosphoproteomic studies identified a large number of novel serine/threonine phosphorylation sites in B. subtilis, including a) two transition phase global gene regulators DegS and AbrB and b) RecA, that plays a major role in double-strand break repair and DNA recombination. .B. subtilis disposes of several putative Ser/Thr kinases like PrkA, YbdM, YabT and a characterizd kinase PrkC, but very few physiological substrates for these have been defined so far. In vitro phosphorylation assays were used to identify which of these kinases were able to phosphorylate DegS, RecA and AbrB. DegS phosphorylation on serine 76 by the kinase YbdM influenced its activity towards DegU both in vitro and in vivo, and expression of DegS S76D( on replacing serine to aspartate) in B. subtilis perturbed cellular processes regulated by the DegS/DegU two component system. This suggests a link between DegS phosphorylation at serine 76 and the level of DegU phosphorylation, establishing this post-translational modification as an additional trigger for this two-component system. At the onset of sporulation, B. subtilis expresses an unusual serine/threonine kinase YabT, which exhibits a septal localization and is activated by non-sequence-specific DNA binding. Activated YabT phosphorylates RecA at the residue serine 2, which in turn promotes the formation of RecA foci at the onset of spore development. On the other hand, non-phosphorylatable RecA or inactivated YabT lead to reduced spore formation in the presence of DNA lesions . This suggests a functional similarity between B. subtilis developmental stage dependent RecA phosphorylation and its eukaryal homologous Rad51 phosphorylation, which leads to its recruitment to the lesion sites. We therefore proposed that RecA phosphorylation serves as an additional signal mechanism that promotes focus formation during spore development. AbrB is phosphorylated by YabT, YbdM and PrkC in vitro and AbrB phosphorylation leads to reduced affinity for its target DNA and abolished binding cooperativity in vitro and in vivo. Expression of the phosphomimetic AbrB-S86D or of the non-phosphorylatable AbrB-S86A mutant protein in B. subtilis disturbed some stationary phase phenomena such as exoprotease production, competence and the onset of sporulation, probably by deregulation of AbrB-target genes and operons. We therefore, proposed that AbrB phosphorylation as an additional regulatory mechanism needed to switch off this ambiactive gene regulator during the transition phase.

Bacillus subtilis est la bactérie modèle des bactéries Gram-positif à bas pourcentage en GC et possède un intérêt marqué en biotechnologie. Par ailleurs, la phosphorylation des protéines est un mécanisme de régulation essentiel chez les bactéries qui reste encore largement à explorer. B. subtilis possède plusieurs ser/thr kinases potentielles (PrkA, YbdM, YabT et PrkC, qui a été déjà largement caractérisée), mais très peu de substrats de ces kinases ont été mis en évidence. Récemment, des études phosphoprotéomiques ont permis d’identifier de nombreux peptides phosphorylés sur des sérines ou des thréonines chez B. subtilis, incluant: a) deux régulateurs globaux de la phase de transition, DegS et AbrB et b) RecA, qui joue un rôle essentiel dans la réparation des cassures double-brin de l’ADN et la recombinaison. Des tests de phosphorylation in vitro nous ont permis d’identifier les ser/thr kinases capables de phosphoryler DegS, RecA et AbrB. La phosphorylation de DegS sur son résidu sérine 76 par la kinase YbdM influence, in vitro et in vivo, son activité kinase vis à vis de son substrat DegU. L’expression chez B. subtilis d’un allèle codant la protéine DegS-S76D (la sérine étant remplacée par un aspartate phosphomimétique) perturbe l’ensemble des processi cellulaires régulés par le système à deux composants DegS/DegU. Ces résultats suggèrent un lien entre la phosphorylation de DegS sur sa sérine 78 et le niveau de phosphorylation de son substrat DegU, cette modification post-traductionnelle représentant un degré supplémentaire de régulation pour ce système à deux composants. Au cours du démarrage de la sporulation, B. subtilis exprime une ser/thr kinase atypique, YabT, qui localise au septum et est activée grâce à la