Browsing by Author "Mohanraj, Ushanandini"

The rapid emergence of antibiotic resistance among many pathogenic bacteria has created a profound need to discover new alternatives to antibiotics. Bacteriophages are viruses which infect bacteria and are able to produce special proteins involved in bacterial lysis. However, for many bacteriophage-encoded gene products, the function is not known, i.e., hypothetical proteins of unknown function (HPUFs). Screening these proteins likely identifies a rich source of leads that will help in the development of novel antibacterial compounds. The current study presents two phage genomics-based screening approaches to identify phage HPUFs with antibacterial activity. Both screening assays are based on inhibition of bacterial growth when a toxic gene is expression cloned into a plasmid vector. The first approach was a luxAB/luxCDE -based luminescence screening assay. The luxCDE genes encoding the luciferase substrate producing enzymes were integrated into an Escherichia coli strain genome as a transcriptional fusion. Also, a vector carrying the luxAB genes, encoding the luciferase enzyme, and a cloning site for the phage HPUF genes, was constructed. Ligation of a toxic gene into the vector would result in few or rare transformants after electroporation while ligation of a non-toxic gene would result in large number of transformants, and the difference in number of transformants will be reflected in the amount of bioluminescence after electroporation. The proof of concept of the approach was verified using the control genes g150 (a structural, thus a non-toxic gene of phage R1-RT) and regB (a known toxic gene of phage T4). The results demonstrated a significant difference in Relative Luminescence Units (RLU) between the g150 and regB electroporation mixtures. The second screening approach was an optimized plating assay producing a significant difference in the number of transformants after ligation of the toxic and non-toxic genes into a cloning vector. This assay was tested and optimized with several known control toxic and non-toxic genes. Using the plating assay approach, in the current study, ninety-four R1-RT HPUFs were screened and ten of them showed toxicity in E. coli. In future, the identified toxic HPUFs of R1-RT could be purified and characterized to identify their bacterial targets. Further, both of these screening assays can be used to screen among HPUFs of other phages, and this should allow the discovery of a wide variety of putative inhibitors for the control of current and emerging bacterial pathogens.