Control of virulence by small RNAs in Streptococcus pneumoniae.
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ABSTRACT:
SUBMITTER: Mann B
PROVIDER: S-EPMC3395615 | biostudies-literature | 2012
REPOSITORIES: biostudies-literature
Publications
Control of virulence by small RNAs in Streptococcus pneumoniae.
Mann Beth B van Opijnen Tim T Wang Jianmin J Obert Caroline C Wang Yong-Dong YD Carter Robert R McGoldrick Daniel J DJ Ridout Granger G Camilli Andrew A Tuomanen Elaine I EI Rosch Jason W JW
PLoS pathogens 20120712 7
Small noncoding RNAs (sRNAs) play important roles in gene regulation in both prokaryotes and eukaryotes. Thus far, no sRNA has been assigned a definitive role in virulence in the major human pathogen Streptococcus pneumoniae. Based on the potential coding capacity of intergenic regions, we hypothesized that the pneumococcus produces many sRNAs and that they would play an important role in pathogenesis. We describe the application of whole-genome transcriptional sequencing to systematically ident ...[more]
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Project description:We report a search for small RNAs (sRNAs) in the low-GC, gram-positive human pathogen Streptococcus pneumoniae. Based on bioinformatic analyses by Livny et al. (J. Livny, A. Brencic, S. Lory, and M. K. Waldor, Nucleic Acids Res. 34:3484-3493, 2006), we tested 40 candidates by Northern blotting and confirmed the expression of nine new and one previously reported (CcnA) sRNAs in strain D39. CcnA is one of five redundant sRNAs reported by Halfmann et al. (A. Halfmann, M. Kovacs, R. Hakenbeck, and R. Bruckner, Mol. Microbiol. 66:110-126, 2007) that are positively controlled by the CiaR response regulator. We characterized 3 of these 14 sRNAs: Spd-sr17 (144 nucleotides [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 cotranscribed 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 Delta ciaR mutants, but attempts to link CcnA to -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.
| S-EPMC2798261 | biostudies-literature
Project description:We report here an experimental search for small non-coding RNAs in a low GC, gram-positive, human pathogenic bacteria Streptococcus pneumoniae. Based on the bioinformatic analyses by Livny et al. (2006, Nucleic Acids Res. 34:3484-93), we tested 36 candidates by Northern analyses and confirmed the expression of 8 novel and one previously-reported sRNAs (CcnA) in the genome of D39 serotype 2 strain. Some of the sRNAs showed differential expression in stationary phase or after treatment with competence stimulatory peptide compared to untreated exponential cells. One sRNA we detected is CcnA, one of five CiaR-controlled non-coding RNA (CcnA to E) reported previously. By ectopically expressing this sRNA in a ΔciaR strain, we provide evidence that CcnA plays important roles in the regulation of growth and chain length of S. pneumoniae. Microarray analysis comparing global gene expression of CcnA-expressing and CcnA-nonexpressing ΔciaR strains showed significant differences in expression of competence related genes and five putative transcriptional regulators. QPCR and transformation assays further confirmed that CcnA sRNA mediates a strong negative effect on competence development. Our findings suggested many of the CiaRH function may be mediated via Ccn sRNAs and that sRNAs play regulatory roles in S. pneumoniae, an organism that lacks Hfq RNA chaperone.
2010-02-03 | GSE14688 | GEO
Project description:We report here an experimental search for small non-coding RNAs in a low GC, gram-positive, human pathogenic bacteria Streptococcus pneumoniae. Based on the bioinformatic analyses by Livny et al. (2006, Nucleic Acids Res. 34:3484-93), we tested 36 candidates by Northern analyses and confirmed the expression of 8 novel and one previously-reported sRNAs (CcnA) in the genome of D39 serotype 2 strain. Some of the sRNAs showed differential expression in stationary phase or after treatment with competence stimulatory peptide compared to untreated exponential cells. One sRNA we detected is CcnA, one of five CiaR-controlled non-coding RNA (CcnA to E) reported previously. By ectopically expressing this sRNA in a ÎciaR strain, we provide evidence that CcnA plays important roles in the regulation of growth and chain length of S. pneumoniae. Microarray analysis comparing global gene expression of CcnA-expressing and CcnA-nonexpressing ÎciaR strains showed significant differences in expression of competence related genes and five putative transcriptional regulators. QPCR and transformation assays further confirmed that CcnA sRNA mediates a strong negative effect on competence development. Our findings suggested many of the CiaRH function may be mediated via Ccn sRNAs and that sRNAs play regulatory roles in S. pneumoniae, an organism that lacks Hfq RNA chaperone. Three independent hybridizations using independent RNA preparations from the strain IU2678 (a ciaR knockout strain with no ccnA expression, reference strain) and strain IU2841 (a ciaR knowckout strain with ccnA expression at an ectopic site) were performed. Dye swap was performed with the reference strain labeled with Cy3 in two hybridizations and Cy5 in the one hybridizations.
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