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Comprehensive mapping of the Helicobacter pylori NikR regulon provides new insights in bacterial nickel responses

View Article: PubMed Central - PubMed

ABSTRACT

Nickel homeostasis is important for pathogenic and ureolytic bacteria, which use this metal ion as enzymatic cofactor. For example, in the human pathogen Helicobacter pylori an optimal balance between nickel uptake and incorporation in metallo-enzymes is fundamental for colonization of the host. Nickel is also used as cofactor to modulate DNA binding of the NikR regulator, which controls transcription of genes involved in nickel trafficking or infection in many bacteria. Accordingly, there is much interest in a systematic characterization of NikR regulation. Herein we use H. pylori as a model to integrate RNA-seq and ChIP-seq data demonstrating that NikR not only regulates metal-ion transporters but also virulence factors, non-coding RNAs, as well as toxin-antitoxin systems in response to nickel stimulation. Altogether, results provide new insights into the pathobiology of H. pylori and contribute to understand the responses to nickel in other bacteria.

No MeSH data available.


Validation of new NikR promoters and internal peaks by DNase I footprinting.(A) Radiolabeled PureA, PhcpC, PmccB and PhopV DNA probes were mixed with 0, 9.7, 29, 97 and 290 nM of the NikR tetramer, without nickel (left side of each panel) or with the addition of 150 μM NiSO4 (right side of each panel), before DNase I cleavage. On the right of each autoradiographic film, the G27 genomic coordinates of DNase I protected regions (black boxes) are reported, with position in brackets with respect to the transcriptional start site (TSS). Low affinity binding sites, if present, are shown as grey boxes surrounded by the same information. On the left, a schematic representation of the promoter is provided, with the TSS (+1, bent arrow) and the −10 region (black box). The position of the consensus sequence is reported with violet boxes, corresponding to the two conserved hemi-operator pentamers linked by a black line (15 nt spacer). In the middle panels a scheme of the corresponding transcriptional unit is shown, together with the normalized tag densities obtained from the ChIP-seq experiments (wt/ni+ in yellow, wt/ni− in orange and ∆nikR/ni+, negative control in green), the predicted peak extension by Homer2 and the DNaseI protected regions. Representation scales of ChIP-seq tracks are indicated on the left in brackets. In the bottom panels, the RNA-seq strand specific tracks of the corresponding genomic locus are visualized for wt/ni+ and wt/ni− samples (plus strand in blue, minus strand in red). P* indicates coordinates mapping on the pHPG27 plasmid. (B) Radiolabeled dapD, exsB, fecD and pcrA DNA probes were mixed with 0, 9.7, 29, 97 and 290 nM of NikR tetramers, without nickel (left side of each panel) or with the addition of 150 μM NiSO4 (right side of each panel). The same elements and information are reported as in panel A.
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f2: Validation of new NikR promoters and internal peaks by DNase I footprinting.(A) Radiolabeled PureA, PhcpC, PmccB and PhopV DNA probes were mixed with 0, 9.7, 29, 97 and 290 nM of the NikR tetramer, without nickel (left side of each panel) or with the addition of 150 μM NiSO4 (right side of each panel), before DNase I cleavage. On the right of each autoradiographic film, the G27 genomic coordinates of DNase I protected regions (black boxes) are reported, with position in brackets with respect to the transcriptional start site (TSS). Low affinity binding sites, if present, are shown as grey boxes surrounded by the same information. On the left, a schematic representation of the promoter is provided, with the TSS (+1, bent arrow) and the −10 region (black box). The position of the consensus sequence is reported with violet boxes, corresponding to the two conserved hemi-operator pentamers linked by a black line (15 nt spacer). In the middle panels a scheme of the corresponding transcriptional unit is shown, together with the normalized tag densities obtained from the ChIP-seq experiments (wt/ni+ in yellow, wt/ni− in orange and ∆nikR/ni+, negative control in green), the predicted peak extension by Homer2 and the DNaseI protected regions. Representation scales of ChIP-seq tracks are indicated on the left in brackets. In the bottom panels, the RNA-seq strand specific tracks of the corresponding genomic locus are visualized for wt/ni+ and wt/ni− samples (plus strand in blue, minus strand in red). P* indicates coordinates mapping on the pHPG27 plasmid. (B) Radiolabeled dapD, exsB, fecD and pcrA DNA probes were mixed with 0, 9.7, 29, 97 and 290 nM of NikR tetramers, without nickel (left side of each panel) or with the addition of 150 μM NiSO4 (right side of each panel). The same elements and information are reported as in panel A.

