A Repetitive DNA Element Regulates Expression of the Helicobacter pylori Sialic Acid Binding Adhesin by a Rheostat-like Mechanism

During persistent infection, optimal expression of bacterial factors is required to match the ever-changing host environment. The gastric pathogen Helicobacter pylori has a large set of simple sequence repeats (SSR), which constitute contingency loci. Through a slipped strand mispairing mechanism, the SSRs generate heterogeneous populations that facilitate adaptation. Here, we present a model that explains, in molecular terms, how an intergenically located T-tract, via slipped strand mispairing, operates with a rheostat-like function, to fine-tune activity of the promoter that drives expression of the sialic acid binding adhesin, SabA. Using T-tract variants, in an isogenic strain background, we show that the length of the T-tract generates multiphasic output from the sabA promoter. Consequently, this alters the H. pylori binding to sialyl-Lewis x receptors on gastric mucosa. Fragment length analysis of post-infection isolated clones shows that the T-tract length is a highly variable feature in H. pylori. This mirrors the host-pathogen interplay, where the bacterium generates a set of clones from which the best-fit phenotypes are selected in the host. In silico and functional in vitro analyzes revealed that the length of the T-tract affects the local DNA structure and thereby binding of the RNA polymerase, through shifting of the axial alignment between the core promoter and UP-like elements. We identified additional genes in H. pylori, with T- or A-tracts positioned similar to that of sabA, and show that variations in the tract length likewise acted as rheostats to modulate cognate promoter output. Thus, we propose that this generally applicable mechanism, mediated by promoter-proximal SSRs, provides an alternative mechanism for transcriptional regulation in bacteria, such as H. pylori, which possesses a limited repertoire of classical trans-acting regulatory factors.


Introduction
A key factor for bacterial pathogens to establish and maintain a persistent infection is the adaptation to host responses and to microenvironmental alterations that occur during pathogenesis.Both stochastic and regulated processes can affect gene expression, and contribute to population heterogeneity.From the plethora of clones, best-fit phenotypes arise to match the current environmental demands.Population heterogeneity can be achieved by epigenetic events, such as DNA methylations; or strictly genetic mechanisms, such as reversible phase variation e.g.homologous recombination or slipped strand mispairing (SSM) of simple sequence repeats (SSRs) [1,2].SSRs create so-called contingency loci, i.e. hypermutable DNA that mediates stochastic genotypic switching, and these regions are often evolutionary conserved [3,4].The role of SSM in regulation of mRNA levels and protein expression is determined by the genetic location of the SSR.Intragenic SSRs cause biphasic translational control and turn protein expression on or off, while intergenic SSRs, may result in altered mRNA levels by different mechanisms [5,6].
Helicobacter pylori infects the human stomach and if left untreated causes chronic gastritis that potentially leads to peptic ulcer disease and gastric cancer [7][8][9].Adhesion is a prerequisite to establish persistent infection.The two dominating carbohydrates targeted by H. pylori in the gastric mucosa are the ABO/Leb blood group and the sialyl Lewis x/a (sLex/sLea) antigens [10][11][12][13][14].In healthy mucosa the ABO/Leb antigens predominate, whereas the sLex/ sLea antigens dominate the inflamed mucosa.H. pylori binds the ABO/Leb-receptors via the blood group antigen binding adhesin BabA, and the sLex/sLea-receptors via the sialic acid binding adhesin SabA.Since the human stomach glycosylation pattern constantly changes, H. pylori needs to adapt its adherence properties accordingly.Expression can efficiently be switched on or off via homologous recombination, or via SSM events [13,[15][16][17][18].The protein expression of the BabA and SabA adhesins also varies between strains [15,16,19,20].
