Browsing by Author "Ward, Lawrence James Henry"

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    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
    (Massey University, 1989) Ward, Lawrence James Henry
    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.
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    Plasmids in Rhizobium phaseoli : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University
    (Massey University, 1982) Ward, Lawrence James Henry
    Fast growing strains of Rhizobium have been divided into four homology groups on the basis of DNA hybridization. Rhizobia from two of these homology groups can form effective nodules with beans (Phaseolus vulgaris). Large plasmids associated with nodulation have been demonstrated in Rhizobium sp. A study was undertaken to examine the plasmids in rhizobia from the two different homology groups capable of nodulating beans. The effectiveness of strains of Rhizobium phaseoli on bean plants were examined. Spontaneous antibiotic resistant mutants which retained the ability to nodulate beans were selected. Antibiotic resistance marked clones were incubated at elevated temperatures to produce an ineffective mutant stain of Rhizobium phaseoli NZP 5492. Methods of extracting large plasmids from Rhizobium phaseoli were developed. Plasmids were visualised by agarose gel electrophoresis and purified by cesium chloride - ethidium bromide density gradient ultracentrifugation. We demonstrated the presence of plasmids of molecular weight range 66 Md to 316 Md in Rhizobium phaseoli strains. Some strains contained a single plasmid while others contained multiple plasmids. Comparisons were made between whole plasmids and restriction endonuclease digests of the plasmids from the two groups. The fragment pattern obtained from Eco RI digests showed differences in fragment numbers and size between plasmids from DNA homology group 1 and DNA homology group 2. Further studies using gel blotting and hybridization techniques are required to ascertain the degree of homology between the plasmids, both within groups and between groups. Rhizobium phaseoli NZP 5479 and NZP 5547 a non-nodulating mutant of strain NZP 5479 were examined. Both strains had plasmids of estimated molecular weights 186 Md and 288 Md. There was no detectable difference in the size of the plasmids in the non-nodulating mutant compared to the effective parent strain. Rhizobium phaseoli NZP 5492 B5/8 (effective) and NZP 5492 B5/1 an ineffective mutant obtained from strain NZP 5492 had plasmids of similar molecular weight. Differences were observed in the Eco RI fragment pattern and possible rearrangements to the DNA to account for these differences are presented.