The Removal of the kdsD Gene and Its Effect on Phage Replication and Growth in Escherichia coli

Abstract

Bacteriophages - viruses that lyse bacteria - are a promising alternative treatment to counter antibiotic resistance that warrants further research. We explored kdsD and kdsC , non-essential genes that contribute to the formation of the outer membrane of E.coli to investigate the effect of an impaired membrane on bacterial growth and viral replication. The kdsD gene is responsible for the formation of the arabinose 5-phosphate isomerase that catalyzes the production of KDO while kdsC turns KDO 8-P to free KDO through phosphorylation, these are able to help form the outer membrane. We employed two-time point phage titer and plaque assays to quantify and compare viral replication in ΔkdsD, ΔkdsC and parent strain. Additionally, lysis curves were conducted to compare viral replication in parent strain and ΔkdsD. We also explored differences in bacterial growth between strains using a growth curve in LB and M9 minimal media. For lysis and growth curves, absorbances were averaged and compared between strains, phages, and media, respectively. The comparative growth curve revealed no noticeable difference between the growth of the parent and knockout strain in LB media. When observing the lysis curve the parent strain and knockout in T4r showed greater lysis than in T2. When comparing the lysis of the parent strain to the ΔkdsD, the parent strain had a higher absorbance value than the knockout when exposed to a bacteriophage environment. This data suggests that the ΔkdsD would increase the viral replication and that the function of kdsD in the KDO synthesis plays a less significant and necessary role. Comparative plaque assay regarding ΔkdsC were also conducted and distinguished that phage grows more abundantly with ΔkdsC than with kdsD. Additional research should be conducted to investigate why viral replication was greater in ΔkdsD than in the parent strain. Further exploration into the bacterial growth and lysis curves of ΔkdsC in comparison to ΔkdsD and parent could be valuable. The ΔkdsD observations indicate that using the gene in phage therapy may be as useful. Testing ΔkdsC and other genes within the KDO pathway could provide insight on the uses of those genes in phage therapy and even more efficient possibilities for phage therapy.