Elucidating the role of RNase G in P. aeruginosa

Pseudomonas aeruginosa (Pae) is an opportunistic human pathogen and a frequent cause of severe hospital acquired infections with significant morbidity and mortality. Natural resistance mechanisms of Pae towards antibiotics alongside the ability of Pae to form biofilms in environments such as the lungs of cystic fibrosis patients, contribute to recalcitrance of Pae towards antimicrobials. Although many studies have been performed in Pae to decipher the molecular basis for virulence and biofilm formation, knowledge of how post-transcriptional gene regulation contributes to pathogenicity remains poorly understood. Indeed, this will be the first study performed regarding RNA stability and processing in Pae. In all organisms RNA degradation is mastered by a rather restricted number of enzymes. One of these enzymes, RNase G, has been shown to regulate a vast array of transcripts in the model organism Escherichia coli. The major objective of the proposed studies is to discover RNase G regulated genes as well as non-coding (nc) RNAs that encode or regulate factors required for virulence and/or biofilm formation on a genome-wide scale.

Pseudomonas aeruginosa (Pae) is an opportunistic human pathogen and a frequent cause of hospital acquired infections with significant morbidity and mortality. Although many studies have been performed in Pae to decipher regulatory circuits required for virulence gene expression and biofilm formation at the level of transcription comparatively little is known about post- transcriptional gene regulatory mechanisms that contribute to pathogenicity. The major objective of these studies was to unravel RNase G regulated genes as well as non-coding (nc) RNAs that regulate factors required for virulence and/or biofilm formation at a genome-wide scale. In order to achieve this, comparative transcriptome, phenotypic and in vitro analyses were performed. By merging differential and global RNAseq and making efficient use of tags, we were able to identify transcriptional start sites and processing sites simultaneously. This allowed to reduce artefacts and bias resulting from running global and differential RNA sequencing separately. While no involvement of RNase G in Pae virulence or biofilm formation can be concluded from our studies, we identified the possible involvement of RNase G in the degradation of the ncRNA crcZ. CrcZ was shown to sequester the RNA chaperone Hfq, which in turn impacts on nutrient selection, virulence and antibiotic resistance. Our results suggest that while conserving a 5 monophosphate sensing ability and playing a role in rRNA processing, RNase G has a targeted regulatory role in Pae, which contrasts with its global regulatory role in other Gram-negative bacteria, where the deletion of the RNase G gene has a direct effect on phenotype(s) and RNA turnover. Furthermore, we investigated the role of the sRNA ReaL in the regulation of rpoS mRNA, encoding the master regulator of stationary phase stress, RpoS (sS). The latter is also involved in the regulation of quorum sensing and several virulence genes. We identified ReaL as the first known bacterial sRNA, which apparently represses rpoS translation by a base-pairing mechanism in a Hfq-dependent manner.