Mentions: The peak regions were identified by comparing the ChIP-seq profiles of nickel-treated wt strain (wt/ni+) with those of the nickel-treated ΔnikR mutant (ΔnikR/ni+), setting the latter as negative control (background) of the whole experiment. Irreproducible Discovery Rate (IDR) analysis outlined the good reproducibility of the replicates (Supplementary Table S1). Consequently, an optimal set of 72 high-quality peaks was defined (Supplementary Table S3). These were mapped with respect to the list of putative H. pylori TSSs, obtained by remapping the H. pylori 26695 primary transcriptome annotation20 onto the H. pylori G27 reference genome, or by de novo 5′-end mapping primer extension analysis. 23 peaks were classified as bona-fide “promoter peaks” because they were centered between position −150/+30 with respect to a TSS. The remaining peaks were subdivided into “intragenic peaks” (41 peaks) and “intergenic peaks” (8 peaks) according to the position of their center respectively within or outside the annotated genes. Consistently, many “promoter peaks” overlap the promoters of known NikR–regulated operons. Moreover, we identified peaks on the promoters of the newly identified nickel–responsive operons: hopV, hopW, hcpC, dvnA and mccB. No peaks mapping to the promoters of hpn, hpn2, hydA and groES genes were detected (Fig. 2A, Figs S2 and S3).


