<p> Sinorhizobium meliloti is an alpha-proteobacterium that forms symbiotic nodules on the roots of Medicago sativa (alfalfa). The ability to catabolize specific compounds available in the soil is one of the best-characterized factors to increase competition for nodulation. In order to successfully attain symbiosis S. meliloti must compete for nutrients in the rhizosphere, which can be done by having a large number of transport systems encoded in its genome. Genes encoding proteins involved in transport constitute the largest (12%) class of genes in the S. meliloti genome. Great interest now lies in determining substrates for the transport systems and their role in the survival and fitness of S. meliloti.</p> <p> An estimated 824 transport genes in the genome of the soil bacterium Sinorhizobium meliloti are predicted to encode 382 transport systems. All of the S. meliloti transporters had been studied under 120 different conditions, including growth on various carbon and nitrogen sources, seed and root exudates and starvation conditions.</p> <p> From this screen of every transport system in S. meliloti, the substrates that induce expression of over 50 transport systems have been identified. We have found putative transporters for amino acids, sugars, sugar alcohols, amino sugars, betaines and other compounds that might be found in the soil. This large scale expression analysis gives insight into the natural environment of S. meliloti by studying those genes that are induced by compounds that would be found in the soil.</p> / Thesis / Master of Science (MSc)

Sinorhizobium meliloti is a model bacterium for the study of symbiotic nitrogen fixation (SNF). It infects the roots of alfalfa as well as some other legumes and differentiates into N2-fixing bacteroids within the plant cells of specialized nodule organs. To understand genes essential for SNF and, in the longer term, to facilitate the manipulation of this SNF process for agricultural purposes, it is highly desirable to construct the minimal genome for SNF in this organism. S. meliloti harbors two replicons required for SNF, a 1.7-Mb chromid (pSymB) and a 1.4-Mb megaplasmid (pSymA). A previous deletion analysis revealed that only four gene regions, accounting for <12% of the total sequences of pSymA and pSymB that, were essential for SNF. In the first part of the thesis, I report the cloning of these two pSymA SNF-essential regions on a plasmid (pTH3255) in Escherichia coli, and the integration of this plasmid into the genome of a ∆pSymA S. meliloti derivative strain (the strain was named as RmP4291 after integration). Plant root dry weight and nitrogenase-catalyzed acetylene reduction assays were carried out on RmP4291 with four host plants, including Medicago sativa, Medicago truncatula, Melilotus alba and Melilotus officinalis. Nodule kinetic assays were also performed on RmP4291 and RmP110(wt). The results showed that the SNF-essential regions from pSymA were sufficient to restore the symbiotic capabilities to the ∆pSymA derivative strain with all the host plants tested, except a significant reduction (~40%) in SNF by RmP4291 was noticed on M. officinalis compared to that by wildtype S. meliloti. A higher alfalfa nodulation efficiency of RmP4291 compared to that of wildtype RmP110 was also discovered. In the second part of the thesis, a histochemical staining method for S. meliloti nodules was developed by integrating the marker genes gusA (β-glucuronidase) and celB (β-glucosidase) into the S. meliloti genome. This staining method was found to be useful in the study of nodule competitiveness. A nodule competition assay was carried out between RmP4291 and RmP110 using the new staining method. RmP4291 was found to be significantly reduced in nodulation competitiveness compared to wildtype S. meliloti. The development of the histochemical staining method for S. meliloti nodules will accelerate the identification of genes required for nodule competitiveness in the organism, which will be of crucial importance to the construction of the minimal genome strains with high SNF efficiency. / Thesis / Master of Science (MSc) / Nitrogen is one of the critical elements for life. Biological nitrogen fixation plays a crucial role in providing fixed nitrogen for the ecosystem on Earth. Our Laboratory has endeavored to establish a minimal symbiotic genome in Sinorhizobium meliloti, a model nitrogen fixing bacterium which forms symbiosis with certain kinds of legumes. Building this minimal symbiotic genome will improve our understanding of the symbiotic nitrogen fixation process in S. meliloti at gene level. It may also help in eventually introducing a nitrogen fixation system into other organisms. In this study, the minimal symbiotic genome of the pSymA replicon in S. meliloti was constructed. In addition, a staining method to detect specific S. meliloti strains in nodules was established. This method is potentially useful in finding genes related to nodule competitiveness, and these are potentially important for augmenting the genes that constitute the minimal symbiotic genome.

