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Free-living and symbiotic characterisation of carbon utilisation mutants in Rhizobium tropici CIAT899

Rogers, Talitha (2016) Free-living and symbiotic characterisation of carbon utilisation mutants in Rhizobium tropici CIAT899. Honours thesis, Murdoch University.

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The reduction of atmospheric nitrogen (N2) to ammonia (N2 fixation) by bacteria (rhizobia) living symbiotically within legume root nodules contributes approximately 50% of the biosphere’s available nitrogen. This association provides the differentiated microsymbiont (the bacteroid) within the root nodule of the host legume a source of carbon in the form of malate. While studies suggest the oxidation of malate through the TCA cycle is essential for effective symbiotic N2 fixation in rhizobia, exactly how bacteroids balance their own biosynthetic requirements with the need to supply ATP and reductant to drive N2 fixation is currently unclear. However, this picture of bacteroid metabolism is limited to the detailed analysis of very few strains. Developing clearer explanations of how bacteroids power N2 fixation therefore requires analysis of a wider range of rhizobia, with particular focus on well-characterised strains with full genome sequences available that are also highly effective in N2 fixation on a legume host.

Rhizobium tropici CIAT899 is a highly effective N2-fixing microsymbiont of the grain legume Phaseolus vulgaris (common bean). R. tropici CIAT899 is the commercial inoculant for P. vulgaris production in many parts of the world and the genome of this strain was recently fully sequenced. Despite its significant role in agriculture, very little is known about the metabolism of R. tropici CIAT899 in symbiotic and free-living conditions, making it an ideal strain for further investigations.

The symbiotic and free-living metabolism of R. tropici CIAT899 was investigated using a combination of site-directed and random mutagenesis approaches. For the site directed approach, inactivation vectors pk19icdA and pJQ200SksucA were successfully created to target genes encoding the TCA cycle enzymes isocitrate dehydrogenase (icdA) and the E1 component of 2-oxogluarate dehydrogenase (sucA), respectively. However, isolation of icdA or sucA mutants was not achieved. In the case of icdA, the pk19icdA construct failed to suicide in CIAT899, making selection of CIAT899 icdA mutants impractical. For pJQ200SKsucA, although transconjugants were successfully isolated, subsequent analysis revealed the vector had most likely integrated non-specifically into the CIAT899 genome, with all transconjugants carrying intact copies of sucA.

For the random mutagenesis approach, a total of 3,200 CIAT899 mutants carrying the mTn5-GNm were screened for growth defects on minimal media with glucose, succinate, arabinose or pyruvate as sole carbon sources. From this, 20 mutants were isolated which were further categorised into six different classes based on their confirmed growth phenotypes. To assess the effect of these mutations on symbiotic performance, 13 mutants were selected and inoculated onto P. vulgaris in a glasshouse trial. Two strains, M2A2 and M4A1 produced significantly reduced mean shoot dry weights compared to wild-type CIAT899 on this host. Nested PCR and subsequent Sanger sequencing revealed the M4A1 mutation to be in the pyrB gene (RTCIAT899_CH07120), encoding the putative aspartate carbamoyltransferase catalysing an integral step pyrimidine biosynthesis. For M2A2, sequencing revealed the insertion to be in phaC (RTCIAT899_RS07985). The phaC gene encodes poly-β- hydroxybutyrate (PHB) synthase (polymerase) catalysing the final step in the synthesis of this polymer.

This is the first study to report a pyrB mutation in a rhizobial species and a phaC mutation resulting in a dramatic reduction in N2 fixation on P. vulgaris. While further work is required to rule-out the involvement of secondary mutations or polar-effects on downstream genes, the results presented here are likely to form the basis of future studies on bacteroid metabolism. In particular, the phaC mutant, if confirmed, may help explain how bacteroids balance their biosynthetic and N2 fixation requirements, leading to a better understanding of the metabolic determinants of symbiotic N2 fixation.

Publication Type: Thesis (Honours)
Murdoch Affiliation: School of Veterinary and Life Sciences
Supervisor: Terpolilli, Jason and Reeve, Wayne
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