A Grad-seq view of RNA and protein complexes inPseudomonas aeruginosaunder standard and bacteriophage predation conditions

Abstract

The Gram-negative rod-shaped bacterium Pseudomonas aeruginosa is not only a major cause of nosocomial infections but also serves as a model species of bacterial RNA biology. While its transcriptome architecture and post-transcriptional regulation through the RNA-binding proteins Hfq, RsmA and RsmN have been studied in detail, global information about stable RNA–protein complexes is currently lacking in this human pathogen. Here, we implement Gradient profiling by sequencing (Grad-seq) in exponentially growing P. aeruginosa cells to comprehensively predict RNA and protein complexes, based on glycerol gradient sedimentation profiles of >73% of all transcripts and ∼40% of all proteins. As to benchmarking, our global profiles readily reported complexes of stable RNAs of P. aeruginosa, including 6S RNA with RNA polymerase and associated pRNAs. We observe specific clusters of non-coding RNAs, which correlate with Hfq and RsmA/N, and provide a first hint that P. aeruginosa expresses a ProQ-like FinO domain containing RNA-binding protein. To understand how biological stress may perturb cellular RNA/protein complexes, we performed Grad-seq after infection by the bacteriophage ΦKZ. This model phage, which has a well-defined transcription profile during host takeover, displayed efficient translational utilization of phage mRNAs and tRNAs, as evident from their increased co-sedimentation with ribosomal subunits. Additionally, Grad-seq experimentally determines previously overlooked phage-encoded non-coding RNAs. Taken together, the Pseudomonas protein and RNA complex data provided here will pave the way to a better understanding of RNA-protein interactions during viral predation of the bacterial cell.IMPORTANCEStable complexes by cellular proteins and RNA molecules lie at the heart of gene regulation and physiology in any bacterium of interest. It is therefore crucial to globally determine these complexes in order to identify and characterize new molecular players and regulation mechanisms. Pseudomonads harbour some of the largest genomes known in bacteria, encoding ∼5,500 different proteins. Here, we provide a first glimpse on which proteins and cellular transcripts form stable complexes in the human pathogen Pseudomonas aeruginosa. We additionally performed this analysis with bacteria subjected to the important and frequently encountered biological stress of a bacteriophage infection. We identified several molecules with established roles in a variety of cellular pathways, which were affected by the phage and can now be explored for their role during phage infection. Most importantly, we observed strong co-localization of phage transcripts and host ribosomes, indicating the existence of specialized translation mechanisms during phage infection. All data are publicly available in an interactive and easy to use browser.

Development of ONT-cappable-seq to unravel the transcriptional landscape of Pseudomonas phages

“…By contrast, the shortest sRNA candidate, sRNA3, seemingly lacks an annotated TSS, suggesting that this fragment is derived from a primary precursor through processing events. Functional sRNA biogenesis through RNase activity has already been demonstrated in various bacteria (64–66), and was recently also suggested to occur in Pseudomonas phage phiKZ (67). In addition, the biological relevance of small non-coding RNAs in the regulation of phage and host development is increasingly being recognized in several other Pseudomonas phages (5, 68).…”

“…Functional sRNA biogenesis through RNase activity has already been demonstrated in various bacteria (64)(65)(66), and was recently also suggested to occur in Pseudomonas phage phiKZ (67). In addition, the biological relevance of small non-coding RNAs in the regulation of phage and host development is increasingly being recognized in several other Pseudomonas phages (5,68).…”