Abstract
Pseudomonads typically carry multiple non-identical alleles of the post-transcriptional regulator rsmA. In Pseudomonas aeruginosa, RsmN is notable in that its structural rearrangement confers distinct and overlapping functions with RsmA. However, little is known about the specificities of RsmN for its target RNAs and overall impact on the biology of this pathogen. We purified and mapped 503 transcripts directly bound by RsmN in P. aeruginosa. About 200 of the mRNAs identified encode proteins of demonstrated function including some determining acute and chronic virulence traits. For example, RsmN reduces biofilm development both directly and indirectly via multiple pathways, involving control of Pel exopolysaccharide biosynthesis and c-di-GMP levels. The RsmN targets identified are also shared with RsmA, although deletion of rsmN generally results in less pronounced phenotypes than those observed for ΔrsmA or ΔrsmArsmNind mutants, probably as a consequence of different binding affinities. Targets newly identified for the Rsm system include the small non-coding RNA CrcZ involved in carbon catabolite repression, for which differential binding of RsmN and RsmA to specific CrcZ regions is demonstrated. The results presented here provide new insights into the intricacy of riboregulatory networks involving multiple but distinct RsmA homologues.
Highlights
Environmental fluctuations drive the development of coordinated sensory responses to facilitate bacterial survival and growth in challenging conditions
To identify genes potentially regulated by RsmN through direct interaction with their corresponding transcripts, Histagged RsmN was expressed in P. aeruginosa PAO1-L and purified bound to its RNA targets
A total of 503 transcripts were enriched above the set threshold in the RNA samples obtained by co-purification with RsmN compared with total RNA (Supplementary Figure S2 and Supplementary Table S2)
Summary
Environmental fluctuations drive the development of coordinated sensory responses to facilitate bacterial survival and growth in challenging conditions. Among the >60 TCSs predicted within the genome of P. aeruginosa, the GacS/GacA TCS can channel signals from a range of sensors so acting as an integrating global regulator of multiple pathways. This enables P. aeruginosa to adopt different free-living or biofilm associated lifestyles, appropriate for establishing acute or chronic infections. A molecular signal(s) that has yet to be chemically characterized induces autophosphorylation of the sensor kinase GacS and the transfer of a phosphate group to its cognate regulator GacA This in turn induces the transcription of the small regulatory RNAs (sRNAs), RsmY and RsmZ. These sRNAs sequester the RNA-binding protein RsmA, a key post-transcriptional regulator that impacts either positively or negatively on the translation rates of multiple transcripts in P. aeruginosa (∼9% of the gene transcripts, [3])
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