Novel regulatory small RNAs in Streptococcus pyogenes

Abstract

Streptococcus pyogenes (Group A Streptococcus or GAS) is a Gram-positive bacterial pathogen that has shown complex modes of regulation of its virulence factors to cause diverse diseases. Bacterial small RNAs are regarded as novel widespread regulators of gene expression in response to environmental signals. Recent studies have revealed that several small RNAs (sRNAs) have an important role in S. pyogenes physiology and pathogenesis by regulating gene expression at the translational level. To search for new sRNAs in S. pyogenes, we performed a genomewide analysis through computational prediction followed by experimental verification. To overcome the limitation of low accuracy in computational prediction, we employed a combination of three different computational algorithms (sRNAPredict, eQRNA and RNAz). A total of 45 candidates were chosen based on the computational analysis, and their transcription was analyzed by reverse-transcriptase PCR and Northern blot. Through this process, we discovered 7 putative novel trans-acting sRNAs. Their abundance varied between different growth phases, suggesting that their expression is influenced by environmental or internal signals. Further, to screen target mRNAs of an sRNA, we employed differential RNA sequencing analysis. This study provides a significant resource for future study of small RNAs and their roles in physiology and pathogenesis of S. pyogenes.

Figure 1. A combination of three computational algorithms was used to predict small regulatory RNAs in S. pyogenes.

A) The scheme of the computational approach for the prediction of small RNAs in S. pyogenes. The rectangles, ovals, and arrow lines represent computational algorithms, input or output data of computational analyses, and data flow, respectively. The processes were performed to run the algorithms, RNAz, eQRNA and sRNAPredict. B) The candidates predicted by any two algorithms at the same time were considered sRNA candidates. Then, putative cis-regulatory sequences located immediately upstream of annotated ORFs and candidates within prophage sequences were removed from the candidate list. The number of final candidates selected in this manner was 45.

Figure 2. Northern blot identified S. pyogenes sRNAs from the candidates predicted by the computational analysis.

Northern blots were performed with RNA (20 µg) extracted from MGAS315 at the exponential growth phase (Optical density at 600 nm, OD₆₀₀, ∼0.5). The names of the candidate RNA molecules are shown at the bottom of each Northern blot as SSRC (Streptococcal Small RNA Candidate) number. The locations of size markers in nucleotides are shown at the left side of each Northern blot. The approximate sizes of SSRCs calculated based on the location of the size markers are shown in nucleotides (nts) below their names.

Figure 3. Sequence analysis of candidate sRNA transcriptional start and stop sites, promoter regions and terminators.

The transcriptional start and stop sites of candidate sRNAs were determined by circular RACE. The sRNA sequences based on the transcriptional start and stop sites are in black. The putative −10 and −35 promoter sequences are colored green, and putative Rho-independent terminators, which are identified by the algorithm ARNold (http://rna.igmors.u-psud.fr/toolbox/arnold/index.php), are underlined. Neighboring sequences of the sRNA sequences are colored in blue. The deleted part in the SSRC21 deletion mutant, ΔSSRC21cat, is italicized. A putative CovR-binding site upstream of SSRC34 is colored in red. The nucleotide coordinates based on the genome sequence of S. pyogenes MGAS315 and sizes of the sRNAs are shown in parenthesis.

Figure 4. The abundance of newly discovered streptococcal small RNA candidates (SSRCs) varied between growth phases.

A) The intracellular abundance of SSRCs at different growth phases. The abundance of each SSRC was determined over the course of growth (exponential phase, EX; early stationary phase, ES; late stationary phase, LS) through Northern blotting. Size markers (S) were run and their sizes are indicated at the left sides of Northern blots. The abundance of 5S RNA (5S) was also determined as a loading control and shown below each Northern blot. B) Abundance of each intracellular sRNA relative to that at the exponential growth phase. Abundance of sRNAs on Northern blots was determined by densitometry, normalized with the abundance of 5S RNA, and expressed relative to the abundance at the exponential growth phase.

Figure 5. Computational prediction of an interaction between the transcript of a putative regulator SpyM3_0113 and SSRC21.

The drawing was generated with the algorithm IntaRNA .