DNA replication initiates at a precise location on the bacterial chromosome, the origin of replication (oriC). This work has localized the origin of DNA replication on the Sinorhizobium meliloti chromosome to a region spanning the hemE gene. A genetic dissection of the locus revealed that a much larger fragment of DNA (1802 bp) is required for a functional oriC than that of the other characterized alpha-proteobacterial chromosome origin from Caulobacter crescentus. Site-directed mutations of predicted DnaA binding sites has identified several essential elements for replication of the plasmid borne oriC. Mutations in these DnaA boxes also reduce transcription of hemE and thus it is likely that transcription of hemE and replication of the S. meliloti chromosome are coupled. The ColEl plasmid pUCP30T can autonomously replicate when the S. meliloti oriC is cloned into the suicide vector (pTH838) and can be efficiently mobilized out of S. meliloti into E. coli. The pTH838 oriC plasmid when transferred into S. meliloti results in both small and large colonies and both of these transconjugant classes take longer to form than the S. meliloti recA::Tn5 recipient. We attributed this phenotype to the very low copy number of the pTH838 plasmid which was determined to be 0.053 - 0.135 copies per chromosome. The DnaA protein responsible for replication initiation in many bacteria has been purified and used in electrophoretic mobility shift assays. The DnaA protein interacts specifically with sequences in the hemE - Y02793 intergenic region and upstream of the repA2 gene on the pSymA megaplasmid. The DnaA protein has also been implicated as a link between DNA replication and cell division in S. meliloti as overexpression of DnaA in both E. coli and S. meliloti results in filamentation. / Thesis / Master of Science (MSc)

Sinorhizobium meliloti est une bactérie du sol Gram- capable d'induire la formation denodosités fixatrices d'azote lors d'une interaction symbiotique avec les plantes de la familledes légumineuses. L'importance de la balance redox au cours de cette interaction a été miseen évidence. Ainsi, des mutants bactériens déficients dans la production du glutathion (GSH),présentent un phénotype altéré d'infection et de fixation de l'azote atmosphérique.Le premier objectif a donc été de déterminer si les phénotypes observés chez les mutants de lavoie de biosynthèse du GSH étaient liés à l'activité des glutarédoxines (GRX). Une analysebioinformatique a révélé la présence de trois gènes codant des GRX chez S. meliloti. Lesmutants, Smgrx1, Smgrx2 et Smgrx3, déficients pour chacune des GRX, ne produisent pasdes phénotypes similaires à ceux observés avec les mutants GSH. Si Smgrx2 présente unphénotype moins marqué, Smgrx1 est plus sévèrement affecté puisqu'il n'est plus capable dese différencier en bactéroïde. L'implication de SmGrx2 dans la régulation du métabolisme dufer et la mise en place des centres Fe-S a, par ailleurs, été mise en évidence.Le second objectif a été de définir s'il existait, chez S. meliloti, une redondance fonctionnelleentre les GRX et les thiorédoxines (TRX). Ainsi, le mutant SmtrxB, dépourvu de thiorédoxineréductase, présente la particularité d'induire la formation d'un plus grand nombre de nodulesque la souche sauvage. Le système TRX de S. meliloti apparaît donc comme un régulateurnégatif de la nodulation. D'autre part, les nodosités formées par ce mutant SmtrxB, ont uneactivité fixatrice d'azote significativement diminuée. Les rôles des TRX et des GRXapparaissent donc, au moins partiellement, distincts.Les résultats obtenus ici apportent des éléments nouveaux sur l'implication du GSH, des GRXet des TRX dans la mise en place d'une nodosité fonctionnelle, et ouvrent de nouvellesperspectives d'études sur les rôles de ces molécules dans le processus de fixation d'azote. / Sinorhizobium meliloti is a soil bacterium Gram- able to induce the formation of nitrogenfixingnodules during a symbiotic interaction with plants of the legume family. Theimportance of redox balance during this interaction has been demonstrated. In this way,bacterial mutants deficient in the production of glutathione (GSH), exhibit an alteredphenotype of infection and fixation of atmospheric nitrogen.The first objective was therefore to determine whether the phenotypes observed in mutants ofthe GSH biosynthesis pathway were related to the activity of glutaredoxins (GRX). Abioinformatic analysis revealed the presence of three genes encoding GRX in S. meliloti. Themutants, Smgrx1, Smgrx2 and Smgrx3, deficient for each of the GRX, do not producephenotypes similar to those observed with the GSH mutants. If Smgrx2 presents a less severephenotype, Smgrx1 is more severely affected since it is incapable of differentiating intobacteroïd. The involvement of SmGrx2 in the regulation of iron metabolism and theestablishment of Fe-S cluster has also been demonstrated.The second objective was to determine if there was, in S. meliloti, a functional redundancybetween GRX and thioredoxin (TRX). Thus, the SmtrxB mutant, devoid of thioredoxinreductase, has the distinctive feature of inducing the formation of more nodules than the wildtype strain. The TRX system of S. meliloti appears to be a negative regulator of nodulation.On the other hand, the nodules formed by this SmtrxB mutant have a significantly decreasednitrogen-fixing activity. Hence, the roles of TRX and GRX appear to be at least partiallydistinct.The results obtained here provide new evidence on the involvement of GSH, the GRX andTRX in the establishment of a functional nodule, and open new perspectives for studying onthe roles of these molecules in the process of nitrogen fixation.

