Silistre, Hazel
(2016)
Riboregulation in Pseudomonas aeruginosa.
PhD thesis, University of Nottingham.
Abstract
The opportunistic human pathogen Pseudomonas aeruginosa controls virulence, production of secondary metabolites, motility, biofilm formation, growth in anaerobic conditions, intracellular and intercellular signalling and the switch from an acute to a chronic mode of infection at the transcriptional and post-transcriptional levels by modulation of the Gac/Rsm system. Cell density-dependent signal accumulation and environmental stimulators such as pH changes and ion limitation activate the GacS/GacA two-component system which in turn triggers transcription of the small regulatory RNAs RsmY and RsmZ. These sRNAs sequester multiple copies of the RNA-binding protein RsmA, antagonising its function. The RsmA/CsrA proteins act as translational repressors by binding to the GGA-motifs in the untranslated region of target mRNAs and blocking ribosome binding. In this study, the biological function of RsmN, an RsmA homologue with a conserved RNA-binding pocket but a distinct protein folding, the predicted autoregulatory mechanism of RsmN, the nature of target transcripts of RsmN, and the cross-regulation between the two Rsm proteins were investigated.
The positive control of proteolytic and elastinolytic activities and swarming motility by RsmN has been demonstrated using single and inducible double deletion mutants of rsmN. Furthermore, rsmN deletion increased microcolony formation during biofilm formation. Regulation by RsmN was most apparent in the absence of RsmA, when rsmN expression was induced via a multicopy plasmid and at temperatures lower than 37°C. The double deletion of rsmA and rsmN affected growth, diminished proteolytic and elastinolytic activities, triggered autolysis and led to the increased secretion of the type VI secretion system protein Hcp1. Moreover, the double deletion of rsmA and rsmN altered the colony morphology of P. aeruginosa. Mutagenesis of the functionally critical, conserved RNA-binding residue which is identified as Arg44 in RsmA and Arg62 in RsmN resulted in the loss of RsmN function.
In a genome-wide analysis by RNASeq, target transcripts were co-purified with RsmN from 37°C and 34°C cultures of a wild-type strain expressing rsmN in multicopy numbers. RNASeq results indicated that small regulatory RNAs such as CrcZ, RsmY and RgsA are common targets of RsmN and RsmA, whereas PhrS is a target of RsmN only. Other common RsmA and RsmN targets included transcriptional regulators, heat shock proteins, proteases, starvation response proteins, components of the denitrification pathway, outer membrane proteins required for pore formation, type III and type VI secretion system proteins and RsmA. Transcripts of heat shock proteins, the tss operon genes and rsmA were enriched by RsmN at 37°C but not at 34°C whereas the lasB transcript was enriched by RsmN at 34°C but not at 37°C.
Based on the list of common targets of RsmA and RsmN and the results obtained from phenotypic assays, induction of the lytic Pf4 prophage, accumulation of alkyl quinolone or c-di-GMP signalling molecules, imbalanced redox state, carbon starvation, increased membrane permeability, and aggregation of misfolded proteins are suggested as possible mechanisms triggering the excessive autolysis of the rsmNind ΔrsmA mutant under uninducing conditions.
The data gathered so far suggests that rsmN is differentially expressed, with increased RsmN activity at temperatures below 37°C in comparison with RsmA, and, RsmA and RsmN collectively contribute to the regulation of secondary metabolite production, motility and microcolony formation in P. aeruginosa.
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