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The necrotrophic plant pathogenic bacterium Dickeya solani emerged in the potato agrosystem in Europe. All isolated strains of D. solani contain several large polyketide synthase/non-ribosomal peptide synthetase gene clusters. Analogy with genes described innother bacteria suggests that the clusters ooc and zms are involved in the production of secondary metabolites of the oocydin and zeamine families, respectively. A third cluster that we named ssm for solani secondary metabolite had an unknown function. In this study, we constructed mutants impaired in each of the three secondary metabolite clusters ssm, ooc, and zms to compare first the phenotype of the D. solani wild-type strain D s0432-1 with its associated mutants. We demonstrated the antimicrobial functions of these three PKS/NRPS clusters against bacteria, yeasts or fungi. The secondary metabolite cluster ssm, conserved in several other Dickeya species, produces a secondary metabolite inhibiting yeasts. Phenotyping and comparative genomics of different D. solani wild-type isolates revealed that the small regulatory RNA ArcZ plays a major role in the control of the clusters ssm and zms. A single-point mutation, conserved in some Dickeya wild-type strains, including the type strain IPO 2222, impairs the ArcZ function by affecting its processing into an active form.

The necrotrophic plant pathogenic bacterium Dickeya solani emerged in the potato agrosystem in Europe. All isolated strains of D. solani contain several large polyketide synthase/non-ribosomal peptide synthetase (PKS/NRPS) gene clusters. Analogy with genes described in other bacteria suggests that the clusters ooc and zms are involved in the production of secondary metabolites of the oocydin and zeamine families, respectively. A third cluster named sol was recently shown to produce an antifungal molecule. In this study, we constructed mutants impaired in each of the three secondary metabolite clusters sol, ooc, and zms to compare first the phenotype of the D. solani wild-type strain D s0432-1 with its associated mutants. We demonstrated the antimicrobial functions of these three PKS/NRPS clusters against bacteria, yeasts or fungi. The cluster sol, conserved in several other Dickeya species, produces a secondary metabolite inhibiting yeasts. Phenotyping and comparative genomics of different D. solani wild-type isolates revealed that the small regulatory RNA ArcZ plays a major role in the control of the clusters sol and zms. A single-point mutation, conserved in some Dickeya wild-type strains, including the D. solani type strain IPO 2222, impairs the ArcZ function by affecting its processing into an active form.

Streptococcus pneumoniae est un pathogène majeur de l'homme qui pose un problème de santé publique du fait de sa variabilité antigénique et de sa capacité à acquérir des gènes de résistance aux antibiotiques. En fait, cette bactérie peut développer un état physiologique transitoire régulé génétiquement (la compétence), lors duquel elle peut internaliser et intégrer dans son chromosome par recombinaison homologue de l'ADN exogène (transformation génétique naturelle). Nous avons montré que la compétence (et donc la transformation) pouvait être induite par des antibiotiques couramment utilisés en thérapie (Prudhomme, Attaiech et al. Science 2006). Ainsi, l'utilisation mal contrôlée d'antibiotiques peut augmenter le pouvoir pathogène de S. pneumoniae en favorisant les transferts génétiques. J'ai ensuite travaillé sur le devenir de l'ADN transformant après son internalisation. J'ai tout d'abord tenté d'identifier la/les nucléase(s) qui peuvent agir sur cet ADN. Les différentes approches utilisées n'ont pas abouti mais parmi les candidats testés, j'ai pu montrer que Pms et CoiA influent sur l'efficacité du système général de réparation des mésappariements (Hex) et j'ai étudié RadC pour laquelle nous avons proposé une révision de l'annotation pfam (Attaiech, et al. J. Bact. 2008). Nous avons ensuite déterminé que la protéine majeure du complexe d'éclipse (forme protégée de l'ADN internalisé) est SsbB (Morrison et al. J. Bact. 2007). Toutefois, en poursuivant la caractérisation de ce complexe, nous avons pu montrer que la méthode de purification utilisée n'est pas adaptée pour connaître sa composition protéique exhaustive (manuscrit en préparation)., During my doctorate, I studied different aspects of genetic transformation in the human pathogen Streptococcus pneumoniae. This bacterium can naturally develop a transient physiological state, named competence, in which it is able to internalize and integrate exogenous DNA into its chromosome by homologous recombination. We showed that antibiotic treatment can induce competence and transformation, thus enabling genetic plasticity and increasing pathogenicity of S. pneumoniae (Prudhomme, Attaiech et al. Science 2006). Then, my work concentrates on the fate of DNA after internalization during the process of transformation. First, I have analysed several mutants with the aim of identifying the nuclease(s) that can degrade internalized DNA. So far, I have been unable to identify this nuclease. However, among the tested candidates, CoiA and Pms have been implied in the mismatch repair process and we showed that, despite the original annotation, there is no evidence of a role of RadC in connection with repair and/or recombination. This permitted a revision of the Pfam annotation (Attaiech et al. J. Bact. 2008). Finally, we focused on the protection of internalized DNA and showed that the major protein component of the eclipse complex (EC, a nucleocomplex in which internalized DNA is protected) is the competence-induced protein SsbB (Morrison et al. J. Bact. 2007). I have done further biochemical experiments on the characteristics of EC and show that the support used during purification exhibits strong affinity for a lot of protein. That implies that the real nature of the EC has to be discovered (Attaiech et al. in prep).