Le monoxyde d'azote (NO) est une molécule gazeuse impliquée dans de nombreux processus biologiques chez les plantes, de la germination de la graine à la mise en place de réponses à des stress abiotiques et biotiques. Dans les interactions plante/ pathogène, le NO fait partie de l'arsenal de défenses de l'hôte. En réponse, les pathogènes ont développé des mécanismes pour contrer les effets du NO. Dans la symbiose fixatrice d'azote entre la légumineuse modèle Medicago truncatula et la bactérie Sinorhizobium meliloti, du NO a été détecté durant toutes les phases de l'interaction. L'équipe avait précédemment montré que la réponse de S. meliloti au NO est nécessaire au maintien de la symbiose puisque des nodules formés par une souche mutée dans le gène hmp (le gène hmp est induit par le NO et code pour une protéine qui dégrade le NO) sénescent prématurément. Au cours de cette thèse, nous avons étudié 3 nouveaux gènes de S. meliloti induits par le NO : nnrS1, nnrS2 et norB. nnrS1 et nnrS2 codent pour deux protéines de fonction inconnue et norB code pour une NO réductase qui dégrade le NO. Nous avons montré que ces 3 protéines participent d'une part à la résistance des bactéries au NO en culture et d'autre part, au maintien de l'interaction symbiotique. Par ailleurs, nous avons montré que ces 3 protéines sont impliquées directement ou indirectement dans la dégradation du NO et des résultats préliminaires suggèrent que NnrS1 présente une activité NO réductase. De plus, nous avons montré que NnrS1 et Hmp n'agissent pas seulement sur les bactéries mais aussi sur les protéines végétales. Il était connu que dans les nodules de M. truncatula, la glutamine synthétase (GS) végétale, une enzyme clé de la symbiose, est inhibée par tyrosine nitration, une modification post-traductionnelle dépendante du NO. Nous avons montré que NnrS1 et Hmp, en modulant le niveau de NO dans les nodules, contrôlent l'activité de la GS. Enfin des expériences préliminaires montrent que d'autres protéines (bactériennes et/ou végétales) pourraient être tyrosine nitratées. / Nitric oxide (NO) is a gaseous molecule involved in a large range of biological processes in plants from the seed germination to abiotic and biotic stress responses. In plant-pathogen interactions, NO is part of the defense systems. In response, pathogens have developed mechanisms in order to counteract the NO effects. In the nitrogen fixing symbiosis between the model leguminous plant Medicago truncatula and the bacterium Sinorhizobium meliloti, NO has been detected at all stages of the symbiosis. The team had previously shown that the S. meliloti response to NO is necessary to maintain the symbiotic interaction since nodules elicited by a strain mutated in the hmp gene (hmp is induced by NO and codes for a flavohemoglobine that degrades NO) senesce prematurely. During this thesis, we have studied 3 new genes of S. meliloti whose expression is induced by NO: nnrS1, nnrS2 and norB. nnrS1 and nnrS2 encode two proteins of unknown function and norB codes for a NO reductase which degrades NO. We have shown that these 3 proteins participate on one hand in bacterial NO resistance in culture and on the other hand in maintaining the symbiotic interaction. Furthermore, we have shown that these 3 proteins are involved, directly or indirectly, in NO degradation and preliminary results suggest that NnrS1 displays a NO reductase activity. Moreover, we have shown that NnrS1 and Hmp are not only dedicated to protect bacteria against NO but also play a role on plant proteins. It was already known that, in M. truncatula nodules, the plant glutamine synthétase (GS), a key enzyme of the symbiosis is inhibited by tyrosine nitration, a NO post-translational modification. We have shown that NnrS1 and Hmp, by modulating NO levels in nodules, control the GS activity. Finally, preliminary experiments suggest that other proteins (from bacterial and/or plant origin), could be tyrosine nitrated.

Les Légumineuses ont développé une interaction symbiotique avec des bactéries du sol, les rhizobia, qui fixent l’azote atmosphérique et le transfèrent à la plante sous forme assimilable.Cette interaction a lieu, au sein des nodosités, des organes racinaires où les bactéries intracellulaires se différencient en bactéroïdes. Chez Medicago truncatula, ces bactéroïdes correspondent à un stade de différentiation terminale corrélée à une endoréplication de leur génome, une augmentation de la taille des cellules, une modification des membranes et une faible capacité à se propager. Cette différentiation est induite par des facteurs de la plante appelés NCR (Nodule-specific Cysteine Rich). Les peptides NCRs ressemblent à des défensines, des peptides antimicrobiens ayant une activité antimicrobienne in vitro, tuant des bactéries. Ainsi, un élément clef dans la différenciation des bactéroïdes est la protéine bactérienne BacA, un transporteur membranaire qui confère une résistance contre l’activité antimicrobienne des peptides. Dans le cadre de ce travail de thèse, j’ai montré que l'expression des NCR est soumise à une régulation stricte et qu’ils sont activés dans trois vagues dans les cellules symbiotiques polyploïdes.Les mécanismes de contrôle par la plante sur les rhizobia intracellulaires demeurent à ce jourpeu connus et le seul modèle étudié, au début de ce travail de thèse, restait l'interaction entre M. truncatula et S. meliloti. Je me suis donc intéressée à la symbiose de certaines Légumineuses tropicales du genre Aeschynomene appartenant au clade des Dalbergoïdes où jemontre qu’ils utilisent une classe différente de peptides riches en cystéine (NCR-like) pour induire la différenciation des bactéroïdes. Ce mécanisme est analogue à celui décrit précédemment chez Medicago qui était jusqu'à présent supposé être limitée aux légumineuses appartenant au clade des IRLC. J’ai également montré que Bradyrhizobium, symbionte d’Aeschynomene possèdent un transporteur de type ABC homologues à BacA de Sinorhizobium nommé BclA. Ce gène permet l'importation d'une