Identification of novel growth phase- and media-dependent small non-coding RNAs in Streptococcus pyogenes M49 using intergenic tiling arrays.
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.
Institute of Medical Microbiology and Hospital Hygiene, University of Rostock, Schillingallee 70, 18057, Rostock, Germany.
Nadja
N
Patenge
André
A
Billion
Peter
P
Raasch
Jana
J
Normann
Aleksandra
A
Wisniewska-Kucper
Julia
J
Retey
Valesca
V
Boisguérin
Thomas
T
Hartsch
Torsten
T
Hain
Bernd
B
Kreikemeyer
D001483:Base Sequence; D015152:Blotting, Northern; D019295:Computational Biology; D021901:DNA, Intergenic / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D016680:Genome, Bacterial / Q000235:genetics*; D008969:Molecular Sequence Data; D058727:RNA, Small Untranslated / Q000235:genetics* / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017385:Sequence Homology; D013045:Species Specificity; D013297:Streptococcus pyogenes / Q000235:genetics* / Q000254:growth & development
D021901:DNA, Intergenic; D058727:RNA, Small Untranslated