Analysis of Integrase and Transposase-Mediated Mechanisms for Transfer of Mobile Genetic Elements

Mobile genetic elements (MGE) are DNA segments most of which are capable of their own self mobilisation through genes either intracellularly (transposase or integrase genes) or intercellularly (conjugation genes), thus both contributing in bacterial evolution. This thesis investigated the functions of two distinct integrase genes in the mobilisation of two metal resistance GIs, G08 and G62, of Acinetobacter baumannii. G8int encodes a novel family of phage-type integrases whereas the G62int gene encodes a classic phage-type integrase. In addition, the role of transposon-mobilised resistance genes have been addressed in a number of Staphylococcus epidermidis clinical isolates, conferring resistance to biocides by way of the metabolic ileS and fabI genes. Real-time PCR demonstrated integrase dependent GI excision, utilising IPTG-inducible integrase genes in plasmid-based mini-GIs in E. coli. In A. baumannii integrase-dependent excision of the original chromosomal GIs could be observed after mitomycin C induction. In both E. coli and A. baumannii the rate of excision and circularisation was found to be dependent on the expression level of the recombinases. Susceptibility testing in A. baumannii strains ATCC 17978, A424 and their respective ΔG62 and ΔG08 mutants confirmed the contribution of the GI-encoded efflux transporters to heavy metal resistance mainly for copper. In S. epidermidis, the distribution and phenotypes of the insertion-sequence mobilised ileS2 gene on mupirocin resistance and sh-fabI gene on tricolsan resistance is described in this study and the novel type of mobilization of the sh-fabI carrying TnSha1 element was investigated. In summary, the data evidence the functionality and essential contribution of two integrases in the mobilisation of the two A. baumannii heavy metal resistance GIs G08 and G62 in E. coli as well as when chromosomally located in their natural host. Moreover, the contribution of additional laterally transferred genes, ileS2 and sh-fabI in reducing the susceptibility to mupirocin and triclosan, respectively was confirmed in the current study. The different transfer mechanisms in both elements result in genomes plasticity participating in the dissemination of resistance determinants in A. baumannii and Staphylococcus species.