variété de peptides comprenant des peptides NCR. En l'absence de ce transporteur, les rhizobiums sont incapables de se différencier et de fixer l'azote.Cette étude a permis d'élargir nos connaissances sur l'évolution de la symbiose en montrant qu’au cours de l’évolution, deux clades de Légumineuses relativement éloignés (IRLC et Dalbergoïdes) aient convergé vers l’utilisation de peptides de l’immunité innée afin de contrôler leur symbionte bactérien et d’en tirer un bénéfice maximal au cours de l’interaction symbiotique. / The ability of legumes to acquire sufficient nitrogen from the symbiosis with Rhizobium relies on the intimate contact between the endosymbiotic, intracellular rhizobia, called bacteroids, and their host cells, the symbiotic nodule cells. A well-studied example is the symbiotic nitrogen fixing bacterium Sinorhizobium meliloti, which nodulates the legume Medicago truncatula. Nodules of M. truncatula produce an enormous diversity of peptides called NCRs which are similar to antimicrobial peptides (AMPs) of innate immune systems. These NCRs are involved in maintaining the homeostasis between the host cells in the nodules and the large bacterial population they contain. Although many NCRs are genuine AMPs which kill microbes in vitro, in nodule cells they do not kill the bacteria but induce them into the terminally differentiated bacteroid state involving cell elongation, genome amplification, membrane fragilization and loss of cell division capacity. Protection against the antimicrobial action of NCRs by the bacterial BacA protein is critical for bacteroid survival in the symbiotic cells and thus for symbiosis. As a part of my PhD thesis, I have shown that the differentiation of the symbiotic cells in M. truncatula is associated with a tremendous transcriptional reprogramming involving hundreds of genes, mainly NCR genes, which are only expressed in these cells. Although the extensive work on the model M. truncatula/S. meliloti, little is known how the plant controls its intracellular population and imposes its differentiation into a functional form, the bacteroids in other symbiotic systems.In my PhD work, I provide several independent pieces of evidence to show that tropical legumes of the Aeschynomene genus which belong to the Dalbergoid legume clade use a different class of cysteine rich peptides (NCR-like) to govern bacteroid differentiation. This mechanism is similar to the one previously described in Medicago which was up to now assumed to be restricted to the advanced IRLC legume clade, to which it belongs. I have also shown that the Bradyrhizobium symbionts of Aeschynomene legumes possess a multidrug transporter, named BclA, which mediates the import of a diversity of peptides including NCR peptides. In the absence of this transporter, the rhizobia do not differentiate and do not fix nitrogen. BclA has a transmembrane domain of the same family as the transmembrane domain of the BacA transporter of Rhizobium and Sinorhizobium species which is known to be required in these rhizobia to respond to the NCR peptides of IRLC legumes. Again this is a mechanism which is analogous to the one described in S. meliloti the symbiont of Medicago.This study broaden our knowledge on the evolution of symbiosis by showing that the modus operandi involving peptides derived from innate immunity used by some legumes to keep their intracellular bacterial population under control is more widespread and ancient than previously thought and has been invented by evolution several times.

Most members of the Rhizobiaceae possess single copies of the poly-3-hydroxybutyrate biosynthesis genes, phbA, phbB and phbC. Analysis of the genome sequence of Bradyrhizobium japonicum reveals the presence of five homologues of the PHB synthase gene phbC as well as two homologues of the biosynthesis operon, phbAB. The presence of multiple, seemingly redundant homologues may suggest a functional importance. Each B. japonicum phbC gene was cloned and used to complement the pleiotropic phenotype of a Sinorhizobium meliloti phbC mutant; this mutant is unable to synthesize PHB, grow on certain PHB cycle intermediates and forms non-mucoid colonies on yeast mannitol medium. Two of the five putative B. japonicum phbC genes were found to complement the S. meliloti phbC mutant phenotype on D-3-hydroxybutyrate although none of them could fully complement the phenotype on acetoacetate. Both complementing genes were also able to restore PHB accumulation and formation of mucoid colonies on yeast mannitol agar to phbC mutants. In-frame deletions were constructed in three of the five phbC open reading frames in B. japonicum, as well as in both phbAB operons, by allelic replacement. One of the phbC mutants was unable to synthesize PHB under free-living conditions; one of the two phbAB operons was shown to be necessary and sufficient for PHB production under free-living conditions. These mutants also demonstrated an exopolysaccharide phenotype that was comparable to S meliloti PHB synthesis mutants. These strains were non-mucoid when grown under PHB-inducing conditions and, in contrast to wild-type B. japonicum, formed a compact pellet upon centrifugation. Interestingly, none of the mutants exhibited carbon-utilization phenotypes similar to those exhibited by S. meliloti PHB mutants. Wild-type B. japonicum accumulates PHB during symbiosis, and plants inoculated with the phbC mutants demonstrate a reproducible reduction in shoot dry mass. Analysis of bacteroid PHB accumulation in the mutant strains suggests that the phbAB operons of B. japonicum are differently regulated relative to growth under free-living conditions; mutants of the second phbAB operon demonstrated a significant reduction in PHB accumulation during symbiosis. These data suggest that the first phbAB operon is required for PHB synthesis only under free-living conditions, but is able to partially substitute for the second operon during symbiosis. Deletion of both phbAB operons completely abolished PHB synthesis in