Molecular insights into the fine-tuning of pH-dependent ArsR-mediated regulation of the SabA adhesin in Helicobacter pylori

Abstract Adaptation to variations in pH is crucial for the ability of Helicobacter pylori to persist in the human stomach. The acid responsive two-component system ArsRS, constitutes the global regulon that responds to acidic conditions, but molecular details of how transcription is affected by the ArsR response regulator remains poorly understood. Using a combination of DNA-binding studies, in vitro transcription assays, and H. pylori mutants, we demonstrate that phosphorylated ArsR (ArsR-P) forms an active protein complex that binds DNA with high specificity in order to affect transcription. Our data showed that DNA topology is key for DNA binding. We found that AT-rich DNA sequences direct ArsR-P to specific sites and that DNA-bending proteins are important for the effect of ArsR-P on transcription regulation. The repression of sabA transcription is mediated by ArsR-P with the support of Hup and is affected by simple sequence repeats located upstream of the sabA promoter. Here stochastic events clearly contribute to the fine-tuning of pH-dependent gene regulation. Our results reveal important molecular aspects for how ArsR-P acts to repress transcription in response to acidic conditions. Such transcriptional control likely mediates shifts in bacterial positioning in the gastric mucus layer.


Figure S1 .
Figure S1.ArsR binding to PsabA DNA occurs at two different sites.Binding of His6-ArsR to PsabA DNA analyzed by EMSA followed by DNase I footprint analysis.A total of 25 nM PsabA DNA (407 bp, the same as in Fig.2A) was mixed with protein storage buffer (lane DNA), 10-20 μM ArsR-nP (lane ArsR-nP), or 10-20 μM ArsR-P (lane ArsR-P) for 30 min at 25°C.After DNase I treatment, samples were run in TBE PAGE, and non-shifted and shifted DNA bands were cut from the gel and purified and then loaded onto a sequencing gel.Transcriptional start site (+1) and Ttract are indicated along the left side, as well as the regions described in Fig.3A.Binding sites are marked by solid lines, and the nucleotide positions of the binding sites are shown along the right side.The image shown is one representative of two independent experiments.Line representations of the protection pattern of ArsR-nP and ArsR-P on PsabA are shown in the diagram to the right.The densities of the DNase footprint bands (indicated by intensity) are plotted as a migration (from the top to bottom of the DNase footprint).Numbers representing nucleotide positions relative to the transcriptional start site (+1) are shown on top of the peaks.The ArsR BS I and BS II found by DNase I footprinting (Fig. 2C-D) are shown as solid black lines below the diagram.

Figure S2 .
Figure S2.Binding of ArsR to the two sites in PsabA DNA is affected by AT-rich DNA sequences.(A) Sequence alignment of region 2-4 of the sabA promoter in 48 different H. pylori strains with annotated sequences (see TableS3).The regions described in Fig.3Aare separated by vertical lines are separated by vertical lines and noted above the alignement, and the solid black lines mark the ArsR BS I and BS II found by DNase I footprinting.(B) Line representation of the protection pattern of ArsR-nP and ArsR-P on PsabA with scrambled region 2*, 3*, 4*, and 3*+4*.The densities of the DNase footprint bands (indicated by intensity) are plotted as a migration (from the top to bottom of the DNase footprint).Each diagram represents different PsabA DNA templates with the DNase I footprint result for DNA alone, ArsR-nP, and ArsR-P as shown in Fig. 3B-C.The positions of ArsR BS I and BS II found by DNase footprinting are shown as solid black lines below the diagrams.

Figure S3 .
Figure S3.Binding of ArsR to PsabA DNA from different strains or different T-tract lengths.DNase I footprint analysis of ArsR binding to PsabA DNA (410 bp PCR; primers 486/485-TET) from (A) G27 (template pAAG265), (B) 17875/sLex (template pAAG266), and (C) the isogenic T18 variant of SMI109 (template pAAG286).Transcriptional start site (+1), T-tract, and the regions described in Fig. 3A are shown to the left of the gel images.Maxam and Gilbert DNA sequencing reaction (lane A+G) shows the sequences of the DNA used.DNA was mixed with buffer (lane DNA), 10 μM ArsR-nP (lane ArsR-nP), or 10 μM ArsR-P (lane ArsR-P).Binding sites are marked by solid lines, and nucleotide positions are shown to the right of each gel image.The images show one representative gel of at least two independent experiments.A line representation of the protection pattern of ArsR is shown in the diagram to the right of each gel image (black, free DNA; orange, ArsR-nP; grey, ArsR-P).The densities of the DNase I footprint bands (indicated by intensity) are plotted as a migration (from the top to bottom of the DNase footprint).Nucleotide positions are written on top of the peaks, and the two ArsR binding sites are marked by solid lines.

