Effect of mutations in E. coli membrane components on bacterial conjugation

Examensarbete för masterexamen
Master's Thesis
Biotechnology (MPBIO), MSc
Ahlstedt, Emelie
The rise of antibiotic resistant bacteria is a major threat to global health care as antibiotics are an important tool in modern medicine. Antibiotic resistance genes can spread between bacteria in a process called conjugation. Conjugation is a form of horizontal gene transfer where a donor cell transfers genetic material to a recipient cell. A possible method to stop the spread of antibiotic resistance genes is to target conjugation. In this project seven different single gene deletions were introduced in the donor. The genes are related to membrane components in Escherichia coli and their effect on conjugation was investigated. The genes of interest were fabF, fabH, lpp, plsX, rfaD, rseA, and wzzE. fabF, fabH, plsX, rfaD, and wzzE were investigated through liquid mating assays while lpp and rseA were investigated by a microfluidics method. However, this microfluidic method is not suited for large scale experiments and was therefore further developed in this project to enable a microfluidics system with four parallel channels. The liquid mating assay with mutant strains showed that ΔfabH and ΔplsX have a small effect on the transfer efficiency of the donor. ΔfabF and ΔwzzE were shown to have a moderate effect on transfer frequency while ΔrfaD showed a large effect on transfer frequency. ΔfabF and ΔrfaD were investigated further by a solid mating assay as these two mutations showed the biggest effect on transfer frequency. The results from the solid mating assay were similar to the liquid mating assay. It is hypothesised that the effects seen for ΔfabF and ΔrfaD could be due to altered membrane fluidity. Thus, the effect of altered membrane fluidity was investigated by treating the donor cells with the membrane softener pentanol in liquid mating assays. Pentanol concentrations of 0 M to 20 mM were examined. The liquid mating assay performed with pentanol showed that increased membrane fluidity decreases the transfer frequency of conjugation. Lastly, the development of the microfluidics method tested different surface modifications of the channels for the best adhesion of cells. A four-channel system was developed but needs further development to be used for conjugation experiments. As the microfluidics method showed problems with, for example, pressure sensitivity and adherence of cells, and issues with fluorescent markers in the mutant strains Δlpp and ΔrseA, conjugation with the two mutants were ultimately not investigated.
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