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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.


Related in: MedlinePlus

Validation of the new NikR-dependent nickel-regulated ncRNAs.Top panels: transcriptional analysis of ncRNAs in wt/ni−, wt/ni+, ∆nikR/ni− and ∆nikR/ni+ conditions. (A) Northern blot of Nrr1 (left) and quantitative RT-qPCR of its transcript levels (right) (see legend 2D for details). (B) Primer extension analysis of the Nrr2 transcript. (C) Northern blot of the IsoB transcript. Middle panels: DNase I footprinting of radiolabeled Nrr1 (A), Nrr2 (B) and IsoB (C) DNA probes, mixed with 0, 9.7 (only panel B), 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). Uncropped blots and gels are provided in the Supplementary Information. Legends and symbols as in Fig. 2.
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f3: Validation of the new NikR-dependent nickel-regulated ncRNAs.Top panels: transcriptional analysis of ncRNAs in wt/ni−, wt/ni+, ∆nikR/ni− and ∆nikR/ni+ conditions. (A) Northern blot of Nrr1 (left) and quantitative RT-qPCR of its transcript levels (right) (see legend 2D for details). (B) Primer extension analysis of the Nrr2 transcript. (C) Northern blot of the IsoB transcript. Middle panels: DNase I footprinting of radiolabeled Nrr1 (A), Nrr2 (B) and IsoB (C) DNA probes, mixed with 0, 9.7 (only panel B), 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). Uncropped blots and gels are provided in the Supplementary Information. Legends and symbols as in Fig. 2.

Mentions: Interestingly, the ChIP-seq analysis mapped a NikR binding site to the promoter of a transcript corresponding to aapA6 (HPnc8050) in strain 2669520. This gene encodes a small ORF-encoding component of a putative class I toxin-antitoxin system. Analysis of the RNA-seq tracks showed a transcript downstream of this putative promoter also in strain G27: the TSS was validated by primer extension analysis, while RNA-blotting showed a single product of 170 nt (Fig. 3A), with only partial sequence similarity to the 5′ region of the aapA6 transcript. In fact, the transcript encoded by the G27 strain lacked any obvious ORF and no antisense transcript was detected on the opposite strand (Fig. 3A). These observations suggest that in strain G27 this locus encodes a non-coding RNA rather than a toxin-antitoxin system. The transcript was accordingly renamed Nrr1, for NikR-regulated sRNA1. The binding of NikR to the nrr1 promoter was confirmed in vitro by DNase I footprinting, with a strong protection occurring at the minimal concentration of protein tested only with the presence of nickel (Fig. 3A). Nrr1 expression levels were assayed by qRT-PCR, with results showing reduced transcript levels in response to nickel, and the loss of regulation in a ΔnikR genetic background, suggesting that NikR represses Nrr1 under nickel-replete conditions (Fig. 3A).


Comprehensive mapping of the Helicobacter pylori NikR regulon provides new insights in bacterial nickel responses
Validation of the new NikR-dependent nickel-regulated ncRNAs.Top panels: transcriptional analysis of ncRNAs in wt/ni−, wt/ni+, ∆nikR/ni− and ∆nikR/ni+ conditions. (A) Northern blot of Nrr1 (left) and quantitative RT-qPCR of its transcript levels (right) (see legend 2D for details). (B) Primer extension analysis of the Nrr2 transcript. (C) Northern blot of the IsoB transcript. Middle panels: DNase I footprinting of radiolabeled Nrr1 (A), Nrr2 (B) and IsoB (C) DNA probes, mixed with 0, 9.7 (only panel B), 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). Uncropped blots and gels are provided in the Supplementary Information. Legends and symbols as in Fig. 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5385501&req=5

f3: Validation of the new NikR-dependent nickel-regulated ncRNAs.Top panels: transcriptional analysis of ncRNAs in wt/ni−, wt/ni+, ∆nikR/ni− and ∆nikR/ni+ conditions. (A) Northern blot of Nrr1 (left) and quantitative RT-qPCR of its transcript levels (right) (see legend 2D for details). (B) Primer extension analysis of the Nrr2 transcript. (C) Northern blot of the IsoB transcript. Middle panels: DNase I footprinting of radiolabeled Nrr1 (A), Nrr2 (B) and IsoB (C) DNA probes, mixed with 0, 9.7 (only panel B), 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). Uncropped blots and gels are provided in the Supplementary Information. Legends and symbols as in Fig. 2.
Mentions: Interestingly, the ChIP-seq analysis mapped a NikR binding site to the promoter of a transcript corresponding to aapA6 (HPnc8050) in strain 2669520. This gene encodes a small ORF-encoding component of a putative class I toxin-antitoxin system. Analysis of the RNA-seq tracks showed a transcript downstream of this putative promoter also in strain G27: the TSS was validated by primer extension analysis, while RNA-blotting showed a single product of 170 nt (Fig. 3A), with only partial sequence similarity to the 5′ region of the aapA6 transcript. In fact, the transcript encoded by the G27 strain lacked any obvious ORF and no antisense transcript was detected on the opposite strand (Fig. 3A). These observations suggest that in strain G27 this locus encodes a non-coding RNA rather than a toxin-antitoxin system. The transcript was accordingly renamed Nrr1, for NikR-regulated sRNA1. The binding of NikR to the nrr1 promoter was confirmed in vitro by DNase I footprinting, with a strong protection occurring at the minimal concentration of protein tested only with the presence of nickel (Fig. 3A). Nrr1 expression levels were assayed by qRT-PCR, with results showing reduced transcript levels in response to nickel, and the loss of regulation in a ΔnikR genetic background, suggesting that NikR represses Nrr1 under nickel-replete conditions (Fig. 3A).

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.


Related in: MedlinePlus