Isolation and DNA sequence analysis of a Rhizobium loti gene required for effective nodulation of Lotus pedunculatus : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology at Massey University, Palmerston North, New Zealand

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A Rhizobium loti gene required for effective nodulation of the host Lotus pedunculatus has been identified by transposon Tn5 mutagenesis. Cosmids from a R. loti gene library which complemented a previously isolated mutant strain, PN239, (Chua et at 1985; J. Bacteriol. 162; 335-343) at this locus were identified by in planta complementation. A physical map of these cosmids was constructed and the site of insertion of the Tn5 was mapped to a 7.5 kb EcoRI fragment common to all cosmids which complemented the mutation. This 7.5 kb EcoRI fragment was subcloned into pBR328 and pLAFR1 and a more detailed physical map constructed. The 7.5 kb EcoRI fragment in pLAFR1 was able to complement the Tn5 mutation when introduced into strain PN239. Further Tn5 mutagenesis of the 7.5 kb EcoRI fragment was carried out in E. coli and the mutations were homogenotised into R. loti NZP2037. Three additional mutations were isolated which caused a Fix- phenotype on Lotus pedunculatus. The Tn5 inserts which caused a Fix- phenotype were mapped to positions adjacent to the position of the original mutation in strain PN239. All other Tn5 insertions isolated in the 7.5 kb EcoRI fragment gave a Fix+ phenotype on Lotus pedunculatus. A region was sequenced which was involved in effective nodulation of Lotus pedunculatus as indicated by the position of the Tn5 insertions. Analysis of the consensus sequence of 2307 bases identified a potential open reading frame (ORF) of 576 base pairs, coding for a putative protein of 21.2 kD. The positions of the Tn5 insertions causing a Fix- phenotype and the adjacent Tn5 insertions which did not affect fixation were determined in the sequence. The position and orientation of the ORF identified was consistent with the sequenced positions of these Tn5 insertions. A fragment containing most of the ORF identified from the sequence was used as a hybridization probe to various strains of rhizobia. Homology was only demonstrated with DNA from other R. loti strains. of loti strains containing Tn5 insertions which were Fix- on Lotus pedunculatus were found to be fully effective on Lotus corniculatus. These observations suggest that the gene characterised in this investigation may be involved in the host specificity of R. loti for Lotus pedunculatus.
Content removed due to copyright restrictions: Ward, L. J., Rockman, E. S., Ball, P., Jarvis, B. D., & Scott, D. B. (1989). Isolation and characterization of a rhizobium loti gene required for effective nodulation of lotus pedunculatus. Molecular Plant-Microbe Interactions : MPMI, 2(5), 224-232.
Rhizobium genetics, Nitrogen-fixing microorganisms, Genetics, Rhizobium loti, Lotus pedunculatus