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Genome-wide identification of regulatory RNAs in the human pathogen Clostridium difficile.

Soutourina OA, Monot M, Boudry P, Saujet L, Pichon C, Sismeiro O, Semenova E, Severinov K, Le Bouguenec C, Coppée JY, Dupuy B, Martin-Verstraete I - PLoS Genet. (2013)

Bottom Line: Expression of 35 sRNAs was confirmed by gene-specific experimental approaches.These RNAs may be important for C. difficile survival in bacteriophage-rich gut communities.Altogether, this first experimental genome-wide identification of C. difficile sRNAs provides a firm basis for future RNome characterization and identification of molecular mechanisms of sRNA-based regulation of gene expression in this emergent enteropathogen.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire Pathogenèse des Bactéries Anaérobies, Institut Pasteur, Paris, France. olga.soutourina@pasteur.fr

ABSTRACT
Clostridium difficile is an emergent pathogen, and the most common cause of nosocomial diarrhea. In an effort to understand the role of small noncoding RNAs (sRNAs) in C. difficile physiology and pathogenesis, we used an in silico approach to identify 511 sRNA candidates in both intergenic and coding regions. In parallel, RNA-seq and differential 5'-end RNA-seq were used for global identification of C. difficile sRNAs and their transcriptional start sites at three different growth conditions (exponential growth phase, stationary phase, and starvation). This global experimental approach identified 251 putative regulatory sRNAs including 94 potential trans riboregulators located in intergenic regions, 91 cis-antisense RNAs, and 66 riboswitches. Expression of 35 sRNAs was confirmed by gene-specific experimental approaches. Some sRNAs, including an antisense RNA that may be involved in control of C. difficile autolytic activity, showed growth phase-dependent expression profiles. Expression of each of 16 predicted c-di-GMP-responsive riboswitches was observed, and experimental evidence for their regulatory role in coordinated control of motility and biofilm formation was obtained. Finally, we detected abundant sRNAs encoded by multiple C. difficile CRISPR loci. These RNAs may be important for C. difficile survival in bacteriophage-rich gut communities. Altogether, this first experimental genome-wide identification of C. difficile sRNAs provides a firm basis for future RNome characterization and identification of molecular mechanisms of sRNA-based regulation of gene expression in this emergent enteropathogen.

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Functional analysis of c-di-GMP riboswitches in C. difficile.(A) Northern blot detection of SQ173 located upstream of the flgB flagella operon. Coding sequences are indicated by blue arrows and SQ173 is indicated by a grey arrow. For Northern blot analysis, RNA samples correspond to those indicated for Figure 2. 5S RNA at the bottom serves as loading control. The arrow on the left shows the detected transcripts with their estimated size. (B) Effect of overexpression of CD1420 (dccA) encoding diguanylate cyclase on motility and biofilm formation. A representative result of motility and biofilm formation assay of strains 630/pdccA (pRPF185-CD1420) and 630/p (pRPF185) performed in the absence or in the presence of ATc (500 ng/mL) inducing the expression of the CD1420 gene. qRT-PCR data show fold change for 630/pdccA/630/p ratio for the flgB gene positively regulated by Cdi1_3 riboswitch, flagellin-encoding fliC gene and CD3513, CD2831 genes negatively regulated by c-di-GMP riboswitches of type II. Northern blot analysis of c-di-GMP-I (C) and c-di-GMP-II (D) riboswitches. RNA samples correspond to those indicated for Figure 6, 630/p and 630/pdccA strain RNA samples were extracted at late exponential growth phase (LE) in the presence of 500 ng/mL ATc. White arrows correspond to target gene full-length transcripts and black arrows to premature terminated transcripts and processed forms (C) or stable spliced forms (D).
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pgen-1003493-g007: Functional analysis of c-di-GMP riboswitches in C. difficile.(A) Northern blot detection of SQ173 located upstream of the flgB flagella operon. Coding sequences are indicated by blue arrows and SQ173 is indicated by a grey arrow. For Northern blot analysis, RNA samples correspond to those indicated for Figure 2. 5S RNA at the bottom serves as loading control. The arrow on the left shows the detected transcripts with their estimated size. (B) Effect of overexpression of CD1420 (dccA) encoding diguanylate cyclase on motility and biofilm formation. A representative result of motility and biofilm formation assay of strains 630/pdccA (pRPF185-CD1420) and 630/p (pRPF185) performed in the absence or in the presence of ATc (500 ng/mL) inducing the expression of the CD1420 gene. qRT-PCR data show fold change for 630/pdccA/630/p ratio for the flgB gene positively regulated by Cdi1_3 riboswitch, flagellin-encoding fliC gene and CD3513, CD2831 genes negatively regulated by c-di-GMP riboswitches of type II. Northern blot analysis of c-di-GMP-I (C) and c-di-GMP-II (D) riboswitches. RNA samples correspond to those indicated for Figure 6, 630/p and 630/pdccA strain RNA samples were extracted at late exponential growth phase (LE) in the presence of 500 ng/mL ATc. White arrows correspond to target gene full-length transcripts and black arrows to premature terminated transcripts and processed forms (C) or stable spliced forms (D).

