Regulation of the P. aeruginosa amidase operon by Hfq

Pseudomonas aeruginosa (PAO1) is an opportunistic pathogen, which is notorious for its resistance towards many commonly used antibiotics. PAO1 causes nosocomial pneumonia, urinary tract infection, and wound- and bloodstream infections. In previous publications we have shown that the inactivation of the PAO1 hfq gene, encoding the Sm-like protein Hfq, resulted in an attenuated virulence in a mouse and an insect model. A transcriptome analysis identified the amidase operon, consisting of 5 genes, to be up-regulated in an hfq- background. Hfq-dependent post-transcriptional regulation usually involves small non-coding RNAs (ncRNAs), which base-pair with target mRNAs, often resulting in repression of translation. The aliphatic amidase of P. aeruginosa catalyses the hydrolysis of amides with short acyl-chains, resulting in the formation of the corresponding acids and ammonia, and contributes to the metabolic versatility of the organism. The major task of this study is to get more insights into the regulation of the PAO1 amidase operon by Hfq and succinate and its possible role in virulence. Succinate was shown to be a strong catabolite repressor of the amidase operon only in the presence of Hfq. It is currently not clear whether Hfq and Pae20 affect amidase expression by interfering with binding to AmiR, the positive regulator of the amidase operon, or by acting directly on the amiE mRNA. Translational lacZ-fusions to Pae20-amiE and in vitro experiments will be used to answer this question. The Pae20 RNA, which had been previously identified as the 5`-RNA leader of the amidase operon, was isolated in a shot-gun cloning approach (RNomics) of small sized RNAs of PAO1 that had been co-immunoprecipitated with Hfq. Proteomics and bioinformatics suggested that Pae20 functions as a global riboregulator. To verify this hypothesis experimentally, I will analyze whether Pae20 affects expression of pcrV and truA, both of which are involved in type III secretion, and thus in virulence. In addition, HeLa cells will be used in a virulence assay to determine whether Pae20 is involved in type III secretion. The significance of this project lies in the characterization of ncRNAs that regulate the metabolic versatility and virulence of P. aeruginosa.