liaison de séquences ADN non spécifiques. YabT activée phosphoryle RecA sur sa sérine 2, ce qui induit la formation de foci RecA. Dans une souche exprimant une protéine RecA non phosphorylable (RecA-S2A) ou inactivée pour yabT, la formation de spores en présence de lésions de l’ADN est diminuée. Ces résultats suggèrent une homologie fonctionnelle au cours du développement entre la phosphorylation de RecA chez B. subtilis et la phosphorylation de son homologue eukaryote Rad51, qui permet leur recrutement sur des lésions de l’ADN. Nous proposons donc que la phosphorylation de RecA serve de signal pour promouvoir la formation de foci au cours de la sporulation. In vitro, le régulateur transcriptionnel AbrB est phosphorylé par les kinases YabT, YbdM et PrkC, L’utilisation de protéines mutées AbrB-S86A (non phosphorylable) et AbrB-S86D (forme phosphomimétique) nous a permis de montrer que la phosphorylation d’AbrB diminue son affinité pour l’ADN cible. L’expression chez B. subtilis des protéines AbrB-S86A et –S86D perturbe des phénomènes mis en place au cours de la phase stationnaire comme la production d’exoprotéases, la compétence et la sporulation via la dérégulation des gènes et opérons AbrB-dépendants correspondants. Nous proposons donc que la phosphorylation d’AbrB par les Hanks-kinases constitue un mécanisme de contrôle supplémentaire nécessaire à l’inactivation de ce régulateur transcriptionnel, qui peut être activateur ou répresseur, pendant la phase de transition. / Bacillus subtilis is the model organism for low GC Gram-positive bacteria and is of great biotechnological interest. Protein phosphorylation is an important regulatory mechanism in bacteria and it has not been extensively studied yet. Recent site-specific phosphoproteomic studies identified a large number of novel serine/threonine phosphorylation sites in B. subtilis, including a) two transition phase global gene regulators DegS and AbrB and b) RecA, that plays a major role in double-strand break repair and DNA recombination. .B. subtilis disposes of several putative Ser/Thr kinases like PrkA, YbdM, YabT and a characterizd kinase PrkC, but very few physiological substrates for these have been defined so far. In vitro phosphorylation assays were used to identify which of these kinases were able to phosphorylate DegS, RecA and AbrB. DegS phosphorylation on serine 76 by the kinase YbdM influenced its activity towards DegU both in vitro and in vivo, and expression of DegS S76D( on replacing serine to aspartate) in B. subtilis perturbed cellular processes regulated by the DegS/DegU two component system. This suggests a link between DegS phosphorylation at serine 76 and the level of DegU phosphorylation, establishing this post-translational modification as an additional trigger for this two-component system. At the onset of sporulation, B. subtilis expresses an unusual serine/threonine kinase YabT, which exhibits a septal localization and is activated by non-sequence-specific DNA binding. Activated YabT phosphorylates RecA at the residue serine 2, which in turn promotes the formation of RecA foci at the onset of spore development. On the other hand, non-phosphorylatable RecA or inactivated YabT lead to reduced spore formation in the presence of DNA lesions . This suggests a functional similarity between B. subtilis developmental stage dependent RecA phosphorylation and its eukaryal homologous Rad51 phosphorylation, which leads to its recruitment to the lesion sites. We therefore proposed that RecA phosphorylation serves as an additional signal mechanism that promotes focus formation during spore development. AbrB is phosphorylated by YabT, YbdM and PrkC in vitro and AbrB phosphorylation leads to reduced affinity for its target DNA and abolished binding cooperativity in vitro and in vivo. Expression of the phosphomimetic AbrB-S86D or of the non-phosphorylatable AbrB-S86A mutant protein in B. subtilis disturbed some stationary phase phenomena such as exoprotease production, competence and the onset of sporulation, probably by deregulation of AbrB-target genes and operons. We therefore, proposed that AbrB phosphorylation as an additional regulatory mechanism needed to switch off this ambiactive gene regulator during the transition phase.