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Genome-wide prediction and validation of σ70 promoters in Lactobacillus plantarum WCFS1

ABSTRACT: Background: In prokaryotes, sigma factors are essential for the targeting of the transcription machinery to promoters. Various sigma factors have been described that recognize and bind to specific DNA sequence motifs in promoter sequences. The canonical sigma factor σ70 is commonly involved in transcription of the cell's general housekeeping genes, which is mediated by the conserved σ70 promoter sequence motifs. This study detects and predicts the general σ70-promoter sequences in Lactobacillus plantarum WCFS1 using genome-wide analysis. The accuracy of the transcriptionally-active part of this promoter prediction was subsequently evaluated by correlating locations of predicted promoters with experimentally detected transcription starts sites (TSSs) using high-resolution tiling array transcriptome datasets. Results: To identify σ70-related promoter sequences, we performed a genome-wide sequence motif scan of the L. plantarum WCFS1 genome focusing on the regions upstream of protein-encoding genes. We obtained several highly conserved motifs including those resembling the conserved σ70-promoter consensus. Position weight matrices-based models of the recovered σ70-promoter sequence motif were employed to identify 4746 motifs with significant similarity (p-value < 10-4) to the model-motif in the L. plantarum genome. Genome-wide transcription start site information deduced from whole genome tiling-array transcriptome datasets revealed 936 TSSs that were employed to validate the transcriptionally active fraction of these predicted promoters. In total, 578 predicted promoters were found in proximity (≤ 100 nucleotides) of the identified TSSs, showing a highly significant co-occurrence of predicted promoter and measured TSS (p-value < 10-23). An additional 224 predicted promoters was validated when the significant similarity to the model-motif was applied less strictly (10-4 ≤ p-value < 10-3). Conclusions: High-resolution tiling arrays provide a suitable source for TSS detection at a genome-wide level, and allow experimental verification of in silico-predicted promoter sequence motifs.

ORGANISM(S): Lactiplantibacillus plantarum WCFS1 Lactiplantibacillus plantarum

PROVIDER: GSE36898 | GEO | 2012/03/29

SECONDARY ACCESSION(S): PRJNA157151

REPOSITORIES: GEO

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Project description:Background: In prokaryotes, sigma factors are essential for the targeting of the transcription machinery to promoters. Various sigma factors have been described that recognize and bind to specific DNA sequence motifs in promoter sequences. The canonical sigma factor M-OM-^C70 is commonly involved in transcription of the cell's general housekeeping genes, which is mediated by the conserved M-OM-^C70 promoter sequence motifs. This study detects and predicts the general M-OM-^C70-promoter sequences in Lactobacillus plantarum WCFS1 using genome-wide analysis. The accuracy of the transcriptionally-active part of this promoter prediction was subsequently evaluated by correlating locations of predicted promoters with experimentally detected transcription starts sites (TSSs) using high-resolution tiling array transcriptome datasets. Results: To identify M-OM-^C70-related promoter sequences, we performed a genome-wide sequence motif scan of the L. plantarum WCFS1 genome focusing on the regions upstream of protein-encoding genes. We obtained several highly conserved motifs including those resembling the conserved M-OM-^C70-promoter consensus. Position weight matrices-based models of the recovered M-OM-^C70-promoter sequence motif were employed to identify 4746 motifs with significant similarity (p-value < 10-4) to the model-motif in the L. plantarum genome. Genome-wide transcription start site information deduced from whole genome tiling-array transcriptome datasets revealed 936 TSSs that were employed to validate the transcriptionally active fraction of these predicted promoters. In total, 578 predicted promoters were found in proximity (M-bM-^IM-$ 100 nucleotides) of the identified TSSs, showing a highly significant co-occurrence of predicted promoter and measured TSS (p-value < 10-23). An additional 224 predicted promoters was validated when the significant similarity to the model-motif was applied less strictly (10-4 M-bM-^IM-$ p-value < 10-3). Conclusions: High-resolution tiling arrays provide a suitable source for TSS detection at a genome-wide level, and allow experimental verification of in silico-predicted promoter sequence motifs. Triplicate hybridizations (technical replicates) of two L. plantarum WCFS1 samples (biological replicates) gathered from a fermentation run at OD600 of 1. Dye swap applied to one of the three technical replicates.

Project description:When performed at single bp resolution, the genome-wide location, occupancy level, and structural organization of DNA binding proteins provides mechanistic insights into genome regulation. Here we use ChIP-exo to provide the first high resolution view of the epigenomic organization of the E. coli transcription machinery and nucleoid structural proteins when cells are growing exponentially and upon rapid reprogramming (acute heat shock). We suggest that indirect readout of DNA shape at the flanks of cognate motifs provide major contributions to site specificity at promoter positions -35/-24 and -10/-12. We examined the site specificity of three sigma factors (RpoD/70, RpoH/32, and RpoN/54), RNA polymerase (RNAP or RpoA, B, C) and two nucleoid proteins (Fis and IHF). Our results confirm and refine reports that RpoD binds most annotated promoters, whereas RpoH and RpoN bind a much smaller subset, each through their cognate motifs. However, only upon heat shock does RpoH becomes active for RNAP recruitment. In contrast, upon heat shock RpoD remains active at its cognate promoters (including at heat shock genes), whereas RpoN remains inactive at its cognate promoters. RpoN binds ~1,000 non-annotated RpoN motifs, which may reflect a large number of condition-specific transcription units. Occupancy patterns of sigma factors and RNAP suggest a common promoter recruitment mechanism that differs from the long-standing views that sigma and RNAP are co-recruited as a complex, and also simultaneously dissociate from promoters. Our findings suggest that sigma factors are recruited and/or maintained at most promoters via an RNAP-independent mechanism. When RNAP arrives, it dwells for a relatively short time before clearing the promoter, leaving sigma behind. Taken together these findings add new dimensions to how sigma factors achieve promoter specificity through DNA sequence and shape and redefine mechanistic steps in regulated promoter assembly in E. coli.

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Genome-wide prediction and validation of σ70 promoters in Lactobacillus plantarum WCFS1

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