The transmission of genes for antibiotic resistance often occurs via plasmid transfer in a bacterium-to-bacterium process called conjugation. The conjugative plasmid pKF3-94 of the IncF group was isolated from Klebsiella pneumoniae strain KF3 by our collaborators in China (Zhao et al., 2010). It contains 144 putative genes of which 69 can be functionally assigned to be involved in transfer conjugation, antibiotic resistance, transposon function and plasmid replication (Ying et al., 2015). This is a plasmid of great significance in the current spread of multi-drug resistant K. pneumoniae across south China.
We sought to address a fundamental question in bacterial cell biology, namely how does a recipient cell contribute to conjugation? Previously, Mendoza et al., (2009) analyzed the individual contribution of each Escherichia coli gene in recipient cells and found that none were absolutely required for conjugation. However, the gene encoding a surface-located protein called OmpA has been shown to play an important, yet ill-defined role in conjugation (Manoil et al., 1982). Based on these two studies we have designed a synthetic lethal screening approach to identify genes determining the recipients’ response to conjugation.
The ompA mutation has been introduced into a library of 3818 E. coli mutants, each one of which lacks a non-essential gene. The library has been arrayed into a format suitable for high-throughput screening with a Singer RoToR robotics platform. The double mutant collection library will be surveyed to select for those mutants that cannot receive plasmid pKF3-94 employing K. pneumoniae as the donor strain. The inhibition of plasmid dissemination, by inhibiting conjugation, might serve to enhance or complement the efficacy of antibiotics and curb the isolation of antibiotic resistant bacterial pathogens. We hope from this study to map out the genes involved in conjugation in the recipient which could identify targets for new antibiotics.