Synergies between RNA degradation and trans-translation in Streptococcus pneumoniae: cross regulation and co-transcription of RNase R and SmpB

Abstract

BackgroundRibonuclease R (RNase R) is an exoribonuclease that recognizes and degrades a wide range of RNA molecules. It is a stress-induced protein shown to be important for the establishment of virulence in several pathogenic bacteria. RNase R has also been implicated in the trans-translation process. Transfer-messenger RNA (tmRNA/SsrA RNA) and SmpB are the main effectors of trans-translation, an RNA and protein quality control system that resolves challenges associated with stalled ribosomes on non-stop mRNAs. Trans-translation has also been associated with deficiencies in stress-response mechanisms and pathogenicity.ResultsIn this work we study the expression of RNase R in the human pathogen Streptococcus pneumoniae and analyse the interplay of this enzyme with the main components of the trans-translation machinery (SmpB and tmRNA/SsrA). We show that RNase R is induced after a 37°C to 15°C temperature downshift and that its levels are dependent on SmpB. On the other hand, our results revealed a strong accumulation of the smpB transcript in the absence of RNase R at 15°C. Transcriptional analysis of the S. pneumoniae rnr gene demonstrated that it is co-transcribed with the flanking genes, secG and smpB. Transcription of these genes is driven from a promoter upstream of secG and the transcript is processed to yield mature independent mRNAs. This genetic organization seems to be a common feature of Gram positive bacteria, and the biological significance of this gene cluster is further discussed.ConclusionsThis study unravels an additional contribution of RNase R to the trans-translation system by demonstrating that smpB is regulated by this exoribonuclease. RNase R in turn, is shown to be under the control of SmpB. These proteins are therefore mutually dependent and cross-regulated. The data presented here shed light on the interactions between RNase R, trans-translation and cold-shock response in an important human pathogen.

Reactive Oxygen Species Contribute to the Bactericidal Effects of the Fluoroquinolone Moxifloxacin in Streptococcus pneumoniae

“…The operon also codes for RNase R, involved in RNA degradation, and SmpB, which mediates transtranslation. This operon is also regulated by stress and is upregulated by cold shock (37). In addition, lepA, the gene coding EF-4, which is involved in trans-translation, is also downregulated (Table 1).…”

“…Group III represented 2.5% to 5% of the affected genes. These included genes involved in pathogenesis (all 7 downregulated); amino acid biosynthesis (4 upregulated, 1 downregulated); genes of two-component systems (TCSs) (3 of TCS12 upregulated, 1 of TCS11 downregulated); DNA metabolism; fatty acid metabolism; cell envelope; and the secG-rnr-smpB (37) operon that is involved in protein secretion (SecG), RNA degradation (RNase R), and trans-translation (SmpB). Group IV represented Ͻ2.5% of the affected genes and included diverse functions, with no clear pattern in responses.…”

Bacterialtrans-Translation: From Functions to Applications

“…In the absence of either tmRNA or SmpB, the half-life of RNase R and its level in exponential-phase cells are increased markedly [84]. In contrast, in Streptococcus pneumoniae, SmpB is regulated by RNase R which is, in turn, under the control of SmpB [85]. These proteins are therefore mutually dependent, confirming the tight crossregulation between trans-translation and RNase R. …”

A new tool for cloning and gene expression in Streptococcus pneumoniae

“…20 ml culture samples were collected, mixed TIGR4 rnr À (Drnr-Cm R ) carrying pIL253 (Ery R ) expressing RNase R Moreira et al (2012) with 1 volume of stop solution (defined above) and harvested by centrifugation (10 min, 2800 Â g, 4°C). The cell pellet was resuspended in 100 ll of TE buffer supplemented with 1 mM PMSF, 0.15% sodium deoxycholate and 0.01% SDS.…”

“…pIL253 carrying the coding sequence of the TIGR4 RNase R inserted into the SmaI/PstI sites of the plasmid MCS (Moreira et al, 2012) was used to assess the ability of pIL253 to stably carry and express a DNA fragment in S. pneumoniae. Expression of RNase R was evaluated by transforming this construct into a S. pneumoniae TIGR4 RNase R À mutant strain (see below).…”