Detailed studies of adhesin expression regulation in H. pylori are scarce.In other eubacteria, RNA polymerase sigma (s) factors and transcriptional regulators control gene expression at the mRNA level.These likely play a diminished role in H. pylori, as only three s-factors (s 80 , s 54 and s 28 ) and few classical trans-acting regulators are present [21][22][23].Thus, fine-tuning of mRNA levels in H. pylori likely involve alternative processes.H. pylori, like other bacteria with small genomes, has a high content of SSRs, primarily in genes encoding outer membrane proteins e.g.alpA, alpB, babA, babB, sabA and sabB [24][25][26].In H. pylori, the impact of SSRs is probably further accentuated by the lack of mismatch repair systems and proof reading deficiency of the DNA polymerase I [27,28].In this context, SSM can rapidly create a large pool of heterogeneous clones and not surprisingly, H. pylori has an extremely high intraspecies genetic variability [29][30][31][32].
A cytosine-thymine dinucleotide (CT) repeat tract in the 59-end of the sabA coding sequence (CDS) causes translational frameshifts and on/off phase variation [13,15].Additionally, a thymine (T) nucleotide repeat tract is found adjacent to the sabA 235 promoter element.The length of this T-tract varies between strains and such length variations have been suggested to influence sabA expression [33,34]; however, the functional mechanism of how the T-tract regulates transcription remains to be elucidated.In this paper, we present data illustrating that the T-tract length, in clones isolated post-infection from different local gastric environments, is variable in vivo.We also demonstrate that the T-tract length controls sabA transcription initiation, and thus SabA expression and functional sLex-receptor binding to gastric mucosa, in a multiphasic manner by affecting binding of the RNA polymerase.We describe in molecular terms how the T-tract length influences the local DNA structure, by changing the axial alignment between the core promoter and UP-like elements, thereby affecting interaction of the RNA polymerase a-subunits to the sabA promoter.In addition, we provide evidence that a similar mechanism controls multiple loci in H. pylori.Therefore, we propose a generally applicable model in which T-or A-tracts located adjacent to 235 promoter elements act by a rheostat-like mechanism, to control transcription initiation in H. pylori.

Results and Discussion
The T-tract fine-tunes sabA expression and consequently binding to the sialyl Lewis x receptor It was previously shown that expression of SabA varies among different clinical isolates and that expression levels match the binding activity to the cognate sialyl Lewis x (sLex) receptor [15,19,20,35].In this study, we set out to scrutinize determinants that cause these differences.A set of five H. pylori strains, representing numerous geographical origins and isolated from patients with different disease symptoms (described in Table 1), were chosen for the analysis.SMI109 (Sweden, GC), J99 (USA, DU), G27 (Italy, GA), 17875/sLex (Australia, GA) and 26695 (UK, GA) were assayed for SabA protein expression by immunedetection, and for receptor binding activity by RadioImmunoAssay (RIA) using 125 I-sLex-receptor conjugates.As expected strain 26695, with a predicted frameshift in the sabA CT-tract, did not express any SabA protein nor could it bind to sLex-receptor conjugates (Fig. 1A and 1C).Strains SMI109 and 17875/sLex displayed highest SabA expression and accordingly cognate sLexreceptor binding activity, whereas strains J99 and G27 displayed intermediate levels of both (Fig. 1A).These results confirmed the significant variation of SabA expression between strains and the link between protein expression and receptor binding activity.
To establish if mRNA levels were related to the SabA protein expression, we analyzed sabA mRNA levels with RT-qPCR in the corresponding H. pylori strains.A clear correlation was observed (Fig. 1B).We also generated transcriptional lacZ reporter fusions of the sabA promoter (PsabA) from the different strains (Fig. S1A) and found transcriptional initiation to vary when measuring promoter activity by b-galactosidase assay in E. coli (Fig. S1B).However, the promoter activities did not correlate with the mRNA levels or SabA protein expression found in the different H. pylori strains.For example, activity of the PsabA from strain 26695 was comparatively high, considering that this strain did not express any detectable cognate sabA mRNA (compare Fig. 1B and S1B).This is likely explained by the correlation between transcriptional and translational processes in H. pylori recently shown [36] and illustrates that downstream effectors, like mRNA stability or H. pylori specific factors, are essential for absolute mRNA levels.Further, this emphasizes the importance of studying expression in an isogenic strain background.
