Hi-GRIL-Seq: Probing the sRNA regulatory landscape of P. aeruginosa: post-transcriptional control of determinants of pathogenicity and antibiotic susceptibility

During environmental adaptation bacteria use small regulatory RNAs (sRNAs) to repress or activate translation of a large fraction of their proteome. Using a modification of the in vivo RNA proximity ligation method, we probed global sRNA interactions with their targets in Pseudomonas aeruginosa. We identified two sRNAs (Sr0161 and ErsA) that interact with the mRNA encoding the major porin OprD responsible for the uptake of carbapenem antibiotics. These two sRNAs base-pair with the 5’ UTR of oprD and prevent initiation of translation, resulting in an increase in resistance of the bacteria to meropenem. Additional proximity ligation experiments and enrichment for Sr0161 targets identified the mRNA for the regulator of type III secretion system. Interaction between the exsA mRNA and Sr0161 lead to a block in the synthesis of a component of the T3SS apparatus and an effector. Another sRNA, Sr006, positively regulates, without Hfq, the expression of PagL, an enzyme responsible for deacylation of lipid A thus reducing its pro-inflammatory property and resulting in polymyxin resistance. Collectively, our work shows that an analysis of global sRNA-mRNA interactions can lead to discoveries of novel pathways controlling gene expression that are likely integrated into larger regulatory networks.