bacteroids. Analysis of the upstream regions of these genes suggest the existence of putative RpoN binding sites, perhaps indicating a potential mode of regulation and highlighting the metabolic complexity that is characteristic of the Rhizobiaceae. PHB metabolism in S. meliloti has been studied in considerable detail with two notable exceptions. No reports of the construction of either a β-ketothiolase (phbA) or a PHB depolymerase (phaZ ) mutant have ever been documented. The phaZ gene, encoding the first enzyme of the catabolic half of the PHB cycle in S. meliloti, was identified and a phaZ mutant strain was generated by insertion mutagenesis. The phaZ mutant demonstrates a Fix+ symbiotic phenotype and, unlike other PHB cycle mutants, does not demonstrate reduced rhizosphere competitiveness. Bacteroids of this strain were shown to accumulate PHB, demonstrating for the first time that S. meliloti is able to synthesize and accumulate PHB during symbiosis. Interestingly, there is no significant difference in shoot dry mass of plants inoculated with the phaZ mutant, suggesting that PHB accumulation does not occur at the expense of nitrogen fixation. The phaZ mutant strain was also used to demonstrate roles for PhaZ in the control of PHB accumulation and exopolysaccharide production. When grown on high-carbon media, this mutant demonstrates a mucoid phenotype characteristic of exopolysaccharide production. Subsequent analyses of a phoA::exoF fusion confirmed elevated transcription levels in the phaZ mutant background. In contrast, mutants of the PHB biosynthesis gene, phbC, have a characteristically dry phenotype and demonstrate reduced exoF transcriptional activity. The phaZ mutant also demonstrates a significant increase in PHB accumulation relative to the wild-type strain. Previous work on phasin mutants in S. meliloti demonstrated that they lack the ability to synthesize PHB. Transduction of the phaZ lesion into the phasin mutant background was used to construct a phaZ-phasin mutant strain. Analysis of the PHB biosynthesis capacity of this strain showed that the lack of PHB synthesis exhibited by S. meliloti phasin mutants is due to loss of PHB biosynthesis activity and not due to an inherent instability in the PHB granules themselves. A recent study suggested that some bacteria may possess an alternate pathway for acetate assimilation that would bypass the need for the glyoxylate cycle in organisms that do not possess the enzyme, isocitrate lyase. In these organisms, acetate is assimilated through the ethylmalonyl-CoA pathway, which has significant overlap with the anabolic half of the PHB cycle, including reliance on the PHB intermediate 3-hydroxybutyryl-CoA. The observation that phbB and phbC mutants of S. meliloti are unable to grow well on acetoacetate -- coupled with previously unexplained data that show a class of mutants (designated bhbA-D) are able to grow on acetate, but not on hydroxybutyrate or acetoacetate -- made it tempting to speculate that an ethylmalonyl-CoA-like pathway might be present in S. meliloti, and that this pathway might overlap with the PHB cycle at the point of 3-hydroxybutyryl-CoA. An in-frame mutation of phbA was constructed by cross-over PCR and allelic replacement. This mutant exhibited a complete abolition of growth on acetoacetate, suggesting that PhbA represents the only exit point for carbon from the PHB cycle and that an alternative ethylmalonyl-CoA-like pathway is not present in this organism. During symbiosis, rhizobial cells are dependent on the provision of carbon from the host plant in order to fuel cellular metabolism. This carbon is transported into the bacteroids via the dicarboxylate transport protein, DctA. Most rhizobia possess single copies of the transporter gene dctA and its corresponding two-component regulatory system dctBD. The completed genome sequence of B. japonicum suggests that it possesses seven copies of dctA. Complementation of Sinorhizobium meliloti dct mutants using the cosmid bank of B. japonicum USDA110 led to the identification a dctA locus and a dctBD operon. Interestingly, the B. japonicum dctABD system carried on the complementing cosmid was not able to complement the symbiotic deficiency of S. meliloti strains carrying individual mutations in either dctA, dctB, or dctD suggesting that the B. japonicum dctBD is unable to recognize either DctB/DctD or the DctB/DctD-independent regulatory elements in S. meliloti. All seven B. japonicum dctA ORFs were cloned and an analysis of their capacity to complement the free-living phenotype of a S. meliloti dctA mutant demonstrated that they all possess some capacity for dicarboxylate transport. Mutants of all seven B. japonicum dctA ORFs were constructed and an analysis of their free-living phenotypes suggested that significant functional redundancy exists in B. japonicum DctA function. Given the large number of potential dctA genes in the genome, coupled with an apparent lack of dctBD regulators, it is tempting to speculate that different DctA isoforms may be used during free-living and symbiotic growth and may be subject to different regulatory mechanisms than those of better-studied systems. A comprehensive analysis of desiccation tolerance and ion sensitivity in S. meliloti was conducted. The results of these analyses suggest that genetic elements on both pSymA and pSymB may play a significant role in enhancing cell survival under conditions of osmotic stress. The S. meliloti expR+ strains SmUW3 and SmUW6 were both shown to exhibit considerably higher desiccation tolerance than Rm1021, suggesting a role for enhanced exopolysaccharide production in facilitating survival under adverse conditions. Furthermore, scanning electron microscopy of inoculated seeds suggests that S. meliloti cells initiate biofilm formation upon application to the surface of seeds. This finding has implications for the analysis of OSS and the development of desiccation assays and may explain some of the variability that is characteristic of desiccation studies.