Comprehensive mapping of the Helicobacter pylori NikR regulon provides new insights in bacterial nickel responses
Validation of new NikR promoters and internal peaks by DNase I footprinting.(A) Radiolabeled PureA, PhcpC, PmccB and PhopV DNA probes were mixed with 0, 9.7, 29, 97 and 290 nM of the NikR tetramer, without nickel (left side of each panel) or with the addition of 150 μM NiSO4 (right side of each panel), before DNase I cleavage. On the right of each autoradiographic film, the G27 genomic coordinates of DNase I protected regions (black boxes) are reported, with position in brackets with respect to the transcriptional start site (TSS). Low affinity binding sites, if present, are shown as grey boxes surrounded by the same information. On the left, a schematic representation of the promoter is provided, with the TSS (+1, bent arrow) and the −10 region (black box). The position of the consensus sequence is reported with violet boxes, corresponding to the two conserved hemi-operator pentamers linked by a black line (15 nt spacer). In the middle panels a scheme of the corresponding transcriptional unit is shown, together with the normalized tag densities obtained from the ChIP-seq experiments (wt/ni+ in yellow, wt/ni− in orange and ∆nikR/ni+, negative control in green), the predicted peak extension by Homer2 and the DNaseI protected regions. Representation scales of ChIP-seq tracks are indicated on the left in brackets. In the bottom panels, the RNA-seq strand specific tracks of the corresponding genomic locus are visualized for wt/ni+ and wt/ni− samples (plus strand in blue, minus strand in red). P* indicates coordinates mapping on the pHPG27 plasmid. (B) Radiolabeled dapD, exsB, fecD and pcrA DNA probes were mixed with 0, 9.7, 29, 97 and 290 nM of NikR tetramers, without nickel (left side of each panel) or with the addition of 150 μM NiSO4 (right side of each panel). The same elements and information are reported as in panel A.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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f2: Validation of new NikR promoters and internal peaks by DNase I footprinting.(A) Radiolabeled PureA, PhcpC, PmccB and PhopV DNA probes were mixed with 0, 9.7, 29, 97 and 290 nM of the NikR tetramer, without nickel (left side of each panel) or with the addition of 150 μM NiSO4 (right side of each panel), before DNase I cleavage. On the right of each autoradiographic film, the G27 genomic coordinates of DNase I protected regions (black boxes) are reported, with position in brackets with respect to the transcriptional start site (TSS). Low affinity binding sites, if present, are shown as grey boxes surrounded by the same information. On the left, a schematic representation of the promoter is provided, with the TSS (+1, bent arrow) and the −10 region (black box). The position of the consensus sequence is reported with violet boxes, corresponding to the two conserved hemi-operator pentamers linked by a black line (15 nt spacer). In the middle panels a scheme of the corresponding transcriptional unit is shown, together with the normalized tag densities obtained from the ChIP-seq experiments (wt/ni+ in yellow, wt/ni− in orange and ∆nikR/ni+, negative control in green), the predicted peak extension by Homer2 and the DNaseI protected regions. Representation scales of ChIP-seq tracks are indicated on the left in brackets. In the bottom panels, the RNA-seq strand specific tracks of the corresponding genomic locus are visualized for wt/ni+ and wt/ni− samples (plus strand in blue, minus strand in red). P* indicates coordinates mapping on the pHPG27 plasmid. (B) Radiolabeled dapD, exsB, fecD and pcrA DNA probes were mixed with 0, 9.7, 29, 97 and 290 nM of NikR tetramers, without nickel (left side of each panel) or with the addition of 150 μM NiSO4 (right side of each panel). The same elements and information are reported as in panel A.
Mentions: The peak regions were identified by comparing the ChIP-seq profiles of nickel-treated wt strain (wt/ni+) with those of the nickel-treated ΔnikR mutant (ΔnikR/ni+), setting the latter as negative control (background) of the whole experiment. Irreproducible Discovery Rate (IDR) analysis outlined the good reproducibility of the replicates (Supplementary Table S1). Consequently, an optimal set of 72 high-quality peaks was defined (Supplementary Table S3). These were mapped with respect to the list of putative H. pylori TSSs, obtained by remapping the H. pylori 26695 primary transcriptome annotation20 onto the H. pylori G27 reference genome, or by de novo 5′-end mapping primer extension analysis. 23 peaks were classified as bona-fide “promoter peaks” because they were centered between position −150/+30 with respect to a TSS. The remaining peaks were subdivided into “intragenic peaks” (41 peaks) and “intergenic peaks” (8 peaks) according to the position of their center respectively within or outside the annotated genes. Consistently, many “promoter peaks” overlap the promoters of known NikR–regulated operons. Moreover, we identified peaks on the promoters of the newly identified nickel–responsive operons: hopV, hopW, hcpC, dvnA and mccB. No peaks mapping to the promoters of hpn, hpn2, hydA and groES genes were detected (Fig. 2A, Figs S2 and S3).

View Article: PubMed Central - PubMed

ABSTRACT

Nickel homeostasis is important for pathogenic and ureolytic bacteria, which use this metal ion as enzymatic cofactor. For example, in the human pathogen Helicobacter pylori an optimal balance between nickel uptake and incorporation in metallo-enzymes is fundamental for colonization of the host. Nickel is also used as cofactor to modulate DNA binding of the NikR regulator, which controls transcription of genes involved in nickel trafficking or infection in many bacteria. Accordingly, there is much interest in a systematic characterization of NikR regulation. Herein we use H. pylori as a model to integrate RNA-seq and ChIP-seq data demonstrating that NikR not only regulates metal-ion transporters but also virulence factors, non-coding RNAs, as well as toxin-antitoxin systems in response to nickel stimulation. Altogether, results provide new insights into the pathobiology of H. pylori and contribute to understand the responses to nickel in other bacteria.

No MeSH data available.