Grad‐seq in a Gram‐positive bacterium reveals exonucleolytic sRNA activation in competence control

Abstract

RNA-protein interactions are the crucial basis for many steps of bacterial gene expression, including post-transcriptional control by small regulatory RNAs (sRNAs). In stark contrast to recent progress in the analysis of Gram-negative bacteria, knowledge about RNAprotein complexes in Gram-positive species remains scarce. Here, we used the Grad-seq approach to draft a comprehensive landscape of such complexes in Streptococcus pneumoniae, in total determining the sedimentation profiles of~88% of the transcripts and~62% of the proteins of this important human pathogen. Analysis of in-gradient distributions and subsequent tag-based protein capture identified interactions of the exoribonuclease Cbf1/YhaM with sRNAs that control bacterial competence for DNA uptake. Unexpectedly, the nucleolytic activity of Cbf1 stabilizes these sRNAs, thereby promoting their function as repressors of competence. Overall, these results provide the first RNA/protein complexome resource of a Gram-positive species and illustrate how this can be utilized to identify new molecular factors with functions in RNA-based regulation of virulence-relevant pathways.

Grad-seq shines light on unrecognized RNA and protein complexes in the model bacteriumEscherichia coli

“…Glycerol gradient fractionation was performed as previously described (19), with the exception of the growth and lysis conditions: 100 ml of E. coli MG1655 wild type were grown to an OD600 of 2, cooled down in an ice-water bath for 15 min and then harvested by centrifugation for 20 min at 4°C and 4,000 rcf. The cells were washed three times in ice-cold 1x TBS, resuspended in 500 µl ice-cold 1x lysis buffer A [20 mM Tris-HCl, pH 7.5, 150 mM KCl, 1 mM MgCl2, 1 mM DTT, 1 mM PMSF, 0.2% Triton X 100, 20 U/ml DNase I (Thermo Fisher), 200 U/ml RNase inhibitor] and lysed by addition of 750 µl of 0.1 mm glass beads (Carl Roth) and 10 cycles of vortexing for 30 s followed by cooling on ice for 15 s. To remove insoluble debris and the glass beads, the lysate was cleared by centrifugation for 10 min at 4°C and 16,100 rcf.…”

“…RNA-seq was performed as described before (19). Briefly, 5 µl of the gradient samples were diluted in 45 µl DEPC-treated H2O.…”

“…We performed Grad-seq on E. coli grown to early stationary phase (OD600 of 2.0) in rich medium. As before (14,19), soluble particles from a lysate were separated on a linear 10%-40% glycerol gradient, followed by RNA-seq and MS analyses of all 20 gradient fractions and the pellet ( Figure 1A). The A260 UV profile of the gradient showed the typical three peaks, one bulk peak around low molecular weight (LMW) fraction 2 and the two peaks representing the small (30S) and large (50S) ribosomal subunits ( Figure 1B) (14,19).…”

“…pneumoniae (19), which permits a cross-species comparison of the sedimentation profiles of selected entries. Comparison of the closely related enterobacteria E. coli and Salmonella whose proteins tend to be generally similar will be useful for a fine-grained analysis of ingradient distributions.…”

“…We describe use cases for this resource to be exploited: a stable association with the 30S ribosomal subunit reveals the seemingly noncoding RyeG RNA of prophage origin as an mRNA that encodes a small toxic protein; the broadly conserved uncharacterized protein YggL is revealed to be a 50S subunit factor in assembled 70S ribosomes. The E. coli Grad-seq data are viewable in an online browser (https://helmholtz-hiri.de/en/datasets/gradseqec/) that not only enables easy access to all recorded RNA and protein gradient sedimentation profiles but also permits cross-species comparison with published Salmonella enterica (14) and Streptococcus pneumoniae (19) datasets.…”