Most members of the Rhizobiaceae possess single copies of the poly-3-hydroxybutyrate biosynthesis genes, phbA, phbB and phbC. Analysis of the genome sequence of Bradyrhizobium japonicum reveals the presence of five homologues of the PHB synthase gene phbC as well as two homologues of the biosynthesis operon, phbAB. The presence of multiple, seemingly redundant homologues may suggest a functional importance. Each B. japonicum phbC gene was cloned and used to complement the pleiotropic phenotype of a Sinorhizobium meliloti phbC mutant; this mutant is unable to synthesize PHB, grow on certain PHB cycle intermediates and forms non-mucoid colonies on yeast mannitol medium. Two of the five putative B. japonicum phbC genes were found to complement the S. meliloti phbC mutant phenotype on D-3-hydroxybutyrate although none of them could fully complement the phenotype on acetoacetate. Both complementing genes were also able to restore PHB accumulation and formation of mucoid colonies on yeast mannitol agar to phbC mutants. In-frame deletions were constructed in three of the five phbC open reading frames in B. japonicum, as well as in both phbAB operons, by allelic replacement. One of the phbC mutants was unable to synthesize PHB under free-living conditions; one of the two phbAB operons was shown to be necessary and sufficient for PHB production under free-living conditions. These mutants also demonstrated an exopolysaccharide phenotype that was comparable to S meliloti PHB synthesis mutants. These strains were non-mucoid when grown under PHB-inducing conditions and, in contrast to wild-type B. japonicum, formed a compact pellet upon centrifugation. Interestingly, none of the mutants exhibited carbon-utilization phenotypes similar to those exhibited by S. meliloti PHB mutants. Wild-type B. japonicum accumulates PHB during symbiosis, and plants inoculated with the phbC mutants demonstrate a reproducible reduction in shoot dry mass. Analysis of bacteroid PHB accumulation in the mutant strains suggests that the phbAB operons of B. japonicum are differently regulated relative to growth under free-living conditions; mutants of the second phbAB operon demonstrated a significant reduction in PHB accumulation during symbiosis. These data suggest that the first phbAB operon is required for PHB synthesis only under free-living conditions, but is able to partially substitute for the second operon during symbiosis. Deletion of both phbAB operons completely abolished PHB synthesis in bacteroids. Analysis of the upstream regions of these genes suggest the existence of putative RpoN binding sites, perhaps indicating a potential mode of regulation and highlighting the metabolic complexity that is characteristic of the Rhizobiaceae. PHB metabolism in S. meliloti has been studied in considerable detail with two notable exceptions. No reports of the construction of either a β-ketothiolase (phbA) or a PHB depolymerase (phaZ ) mutant have ever been documented. The phaZ gene, encoding the first enzyme of the catabolic half of the PHB cycle in S. meliloti, was identified and a phaZ mutant strain was generated by insertion mutagenesis. The phaZ mutant demonstrates a Fix+ symbiotic phenotype and, unlike other PHB cycle mutants, does not demonstrate reduced rhizosphere competitiveness. Bacteroids of this strain were shown to accumulate PHB, demonstrating for the first time that S. meliloti is able to synthesize and accumulate PHB during symbiosis. Interestingly, there is no significant difference in shoot dry mass of plants inoculated with the phaZ mutant, suggesting that PHB accumulation does not occur at the expense of nitrogen fixation. The phaZ mutant strain was also used to demonstrate roles for PhaZ in the control of PHB accumulation and exopolysaccharide production. When grown on high-carbon media, this mutant demonstrates a mucoid phenotype characteristic of exopolysaccharide production. Subsequent analyses of a phoA::exoF fusion confirmed elevated transcription levels in the phaZ mutant background. In contrast, mutants of the PHB biosynthesis gene, phbC, have a characteristically dry phenotype and demonstrate reduced exoF transcriptional activity. The phaZ mutant also demonstrates a significant increase in PHB accumulation relative to the wild-type strain. Previous work on phasin mutants in S. meliloti demonstrated that they lack the ability to synthesize PHB. Transduction of the phaZ lesion into the phasin mutant background was used to construct a phaZ-phasin mutant strain. Analysis of the PHB biosynthesis capacity of this strain showed that the lack of PHB synthesis exhibited by S. meliloti phasin mutants is due to loss of PHB biosynthesis activity and not due to an inherent instability in the PHB