Perennial clovers are being evaluated for their potential to reduce groundwater levels in Australian cropping zones where many soils are considered too acidic for reliable lucerne nodulation. However, the release of effective inocula for perennial clovers into such areas where sub clover is the predominant legume, could potentially compromise nitrogen fixation from this valuable annual clover if the symbiosis between the new inoculants and sub clover is not optimal. Studies were therefore designed to increase our understanding of these symbiotic interactions to optimise the management of legume-rhizobia interactions to extend (rather than restrict) the use of legumes in new environments. To assist the understanding of interactions between clovers and their microsymbionts, a glasshouse-based study of the cross-inoculation characteristics of 38 strains of Rhizobium leguminosarum biovar trifolii (R. l. trifolii) associated with 38 genotypes of annual and perennial Trifolium spp. from world centres of diversity was undertaken. Rhizobial isolates and clovers were assembled from South and equatorial Africa, North and South America and the Euro-Mediterranean regions. There was substantial specificity amongst the African clovers for effective nodulation. No strain of rhizobia from the South American perennial T. polymorphum, or from the Ethiopian clovers, was able to nodulate sub clover effectively, whilst less than 33% of the 18 strains from these regions could form nodules with the less promiscuous Mediterranean annual T. glanduliferum. Seventy of 476 cross-inoculation treatments examined did not nodulate, whilst 81 treatments clearly demonstrated effective nodulation. The remainder of the crossinoculation pairings revealed only partially effective or ineffective nodulation. Two barriers to effective nodulation were identified from the cross- inoculation study: a geographic barrier representing the broad centres of clover diversity, across which few host- strain combinations were effective; and within each region, a significant barrier to effective nodulation between an isolate from an annual host on a perennial host, or vice versa. Clovers and their rhizobia from within the Euro-Mediterranean region of diversity were more able to overlap the annual/perennial barrier than genotypes from the other regions. The data indicate that it will be a substantial challenge to develop inocula for perennial clovers that do not adversely affect nitrogen fixation by sub clover and other annual clovers in commerce, especially if the perennial clovers originate from Africa or America. To investigate the management of legume-rhizobia interactions when introducing legumes into new environments, a study was initiated in Uruguay (Mediterranean annual clovers were introduced into a predominantly perennial clover setting) that could be considered opposite to the situation emerging within southern Australia (perennial clovers evaluated in a predominantly annual clover setting). The Uruguayan grasslands contain populations of indigenous R. l. trifolii that nodulate endemic T. polymorphum but form ineffective nodules on clovers originating from the Mediterranean region. Importantly in the Uruguayan setting, Government policy has facilitated the introduction of numerous varieties of annual Mediterranean clovers with the aim of improving overall winter production in their naturally managed grasslands. In an attempt to understand the rhizobial ecology of this scenario, a cross-row experiment was set-up in 1999 in a basaltic, acid soil in Glencoe, Uruguay, to follow the survival and symbiotic performance of nine exotic strains of R. l. trifolii. In this thesis I report on the ability of the introduced strains to compete for nodule occupancy of Mediterranean clover hosts and show the impacts of the introduced strains on the productivity of the indigenous Uruguayan clover, T. polymorphum. Of the introduced strains, WSM1325 was a superior inoculant and remained highly persistent and competitive in forming effective symbioses with the Mediterranean hosts, T. purpureum and T. repens, in the Uruguayan environment over a 3 year period. T. purpureum and T. repens, when inoculated with the introduced strains, did not nodulate with any indigenous R. l. trifolii as typed from nodules of T. polymorphum. Conversely, there were no nodules on the Uruguayan host T. polymorphum that contained the introduced R. l. trifolii. These results revealed that there were effective symbioses between strains of R. l. trifolii and clovers, even though the soil contained ineffective R. l. trifolii for all hosts. This represents the first reported example of selective nodulation for an effective symbiosis in situ with annual and perennial clovers in acid soils. This phenomenon raised the question of whether this was restricted to the particular edaphic scenario in Glencoe, Uruguay. Glasshouse-based experiments in Australia were conducted to further understand the selection phenomenon. Two strains were selected for comparisons; strain WSM1325 isolated from an annual clover in the Mediterranean and WSM2304 isolated from