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Transcriptomic profiling of Yersinia pseudotuberculosis reveals reprogramming of the Crp regulon by temperature and uncovers Crp as a master regulator of small RNAs.

Nuss AM, Heroven AK, Waldmann B, Reinkensmeier J, Jarek M, Beckstette M, Dersch P - PLoS Genet. (2015)

Bottom Line: We identified 1151 individual transcription start sites, multiple riboswitch-like RNA elements, and a global set of antisense RNAs and previously unrecognized trans-acting RNAs.To elucidate the regulatory architecture linking nutritional status to virulence we also refined the CRP regulon.We identified a massive remodelling of the CRP-controlled network in response to temperature and discovered CRP as a transcriptional master regulator of numerous conserved and newly identified non-coding RNAs which participate in this process.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Infection Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany.

ABSTRACT
One hallmark of pathogenic yersiniae is their ability to rapidly adjust their life-style and pathogenesis upon host entry. In order to capture the range, magnitude and complexity of the underlying gene control mechanisms we used comparative RNA-seq-based transcriptomic profiling of the enteric pathogen Y. pseudotuberculosis under environmental and infection-relevant conditions. We identified 1151 individual transcription start sites, multiple riboswitch-like RNA elements, and a global set of antisense RNAs and previously unrecognized trans-acting RNAs. Taking advantage of these data, we revealed a temperature-induced and growth phase-dependent reprogramming of a large set of catabolic/energy production genes and uncovered the existence of a thermo-regulated 'acetate switch', which appear to prime the bacteria for growth in the digestive tract. To elucidate the regulatory architecture linking nutritional status to virulence we also refined the CRP regulon. We identified a massive remodelling of the CRP-controlled network in response to temperature and discovered CRP as a transcriptional master regulator of numerous conserved and newly identified non-coding RNAs which participate in this process. This finding highlights a novel level of complexity of the regulatory network in which the concerted action of transcriptional regulators and multiple non-coding RNAs under control of CRP adjusts the control of Yersinia fitness and virulence to the requirements of their environmental and virulent life-styles.

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CRP-dependent non-coding RNAs of Y. pseudotuberculosis.(A) Northern blot analyses of selected CRP-regulated non-coding RNAs. RNA samples were prepared from YPIII and YP89 (YPIII Δcrp) bacteria grown to stationary growth phase (stat) at 25°C and 37°C. 5S rRNA served as loading control. The size marker (nt) is indicated. (B) Interaction of CRP with the regulatory regions of selected CRP-regulated sRNA genes. Individual DNA fragments with the predicted CRP-binding site(s) (yellow boxes; S3 Dataset) used for electrophoretic mobility shift assays are illustrated. An individual sRNA promoter fragment (ysr204) for which no CRP-binding site was predicted was included as negative control. Respective DNA fragments were incubated with increasing concentrations of CRP and 0.2 mM cAMP. As a negative control, cAMP was omitted in samples with the highest CRP concentration (right lane). The CRP-DNA complexes were separated on 4% polyacrylamide gels. The position of specific higher molecular weight complexes is marked with an asterisk. A molecular weight standard (M) was loaded, and the corresponding molecular weights are indicated. A csiD PCR fragment amplified from E. coli served as an internal negative control.
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pgen.1005087.g005: CRP-dependent non-coding RNAs of Y. pseudotuberculosis.(A) Northern blot analyses of selected CRP-regulated non-coding RNAs. RNA samples were prepared from YPIII and YP89 (YPIII Δcrp) bacteria grown to stationary growth phase (stat) at 25°C and 37°C. 5S rRNA served as loading control. The size marker (nt) is indicated. (B) Interaction of CRP with the regulatory regions of selected CRP-regulated sRNA genes. Individual DNA fragments with the predicted CRP-binding site(s) (yellow boxes; S3 Dataset) used for electrophoretic mobility shift assays are illustrated. An individual sRNA promoter fragment (ysr204) for which no CRP-binding site was predicted was included as negative control. Respective DNA fragments were incubated with increasing concentrations of CRP and 0.2 mM cAMP. As a negative control, cAMP was omitted in samples with the highest CRP concentration (right lane). The CRP-DNA complexes were separated on 4% polyacrylamide gels. The position of specific higher molecular weight complexes is marked with an asterisk. A molecular weight standard (M) was loaded, and the corresponding molecular weights are indicated. A csiD PCR fragment amplified from E. coli served as an internal negative control.

