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Title: Molecular analysis of lipopolysaccharide and membrane associated proteins in Rhizobium leguminosarum.
Author: Sindhu, Satyavir Singh.
ISNI:       0000 0001 3414 502X
Awarding Body: University of East Anglia
Current Institution: University of East Anglia
Date of Award: 1990
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This study describes the use of monoclonal antibodies to investigate molecular components of the Rhizobium cell surface that might be important for symbiotic interactions with the host legume. Components that have been identified include lipopolysaccharide and both membrane-associated and secreted proteins. Differences were observed in the structure and antigenicity of lipopolysaccharide (LPS) from free-living rhizobia compared with that of endosymbiotic bacteroids. Culture pH, oxygen concentration and carbon source were all found to be important factors that could affect the expression of LPS antigens within the nodule. Mutants with altered LPS demonstrated that complete LPS structures are necessary for effective symbiosis. Monoclonal antibody nM 25 identified a protease-sensitive epitope that appeared to be attached to a particular species of LPS macromolecule (identified by MAC 114 antibody). Immunocytochemical localization studies of pea nodule sections revealed that JIM 25 antigen, present on the cell surface, was expressed in infection threads but its expression was low in the symbiotic zone containing mature bacteroids. A 38kDa secreted protein was identified by JIM 24 antibody. Fractionation of nodule extracts by differential centrifugation suggested that the protein was present in the peribacteroid space. A 55kDa membrane protein recognized by MAC 115 antibody proved to be a species-specific marker for R. leguminosarum. The structural gene(s) for this protein are encoded on a 2.8kb EcoRI fragment. Localised mutagenesis of this DNA region with the transposon TnPhoA (which carries a promoter-less gene for alkaline phosphatase) provided evidence that the 55kDa protein was' membraneassociated. However, attempts to "marker-exchange" the transposon-induced mutations from cosmid DNA into the Rhizobium genome were unsuccessful, suggesting that the 55kDa protein is essential for growth of free-living rhizobia. In a DNA region adjoining this 2.8kb EcoRI fragment that encodes the 55kDa protein, a new gene has been described, termed muc. When present on a cosmid the muc gene from R. leguminosarum conferred non-mucoid colony morphology on R. meliloti strain B287. "Marker-exchange" of muc::Tn5 mutations from the plasmid to R. ieguminosarum 8002 (bv. phaseo/i) resulted in derivatives that had lost the ability to nodulate Phaseolus beans. However, marker-exchange into R. leguminosarum B556 (bv. viciae) resulted in mutants that showed no abnormal symbiotic phenotypes on peas. The cosmid carrying the muc and 55kDa protein determinants (pIJ1639) was subjected to saturation transposon mutagenesis with TnPhoA This study revealed several new genes that probably encode membrane-associated or secreted proteins. In some cases gene transcription was dependent on the presence of hesperitin [which is known to be an activator of Rhizobium nodulation (nod) genes]. A 4.6kb EcoRI fragment adjacent to the 2.8kb fragment described above was also found to encode essential functions that prevented the construction of chromosomal mutants by marker-exchange.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Genetics