Universally conserved protein Gcp is essential for Pseudomonas aeruginosa viability

The significant increase in the occurrence of Pseudomonas aeruginosa (multi)-drug resistant strains urgently calls for the discovery of novel essential genes or pathways, not yet targeted by antibiotics. For this purpose we set up a genome-wide identification of “essential-for-growth” genes in P. aeruginosa via construction and screening of shotgun antisense libraries. Among several inserts impairing PAO1 growth, we focused on one that targets locus PA0580 encoding for the 341 aa protein gcp, belonging to the YgjD/Kae1 family on the top-10 list of universally conserved proteins. It has been recently shown that this family is involved in the biosynthesis of N6-threonylcarbamoyladenosine (t6A), a universal modification found at position 37 of tRNAs decoding ANN codons. YeaZ, an essential YgjD paralog conserved uniquely in bacteria, interacts with and is required for YgjD function in vivo. We validated the gcp essential role in P. aeruginosa both by insertional and conditional mutagenesis. In addition, we purified gcp through affinity chromatography. This approach will allow us to assess biochemical activity and identify potential functional interactors.