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Characterization of the Escherichia coli σ(S) core regulon by Chromatin Immunoprecipitation-sequencing (ChIP-seq) analysis.

Peano C, Wolf J, Demol J, Rossi E, Petiti L, De Bellis G, Geiselmann J, Egli T, Lacour S, Landini P - Sci Rep (2015)

Bottom Line: Eσ(S) binding did not always correlate with an increase in transcription level, suggesting that, at some σ(S)-dependent promoters, Eσ(S) might remain poised in a pre-initiation state upon binding.In particular, Eσ(S) appears to contribute significantly to transcription of genes encoding proteins involved in LPS biosynthesis and in cell surface composition.Finally, our results highlight a direct role of Eσ(S) in the regulation of non coding RNAs, such as OmrA/B, RyeA/B and SibC.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomedical Technologies, National Research Council (ITB-CNR), Segrate (MI), Italy.

ABSTRACT
In bacteria, selective promoter recognition by RNA polymerase is achieved by its association with σ factors, accessory subunits able to direct RNA polymerase "core enzyme" (E) to different promoter sequences. Using Chromatin Immunoprecipitation-sequencing (ChIP-seq), we searched for promoters bound by the σ(S)-associated RNA polymerase form (Eσ(S)) during transition from exponential to stationary phase. We identified 63 binding sites for Eσ(S) overlapping known or putative promoters, often located upstream of genes (encoding either ORFs or non-coding RNAs) showing at least some degree of dependence on the σ(S)-encoding rpoS gene. Eσ(S) binding did not always correlate with an increase in transcription level, suggesting that, at some σ(S)-dependent promoters, Eσ(S) might remain poised in a pre-initiation state upon binding. A large fraction of Eσ(S)-binding sites corresponded to promoters recognized by RNA polymerase associated with σ(70) or other σ factors, suggesting a considerable overlap in promoter recognition between different forms of RNA polymerase. In particular, Eσ(S) appears to contribute significantly to transcription of genes encoding proteins involved in LPS biosynthesis and in cell surface composition. Finally, our results highlight a direct role of Eσ(S) in the regulation of non coding RNAs, such as OmrA/B, RyeA/B and SibC.

No MeSH data available.


Related in: MedlinePlus

Regulation of small non-coding RNAs by σS.A. Northern blot hybridization. RNA were extracted at the onset of stationary phase (OD600nm of 3) from bacteria grown in LB at either 28 °C or 37 °C and probed for SibC, OmrA, and RyeA transcript levels (left to right). Numbers on the right side of each panel indicate the size of the respective ncRNA. The gels were probed for the genes of interest, then the probe was removed by washing and the gels were re-probed for 5S RNA, which was used as internal control. B. Relative fluorescence of transcriptional fusions of the omrA and omrB promoters to the GFP reporter gene. The promoter activity (solid line) is expressed as ratio between the fluorescence and the absorbance of the culture (dashed line) after background correction (RFU/OD600 nm). C. Effects of the substitution of the −12C to a T nucleotide in the omrA promoter region. Data were taken from overnight cultures and are the average of four independent experiments.
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f5: Regulation of small non-coding RNAs by σS.A. Northern blot hybridization. RNA were extracted at the onset of stationary phase (OD600nm of 3) from bacteria grown in LB at either 28 °C or 37 °C and probed for SibC, OmrA, and RyeA transcript levels (left to right). Numbers on the right side of each panel indicate the size of the respective ncRNA. The gels were probed for the genes of interest, then the probe was removed by washing and the gels were re-probed for 5S RNA, which was used as internal control. B. Relative fluorescence of transcriptional fusions of the omrA and omrB promoters to the GFP reporter gene. The promoter activity (solid line) is expressed as ratio between the fluorescence and the absorbance of the culture (dashed line) after background correction (RFU/OD600 nm). C. Effects of the substitution of the −12C to a T nucleotide in the omrA promoter region. Data were taken from overnight cultures and are the average of four independent experiments.