the perennial clover T. polymorphum in Uruguay, South America. Variables that may have been specific to Glencoe were investigated. Thus, the effect of cell density and strain ratio at the time of inoculation, as well as soil pH, were examined on the two hosts (T. purpureum and T. polymorphum). Each was exposed to the same effective and ineffective micro-symbionts. In co-inoculation experiments at a cell density of 104 cells mL-1, each host nodulated solely with its effective strain, even when this strain was out-numbered 100:1 by the ineffective strain. However, the selection process ceased when the effective strain was out-numbered 1000:1. At higher basal cell concentrations of 105 - 108 cells mL-1, selection for WSM1325 to form effective nodules on T. purpureum was evident, but was significantly reduced as the ratio of ineffective cells in the inoculum increased above 4-fold. These results indicate that the selection mechanism is highly dependent upon the basal rhizobial cell density. Soil pH did not significantly alter the process, which could not be simply explained by the rate of strain growth, or extent of nodulation. Greater precision was sought in the terminology applied to nodulation outcomes where legumes have a choice of micro-symbiotic partners from within the same species of root-nodule bacteria. The nominated preferred terms are “nonselective”, “exclusive”, and “selective” nodulation. In view of the difference in host range between WSM1325 and WSM2304 and the selective nodulation process, a preliminary investigation into the genetic backgrounds of WSM1325 and WSM2304 was conducted. A selected range of gene regions were amplified by PCR from each strain and sequenced. Comparative analysis of the nucleotide sequences revealed that although the 16S rRNA sequences were identical, the atpD, GSII and nodD sequences contained distinct differences revealing disparity between the pSym replicons and between the chromosomal replicons of these strains. Of the genes sequenced, the highest degree of divergence was noted for the symbiotic NodD protein products, which are known to be critical determinants in the nodulation of specific hosts. An examination of the nodD gene region of WSM1325 and WSM2304 revealed a further contrasting feature; the regulatory gene nodR was present in the nodD gene region of WSM1325 but absent in WSM2304. Since NodR is known to be required for adding highly unsaturated fatty acyl groups onto the Nod-factor backbone, I could now hypothesise that the nodulation incompatibility observed between Trifolium hosts and micro-symbionts obtained from different geographical locations may result from differences in Nod-factor decoration. With the full genome sequence of the two strains WSM1325 and WSM2304 soon to be available, the role of nodR and any link to the selection phenomenon described in this thesis can be addressed.

Lactococcus lactis est une espèce appartenant au groupe des bactéries lactiques, largement utilisées dans l’industrie pour leur capacité à produire de l’acide lactique au cours de la fabrication des produits laitiers fermentés. L’étude de la diversité globale de L. lactis ssp. lactis a été entreprise par l’intégration de données biologiques obtenues à partir d’analyses génétiques, génomiques, physiologiques, transcriptomiques et métaboliques. L’accès à la phylogénie de l’espèce par l’étude de la variabilité génétique du génome cœur par MLST (MultiLocus Sequence Typing) a permis de décrire deux groupes de souches : les souches environnementales, génétiquement très diverses, isolées de laits crus, de plantes ou d’animaux et les souches domestiquées, génétiquement très proches, isolées des levains utilisés dans l’industrie laitière. Malgré la perte de diversité génétique observée dans les souches domestiquées probablement associée à un processus de spécialisation à un environnement technologique, l’approche intégrative a permis de montrer que ce groupe présente une diversité génomique et fonctionnelle aussi importante que les souches environnementales. L’investigation des génomes de la sous-espèce lactis par la mesure de la taille des chromosomes et la caractérisation en nombre et en taille du contenu plasmidique, a révélé une variabilité de plus de 300 kb en capacité de codage génétique des souches domestiquées et environnementales. D’autre part, les souches domestiquées appartenant au biovar Diacetylactis ont montré des physiologies et des régulations métaboliques différentes, résultant en une production d’arômes de type diacetyl ou acétoïne variable selon la souche. Enfin, le séquençage du génome de la souche environnementale A12 isolée d’un levain de panification, et sa comparaison avec les 4 génomes actuellement séquencés de L. lactis a révélé un pangénome (ensemble des gènes de l’espèce) étendu, montrant que cette espèce offre un grand réservoir de diversité. Environ 20 % des gènes spécifiques de souches ont été mis en évidence témoignant des grandes capacités adaptatives de la sous-espèce. L’étude approfondie de la souche A12 par une analyse transcriptomique a permis de rendre compte des mécanismes impliqués