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Creation and characterisation of genetically-marked Mesorhizobium integrative and conjugative elements

Stagg, Georgina (2018) Creation and characterisation of genetically-marked Mesorhizobium integrative and conjugative elements. Honours thesis, Murdoch University.

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Rhizobia are soil-dwelling bacteria capable of forming symbiotic associations with legumes, where they reduce atmospheric dinitrogen (N2) into ammonia (NH3), providing the host plant with a bioavailable nitrogen source. For rhizobia in the genus Mesorhizobium, genes essential to the establishment (nod) and maintenance of nitrogen fixing-legume symbioses (nif and fix) are chromosomally-encoded on mobile segments of DNA known as symbiosis integrative and conjugative elements (ICEs). Symbiosis ICEs are capable of excising from the host chromosome and transferring to a recipient ICE-free cell via bacterial conjugation. In the recipient cell, the invading ICE integrates into the recipient chromosome at conserved sites known as attachment sites. As well as encoding genes for excision, conjugation and integration, symbiosis ICEs often encode genes for vitamin biosynthesis, such as those for the synthesis of nicotinate, biotin and thiamine.

Symbiosis ICEs can be monopartite or tripartite in nature and it appears that these two conformations of elements may be evolutionarily related, with the more complex tripartite ICE hypothesised to have evolved from two recombination reactions between three independent monopartite ancestral ICEs that co-existed in an ancestral bacterial cell. Testing this hypothesis for tripartite ICE evolution requires conjugation experiments between two strains that harbour different symbiosis ICEs, which has not previously been experimentally attempted. Symbiosis ICE transfer studies have been conducted between an ICE donor strain and an ICE-devoid vitamin auxotrophic recipient strain, with ICE transfer exconjugants selected on the basis of vitamin prototrophy. However, this method of selecting exconjugants is ineffective for conjugation experiments between two ICE-harbouring strains, as both usually harbour the operons for nicotinate, biotin and thiamine biosynthesis. Genetically-marking two structurally similar, yet phenotypically distinct monopartite ICEs with a selectable marker, such as an antibiotic resistance gene would allow for screening exconjugants solely on the presence of the selectable marker. Two such ICEs are ICEMcSym1192 from the genome of the Cicer arietinum (chickpea) nodulating microsymbiont Mesorhizobium ciceri CC1192 and ICEMlSymR7A from the genome of the Lotus-nodulating M. loti R7A. Therefore, the aims of this thesis were to first genetically mark the symbiosis ICEs ICEMcSym1192 and ICEMlSymR7A, with antibiotic resistance genes that could facilitate selection of exconjugants in ICE transfer studies. Second, to investigate the free-living growth and symbiotic phenotype of marked symbiosis ICEs. Finally, to test the in vitro mobility of these genetically marked elements.

Using a site-directed mutagenesis approach, M. ciceri CC1192 ICEMcSym1192 was successfully marked within an intergenic region of the symbiosis ICE, with a gene encoding resistance to neomycin/kanamycin (nptII), yielding ICEMcSym1192::nptII. The free-living phenotype of two M. ciceri ICEMcSym1192::nptII derivatives, MCC91 and MCC92, was assessed alongside wild-type CC1192, in a bacterial growth experiment to compare the mean generation times of all three strains cultured in both rich (TY) and minimal (AMS with either glucose or succinate as the sole carbon source) media. Mean generation times were not significantly different between these strains (p > 0.05) in all media tested. Additionally, the symbiotic phenotype of the ICEMcSym1192::nptII derivatives did not differ significantly (p > 0.05) to wild-type CC1192, as measured by mean nodule number, nodule weight and shoot dry weight of inoculated C. arietinum. Using a similar site-directed approach, M. loti R7A ICEMlSymR7A was also marked within an intragenic region, however, with Ω-aadA encoding resistance to spectinomycin/streptomycin, yielding ICEMlSymR7A::Ω-aadA. Two ICEMlSymR7A::Ω-aadA derivatives, MCC93 and MCC94, were completed towards the end of this Honours project, therefore, the free-living and symbiotic phenotype could not be achieved within the scope of this project.

The transfer of ICEMcSym1192::nptII in MCC91 and MCC92, into the ICE-devoid recipient strain, R7ANS, was tested with conjugation mixtures plated onto three different sets of media to select for R7ANS (ICEMcSym1192::nptII) exconjugants. Integration of ICEMcSym1192::nptII in R7ANS should allow exconjugants to be selected solely on the presence of the antibiotic marker conferring neomycin resistance (encoded on ICEMcSym1192::nptII) and tetracycline resistance (encoded on R7ANS plasmid, pFAJ1700). The three selection conditions included rich or minimal media, with reduced tetracycline concentrations and the addition of the vitamin thiamine. While, putative exconjugants were extracted from two selection conditions, PCR screening confirmed these were not R7ANS exconjugants but instead were most likely spontaneous CC1192 tetracycline resistant isolates. The inability to isolate R7ANS (ICEMcSym1192::nptII) exconjugants could be due to a number of reasons, including the use of HEPES-buffered, rather than phosphate-buffered, minimal media, the combination of neomycin and tetracycline in the selection media attenuating the rate of ICE excision and/or transfer or the insertion of the nptII cassette in ICEMcSym1192::nptII affecting ICE mobility. Nevertheless, the marked strains generated in this thesis and the assessment performed on them to date, provides a solid foundation for subsequent experiments to further characterise the mobility of these derivatives.

The discovery that Mesorhizobium symbiosis ICEs can exist in both monopartite and tripartite configurations raises many questions about ICE stability and persistence. The genetically-marked ICEMcSym1192::nptII and ICEMlSymR7A::Ω-aadA produced in this thesis represent an important set of experimental tools necessary to further investigate the evolution of these symbiotic elements.

Item Type: Thesis (Honours)
Murdoch Affiliation: School of Veterinary and Life Sciences
United Nations SDGs: Goal 12: Responsible Consumption and Production
Supervisor(s): Terpolilli, Jason, O'Hara, Graham and Ramsay, Joshua
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