Mentions: Results of ChIP-seq analysis indicate that three EσS binding sites are positioned in the proximity of genes encoding regulatory RNAs. A putative EσS binding site was identified upstream of the 88 nt-long regulatory RNA omrA, which controls expression of genes involved in flagellar motility, iron uptake, adhesion factors and various outer membrane proteins25. The omrA gene lies next to omrB, which codes for a highly similar small RNA and also regulates some of the targets for omrA2526. The other two EσS binding sites were found in proximity of two complex loci: the ryeA/ryeB locus, which includes two small RNAs overlapping in antisense directions27, and the sibC/ibsC locus, in which a non coding RNA (sibC) overlaps a small ORF, ibsC, reading in the opposite direction, and encoding a toxic peptide28. The location and extension of the three ChIP-seq peaks suggest that EσS might bind the promoter regions of omrA (but not omrB), and of ryeB and sibC, rather than ryeA and ibsC (Fig. 2B), consistent with recent observations that omrA and ryeB are rpoS-dependent in Salmonella enterica2930. To confirm this result, we performed northern blots comparing small RNA levels in the wild type versus the rpoS mutant strain of E. coli (Fig. 5). In addition to standard growth conditions (LB medium at 37 °C), we also carried out northern blot experiments at 28 °C, since low growth temperature favors σS accumulation and positively affects stability of some small RNA31. Due to difficulties in obtaining a clean result with a probe for RyeB, we measured the relative amounts of RyeA, which upon pairing with RyeB, is degraded in an RNaseIII-dependent fashion and shows therefore transcript levels inversely proportional to ryeB2729. Inactivation of the rpoS gene almost abolished omrA transcription, while strongly increasing RyeA transcript levels (Fig. 5A), consistent with rpoS-dependence of transcription of the omrA and ryeB genes. Interestingly, the OmrA and RyeA transcripts also displayed opposite temperature-dependence, with OmrA being more expressed at 28 °C and RyeA at 37 °C. As further confirmation that rpoS-dependent regulation specifically targets omrA, but not omrB, we performed gfp reporter assays. Reporter genes experiments clearly showed very different effects of rpoS inactivation on transcription of the two genes, with omrA showing almost complete rpoS-dependence, while omrB expression was actually slightly increased in the rpoS mutant background (Fig. 5B). Interestingly, the first nucleotide of the −10 region of omrA is a −12C (Supplementary Table S3), a feature favouring specific promoter opening by EσS but not by Eσ7032, while at the omrB promoter, such a selective determinant is replaced by a canonical −12T for Eσ70 and might explain lack of preferential binding by EσS. Substitution of the −12C nucleotide by a −12T in the omrA −10 promoter element increases promoter strength by more than 10-fold and almost completely overcomes its dependence on rpoS (Fig. 5C), suggesting that the −12C act as a determinant for EσS specificity in the omrA promoter. A more complex picture emerged from analysis of the SibC transcript, which, like RyeA, showed increased expression at 37 °C than at 28 °C. At the latter temperature, SibC was transcribed in an rpoS-dependent manner; however, the effect of the rpoS mutation was reversed at 37 °C, possibly suggesting additional regulatory mechanism affecting SibC expression at this temperature (Fig. 5A). The complexity of SibC regulation is also suggested by the presence of two transcripts, either due to the presence of multiple promoters or to RNA processing as already described28.


Characterization of the Escherichia coli σ(S) core regulon by Chromatin Immunoprecipitation-sequencing (ChIP-seq) analysis.

Peano C, Wolf J, Demol J, Rossi E, Petiti L, De Bellis G, Geiselmann J, Egli T, Lacour S, Landini P - Sci Rep (2015)