Sequencing of the PsabA region from the different strains revealed scarce nucleotide variations scattered across the promoter.Some exchanges in the 210 and 235 promoter elements were observed, as well as length variations in the T-tract located adjacent to the 235 element (Fig. 1C).sabA from strain G27 shows a nearly perfect extended 210 promoter element (TGnTAAAAT vs TGnTATAAT in E. coli), which explains the high promoter activity observed with lacZ fusions in E. coli for this promoter (Fig. S1B).Analysis of a larger set of PsabA sequences revealed unusual high homology, except for a major discrepancy in the length of the T-tract (Fig. S2).If the T-tract could play a role in regulating sabA expression, we reasoned that the T-tract length might vary extensively between H. pylori strains to match the present sLex-receptor availability in each infected individual.Therefore, we compared the T-tract length of forty-nine published H. pylori genome sequences and sequenced the PsabA of twelve additional strains.In total, we found fifty-one strains to encode a sabA gene.As assumed, there was a wide distribution of T-tract lengths, ranging from T 5 to T 22 , where T 13 to T 17 being the most common variants (Fig. 1D and Table S1).In a collection of 115 clinical

Author Summary
During persistent H. pylori infection, the local gastric milieu is constantly altered by host responses and inflammation fluxes.As adhesion is crucial to maintain infection, appropriate adaptation of bacterial adherence properties is required to meet these environmental fluctuations.H. pylori uses the SabA protein to bind glycan receptors present on inflamed stomach mucosa.SabA expression can be turned on or off via known genetic mechanisms; however, how fine-tuning of SabA expression occurs to match changes in receptor levels is still unknown.The H. pylori genome encodes few trans-acting regulators but has numerous simple sequence repeats (SSR), i.e. hypermutable DNA segments.Here, we have deciphered a mechanism where a T-repeat tract, located in the sabA promoter region, affects SabA expression.The mechanism involves structural alterations of the promoter DNA that affects interaction of the RNA polymerase, without input from known trans-acting regulators.This mechanism is likely not unique for SabA or to H. pylori, but also applicable to other pathogens with high abundance of SSRs and limited set of transcription factors.Our findings contribute to understanding of the important bacterial-host interplay, and to mechanisms that generate heterogeneous populations of best-fit clones, i.e. stochastic switching.
Taiwanese isolates, Kao et al [34] showed that the T-tract length varied from T 10 to T 28 , with the most common variants being T 14 to T 19 .In further support of the idea for individual selection, we could not find any obvious geographic correlations of T-tract lengths, as exemplified by the extensive T-tract variation (T 5 to T 16 ) in the different Peruvian strains analyzed (Cuz20, PeCan4, PeCan18, Puno120, Puno135, Sat464, Shi112, Shi169, Shi417, Shi470, SJM180; Table S1).
Since a number of factors affect transcription, at different regulatory levels, we generated transcriptional PsabA::lacZ reporter fusions to determine if the T-tract length could impact transcription initiation in a given strain.The PsabA::lacZ fusions were based on the PsabA of the SabA high-expressing strain SMI109, but with varying T-tract lengths comparable to the T-length distribution described in the preceding section (Fig. 1D, T 1 to T 21 ).The bgalactosidase assays were performed in E. coli.First, we used 59   pKD4 Used as template for kanamycin resistance cassette [97] rapid amplification of cDNA ends (59-RACE) and determined the transcriptional start site of sabA in SMI109 to be located at a cytosine, 66 nt upstream of ATG, the same transcriptional start site as previously published for J99 [34].We also verified that an identical transcriptional start site was used in E. coli as in H. pylori by primer extension analysis (data not shown).The b-galactosidase assays revealed that the promoter activity of the PsabA::lacZ fusions with varying T-tract length was gradually multiphasic: high in T 5 , low in T 9 , intermediate in the T 13 (wt) and high in T 18 (Fig. S1C).These results suggested that the T-tract length affects promoter activity.To further analyze this in H. pylori, we decided to explore the role of the T-tract in otherwise isogenic variants of strain SMI109, with