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Title: Mass Spectrometric Studies Of The Rhizobium-Legume Symbiosis
Author: Sumpton, David
ISNI:       0000 0001 3489 9374
Awarding Body: University of York
Current Institution: University of York
Date of Award: 2007
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Abstract:
The highly specific symbiotic interaction between leguminous plants and rhizobial bacteria culminates in the fixation of atmospheric nitrogen which is made available to the host plant and ultimately replenishes the soil. This has the potential to act as a natural means to the benefits now frequently derived through the use of fertilizers Within agriculture. We have been investigating this symbiosis for soybean cultivars cropped in China, where the level of soluble nitrogen often limits crop yields. Through the use of classic carbohydrate isolation methodologies and' state-of-the-art mass spectrometric approaches, the profiling and structural characterisation of key glycoconjugate mediators ofthe symbiosis was performed. The LPS profile fromMesorhizobium tianshanenese, a rhizobium strain which has surprising symbiotic properties, was established and compared to the LPS profiles of other strains of rhizobia. The isolation and the structural characterisation of the capsular polysaccharide from 'the efficient soybean nodulator S. fredii SMHI2, was attempted. It was shown that SMH12 does not produce KPS which resembles any of the other previously identified rhizobial KPSs. The capsular polysaccharide of a Sinorhizobium fredii HH103 mutant (HH103rif ) was also successfully isolated and its structure was shown to be identical to that ofthe HHI03 strain. The structures of the periplasmic and secreted forms ofthe ~-cycloglucan from S. fredii HHI03 were characterised. The degree of polymerisation of the secreted form was found to vary between GlcI8 and Glc26 glucose residues. The periplasmic form was found to vary between 21 and 23 glucoses, and was substituted with three phosphoglycerol moieties. The structures of the major Nod Factor species secreted from two Mesorhizobium loti nod gene mutants (nodZ, nolL) which had shown phenotypic variation in their nodulation behavior, were profiled. Through the analysis ofanother nod mutant ofm. loti, the minimum nodgenes required forM loti to be able to nodulate L. japonicus were identified, namely nodDI, nodD2, nodA and node. Modifications to the structure of rhizobial membrane lipids can significantly compromise the successful outcome of the symbiosis. The /px02 gene is involved in .the synthesis of lipids in Burkholderia cenocepacia and is thought to be important for pathogenesis. In order to investigate the activity of the B. cenocepacia lpx02 gene, the extractable lipids of B. enocepacia and a B. cenocepacia mutant in which extra copies of the lpx02 gene had been introduced were identified using mass spectrometry. The study focused on identifying the site of lipid hydroxylation catalysed by the Ipx02 gene product. The structures of a range of lipid classes (pG, CL, PE, PE-OR, OL, and OL-OR) were profiled in both the wild type and mutant strain. Introduction of extra copies of lpx02 into B. cenocepacia resuIt~d in two additional lipid species, the first of which was consistent with a hydroxylated ornithine lipid structure, hydroxylation being on the amide linked acyl chain. The second species was hydroxylated, on both fatty acid residues. These results suggest that the lpx02 gene is capable of hydroxylating both ornithine lipids and hydroxylated ornithine lipids. Several targeted studies ofthe proteomes ofthe two symbiotic partners were undertaken. Due to the inherent problems associated with plant proteomics, especially the recalcitrant nature of the sample, alternative monolithic LC-MS/MS based methods were developed that allowed the successful analysis of selected sub-proteomes, where classical approaches failed. These approaches were evaluated through the analysis of several different protein mixtures ranging in complexity and generated during the study of the rhizobium-legume symbiosis. These enabled the identification of two new proteins secreted via a type III secretion mechanism, by the beneficial soybean nitrogen fixing micro-symbiont S.fredii HHI03.
Supervisor: Not available Sponsor: Not available
Qualification Name: University of York, 2007 Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.485143  DOI: Not available
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