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Subject: search.filters.subject.Rhizobium loti

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    Characterisation of a Rhizobium loti nodulation mutant : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Molecular Genetics at Massey University
    (Massey University, 1991) Collins-Emerson, Julie Marie
    The aim of the project was to characterise the Rhizobiurn loti Nod- Tn5 mutant strain, PN233. The Tn5 insertion had been previously localised to a 7.1 kb Eco RI chromosomal fragment. This fragment was sub-cloned and a Bam HI/Sal I endonuclease restriction map for the region was determined. Hind III digests were utilised to identify the approximate location of the Tn5 233 insertion and those of four other Tn5 insertions (4016, 4019, 4047 and 4053) in the 7.1 kb region. The 233 mutation was found to map to a 1.45 kb Sal I fragment and that of an overlapping 2.8 kb Barn HI fragment. The 7.1 kb Eco RI fragment and a larger 22.7 kb fragment that encompassed this region, had been cloned into pLAFRl. The construct carrying the 22.7 kb fragment (pPN305) was crossed into four R.l. bv. trifolii strains, each mutant in one of the four common nod genes, A,B,C, and D. The construct was able to complement the nodC mutation indicating the presence of a nodC gene somewhere on the 22.7 kb region. The mutations 4047 and 4053 had been found to map to either side of the 233 Tn5 insertion. Both insertions affected nodule formation and were thus included in further plant complementation tests. These experiments involved crossing both the pPN305 and a construct bearing the smaller 7.1 kb Eco RI fragment (pPN25) into the R. loti and R.l. bv. trifolii Tn5 mutants. What was unusual about the results was that, while the 7.1 kb fragment was able to complement the mutations, the larger 22.7 kb fragment which encompasses that region could complement PN4047 and PN4053 but was unable to complement the PN233 mutant. The 2.8 kb Barn HI and 1.45 kb Sal I fragments, to which the 233 insertion was mapped, and that of an adjacent 1.2 kb Sal I fragment, were sub-cloned and then Bal 31 digested in both orientations to create a series of overlapping fragments. These fragments were then sequenced. The data revealed that the 233 Tn5 had inserted into the R. loti node gene. It was determined that the 4047 Tn5 was also located in this gene, slightly upstream of 233, while 4053 had inserted into the 5'-region of nodI which is downstream of nodC. Nod.A was identified upstream of nodC indicating an arrangement of common nod genes different from the conventional nod.ABeIJ found in other rhizobia. The promoter for these nod genes, the nod box, was located upstream of the nodA gene. A particularly puzzling aspect of the results is that, while PN4047 is complemented by both pPN305 and pPN25, PN233, which has an insertion in the same gene, could only be complemented by the smaller fragment carried by the pPN25 construct. To explain this result, it is proposed that PN233 is producing a mutant NodC protein and that this, in combination with doubled copies of a gene or genes present elsewhere on the 22.7 kb fragment, is responsible for interfering with complementation in this mutant. Alternatively, it may be that the imbalance of doubled copies of downstream, co-transcribed genes in the presence of one copy of a functional node gene causes complementation failure.
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    The isolation and characterization of Rhizobium loti exopolysaccharide mutants : a thesis presented in partial fulfilment of the requirements for the Degree of Doctor of Philosophy in Molecular Genetics at Massey University, Palmerston North, New Zealand
    (Massey University, 1991) Hotter, Grant Spencer
    PN1 84, a streptomycin resistant derivative of the broad host range Rhizobium loti strain NZP2037, was shown to be Calcofluor-bright due to the production of a Calcofluor-binding exopolysaccharide (EPS) with both high and low molecular weight forms, the low molecular weight form being predominant Eight Calcofluor-dark EPS mutants (three smooth and five rough) were generated by Tn5 mutagenesis of PN1 84. Each mutant was shown to carry a single , independent Tn5 insertion. Cosmids that complemented the mutation carried by each of the rough, PN1 84-derived EPS mutants were isolated from a pLAFRl gene library to NZP2037 by complementation of the Calcofluor-dark phenotype. The genetic regions identified were shown to be located on the chromosome, and were not closely linked. The mutants were divided into three (complementation) groups. While the rough, PN1 84-derived EPS mutants failed to synthesize EPS, the smooth, PN1 84-derived EPS mutants were found to synthesize an EPS which failed to bind Calcofluor, and which was shown, by 1 H-NMR spectroscopy, to be significantly less acetylated than the EPS produced by PN1 84. Furthermore, PN1 177, one of the smooth, PN1 84-derived EPS mutants, was shown to produce only a small amount of high molecular weight EPS compared to PN1 84. All the PN1 84-derived EPS mutants induced the formation of fully effective (Nod+Fix+) nodules on Lotus pedunculatus, a determinate nodulating host legume, but, in contrast, induced the formation of ineffective (Nod+Fix-) nodules on Leucaena leucocephala, an indeterminate nodulating host legume. Each rough, PN1 84-derived EPS mutant, c arrying its complementing cosmid, was fully effective on L. leucocephala. PN4 1 15, a streptomycin resistant derivative of the restricted, effective host range R . loti strain NZP2213, was shown to be Calcofluor-dark. PN4 1 1 5 was shown to produce an EPS, which fails to bind Calcofluor, that is acetylated to approximately the same extent as the EPS produced by PN1 84. Like PN1 1 77, PN4 1 15 was shown to produce only a small amount of high molecular weight EPS. Examination of 1 H-NMR spectra of EPS from PN4 1 1 5 and the smooth, PN1 84-derived EPS mutants suggests that these strains produce an EPS of similar structure, with the exception of the degree of 0- acetylation. Three non-mucoid, Calcofluor-bright, EPS mutants were generated by Tn5 mutagenesis of PN4 1 15. Each mutant was shown to carry a single, independent Tn5 insertion. Cosmids could not be isolated which stably complemented the mutation carried by each mutant. None of the mutants produced EPS, but all three mutants produce a Calcofluor-binding EPS, possibly cellulose. All three PN4 1 15-derived EPS mutants induced the formation of fully effective nodules on Lotus corniculatus, a determinate nodulating host legume. On L. leucocephala, PN4 1 15 induced the formation of both small, ineffective, nodular swellings and large, ineffective, tumour-like structures. Occasionally, a low level of nitrogen fixation was observed. In contrast, the PN41 15-derived EPS mutants all induced the formation of only small, ineffective, nodular swellings. These results, obtained in isogenic Rhizobium backgrounds, support suggestions that EPS is required for effective nodulation of indeterminate nodulating legumes, but is not required for effective nodulation of determinate nodulating legumes.
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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.