Regulation of small non-coding RNAs by σS.A. Northern blot hybridization. RNA were extracted at the onset of stationary phase (OD600nm of 3) from bacteria grown in LB at either 28 °C or 37 °C and probed for SibC, OmrA, and RyeA transcript levels (left to right). Numbers on the right side of each panel indicate the size of the respective ncRNA. The gels were probed for the genes of interest, then the probe was removed by washing and the gels were re-probed for 5S RNA, which was used as internal control. B. Relative fluorescence of transcriptional fusions of the omrA and omrB promoters to the GFP reporter gene. The promoter activity (solid line) is expressed as ratio between the fluorescence and the absorbance of the culture (dashed line) after background correction (RFU/OD600 nm). C. Effects of the substitution of the −12C to a T nucleotide in the omrA promoter region. Data were taken from overnight cultures and are the average of four independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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f5: Regulation of small non-coding RNAs by σS.A. Northern blot hybridization. RNA were extracted at the onset of stationary phase (OD600nm of 3) from bacteria grown in LB at either 28 °C or 37 °C and probed for SibC, OmrA, and RyeA transcript levels (left to right). Numbers on the right side of each panel indicate the size of the respective ncRNA. The gels were probed for the genes of interest, then the probe was removed by washing and the gels were re-probed for 5S RNA, which was used as internal control. B. Relative fluorescence of transcriptional fusions of the omrA and omrB promoters to the GFP reporter gene. The promoter activity (solid line) is expressed as ratio between the fluorescence and the absorbance of the culture (dashed line) after background correction (RFU/OD600 nm). C. Effects of the substitution of the −12C to a T nucleotide in the omrA promoter region. Data were taken from overnight cultures and are the average of four independent experiments.
Mentions: Results of ChIP-seq analysis indicate that three EσS binding sites are positioned in the proximity of genes encoding regulatory RNAs. A putative EσS binding site was identified upstream of the 88 nt-long regulatory RNA omrA, which controls expression of genes involved in flagellar motility, iron uptake, adhesion factors and various outer membrane proteins25. The omrA gene lies next to omrB, which codes for a highly similar small RNA and also regulates some of the targets for omrA2526. The other two EσS binding sites were found in proximity of two complex loci: the ryeA/ryeB locus, which includes two small RNAs overlapping in antisense directions27, and the sibC/ibsC locus, in which a non coding RNA (sibC) overlaps a small ORF, ibsC, reading in the opposite direction, and encoding a toxic peptide28. The location and extension of the three ChIP-seq peaks suggest that EσS might bind the promoter regions of omrA (but not omrB), and of ryeB and sibC, rather than ryeA and ibsC (Fig. 2B), consistent with recent observations that omrA and ryeB are rpoS-dependent in Salmonella enterica2930. To confirm this result, we performed northern blots comparing small RNA levels in the wild type versus the rpoS mutant strain of E. coli (Fig. 5). In addition to standard growth conditions (LB medium at 37 °C), we also carried out northern blot experiments at 28 °C, since low growth temperature favors σS accumulation and positively affects stability of some small RNA31. Due to difficulties in obtaining a clean result with a probe for RyeB, we measured the relative amounts of RyeA, which upon pairing with RyeB, is degraded in an RNaseIII-dependent fashion and shows therefore transcript levels inversely proportional to ryeB2729. Inactivation of the rpoS gene almost abolished omrA transcription, while strongly increasing RyeA transcript levels (Fig. 5A), consistent with rpoS-dependence of transcription of the omrA and ryeB genes. Interestingly, the OmrA and RyeA transcripts also displayed opposite temperature-dependence, with OmrA being more expressed at 28 °C and RyeA at 37 °C. As further confirmation that rpoS-dependent regulation specifically targets omrA, but not omrB, we performed gfp reporter assays. Reporter genes experiments clearly showed very different effects of rpoS inactivation on transcription of the two genes, with omrA showing almost complete rpoS-dependence, while omrB expression was actually slightly increased in the rpoS mutant background (Fig. 5B). Interestingly, the first nucleotide of the −10 region of omrA is a −12C (Supplementary Table S3), a feature favouring specific promoter opening by EσS but not by Eσ7032, while at the omrB promoter, such a selective determinant is replaced by a canonical −12T for Eσ70 and might explain lack of preferential binding by EσS. Substitution of the −12C nucleotide by a −12T in the omrA −10 promoter element increases promoter strength by more than 10-fold and almost completely overcomes its dependence on rpoS (Fig. 5C), suggesting that the −12C act as a determinant for EσS specificity in the omrA promoter. A more complex picture emerged from analysis of the SibC transcript, which, like RyeA, showed increased expression at 37 °C than at 28 °C. At the latter temperature, SibC was transcribed in an rpoS-dependent manner; however, the effect of the rpoS mutation was reversed at 37 °C, possibly suggesting additional regulatory mechanism affecting SibC expression at this temperature (Fig. 5A). The complexity of SibC regulation is also suggested by the presence of two transcripts, either due to the presence of multiple promoters or to RNA processing as already described28.

Bottom Line: Eσ(S) binding did not always correlate with an increase in transcription level, suggesting that, at some σ(S)-dependent promoters, Eσ(S) might remain poised in a pre-initiation state upon binding.In particular, Eσ(S) appears to contribute significantly to transcription of genes encoding proteins involved in LPS biosynthesis and in cell surface composition.Finally, our results highlight a direct role of Eσ(S) in the regulation of non coding RNAs, such as OmrA/B, RyeA/B and SibC.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomedical Technologies, National Research Council (ITB-CNR), Segrate (MI), Italy.

ABSTRACT
In bacteria, selective promoter recognition by RNA polymerase is achieved by its association with σ factors, accessory subunits able to direct RNA polymerase "core enzyme" (E) to different promoter sequences. Using Chromatin Immunoprecipitation-sequencing (ChIP-seq), we searched for promoters bound by the σ(S)-associated RNA polymerase form (Eσ(S)) during transition from exponential to stationary phase. We identified 63 binding sites for Eσ(S) overlapping known or putative promoters, often located upstream of genes (encoding either ORFs or non-coding RNAs) showing at least some degree of dependence on the σ(S)-encoding rpoS gene. Eσ(S) binding did not always correlate with an increase in transcription level, suggesting that, at some σ(S)-dependent promoters, Eσ(S) might remain poised in a pre-initiation state upon binding. A large fraction of Eσ(S)-binding sites corresponded to promoters recognized by RNA polymerase associated with σ(70) or other σ factors, suggesting a considerable overlap in promoter recognition between different forms of RNA polymerase. In particular, Eσ(S) appears to contribute significantly to transcription of genes encoding proteins involved in LPS biosynthesis and in cell surface composition. Finally, our results highlight a direct role of Eσ(S) in the regulation of non coding RNAs, such as OmrA/B, RyeA/B and SibC.

No MeSH data available.


Related in: MedlinePlus