Identification of RNA-binding proteins in Pseudomonas aeruginosa

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Abstract Pseudomonas aeruginosa is an important opportunistic pathogen of humans. It is the principal cause of morbidity and mortality in Cystic Fibrosis patients and a major cause of hospital-acquired pneumonia. Central to the ability of P. aeruginosa to cause disease are the activities of two post-transcriptional regulators RsmA and Hfq, each of which influences the translation and/or abundance of hundreds of target mRNA species. However, apart from RsmA and Hfq, little is known about the repertoire of RNA-binding proteins (RBPs) in P. aeruginosa, or the potential regulatory roles they play. We have found that in P. aeruginosa many putative or predicted RBPs (including RsmA) copurify with the cold shock protein CspD, itself an RBP, in an RNase sensitive manner, implying that they are co-bound to subsets of the same RNA species that are bound by CspD. In Aim 1, we will employ this affinity co-capture approach with four additional RBPs in P. aeruginosa to obtain a more comprehensive picture of the RBPs that are found in this important opportunistic pathogen. In Aim 2, we will focus on one protein (PhaF) that co-purifies with CspD in an RNase-sensitive manner, a known regulator whose mechanism of action is poorly understood. Specifically, we propose to identify RNA species that PhaF binds, determine whether the unusual nucleic acid binding region of PhaF represents a novel RNA- binding determinant, and better define the PhaF regulon. Our studies will provide a compendium of RBPs in P. aeruginosa and have the potential to illuminate the regulatory roles and mechanism of action of a novel RNA- binding protein in this organism.