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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

RT-PCR analysis.The Relative expression ratio between WT and rpoS mutant indicated in the graph are the average of at least four experiments (two repeats, each performed on duplicate samples, from two independent RNA extractions), and standard deviations are shown. The asterisks denote significant differences (*=p < 0.05; **= p < 0.01 Tukey multigroup analysis). The dashed line indicates a WT/rpoS mutant expression ratio=1.
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f3: RT-PCR analysis.The Relative expression ratio between WT and rpoS mutant indicated in the graph are the average of at least four experiments (two repeats, each performed on duplicate samples, from two independent RNA extractions), and standard deviations are shown. The asterisks denote significant differences (*=p < 0.05; **= p < 0.01 Tukey multigroup analysis). The dashed line indicates a WT/rpoS mutant expression ratio=1.

Mentions: Results of the qRT-PCR experiments (Fig. 3) could demonstrate rpoS-dependent gene expression for dps, ycgB, ybiI and ydbK, suggesting that the latter two are yet unidentified members of the rpoS regulon. In contrast, the expression of the remaining genes was not affected by the lack of a functional rpoS gene, at least in the conditions tested. To further investigate whether these genes showed any kind of dependence on σS, we tested their expression levels in a rpoS-overexpressing strain (MG1655/pBADrpoS) grown to early stationary phase in LB medium supplemented with 0.1% arabinose. Although intracellular σS amounts were almost 10-fold higher in the pBADrpoS-bearing strains compared to MG1655, no significant changes in relative expression levels were detected for any of the genes tested (data not shown).


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)

RT-PCR analysis.The Relative expression ratio between WT and rpoS mutant indicated in the graph are the average of at least four experiments (two repeats, each performed on duplicate samples, from two independent RNA extractions), and standard deviations are shown. The asterisks denote significant differences (*=p < 0.05; **= p < 0.01 Tukey multigroup analysis). The dashed line indicates a WT/rpoS mutant expression ratio=1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4447067&req=5

f3: RT-PCR analysis.The Relative expression ratio between WT and rpoS mutant indicated in the graph are the average of at least four experiments (two repeats, each performed on duplicate samples, from two independent RNA extractions), and standard deviations are shown. The asterisks denote significant differences (*=p < 0.05; **= p < 0.01 Tukey multigroup analysis). The dashed line indicates a WT/rpoS mutant expression ratio=1.
Mentions: Results of the qRT-PCR experiments (Fig. 3) could demonstrate rpoS-dependent gene expression for dps, ycgB, ybiI and ydbK, suggesting that the latter two are yet unidentified members of the rpoS regulon. In contrast, the expression of the remaining genes was not affected by the lack of a functional rpoS gene, at least in the conditions tested. To further investigate whether these genes showed any kind of dependence on σS, we tested their expression levels in a rpoS-overexpressing strain (MG1655/pBADrpoS) grown to early stationary phase in LB medium supplemented with 0.1% arabinose. Although intracellular σS amounts were almost 10-fold higher in the pBADrpoS-bearing strains compared to MG1655, no significant changes in relative expression levels were detected for any of the genes tested (data not shown).

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