granules themselves. A recent study suggested that some bacteria may possess an alternate pathway for acetate assimilation that would bypass the need for the glyoxylate cycle in organisms that do not possess the enzyme, isocitrate lyase. In these organisms, acetate is assimilated through the ethylmalonyl-CoA pathway, which has significant overlap with the anabolic half of the PHB cycle, including reliance on the PHB intermediate 3-hydroxybutyryl-CoA. The observation that phbB and phbC mutants of S. meliloti are unable to grow well on acetoacetate -- coupled with previously unexplained data that show a class of mutants (designated bhbA-D) are able to grow on acetate, but not on hydroxybutyrate or acetoacetate -- made it tempting to speculate that an ethylmalonyl-CoA-like pathway might be present in S. meliloti, and that this pathway might overlap with the PHB cycle at the point of 3-hydroxybutyryl-CoA. An in-frame mutation of phbA was constructed by cross-over PCR and allelic replacement. This mutant exhibited a complete abolition of growth on acetoacetate, suggesting that PhbA represents the only exit point for carbon from the PHB cycle and that an alternative ethylmalonyl-CoA-like pathway is not present in this organism. During symbiosis, rhizobial cells are dependent on the provision of carbon from the host plant in order to fuel cellular metabolism. This carbon is transported into the bacteroids via the dicarboxylate transport protein, DctA. Most rhizobia possess single copies of the transporter gene dctA and its corresponding two-component regulatory system dctBD. The completed genome sequence of B. japonicum suggests that it possesses seven copies of dctA. Complementation of Sinorhizobium meliloti dct mutants using the cosmid bank of B. japonicum USDA110 led to the identification a dctA locus and a dctBD operon. Interestingly, the B. japonicum dctABD system carried on the complementing cosmid was not able to complement the symbiotic deficiency of S. meliloti strains carrying individual mutations in either dctA, dctB, or dctD suggesting that the B. japonicum dctBD is unable to recognize either DctB/DctD or the DctB/DctD-independent regulatory elements in S. meliloti. All seven B. japonicum dctA ORFs were cloned and an analysis of their capacity to complement the free-living phenotype of a S. meliloti dctA mutant demonstrated that they all possess some capacity for dicarboxylate transport. Mutants of all seven B. japonicum dctA ORFs were constructed and an analysis of their free-living phenotypes suggested that significant functional redundancy exists in B. japonicum DctA function. Given the large number of potential dctA genes in the genome, coupled with an apparent lack of dctBD regulators, it is tempting to speculate that different DctA isoforms may be used during free-living and symbiotic growth and may be subject to different regulatory mechanisms than those of better-studied systems. A comprehensive analysis of desiccation tolerance and ion sensitivity in S. meliloti was conducted. The results of these analyses suggest that genetic elements on both pSymA and pSymB may play a significant role in enhancing cell survival under conditions of osmotic stress. The S. meliloti expR+ strains SmUW3 and SmUW6 were both shown to exhibit considerably higher desiccation tolerance than Rm1021, suggesting a role for enhanced exopolysaccharide production in facilitating survival under adverse conditions. Furthermore, scanning electron microscopy of inoculated seeds suggests that S. meliloti cells initiate biofilm formation upon application to the surface of seeds. This finding has implications for the analysis of OSS and the development of desiccation assays and may explain some of the variability that is characteristic of desiccation studies.

The effect of Ascophyllum nodosum extracts on the nodulation and growth of alfalfa was investigated. Plant growth assay revealed that alfalfa treated with 2 g L-1 ANE exhibited a significant increase in leaf area. Under salt stress, alfalfa treated with 0.5 g L-1 ANE exhibited a significant increase in total length compared to controls. A root hair deformation assay indicated that ANE 0.5 g L-1 stimulated the synthesis of Nod factors secreted by rhizobia thus accelerate root hair deformation of alfalfa. Similarly, ANE 0.5 g L-1 caused an increase in nodC gene expression suggesting that ANE may act similarly to flavonoids in the rhizobium-legume symbiosis. Under field conditions, ANE increased the total number of functional nodules, total root length and total leaf area. Taken together, the results suggest that ANE may contain compound(s) that promote specific metabolic pathway both in alfalfa and bacterium thus enhance the symbiotic relationship.