dans l’adaptation d’une souche à un écosystème complexe / The Lactococcus lactis species belong to lactic acid bacteria group widely used for their ability to produce lactic acid in fermented dairy products. The study of the global diversity of L. lactis ssp. lactis was carry out by the integration of biological data obtained from genetic, genomic, physiological, transcriptomic and metabolic analyses. The genetic variability investigated by MLST (MultiLocus Sequence Typing) describe two strains groups according to their phylogeny : the “environmental” strains, displaying high genetic diversity and isolated from different natural environments such as raw milks, plants and animals and the “domesticated” strains, genetically closely related, isolated from starters in dairy industries. Despite the lost of genetic diversity in domesticated strains, probably associated to a specialisation process, the integrative approach showed a genomic and functional diversity as huge as in environmental strains. The characterization of chromosome size and plasmidic content of the lactis subspecies revealed a variation higher than 300 kb in genetic coding capacity for domesticated and environmental strains. Moreover, the domesticated strains belonging to the biovar Diacetylactis showed different physiologies and metabolic regulations resulting in variable amount of aroma produced according to the strains. Finally, the genome sequencing of the A12 strain isolated from sourdough bread and its comparison with 4 other L. lactis genomes already sequenced revealed a spread pangenome (all the genes of a species). Approximately 20 % of each genome correspond to strain specific genes, showing large adaptive capacities of the subspecies. The in-depth study of the A12 strain by transcriptomic analysis allows to highlight mechanisms involved in the adaptation of a strain to a complex ecosystem

Corynebacterium pseudotuberculosis est une bactérie pathogène intracellulaire facultative, divisée en deux biovars : C. pseudotuberculosis ovis, agent de la lymphadénite caséeuse (petits ruminants), et C. pseudotuberculosis equi, responsable de lymphangites ulcéreuses (chevaux), mammites (bovins) et d’œdèmes cutanés (buffles). La génomique fonctionnelle a pour but d'élucider le rôle que joue chaque gène dans l'organisme, ainsi que l'interaction de ces gènes dans un réseau biologique. Au cours de ce travail de thèse, différentes stratégies protéomiques ont été adoptées pour l’étude fonctionnelle du génome de C. pseudotuberculosis : i) l’analyse du protéome extracellulaire des souches 1002_ovis et C231_ovis a permis la caractérisation totale de 60 protéines différentes de C. pseudotuberculosis dont 18 protéines sont régulées différemment ;ii) le protéome de la souche 1002_ovis été analysé en réponse au stress nitrosant révélant 58 protéines différentiellement régulées et impliquées dans différents processus biologiques susceptibles de favoriser la survie à ce stress ; iii) les souches 1002_ovis et 258_equi ont été utilisées pour induire des infections expérimentales sur modèle souris. L’analyse de leur protéome extracellulaire avant et après passage en série sur souris a permis d’identifier 250 protéines différentiellement régulées touchant diverses fonctions. Pour conclure, ce travail a permis de générer une base de données de protéines et de valider la fonctionnalité de différents gènes jusqu’alors prédits in silico seulement et des informations importantes sur les facteu / Corynebacterium pseudotuberculosis is a facultative intracellular pathogenic bacterium, subdivided into two biovars: C. pseudotuberculosis ovis, agent of caseous lymphadenitis (small ruminants), and C. pseudotuberculosis equi, which causes ulcerative lymphangitis (equines), mastitis (bovines), and oedematous skin disease (buffalos). Functional genomics aims to elucidate the role that each gene plays in organism, as well as the interactions of these genes into a biological network. In this PhD work, various proteomic approaches were used to evaluate the functional genome of C. pseudotuberculosis: i) the analysis of the exoproteome of strains 1002_ovis and C231_ovis enabled the characterization of 60 proteins of C. pseudotuberculosis of which 18 were differentially regulated; ii) the proteome of the strain 1002_ovis was analyzed after a nitrosative stress revealing 58 proteins differentially regulated when compared to the control conditionsThese 58 proteins are involved in different biological processes which may favor the survival of this pathogen under exposition to nitrosative stress; iii) the strains 1002_ovis and 258_equi were used in a murine model of experimental infection. A comparative analysis of their extracellular proteomes before and after passage in mice revealed that 250 proteins ibnvolved in various functions were differentially regulated in C. pseudotuberculosis. Altogether, this PhD work allowed the generation of a protein data base, as well as the validation of several genes previously predicted in silico, and generated information about factors that fa

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