Identification of genes for small non-coding RNAs that belong to the regulon of the two-component regulatory system CiaRH in Streptococcus

Abstract

BackgroundPost-transcriptional regulation by small RNAs (sRNAs) in bacteria is now recognized as a wide-spread regulatory mechanism modulating a variety of physiological responses including virulence. In Streptococcus pneumoniae, an important human pathogen, the first sRNAs to be described were found in the regulon of the CiaRH two-component regulatory system. Five of these sRNAs were detected and designated csRNAs for cia-dependent small RNAs. CiaRH pleiotropically affects β-lactam resistance, autolysis, virulence, and competence development by yet to be defined molecular mechanisms. Since CiaRH is highly conserved among streptococci, it is of interest to determine if csRNAs are also included in the CiaRH regulon in this group of organisms consisting of commensal as well as pathogenic species. Knowledge on the participation of csRNAs in CiaRH-dependent regulatory events will be the key to define the physiological role of this important control system.ResultsGenes for csRNAs were predicted in streptococcal genomes and data base entries other than S. pneumoniae by searching for CiaR-activated promoters located in intergenic regions that are followed by a transcriptional terminator. 61 different candidate genes were obtained specifying csRNAs ranging in size from 51 to 202 nt. Comparing these genes among each other revealed 40 different csRNA types. All streptococcal genomes harbored csRNA genes, their numbers varying between two and six. To validate these predictions, S. mitis, S. oralis, and S. sanguinis were subjected to csRNA-specific northern blot analysis. In addition, a csRNA gene from S. thermophilus plasmid pST0 introduced into S. pneumoniae was also tested. Each of the csRNAs was detected on these blots and showed the anticipated sizes. Thus, the method applied here is able to predict csRNAs with high precision.ConclusionsThe results of this study strongly suggest that genes for small non-coding RNAs, csRNAs, are part of the regulon of the two-component regulatory system CiaRH in all streptococci.

Streptococcus pneumoniae Senses a Human-like Sialic Acid Profile via the Response Regulator CiaR

“…CiaRH is involved in competence, cefotaxime susceptibility, autolysis, bacteriocin production, resistance to oxidative stress and plays a role in pneumococcal virulence (Dagkessamanskaia et al, 2004; Ibrahim et al, 2004a; Mascher et al, 2006; Mascher et al, 2003; Throup et al, 2000). The RR CiaR directly controls a number of promoters and genes, including the high-temperature requirement A gene htrA, enhancing resistance to oxidative stress, and has been shown to affect transcription of several small non-coding RNAs with different down-stream effects (Halfmann et al, 2007; Marx et al, 2010). Phosphorylation of CiaR is required for its regulatory activity, but internal acetyl phosphate (AcPh) may serve as phosphor-donor besides CiaH (Marx et al, 2014).…”

Small regulatory RNAs from low-GC Gram-positive bacteria

“…Curiously, Hungarian S. pneumoniae serotype 19A isolates carry and express longer versions of csRNA5, which apparently arose by internal sequence duplication (R. Brückner, unpublished observations). Expression of CiaR-controlled csRNAs was also confirmed in Streptococcus mitis , Streptococcus oralis, Streptococcus sanguinis, 83 and Streptococcus pyogenes 84 . The presence of multiple csRNAs genes could be predicted in all streptococcal genomes 83 suggesting that they serve an important function in this group of organisms.…”

Small RNAs in streptococci

“…CiaRH is present in all streptococci and searching for CiaR-activated promoters in the genomes of 14 species revealed 61 csRNA candidates of 51 to 202 nt that were further classified into 40 different csRNA types. 74 All streptococcal genomes harbor from two to six csRNAs and the expression of some of them were validated experimentally in Streptococcus mitis, Streptococcus oralis, Streptococcus sanguinis and Streptococcus thermophilus. 74 Five pneumococcal csRNAs [csRNA1-5 transcribed from ccnA-E genes (ccn, cia-controlled non-coding RNA)] are described as non-protein coding sRNAs that are transcribed from the five strongest promoters of the CiaR regulon.…”

“…74 All streptococcal genomes harbor from two to six csRNAs and the expression of some of them were validated experimentally in Streptococcus mitis, Streptococcus oralis, Streptococcus sanguinis and Streptococcus thermophilus. 74 Five pneumococcal csRNAs [csRNA1-5 transcribed from ccnA-E genes (ccn, cia-controlled non-coding RNA)] are described as non-protein coding sRNAs that are transcribed from the five strongest promoters of the CiaR regulon. 37 The csRNAs share a high degree of sequence similarity particularly in the unpaired region of stem-loop structures that are characteristic of these sRNAs.…”

“…However, given the complementarity of their unpaired sequences to translation initiation regions (TIRs), it is predicted that the csRNAs act in trans on target mRNAs to prevent translation initiation. 37,74 It would be of interest to compare the targets, molecular mechanisms and phenotypes of this class of sRNAs in diverse streptococcal species.…”