The conversion of atmospheric dinitrogen (N2) into plant available nitrogen (N), by legumes and their prokaryotic microsymbionts, is an integral component of sustainable farming. A key constraint to increasing the amount of N2 fixed in agricultural systems is the prevalence of symbioses which fix little or no N. The biotic factors leading to this suboptimal N2 fixation have not been extensively analysed. Using the widely studied and cultivated perennial legume Medicago sativa and the model indeterminate annual legume Medicago truncatula with the sequenced bacterial microsymbiont Sinorhizobium meliloti 1021 (Sm1021) as a basis, the work presented in this thesis examined the effectiveness of N2 fixation in these associations and in other comparable systems and investigated factors which lead to the establishment of suboptimally effective symbioses. The ability of Sm1021, S. medicae WSM419 and the uncharacterised Sinorhizobium sp. WSM1022 to fix N with M. truncatula A17, M. sativa cv. Sceptre and a range of other Medicago spp. was evaluated in N-limited conditions. As measured by plant shoot dry weights and N-content, Sm1021 was partially effective with M. truncatula A17 whereas WSM1022 and WSM419 were both effective with this host in comparison to nitrogen-fed (N-fed) control plants. In contrast, Sm1021 and WSM1022 were effective with M. sativa while WSM419 was only partially effective. Nodules induced by Sm1021 on M. truncatula A17 were more numerous, paler, smaller in size and more widely distributed over the entire root system than in the two effective symbioses with this host. On the contrary, nodule number, size and distribution did not differ between these three strains on M. sativa. WSM1022 was effective on M. littoralis, M. tornata and two other cultivars of M. truncatula (Jemalong and Caliph) but Sm1021 was only partially effective on these hosts. These data indicate that the model indeterminate legume symbiosis between M. truncatula and Sm1021 is not optimally matched for N2 fixation and that Sm1021 possesses broader symbiotic deficiencies. In addition, the interaction of WSM1022 with M. polymorpha (small white nodules but does not fix N), M. murex (does not nodulate), M. arabica (partially effective N2 fixation) and M. sphaeorcarpus (partially effective N2 fixation), and the sequence of the 16S rDNA, are all consistent with this isolate belonging to the species S. meliloti. The colony morphology of TY, half-LA and YMA agar plate cultures of Sm1021, WSM419 and WSM1022 suggested differences in EPS profiles between these strains. Sm1021 is very dry, compared to the mucoid WSM419 and extremely mucoid WSM1022. Sm1021 is known to carry an insertion in expR rendering the gene non-functional and resulting in the dry colony phenotype. WSM419 has an intact copy of expR, while the expR status of WSM1022 is not known. Rm8530, a spontaneous mucoid derivative of Sm1021 with an intact expR, was significantly less effective with M. truncatula than Sm1021, but there was no difference in effectiveness between these strains on M. sativa. The effectiveness of Sm1021, when complemented with a plasmid-borne copy of expR from Rm8530, was significantly reduced on M. truncatula but not M. sativa, implicating a functional expR as being the cause of reduced N2 fixation observed with Rm8530 on M. truncatula. ExpR could reduce the effectiveness of Rm8530 by acting as a negative regulator of genes essential for symbiosis with M. truncatula, or by altering the quantity or structure of succinoglycan and/or galactoglucan produced. These data support the emerging view of ExpR being a central regulator of numerous cellular processes. The timing of nodulation between Sm1021 and WSM419 on M. truncatula and M. sativa was investigated. Compared to the other symbioses analysed, the appearance of nodule initials and nodules was delayed when M. truncatula was inoculated with Sm1021 by 3 and 4 days, respectively. To explore whether events during early symbiotic signalling exchange could account for these observed delays, leading to the establishment of a suboptimal N2-fixing symbiosis, a novel system was developed to compare the response of the Sm1021 transcriptome to roots and root exudates of M. truncatula A17 and M. sativa cv. Sceptre. This system consisted of a sealed 1 L polycarbonate chamber containing a stainless steel tripod with a wire mesh platform on which surface-sterilised seeds could be placed and allowed to germinate through the mesh, into a hydroponic medium below. After germination, Sm1021 cells were inoculated into the hydroponic solution, exposed to the roots and root exudates for 16 h, harvested and their RNA extracted. Comparison of Sm1021 mRNA from systems exposed to M. truncatula or M. sativa revealed marked differences in gene expression between the two. Compared to the no plant control, when M. sativa was the host plant, 23 up-regulated and 40 down-regulated Sm1021 genes were detected, while 28 up-regulated and 45 down-regulated genes were detected with M. truncatula as the host. Of these, 12 were up-regulated and 28 were down-regulated independent of whether M. truncatula or M. sativa was the host. Genes expressed differently when exposed to either M. truncatula or M. sativa included nex18, exoK, rpoE1 and a number of other genes coding for either hypothetical proteins or proteins with putative functions including electron transporters and ABC transporters. Characterisation of these differentially expressed genes along with a better understanding of the composition of M. truncatula root exudates would yield a clearer insight into the contribution of early signal exchange to N2 fixation. Comparison of the regulation of nodule number between Sm1021 and WSM419 on M. truncatula and M. sativa revealed nodule initials at 42 days post-inoculation (dpi) on M. truncatula inoculated with Sm1021. In contrast, no new nodule initials were present 21 dpi on any of the other interactions examined. Moreover, analysis of nodule sections revealed that the number of infected cells in M. truncatula-Sm1021 nodules was less than for comparable symbioses. These data suggest that nodule number is not tightly controlled in the M. truncatula-Sm1021 association, probably due to N2 fixation being insufficient to trigger the down regulation of nodulation. Quantification of N2 fixation activity in this and other more effective symbioses is required. The poor effectiveness of the M. truncatula-Sm1021 symbiosis makes these organisms unsuitable as the model indeterminate interaction and the implications for legume research are discussed. The recently sequenced WSM419 strain, revealed here to fix N2 more effectively with M. truncatula than Sm1021, may be a better model microsymbiont, although WSM419 is only partially effective for N2 fixation with M. sativa. The sequencing of S. meliloti WSM1022, a highly effective strain with both M. truncatula and M. sativa, would provide a valuable resource in indentifying factors which preclude the establishment of effective symbioses.