PsabA T-tracts spanning from T 1 to T 21 , the same set of T-variants that were analyzed in E. coli.Such variants were Figure 1.Interstrain variation of sabA mRNA levels, SabA protein expression and functional sLex-receptor binding.A) Analysis of SabA expression and sLex-receptor binding activity in a set of five H. pylori strains.Image shows one representative immunoblot analysis with a-SabA antibodies and the numbers above represents SabA expression quantification, with expression in strain SMI109 set to 1. Equal amounts of crude protein extracts were loaded in each lane (Fig. S10A).The graph shows binding to soluble 125 I-sLex-receptor conjugate of the same strains as analyzed in the immunoblot.Bacteria were grown on plate as described in Materials and Methods prior to the analysis.Average and standard deviations are calculated from at least two independent experiments and duplicate samples/analysis of each strain.B) RT-qPCR analysis of sabA mRNA levels in the same set of strains as in Fig. 1A.The sabA mRNA levels were normalized to a set of reference genes and data is presented as relative, with the levels in strain SMI109 set to 1. Images show one representative semi-quantitative PCR analysis, using the same primers as in the RT-qPCR analysis; sabA-2 and ppk- obtained by exchanging the PsabA region in SMI109 using a method involving contraselection in combination with in vitro mutagenesis (see Materials and Methods).First, SabA protein expression and sLex-receptor binding activity were analyzed in these variants.This revealed an even more pronounced multiphasic appearance than in E. coli, although in H. pylori the T 13 variant exhibited high and the T 18 variant intermediate protein expression and sLex-receptor binding activity (Fig. 2A-B).We also determined the sabA mRNA levels in the T-tract variants T 5 to T 18 with RT-qPCR (Fig. 2C).The mRNA level was likewise gradually multiphasic and closely correlated to the protein expression and receptor activity (Fig. 2).Interestingly, the max/min protein and mRNA levels were observed with T-tract length intervals of approximately ten base pairs, the same distance as one turn of the DNA helix.
To mimic H. pylori adhesion during in vivo conditions and the presentation of the sLex-receptor on epithelial cells, we analyzed SabA-mediated adhesion to human gastric tissue sections.Gastric tissue sections were probed with fluorescently labeled H. pylori of varying T-tract lengths, which displayed different sLex binding activity.The SabA high-expressing T 13 variant clearly exhibited more binding to the tissue sections than the low-expressing T 9 and intermediate-expressing T 18 variants (Fig. 2D).In contrast, neuraminidase-treated mucosa, where the sialic acid moieties had been removed, showed only background binding (data not shown).Likewise, a DsabA mutant derivative of SMI109 exhibited no binding to the tissue sections (Fig. 2D).Thus, our results demonstrate that variations in the T-tract length, in otherwise isogenic strains, affect PsabA activity.This induces multiphasic alterations of sabA mRNA levels and thereby SabA protein Image shows one representative immunoblot with a-SabA antibodies used for the quantification.Expression levels were normalized to expression of the AlpB protein before comparison (Fig. S10B).Data are presented in the bar diagram, as described in Fig. 1A, with the expression in the T 13 (wt) variant set to 1. Stars indicate significant differences from T 13 -variant, * p,0.05, ** p,0.01, ns = non significant.B) Binding to soluble 125 I-sLex-receptor conjugates of the same set of T-variants as in Fig. 2A.The data are presented as in Fig. 1A, with the binding of the T 13 -variant set to 1. Stars mark significant differences from T 13 -variant, see Fig. 2A.C) RT-qPCR analysis of sabA mRNA levels in T-variants of SMI109.Data are presented as in Fig. 1B, with the mRNA levels in the T 13 -variant (wt) set to 1.The upper images show result from one semi-quantitative PCR analysis using primers for sabA-1 and rrnA-2.D) Binding of FITC-labeled SMI109 T-variants (T 9 , T 13 and T 18 ) to human gastric tissue sections.SMI109 DsabA mutant was included as a negative control.Images were taken with 1006magnification.For all analyses in Fig. 2, bacteria were grown on plate prior to the experiment, as described in Materials and Methods.doi:10.1371/journal.ppat.1004234.g002expression levels which adjust the sLex-receptor binding activity and binding to human gastric mucosa.

