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Initially, this thesis investigates patterns of intragenomic codon usage within the genome of the Delta Proteobacterium Bdellovibrio bacteriovorus. Correspondence analyses revealed the primary factor influencing codon usage within this genome to be related to translational selection. The relationship between the degree of codon usage adaptation (as given by the ‘frequency of optimal codons’ statistic) and putative gene expression level was used to look for genes with unusually high or low expression levels in B. bacteriovorus, in comparison to Escherichia coli, in order to gain further insight into the unusual lifestyle of this Delta Proteobacterium. The scope was then broadened to explore intergenomic patterns of codon usage and initially extend a study measuring the strength of selected codon usage bias across bacterial genomes (Sharp et al. 2005). A dataset of 160 fully sequenced bacterial genomes was used and the strength of selected codon usage bias was seen to vary greatly between species. A correlation was observed between (log of) generation time and the strength of selected codon usage bias with fast growing bacteria showing a higher degree of selected codon usage bias than slow growing bacteria. In bacterial species exhibiting significant levels of selected codon usage bias optimal codon choice was examined. It was observed that optimal codon choice is not always conserved across all bacterial genomes under selection but broad trends in optimal codon choice were seen to be associated with particular bacterial clades. In general, optimal codon choice was seen to be linked with differences in mutational biases among the clades, as seen by a correlation between optimal codon choice in particular clades and the G+C content of their genomes. Clades that were A+U rich (Firmicutes, Gamma Proteobacteria main clade) were seen to largely prefer codons of the form NNA/U whilst G+C rich clades (Alpha Proteobacteria, Actinobacteria and Xanthomonas species) showed preference for codons of the form NNG/C in their highly expressed genes. Finally the relationship between optimal codon usage and tRNA abundances was explored. Changes in tRNA abundances were seen to coincide with switches in optimal codon usage. Therefore, switches in codon usage and tRNA abundance are thought to be influenced by changing mutational bias within the genome as reflected by the correlation between optimal codon choice, tRNA gene complements and genomic G+C content.

La découverte d'un contexte génétique chez les streptocoques – codant un petit peptide hydrophobe (SHP) et un régulateur transcriptionnel appartenant à la famille Rgg –, suivi de l'étude d'un de ces loci chez S. thermophilus LMD-9, a conduit à l'hypothèse que les protéines régulatrices Rgg en association avec une phéromone putative SHP pourraient intervenir dans un mécanisme de type quorum-sensing (QS) chez les bactéries à Gram positif. La première partie de ma thèse a consisté à confirmer cette hypothèse sur le locus shp/rgg1358 de S. thermophilus LMD-9, espèce contenant le plus grand nombre de systèmes SHP/Rgg dans son génome. Pour ceci, les étapes impliquées dans un mécanisme de QS ont été étudiées : la sécrétion, la maturation et la détection à une concentration seuil de la phéromone, sa réimportation à l'intérieur de la cellule, son interaction avec un régulateur transcriptionnel et enfin l'interaction de la protéine régulatrice à l'ADN. Par l'utilisation d'approches génétiques et biochimiques, nous avons démontré l'existence d'un nouveau mécanisme de QS impliquant pour la première fois un régulateur transcriptionnel Rgg et une phéromone SHP, importée à l'intérieur de la cellule par le transporteur d'oligopeptides AmiCDEF. Le rôle de la protéase membranaire, Eep, a également été démontré dans la maturation de la phéromone, dont la forme mature a été déterminée par spectrométrie de masse et validée in vivo. Dans un second temps, nous avons exploré la fonctionnalité de ce nouveau mécanisme sur d'autres loci shp/rgg, dans le but d’étudier l'existence d’éventuels phénomènes de cross-talk entre les bactéries. L'étude de nouveaux loci, en système hétérologue chez S. thermophilus LMD-9, a permis d'étendre la fonctionnalité du mécanisme à deux systèmes SHP/Rgg de streptocoques