Importance of Riboswitch-regulated Genes for Pseudomonas aeruginosa

Riboswitches offer a promising avenue as targets for the development of novel bacterial antibiotics. For this approach to work, it requires that target riboswitches control the expression of essential genes for bacterial survival. However, the small number of essential riboswitch-regulated genes restrains the output of viable novel antimicrobial agents. Here, in an effort to increase the number of riboswitch potential targets, we have assessed the essentiality of Pseudomonas aeruginosa riboswitch-controlled genes in nutrient-limited growth conditions. Using a P. aeruginosa library containing inactivating transposons in riboswitch-controlled genes, we find that the majority of these genes are not required for growth in any tested conditions. However, we find that the inactivation of the S-adenosyl-homocysteine (SAH) hydrolase gene results in the complete lack of P. aeruginosa growth in nutrient-limited conditions. Importantly, bacterial growth is rescued when supplementing S-adenosyl-methionine, which is the precursor of SAH in catalyzed methylation processes. RT-qPCR assays show that although the SAH riboswitch is predicted to control translation initiation, mRNA levels are increased in the presence of SAH, suggesting an additional level of control. Lastly, in vitro structural analyses indicate that the riboswitch undergoes significant rearrangement upon SAH binding and exhibits an affinity toward SAH in the micromolar range. Together, our results provide an approach to discover novel riboswitch targets in nutrient-limited growth conditions and suggest that the SAH-sensing riboswitch constitutes a potential target to inhibit the growth of P. aeruginosa.