Conjugative transfer and expression of an integrative and conjugative element in Pectobacterium atrosepticum : A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy at Lincoln University

The rapid evolution of bacteria can be credited to the almost ubiquitous association to Mobile Genetic Elements, the means of Horizontal Gene Transfer enabling rapid exchange of genetic information between bacterial populations. Integrative and Conjugative Elements (ICEs) are Mobile Genetic Elements (MGEs) that are transferred largely by conjugation between two bacterial cells and able to minimise the impact of their acquisition by integration into the host chromosome. The scarce number of studies concerning the frequency of transfer have been predominantly conducted on highly similar strains or non-related bacterial species, impeding the comprehension of its ecological significance. In this study, the conjugative transfer of Horizontally Acquired Island 2 (HAI2), an ICE that enhances virulence of the potato pathogen Pectobacterium atrosepticum SCRI1043, was investigated by assessing frequency of acquisition into strains of SCRI1043 in which HAI2 had been removed. HAI2 was transferred into engineered recipients confirming it as indeed a functional ICE. Further functional analysis of the island using mutants with transposon insertions in selected core genes of HAI2 demonstrated that the process of conjugation is decoupled from the initial excision of the island from the host genome. HAI2 requires a group of genes related to cell partitioning systems and a DNA helicase for effective transfer by conjugation. To understand whether HAI2 was likely to be transferred to related bacteria within the ecological niche of Pectobacterium atrosepticum SCRI1043, conjugation assays were conducted in vitro using plant pathogenic Pectobacteriaceae strains belonging to the genera Pectobacterium and Dickeya from New Zealand and overseas. Thirty-three PCR-positive isolates for at least one HAI2-compatible bacterial attachment (attB) site were selected. Surprisingly, HAI2 was not acquired by any of the selected isolates. A screening of the genome sequences of these potential recipients and other publicly available Pectobacteriaceae genome sequences revealed that the necessary attachment sites were almost always occupied. Several different mobile elements are seemingly integrated at these spots in their chromosomes, suggesting that horizontal transfer of HAI2 may be restricted by previous acquisition of mobile elements. The influence of HAI2 excision as a strategy to manipulate the cell regulatory network has also been investigated. Transcriptome analysis comparing the wild-type and mutant strainslacking the expression of HAI2 core genes involved in DNA topology and processing prior conjugation was performed in vitro and in planta. A topoisomerase IIIβ (ECA0525) and a relaxase (ECA0613) able to induce hyper-excision of the island, and an integrase (ECA0614) and an engineered islandless mutant (ΔHAI2) with no detectable excision were assessed. Genome-wide differential expression was observed, with HAI2 as the most affected portion of the chromosome. The transcriptome profiles were strongly correlated to the excision status of HAI2 rather than to a particular mutation. Hyper-excision was associated with upregulation of HAI2 core genes and genes related to plasmid stability. Absence of HAI2 excision induced downregulation of transcription pathways energy use. Changes in HAI2 excision impacted the expression of HAI5, HAI6 and HAI10, MGEs also integrated in Pba SCRI1043 chromosome. In summary, we provided evidence to confirm that HAI2 is a fully functional and active ICE, able to control its expression and influence the expression of its host. The important association of HAI2 to P. atrosepticum does not seem to be extended to other Pectobacteriaceae by conjugative transfer to environmental strains because of intense competition for attachment sites in the past.