Mentions: The accumulation of regulatory RNAs during stationary phase suggested that they might also be part of the CRP regulon. Our investigation of transcriptomic changes revealed 53 trans-encoded RNAs of Y. pseudotuberculosis (65%) which are up- or down-regulated in the absence of crp at 25°C and/or 37°C (S3 and S5 Datasets). Many conserved trans-encoded RNAs, but also multiple newly identified sRNAs were identified as members of the CRP regulon, and their intracellular level is strongly affected by temperature. CRP-dependent regulation of all 15 randomly selected trans-encoded sRNAs was confirmed by Northern blotting (Fig. 5A). Furthermore, we validated one newly identified trans-encoded RNA Ysr206, as well as two antisense RNAs Ysr232 and Ysr114, which were exclusively detected in the crp mutant (Fig. 5A). Both antisense RNAs constitute the most abundant antisense RNAs identified by our approach (S3 Dataset). The huge subset of CRP-regulated non-coding RNAs strongly indicates that CRP is a master regulator of regulatory RNAs in Y. pseudotuberculosis. Recent identification of four CRP-dependent riboregulators in Y. pestis (CyaR, sR065, sR066, sR084 encoded on pPCP [20]) further indicate that this might also account for other Yersinia species. Furthermore, CRP-mediated control seems to account for the accumulation of many sRNAs during stationary phase, and it also explains downregulation of a subset of sRNAs (e.g. GcvB), which are negatively affected by the regulator (Figs. 3A, 5A).


Transcriptomic profiling of Yersinia pseudotuberculosis reveals reprogramming of the Crp regulon by temperature and uncovers Crp as a master regulator of small RNAs.

Nuss AM, Heroven AK, Waldmann B, Reinkensmeier J, Jarek M, Beckstette M, Dersch P - PLoS Genet. (2015)

CRP-dependent non-coding RNAs of Y. pseudotuberculosis.(A) Northern blot analyses of selected CRP-regulated non-coding RNAs. RNA samples were prepared from YPIII and YP89 (YPIII Δcrp) bacteria grown to stationary growth phase (stat) at 25°C and 37°C. 5S rRNA served as loading control. The size marker (nt) is indicated. (B) Interaction of CRP with the regulatory regions of selected CRP-regulated sRNA genes. Individual DNA fragments with the predicted CRP-binding site(s) (yellow boxes; S3 Dataset) used for electrophoretic mobility shift assays are illustrated. An individual sRNA promoter fragment (ysr204) for which no CRP-binding site was predicted was included as negative control. Respective DNA fragments were incubated with increasing concentrations of CRP and 0.2 mM cAMP. As a negative control, cAMP was omitted in samples with the highest CRP concentration (right lane). The CRP-DNA complexes were separated on 4% polyacrylamide gels. The position of specific higher molecular weight complexes is marked with an asterisk. A molecular weight standard (M) was loaded, and the corresponding molecular weights are indicated. A csiD PCR fragment amplified from E. coli served as an internal negative control.
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Related In: Results  -  Collection

