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Discovery and characterisation of tripartite Integrative & Conjugative Elements

Haskett, Timothy L. (2018) Discovery and characterisation of tripartite Integrative & Conjugative Elements. PhD thesis, Murdoch University.

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Bacterial integrative & conjugative elements (ICEs) are chromosomally-integrated DNA islands that excise to form circular molecules capable of horizontal self-transmission via conjugation (cell-to-cell contact). Symbiosis ICEs, such as ICEMlSymR7A of Mesorhizobium loti, are a group of ICEs that carry genes enabling rhizobial bacteria to engage in N2-fixing symbioses with leguminous plants. Transfer of symbiosis ICEs can convert non-symbiotic rhizobia into legume symbionts in a single evolutionary step.

In this thesis, a novel form of “tripartite” ICE (ICE3) is reported that exists as three entirely separated regions of DNA residing in the chromosomes of genetically diverse N2-fixing Mesorhizobium spp. These ICE3 regions did not excise independently, rather through multiple recombinations with the host chromosome they formed a single contiguous region of DNA prior to excision and conjugative transfer. Upon integration into a recipient chromosome, the ICE3 recombined the recipient chromosome to disassemble into the tripartite form. These recombination reactions were catalysed by three Integrase proteins IntG, IntM, and IntS, acting on three associated integrase attachment sites. The “excisive” recombination reactions (i.e. assembly and excision) were stimulated by three recombination directionality factors RdfG, RdfM, and RdfS. Expression of ICE3 transfer and conjugation genes were found to be induced by quorum-sensing. Quorum-sensing activated expression of rdfS, and in turn RdfS stimulated transcription of both rdfG and rdfM. Therefore, RdfS acts as a “master controller” of ICE3 assembly and excision. A model for ICE3 recombination and transfer is presented in this thesis.

The conservation of gene content between symbiosis ICE and ICE3 indicated that these elements share a common evolutionary history. However, the persistence of ICE3 structure in diverse mesorhizobia is perplexing due to its seemingly unnecessary complexity. Bioinformatic comparisons of ICE and ICE3 indicated that the tripartite configuration itself may provide selective benefits to the element, including enhanced host range, host stability and resistance to destabilization by tandem insertion of competing integrative elements.

In congruency with ICEMlSymR7A, ICE3 acquisition can convey upon recipients the ability to form N2-fixing symbiosis with the host-legume of the ICE3 donor. Interestingly, the effectiveness of N2-fixation may be impaired. The consequences of the emergence of sub-optimal N2-fixing strains following ICE3 transfer in agriculture is discussed. If ICE3 transfer poses a barrier for future inoculation success, the elucidation of the mechanism of ICE3 assembly, excision, and transfer will allow for the development of strategies for management.

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