JB Accepts, published online ahead of print on 23 October 2009

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J. Bacteriol. doi:10.1128/JB.01204-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Identification and Characterization of Non-Coding Small RNAs in Streptococcus pneumoniae Serotype 2 Strain D39

Ho-Ching Tiffany Tsui, Dhriti Mukherjee, Valerie A. Ray, Lok-To Sham, Andrew L. Feig, and Malcolm E. Winkler^*

Department of Biology, Indiana University-Bloomington, Bloomington, IN 47405, U.S.A.; Department of Chemistry, Wayne State University, 5101 Cass Ave., Detroit, MI 48202, U.S.A.

^* To whom correspondence should be addressed. Email: mwinkler{at}bio.indiana.edu.

We report a search for small RNAs in the low GC, gram-positive, human pathogen, Streptococcus pneumoniae. Based on bioinformatic analyses of Livny et al. (2006, Nucleic Acids Res. 34:3484), we tested 40 candidates by Northern blotting and confirmed the expression of 9 new and one previously reported (CcnA) sRNAs in strain D39. CcnA is one of five redundant sRNAs reported by Halfmann et al. (2007, Mol. Microbiol. 66:100) that are positively controlled by the CiaR response regulator. We characterized three of these 14 sRNAs: Spd-sr17 (144 nt; decreased in stationary phase); Spd-sr37 (80 nt; strongly expressed in all growth phases); and CcnA (93 nt; induced by competence stimulatory peptide). Spd-sr17 and CcnA likely fold into structures containing single-stranded regions between hairpin structures, whereas Spd-sr37 forms a base-paired structure. Primer extension mapping and ectopic expression in deletion/insertion mutants confirmed the independent expression of the three sRNAs. Microarray analyses indicated that insertion/deletion mutants in spd-sr37 and ccnA exerted strong cis-acting effects on the transcription of adjacent genes, indicating that these sRNA regions are also co-transcribed in operons. Deletion or overexpression of the three sRNAs did not cause changes in growth, certain stress responses, global transcription, or virulence. Constitutive ectopic expression of CcnA reversed some phenotypes of D39 ciaR mutants, but attempts to link CcnA-E to comC as a target were inconclusive in ciaR⁺ strains. These results show that S. pneumoniae, which lacks known RNA chaperones, expresses numerous sRNAs, but three of these sRNAs do not strongly affect common phenotypes or transcription patterns.