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pgen.1005087.g005: CRP-dependent non-coding RNAs of Y. pseudotuberculosis.(A) Northern blot analyses of selected CRP-regulated non-coding RNAs. RNA samples were prepared from YPIII and YP89 (YPIII Δcrp) bacteria grown to stationary growth phase (stat) at 25°C and 37°C. 5S rRNA served as loading control. The size marker (nt) is indicated. (B) Interaction of CRP with the regulatory regions of selected CRP-regulated sRNA genes. Individual DNA fragments with the predicted CRP-binding site(s) (yellow boxes; S3 Dataset) used for electrophoretic mobility shift assays are illustrated. An individual sRNA promoter fragment (ysr204) for which no CRP-binding site was predicted was included as negative control. Respective DNA fragments were incubated with increasing concentrations of CRP and 0.2 mM cAMP. As a negative control, cAMP was omitted in samples with the highest CRP concentration (right lane). The CRP-DNA complexes were separated on 4% polyacrylamide gels. The position of specific higher molecular weight complexes is marked with an asterisk. A molecular weight standard (M) was loaded, and the corresponding molecular weights are indicated. A csiD PCR fragment amplified from E. coli served as an internal negative control.
Mentions: The accumulation of regulatory RNAs during stationary phase suggested that they might also be part of the CRP regulon. Our investigation of transcriptomic changes revealed 53 trans-encoded RNAs of Y. pseudotuberculosis (65%) which are up- or down-regulated in the absence of crp at 25°C and/or 37°C (S3 and S5 Datasets). Many conserved trans-encoded RNAs, but also multiple newly identified sRNAs were identified as members of the CRP regulon, and their intracellular level is strongly affected by temperature. CRP-dependent regulation of all 15 randomly selected trans-encoded sRNAs was confirmed by Northern blotting (Fig. 5A). Furthermore, we validated one newly identified trans-encoded RNA Ysr206, as well as two antisense RNAs Ysr232 and Ysr114, which were exclusively detected in the crp mutant (Fig. 5A). Both antisense RNAs constitute the most abundant antisense RNAs identified by our approach (S3 Dataset). The huge subset of CRP-regulated non-coding RNAs strongly indicates that CRP is a master regulator of regulatory RNAs in Y. pseudotuberculosis. Recent identification of four CRP-dependent riboregulators in Y. pestis (CyaR, sR065, sR066, sR084 encoded on pPCP [20]) further indicate that this might also account for other Yersinia species. Furthermore, CRP-mediated control seems to account for the accumulation of many sRNAs during stationary phase, and it also explains downregulation of a subset of sRNAs (e.g. GcvB), which are negatively affected by the regulator (Figs. 3A, 5A).

Bottom Line: We identified 1151 individual transcription start sites, multiple riboswitch-like RNA elements, and a global set of antisense RNAs and previously unrecognized trans-acting RNAs.To elucidate the regulatory architecture linking nutritional status to virulence we also refined the CRP regulon.We identified a massive remodelling of the CRP-controlled network in response to temperature and discovered CRP as a transcriptional master regulator of numerous conserved and newly identified non-coding RNAs which participate in this process.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Infection Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany.

ABSTRACT
One hallmark of pathogenic yersiniae is their ability to rapidly adjust their life-style and pathogenesis upon host entry. In order to capture the range, magnitude and complexity of the underlying gene control mechanisms we used comparative RNA-seq-based transcriptomic profiling of the enteric pathogen Y. pseudotuberculosis under environmental and infection-relevant conditions. We identified 1151 individual transcription start sites, multiple riboswitch-like RNA elements, and a global set of antisense RNAs and previously unrecognized trans-acting RNAs. Taking advantage of these data, we revealed a temperature-induced and growth phase-dependent reprogramming of a large set of catabolic/energy production genes and uncovered the existence of a thermo-regulated 'acetate switch', which appear to prime the bacteria for growth in the digestive tract. To elucidate the regulatory architecture linking nutritional status to virulence we also refined the CRP regulon. We identified a massive remodelling of the CRP-controlled network in response to temperature and discovered CRP as a transcriptional master regulator of numerous conserved and newly identified non-coding RNAs which participate in this process. This finding highlights a novel level of complexity of the regulatory network in which the concerted action of transcriptional regulators and multiple non-coding RNAs under control of CRP adjusts the control of Yersinia fitness and virulence to the requirements of their environmental and virulent life-styles.

Show MeSH
Related in: MedlinePlus