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Characterisation of the symbiosis ICE and accessory plasmid of Mesorhizobium ciceri

Bonello, Emma (2018) Characterisation of the symbiosis ICE and accessory plasmid of Mesorhizobium ciceri. Honours thesis, Murdoch University.

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The symbiosis between soil bacteria (rhizobia) and legumes is an integral component of sustainable agricultural practice, providing a source of bioavailable (or fixed) nitrogen to farming systems without supplementation with industrially synthesised fertiliser. Critical to the success of this association are rhizobial nodulation (nod) and nitrogen fixation (nif and fix) genes, that collectively allow for establishment and maintenance of nitrogen-fixing rhizobia-legume symbioses. In the Mesorhizobium genus symbiosis genes are chromosomally-encoded on Integrative and Conjugative Elements (ICEs). ICEs are a form of mobile genetic elements, capable of excising from the host chromosome through the action of the ICE-encoded integrase (IntS) and recombination and directionality factor (RdfS). The excised ICE can then transfer via conjugation into the recipient cell, where the ICE integrates into the host chromosome at specific attachment sites, usually adjacent to amino-acyl tRNA genes. Although recipients receive the complete symbiosis ICE from the donor strain and are subsequently able to nodulate a target legume, these exconjugants frequently fix nitrogen sub-optimally, indicating that a strong interaction between chromosomal and ICE-encoded genes exists. Understanding how different Mesorhizobium chromosomes and ICEs interact requires an array of ICE-devoid Mesorhizobium recipient strains. However currently, there is only such strain available, M. loti R7ANS, produced from the ICE-containing parent strain M. loti R7A. Furthermore, Mesorhizobium spp. may also harbour an accessory plasmid, which may have a role in nitrogen fixation.

Recently, the genome of Mesorhizobium ciceri CC1192, the commercial inoculant for Cicer arietinum (chickpea) was reported. The genome of CC1192 consists of 6.29-Mb chromosome with a symbiosis ICE (ICEMcSym1192) of 419-kb, as well as a 648-kb accessory plasmid, pMc1192. Although the strain has been used as an inoculant for C. arietinum for more than 40 years, very little is known about the symbiosis ICE of this strain or the plasmid that it harbours. Therefore, the aims of this thesis were to determine the symbiotic role of the accessory plasmid, pMc1192 in M. ciceri CC1192 and to investigate means of producing an ICE-devoid M. ciceri strain by curing ICEMcSym1192.

Bioinformatic analysis of pMc1192 revealed it harboured several unique symbiosis genes (fixLJ, fixK and fixS) and several symbiosis genes (fixNOQP and fixGHI) found on the chromosome. All of these genes, apart from fixS, has been shown to be essential for symbiosis. Plasmid pMc1192 was removed through a plasmid incompatibility approach, and loss was confirmed through PCR screening, Eckhardt gel analysis and sequencing. The symbiotic phenotypes of two plasmid-cured strains, MCC69 and MCC70, were assessed alongside the wild type CC1192 with C. arietinum. Mean shoot dry weight, nodule number and nodule weight were not significantly different between the three strains (p > 0.05), indicating that the plasmid-encoded genes were dispensable for symbiosis with C. arietinum.

In an attempt to remove ICEMcSym1192 from CC1192, RdfS which is the protein that stimulates ICE excision in R7A, and for which a homolog exists in CC1192, were individually overexpressed in CC1192. PCR screening of attachment sites formed upon ICEMcSym1192 integration and excision in CC1192 overexpressing strains, as well as replica patching of 1,044 colonies demonstrated that ICEMcSym1192 was not lost upon overexpression of either rdfS, despite the increased proportion of cells excising the ICE when the CC1192 rdfS was overexpressed. An alternate approach to remove ICEMcSym1192 is through the replacement of the entire 419-kb region with an antibiotic marker through homologous recombination. To achieve this, the integrase (intS), responsible for catalysing the excision and integration of the ICE, was first inactivated by a replacement with Ω-aadA to prevent re-integration of ICEMcSym1192 from the chromosome during later deletion of the ICE. PCR screening of the attachment sites in the IntS-deleted strain, MCC86, indicated that the ICE had become immobile, and therefore stabilised in the CC1192 chromosome. The ICE inactivation vector (pMCC8) was also successfully synthesised, providing a tool for future deletion of ICEMcSym1192 from CC1192.

The removal of pMc1192 as well as the attempted deletion of ICEMcSym1192 are steps towards the creation of a minimal M. ciceri CC1192 genome devoid of both ICE and accessory plasmids. This strain will prove a valuable research tool to better understand how the interaction between chromosomal and ICE-encoded genes in strains that have recently acquired symbiosis ICEs lead to sub-optimal nitrogen fixation.

Item Type: Thesis (Honours)
Murdoch Affiliation(s): School of Veterinary and Life Sciences
Supervisor(s): Terpolilli, Jason, O'Hara, Graham and Ramsay, Joshua
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