Abstract
BackgroundSmall non-coding RNAs (sRNAs) have attracted attention as a new class of gene regulators in both eukaryotes and bacteria. Genome-wide screening methods have been successfully applied in Gram-negative bacteria to identify sRNA regulators. Many sRNAs are well characterized, including their target mRNAs and mode of action. In comparison, little is known about sRNAs in Gram-positive pathogens. In this study, we identified novel sRNAs in the exclusively human pathogen Streptococcus pyogenes M49 (Group A Streptococcus, GAS M49), employing a whole genome intergenic tiling array approach. GAS is an important pathogen that causes diseases ranging from mild superficial infections of the skin and mucous membranes of the naso-pharynx, to severe toxic and invasive diseases.ResultsWe identified 55 putative sRNAs in GAS M49 that were expressed during growth. Of these, 42 were novel. Some of the newly-identified sRNAs belonged to one of the common non-coding RNA families described in the Rfam database. Comparison of the results of our screen with the outcome of two recently published bioinformatics tools showed a low level of overlap between putative sRNA genes. Previously, 40 potential sRNAs have been reported to be expressed in a GAS M1T1 serotype, as detected by a whole genome intergenic tiling array approach. Our screen detected 12 putative sRNA genes that were expressed in both strains. Twenty sRNA candidates appeared to be regulated in a medium-dependent fashion, while eight sRNA genes were regulated throughout growth in chemically defined medium. Expression of candidate genes was verified by reverse transcriptase-qPCR. For a subset of sRNAs, the transcriptional start was determined by 5′ rapid amplification of cDNA ends-PCR (RACE-PCR) analysis.ConclusionsIn accord with the results of previous studies, we found little overlap between different screening methods, which underlines the fact that a comprehensive analysis of sRNAs expressed by a given organism requires the complementary use of different methods and the investigation of several environmental conditions. Despite a high conservation of sRNA genes within streptococci, the expression of sRNAs appears to be strain specific.
Highlights
Small non-coding RNAs have attracted attention as a new class of gene regulators in both eukaryotes and bacteria
We found very little overlap between the different screening methods, which underlines the importance of using several complementary methods, as well as several environmental conditions, to attain a comprehensive analysis of bacterial sRNAomes
Identification of sRNAs in the intergenic regions of GAS M49 Custom intergenic tiling arrays representing the genome of S. pyogenes NZ131 (NCBI accession number: NC_011375) were designed to detect the expression of potential sRNAs
Summary
Small non-coding RNAs (sRNAs) have attracted attention as a new class of gene regulators in both eukaryotes and bacteria. The role of small non-coding RNAs (sRNAs) in regulation of bacterial gene expression has become more evident; the large number of sRNAs identified in different bacterial species was unexpected [1,2,3]. With sRNAs representing a whole new level of post-transcriptional regulation, it is no surprise that these molecules play an important role in the tightly controlled expression of virulence factors in many pathogens [11,12]. GAS expresses a large number of virulence factor genes coding for a variety of proteins, including surface components, lytic enzymes, proteinases, cytotoxins, superantigens, and immunoprotective proteins, that are controlled at least partially by the 30 stand-alone transcriptional regulators and 13 two-component systems identified to date in GAS [15]. Virulence factor expression in GAS is highly responsive to environmental conditions and greatly depends on the growth phase
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