Impact of 2-isopropylmalate synthase (LeuA) Knockout on Escherichia coli Growth and Bacteriophage Replication

Abstract

With antibiotic resistance on the rise, researchers are looking at phage therapy to fight antibiotic-resistant bacteria. Bacteriophages target and hijack bacterial cells, using their macromolecules to replicate themselves and ultimately kill the host cell. While the function of bacteriophages is understood, researchers are still unaware of how certain metabolic pathways are manipulated during bacteriophage replication. In our research, we explore how the knockout of the leuA gene, which is crucial to the leucine synthesis pathway in E. coli, affects bacteriophage growth. We compared the growth and phage susceptibility of a leuA knockout E. coli strain to its wild-type parent by generating growth curves, lysis curves, and phage titers under varying leucine conditions. Data were collected using spectrophotometry, plaque assays, and time-point phage quantification, and analyzed to determine how leucine availability and leuA deletion affect bacterial fitness and phage replication. Our growth curves showed that the knockout grew slightly better than the parent strain in LB media, which contains leucine, but both strains grew similarly in M9 media, which does not contain leucine. The lysis curves, which essentially measure bacteriophage replication, showed almost no difference between the parent and knockout strains, indicating that bacteriophage replication is not affected by the LeuA gene. Our plaque assay plates also showed similar levels of bacteriophage growth, corroborating our results for the growth and lysis curves. However, our two-time point titer test showed that bacteriophage grew significantly better in the parent strain than in the knockout strain, which contradicts our data for the other experiments we conducted. That being said, it may also suggest a role leucine has in phage replication. Our results demonstrate that knockout of the LeuA gene has minimal to no impact on the L-Leucine biosynthesis pathway within E. coli, and subsequently E. coli and bacteriophage growth. Future work will involve the knockout of additional genes within the L-Leucine biosynthesis pathway to assess whether or not the observed effects of LeuA are unique. We will also repeat previous experiments to address potential error, and ensure redundancy.