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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

NikR consensus sequence.(A) Weblogo of the NikR consensus sequence elaborated by GLAM2 considering all validated NikR operators within gene promoters. (B) List of the DNA sequences aligned by GLAM2 to generate the consensus sequence, with the strand used for the alignment and the scores resulting from re-alignement of each sequence to the consensus. Homologous regions in G27 were used if the operator was originally characterized in a different strain. *The NikR operator on PceuE was re-mapped accordingly to the Maxam–Gilbert G + A reaction reported in ref. 14. (C) Proposed NikR consensus sequence and comparison with the published ones.
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f4: NikR consensus sequence.(A) Weblogo of the NikR consensus sequence elaborated by GLAM2 considering all validated NikR operators within gene promoters. (B) List of the DNA sequences aligned by GLAM2 to generate the consensus sequence, with the strand used for the alignment and the scores resulting from re-alignement of each sequence to the consensus. Homologous regions in G27 were used if the operator was originally characterized in a different strain. *The NikR operator on PceuE was re-mapped accordingly to the Maxam–Gilbert G + A reaction reported in ref. 14. (C) Proposed NikR consensus sequence and comparison with the published ones.

Mentions: Finally, we used GLAM221, to investigate the consensus for NikR binding (Fig. 4A). Employing the promoter sequences protected by NikR in the footprinting experiments (Fig. 4B, this work and previously reported sequences10111316), we obtained the conserved pentamers TRTTA and TAWTA, positioned 15 nt apart from each other, with a relevant but less conserved TY element in between (Fig. 4A). This consensus sequence closely matches a TRWYA dyad motif predicted by bioinformatic analyses22. The 20 nt distance between the center of the 2 pentamers is in accordance with binding of one NikR tetramer to two hemi-operator regions separated by exactly two DNA helix turns2324 (Fig. 4C). Moreover, the two half-sites of the consensus motif appear to be almost completely conserved among different H. pylori strains, hinting at a conserved operator readout mechanism (Supplementary Table S5). Interestingly, the first thymine of the second repeat is highly conserved among the NikR-bound promoters, since all the sequences used to generate the consensus have a T in that position, with the exception of PnikR, bound by NikR with lower affinity12 (see also Supplemenatry Table S6). Previous analysis of the sequence determinants for a tight DNA-protein binding has identified this position as an essential element for a low KD25. Coherently, the operators encompassing a thymine in this position, exhibit high binding affinity of NikR in our footprinting experiments (Supplementary Table S6). On the contrary, the presence of a cytosine characterizing one of the hemi-operator motifs of the 26695 ureA operator25, appears not to be a pre-requisite for high affinity binding of NikR.


Comprehensive mapping of the Helicobacter pylori NikR regulon provides new insights in bacterial nickel responses
NikR consensus sequence.(A) Weblogo of the NikR consensus sequence elaborated by GLAM2 considering all validated NikR operators within gene promoters. (B) List of the DNA sequences aligned by GLAM2 to generate the consensus sequence, with the strand used for the alignment and the scores resulting from re-alignement of each sequence to the consensus. Homologous regions in G27 were used if the operator was originally characterized in a different strain. *The NikR operator on PceuE was re-mapped accordingly to the Maxam–Gilbert G + A reaction reported in ref. 14. (C) Proposed NikR consensus sequence and comparison with the published ones.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: NikR consensus sequence.(A) Weblogo of the NikR consensus sequence elaborated by GLAM2 considering all validated NikR operators within gene promoters. (B) List of the DNA sequences aligned by GLAM2 to generate the consensus sequence, with the strand used for the alignment and the scores resulting from re-alignement of each sequence to the consensus. Homologous regions in G27 were used if the operator was originally characterized in a different strain. *The NikR operator on PceuE was re-mapped accordingly to the Maxam–Gilbert G + A reaction reported in ref. 14. (C) Proposed NikR consensus sequence and comparison with the published ones.
Mentions: Finally, we used GLAM221, to investigate the consensus for NikR binding (Fig. 4A). Employing the promoter sequences protected by NikR in the footprinting experiments (Fig. 4B, this work and previously reported sequences10111316), we obtained the conserved pentamers TRTTA and TAWTA, positioned 15 nt apart from each other, with a relevant but less conserved TY element in between (Fig. 4A). This consensus sequence closely matches a TRWYA dyad motif predicted by bioinformatic analyses22. The 20 nt distance between the center of the 2 pentamers is in accordance with binding of one NikR tetramer to two hemi-operator regions separated by exactly two DNA helix turns2324 (Fig. 4C). Moreover, the two half-sites of the consensus motif appear to be almost completely conserved among different H. pylori strains, hinting at a conserved operator readout mechanism (Supplementary Table S5). Interestingly, the first thymine of the second repeat is highly conserved among the NikR-bound promoters, since all the sequences used to generate the consensus have a T in that position, with the exception of PnikR, bound by NikR with lower affinity12 (see also Supplemenatry Table S6). Previous analysis of the sequence determinants for a tight DNA-protein binding has identified this position as an essential element for a low KD25. Coherently, the operators encompassing a thymine in this position, exhibit high binding affinity of NikR in our footprinting experiments (Supplementary Table S6). On the contrary, the presence of a cytosine characterizing one of the hemi-operator motifs of the 26695 ureA operator25, appears not to be a pre-requisite for high affinity binding of NikR.

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