Figure S4 .
Figure S4.Expression of control genes in SMI109 wt and in strains with scrambled regions of the sabA promoter or in SMI109 hup strains.(A) DNase I footprint analysis or Hup-His6 and/or His6-ArsR binding to PsabA DNA from SMI109.A total of 25 nM DNA (407 bp PCR; primers 486/485-TET; template pAAG264) was mixed with protein storage buffer (lane DNA), 5 μM Hup-His (lane Hup), and 10 μM ArsR-nP (lane Hup + ArsR-nP) or 10 μM ArsR-P (lane Hup + ArsR-P).Transcriptional start site (+1) and T-tract are marked on top of the gel image, as well as the regions described in Fig. 3A.The Maxam and Gilbert DNA sequencing reaction (lane A+G) shows the sequence of the DNA used.Binding sites are marked by solid lines, and the nucleotide positions of the binding sites are shown below the gel image.The image shows one representative gel of at least two independent experiments.Line representations of the protection pattern of Hup alone, Hup and ArsR-nP or Hup and ArsR-P are shown below the image.The densities of the DNase footprint bands (indicated by intensity) are plotted as a migration (from the top to bottom of the DNase footprint).Nucleotide positions are written on top of the peaks in each diagram.The ArsR binding sites I and II are shown as solid black lines.(B) Expression of sabA analyzed by RT-qPCR after growth on a plate or in broth in SMI109 wt and Δhup strains.Samples were collected after overnight growth on blood agar plates or from bacteria grown in Brucella broth to an OD600 of 0.3.Data from two biological replicates and two technical replicates for each biological replicate were used for the quantification.The number of data points for each average are written on the bars of the diagram.Statistical analysis was performed with Mann-Whitney U-tests (p < 0.01, **; p > 0.05, ns).(C-D) The effect of acid stress on ureA mRNA levels as analyzed by RT-qPCR.SMI109 wt, Δhup, and Δhup ΔarsS (C) or isogenic variants of SMI109 with scrambled PsabA DNA regions 3-4 (D).Cultures were grown in Brucella broth to an OD600 of 0.3 before the shift to pH 5 or grown at pH 7 as a control.Samples were collected 24 h after the pH shift for mRNA level analysis.Data from at least two biological replicates, each including two technical replicates, were used for the quantifications.The number of samples included are shown as separate dots in the diagram.Statistical analysis was performed using Mann-Whitney U-tests (p < 0.0001, ****; p < 0.01, **; p > 0.05, ns).

Figure S5 .
Figure S5.Binding of RNAP to PsabA DNA of different lengths and together with Hup-His6 and/or His6-ArsR.(A) Binding of RNAP to PsabA DNA of different lengths was analyzed by DNase I footprinting.A total of 25 nM TET-labelled PsabA DNA was mixed with protein storage buffer (lane DNA) or 300 nM E. coli σ 70 -RNAP (lane RNAP).PCR-generated DNA was used at two different lengths: long (407 bp, 486/485-TET primers, blue) or short (298 bp, SabA-5/485-TET primers, green).Maxam and Gilbert DNA sequencing reaction (lane A+G) shows the sequences of the DNA used.A line representation of the protection pattern of RNAP on PsabA is shown in the diagram; DNA (black), RNAP + long DNA (blue), and RNAP + short DNA (green).The densities of the DNase footprint bands (indicated by intensity) are plotted as a migration (from the top to bottom of the DNase footprint).Nucleotide positions are written on top of the peaks in the diagram.The RNAP CTD and  70 -subunit binding sites are shown as solid lines, and nucleotide positions are written on the gel.(B) Binding of RNAP together with Hup-His6 and/or His6-ArsR to PsabA DNA from SMI109 was analyzed by DNase I footprinting.A total of 25 nM DNA

Figure S6 .
Figure S6.Binding of RNAP to PsabA DNA from different strains with different T-tract lengths and scrambled region 3* and 4*.Line representations of the protection pattern of ArsR-P or RNAP on PsabA from the SMI109 T13 (wt) and SMI109 T18 variant, G27, and 17875/sLex or region 4* and region 3*.The densities of the DNase footprint bands (indicated by intensity) are plotted as a migration (from the top to bottom of the DNase footprint).Each diagram represents different PsabA DNA templates with the DNase I footprint result for DNA alone, ArsR-P, or RNAP as shown in Fig. 8A-C.The sequences of the DNAs that were used are shown in Fig. 3A.Nucleotide positions are written on top of the peaks in each diagram.The ArsR binding sites I and II are shown as solid black lines, and the binding sites of RNAP are indicated by red lines below each diagram.

Table S1 .
Strains and plasmids used in this study

Table S2 .
Oligonucleotides used in this study

Table S3 .
Strains used for sequence alignment of region 2-4 of PsabA shown in Fig. S2A.