Project Outline

The goal of this project is to investigate the genes required for the efficient spread of conjugative plasmids between bacteria. Widespread antimicrobial resistance (AMR) in pathogenic bacteria is a growing threat to health of both animals and humans. Genes that confer resistance to antibiotics and other antimicrobial chemicals are frequently found on R plasmids that can be transferred to antibiotic-sensitive bacteria by conjugation, causing them to become resistant. The plasmid-born genes required for conjugation and propagation of one important family of R plasmids, the IncP group, have been very well investigated. However, much less is known about how factors encoded by the bacterial host genome affect R-plasmid spread.

This project will use a high-throughput genetic method, known as TraDIS, to identify bacterial genes that influence plasmid transfer and maintenance (both positive and negative) and plasmid-born genes that mitigate bacterial factors that counter plasmid transfer (e.g. bacterial restriction/modification systems). To this end, the student will create libraries of transposon mutations in the donor plasmids and the donor and recipient bacteria genomes. The student will then identify all of the mutations in the library simultaneously using high-throughput sequencing and bioinformatic analysis. Finally, the the student will use molecular genetic methods to investigate the mechanism of action of the genes or mutations identified.

References

Goodall ECA, Robinson A, Johnston IG, Jabbari S, Turner KA, Cunningham AF, Lund PA, Cole JA, Henderson IR. The Essential Genome of Escherichia coli K-12. mBio. 2018 Feb 20;9(1):e02096-17. doi: 10.1128/mBio.02096-17. PMID: 29463657; PMCID: PMC5821084

Techniques

Transposon mutagenesis, preparation of DNA libraries for Illumina sequencing, bioinformatic analysis, molecular cloning, molecular biology, genetic analysis of bacteria.