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Open Access Highly Accessed Research article

Identification of novel growth phase- and media-dependent small non-coding RNAs in Streptococcus pyogenes M49 using intergenic tiling arrays

Nadja Patenge1, André Billion2, Peter Raasch3, Jana Normann1, Aleksandra Wisniewska-Kucper1, Julia Retey4, Valesca Boisguérin5, Thomas Hartsch4, Torsten Hain2 and Bernd Kreikemeyer1*

Author affiliations

1 Institute of Medical Microbiology and Hospital Hygiene, University of Rostock, Schillingallee 70, 18057, Rostock, Germany

2 Institute of Medical Microbiology, Genome Research, Justus-Liebig-University, Frankfurter Strasse 107, 35392, Giessen, Germany

3 Systems Biology and Bioinformatics Group, University of Rostock, 18051, Rostock, Germany

4 Genedata AG, Margarethenstrasse 38, 4053, Basel, Switzerland

5 febit biomed GmbH, Im Neuenheimer Feld 519, 69120, Heidelberg, Germany

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Citation and License

BMC Genomics 2012, 13:550  doi:10.1186/1471-2164-13-550

Published: 13 October 2012

Abstract

Background

Small 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.

Results

We 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.

Conclusions

In 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.

Keywords:
Streptococcus pyogenes; Small noncoding RNAs; Virulence; Transcriptional regulation; Pathogenesis