Mentions: Among the 185 C. difficile 630Δerm sRNAs identified by deep sequencing, 35 out of 40 candidates assayed were detected by Northern blotting of 630Δerm strain RNA samples, while 23 were detected in RNA prepared from the R20291 strain samples. Transcript lengths agreed well with the sizes deduced from RNA-seq approach (Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 7, Figure 8; Figures S2, S3, S4, S5) and were confirmed by independent 5′/3′RACE analysis for 4 selected RNAs (Table S4). 5′RACE experiments unambiguously identified TSSs for eight potential sRNAs and were in complete agreement with the TSSs identified by 5′-end RNA-seq (Table S4).


Genome-wide identification of regulatory RNAs in the human pathogen Clostridium difficile.

Soutourina OA, Monot M, Boudry P, Saujet L, Pichon C, Sismeiro O, Semenova E, Severinov K, Le Bouguenec C, Coppée JY, Dupuy B, Martin-Verstraete I - PLoS Genet. (2013)

Functional analysis of c-di-GMP riboswitches in C. difficile.(A) Northern blot detection of SQ173 located upstream of the flgB flagella operon. Coding sequences are indicated by blue arrows and SQ173 is indicated by a grey arrow. For Northern blot analysis, RNA samples correspond to those indicated for Figure 2. 5S RNA at the bottom serves as loading control. The arrow on the left shows the detected transcripts with their estimated size. (B) Effect of overexpression of CD1420 (dccA) encoding diguanylate cyclase on motility and biofilm formation. A representative result of motility and biofilm formation assay of strains 630/pdccA (pRPF185-CD1420) and 630/p (pRPF185) performed in the absence or in the presence of ATc (500 ng/mL) inducing the expression of the CD1420 gene. qRT-PCR data show fold change for 630/pdccA/630/p ratio for the flgB gene positively regulated by Cdi1_3 riboswitch, flagellin-encoding fliC gene and CD3513, CD2831 genes negatively regulated by c-di-GMP riboswitches of type II. Northern blot analysis of c-di-GMP-I (C) and c-di-GMP-II (D) riboswitches. RNA samples correspond to those indicated for Figure 6, 630/p and 630/pdccA strain RNA samples were extracted at late exponential growth phase (LE) in the presence of 500 ng/mL ATc. White arrows correspond to target gene full-length transcripts and black arrows to premature terminated transcripts and processed forms (C) or stable spliced forms (D).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3649979&req=5