pathogènes, à savoir S. agalactiae et S. mutans. En parallèle à ce travail de caractérisation, l'identification des régulons des systèmes SHP/Rgg a été entreprise. La construction d'un arbre phylogénétique des protéines Rgg-like a permis d'identifier 68 systèmes SHP/Rgg, que nous avons classés en trois groupes. L'analyse des régions promotrices des gènes shp a conduit à l'identification d'un site putatif de liaison des protéines Rgg à l'ADN spécifiques de chaque groupe SHP/Rgg. Une approche in silico a ensuite été menée afin de rechercher, dans les génomes séquencés de streptocoques, les gènes cibles putatifs. Alors que des cibles proximales ont été détectées pour les groupes II et III, des cibles distales ont été identifiées dans les groupes I et II. Actuellement, la validation de certaines cibles est en cours au laboratoire. A l'avenir, ce travail pourrait permettre le développement de petits peptides permettant d'optimiser l'utilisation de S. thermophilus en industries laitières et de réduire la virulence des streptocoques pathogènes. / The discovery of a genetic context – encoding a small hydrophobic peptide (SHP) and a transcriptional regulator belonging to the Rgg family (in nearly all streptococcal genomes) –, following by the study of one of this loci in S. thermophilus LMD-9, led to the hypothesis that the regulatory proteins Rgg in association with a putative pheromone SHP could define a novel quorum-sensing (QS) regulatory mechanism in Gram-positive bacteria. The first part of my PhD consisted to validate this hypothesis. For this purpose, we analyzed the SHP/Rgg system in all the steps that are commonly involved in QS mechanisms: (i) secretion of the putative pheromone, (ii) maturation of the pheromone, (iii) capture of the pheromone from the external environment at a threshold concentration, (iv) importation of the pheromone inside the cell and (v) interaction of the transcriptional regulator to the promoter regions of targeted genes. Experimentally, we focused on the so-called shp/rgg1358 locus of S. thermophilus LMD-9, which is the streptococcal species containing the largest number of shp/rgg pairs in its genome. By using genetic and biochemistry approaches, we uncovered a new QS mechanism that involves the pheromone SHP, the oligopeptide transporter AmiCDEF for the uptake of the pheromone and the transcriptional regulator Rgg for the control of target gene expression. Furthermore, we showed that the membrane protease Eep participates in the production of the mature pheromone, which has been identified by mass spectrometry. Once characterized, the second part of my PhD was to explore the functionality of this new QS system in other streptococcal strain or species, in order to determine if cross-reactivity phenomenon between streptococci can occur. By using heterologous expression in S. thermophilus LMD-9, we extended the functionality of the SHP/Rgg system to two pathogenic streptococcal species, i.e. S. agalactiae and S. mutans. The last part of my PhD consisted in identifying the regulon of all SHP/Rgg systems. Following the construction of a phylogenetic tree of the Rgg-like proteins in low GC Gram-positive bacteria, we identified 68 SHP/Rgg systems that we classified in three groups. Analyzing the promoter regions of all shp genes led to the identification of a putative Rgg DNA binding site specific to each SHP/Rgg group. An in silico approach was used to scan all sequenced streptococcal genomes for the three identified patterns. Whereas proximal target genes were detected for groups II and III, distal target genes were found in groups I and II. In addition, we uncovered that putative Rgg DNA binding sites can be localized in coding or non-coding region. Currently, validations are in progress. To sum-up, my PhD studies provided evidences that the Rgg proteins in association with small peptide pheromones define a new QS mechanism that seems to regulate the expression of distal and proximal genes in a species-dependent manner. Important insights should be obtained concerning a putative crosstalk among streptococci that involves the SHP/Rgg QS system. My studies may constitute a basis for the development of small peptides to optimize the use of S. thermophilus in dairy factories and reduce the virulence of pathogenic streptococci.