pgen-1003493-g007: Functional analysis of c-di-GMP riboswitches in C. difficile.(A) Northern blot detection of SQ173 located upstream of the flgB flagella operon. Coding sequences are indicated by blue arrows and SQ173 is indicated by a grey arrow. For Northern blot analysis, RNA samples correspond to those indicated for Figure 2. 5S RNA at the bottom serves as loading control. The arrow on the left shows the detected transcripts with their estimated size. (B) Effect of overexpression of CD1420 (dccA) encoding diguanylate cyclase on motility and biofilm formation. A representative result of motility and biofilm formation assay of strains 630/pdccA (pRPF185-CD1420) and 630/p (pRPF185) performed in the absence or in the presence of ATc (500 ng/mL) inducing the expression of the CD1420 gene. qRT-PCR data show fold change for 630/pdccA/630/p ratio for the flgB gene positively regulated by Cdi1_3 riboswitch, flagellin-encoding fliC gene and CD3513, CD2831 genes negatively regulated by c-di-GMP riboswitches of type II. Northern blot analysis of c-di-GMP-I (C) and c-di-GMP-II (D) riboswitches. RNA samples correspond to those indicated for Figure 6, 630/p and 630/pdccA strain RNA samples were extracted at late exponential growth phase (LE) in the presence of 500 ng/mL ATc. White arrows correspond to target gene full-length transcripts and black arrows to premature terminated transcripts and processed forms (C) or stable spliced forms (D).
Mentions: Among the 185 C. difficile 630Δerm sRNAs identified by deep sequencing, 35 out of 40 candidates assayed were detected by Northern blotting of 630Δerm strain RNA samples, while 23 were detected in RNA prepared from the R20291 strain samples. Transcript lengths agreed well with the sizes deduced from RNA-seq approach (Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 7, Figure 8; Figures S2, S3, S4, S5) and were confirmed by independent 5′/3′RACE analysis for 4 selected RNAs (Table S4). 5′RACE experiments unambiguously identified TSSs for eight potential sRNAs and were in complete agreement with the TSSs identified by 5′-end RNA-seq (Table S4).

Bottom Line: Expression of 35 sRNAs was confirmed by gene-specific experimental approaches.These RNAs may be important for C. difficile survival in bacteriophage-rich gut communities.Altogether, this first experimental genome-wide identification of C. difficile sRNAs provides a firm basis for future RNome characterization and identification of molecular mechanisms of sRNA-based regulation of gene expression in this emergent enteropathogen.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire Pathogenèse des Bactéries Anaérobies, Institut Pasteur, Paris, France. olga.soutourina@pasteur.fr

ABSTRACT
Clostridium difficile is an emergent pathogen, and the most common cause of nosocomial diarrhea. In an effort to understand the role of small noncoding RNAs (sRNAs) in C. difficile physiology and pathogenesis, we used an in silico approach to identify 511 sRNA candidates in both intergenic and coding regions. In parallel, RNA-seq and differential 5'-end RNA-seq were used for global identification of C. difficile sRNAs and their transcriptional start sites at three different growth conditions (exponential growth phase, stationary phase, and starvation). This global experimental approach identified 251 putative regulatory sRNAs including 94 potential trans riboregulators located in intergenic regions, 91 cis-antisense RNAs, and 66 riboswitches. Expression of 35 sRNAs was confirmed by gene-specific experimental approaches. Some sRNAs, including an antisense RNA that may be involved in control of C. difficile autolytic activity, showed growth phase-dependent expression profiles. Expression of each of 16 predicted c-di-GMP-responsive riboswitches was observed, and experimental evidence for their regulatory role in coordinated control of motility and biofilm formation was obtained. Finally, we detected abundant sRNAs encoded by multiple C. difficile CRISPR loci. These RNAs may be important for C. difficile survival in bacteriophage-rich gut communities. Altogether, this first experimental genome-wide identification of C. difficile sRNAs provides a firm basis for future RNome characterization and identification of molecular mechanisms of sRNA-based regulation of gene expression in this emergent enteropathogen.

Show MeSH
Related in: MedlinePlus