Variations in T-tract length during infection
At the H. pylori infection site, a local inflammation develops as a result of the host cell responses and release of effector molecules.Earlier experimental H. pylori infections in Rhesus monkeys showed variable reciprocal changes of both fucosylated ABO/ Leb and sialylated sLex/a receptor expression during infection [14,37,38].These alterations need to be accompanied by changes of BabA and SabA adhesin expression to maintain infection.Consequently, there is a delicate balance to cycle between nonadherent and adherent states, and to adjust expression levels at the adherent state.We have previously shown that Mongolian gerbils infected with H. pylori strains, expressing different levels of BabA adhesin, display alterations in the infection load and host cell responses, as well as phenotype modifications of the infected strain [39].This scenario likely mirrors the host-pathogen interplay and post-experimental output clones are thus the result of a hostbiopanning procedure that selects for clones with best-fit phenotype, i.e. stochastic switching.
Since our results suggest that the T-tract length ultimately controls sLex-receptor binding activity, selection for certain Ttract variants likely occurs as SabA expression is optimized to the receptor availability in a respective host.As outlined in the preceding section, we propose that the T-tract length is variable over time and under different selection conditions.To study Ttract length variations, and the corresponding functional alteration in sLex-receptor binding over time, we subjected strain SMI109 to a series of in vitro passages for three months.sLex-receptor binding post-passages revealed sub-populations that displayed lower sLexreceptor binding activity (sLex-low) relative to the other subpopulations (sLex-high, data not shown).Single clones were isolated from both sub-populations, and the cognate PsabA regions were sequenced.This analysis revealed that the sLex-high clones had a T 13 tract (wt), while the sLex-low clones had T 12 tract in the PsabA region (Fig. S3A).A similar experiment was also performed with strain J99 and comparable results were observed.Here the sLex-high clones had a T 18 tract (wt), while the sLex-low clones had T 19 tract in the PsabA region (Fig. S3B).
Next, we used a mouse-adapted sLex-negative clone of strain J99 StrR (sabA T 17 and CT 8 -off) to infect five Lewis b (Leb) transgenic FVB/N mice, since we did not succeed to infect mice with strain SMI109 [40].After two months, the mice were terminated and the output bacteria (pools) were scored for recovery of sLex-receptor binding activity.Bacterial pools from three out of five mice had changed their adherence phenotype and were now positive for sLex-receptor binding (Fig. 3A, bar diagram).Sequencing of output pools often generates mixed peaks, likely due to population heterogeneity and loss of signal after repetitive DNA tracts.Therefore, to estimate changes in Tand CT-tract lengths, we used fragment length analysis (FLA) to discriminate length variations of PCR-amplicons, down to one nucleotide.Since FLA of repetitive DNA gives rise to additional ''stutter'' peaks [41], we obtained reference spectra of genomic DNA isolated from the otherwise isogenic T 18 or T 19 variants of SMI109, and correlated our results to these (Fig. S4).To confirm that changes in the CT-tract length caused the alterations in on/ off sLex-receptor binding appearance of the output pools, we ran FLA with primers including part of the sabA coding sequence (CDS).This was exemplified by using genomic DNA isolated from one sLex-negative output pool (mouse 2) and one sLex-positive output pool (mouse 4), and also the sLex-negative input strain as reference.As expected, our analysis revealed two nucleotides shorter PCR-amplicons from output pools of mouse 4, corresponding to a change in the CT-tract length from eight to seven repeats, thereby placing translation in the right open reading frame.The PCR-amplicons of output pools of mouse 2 displayed the same length as the input strain (data not shown).
In order to only analyze changes in the T-tract length, we ran FLA with primers excluding the CT-tract.The results showed that bacterial pools from mouse 3 and 5 had a left-shifted curve pattern indicative of a larger subpopulation with one nucleotide shorter Ttract, which contrasts the unchanged curve pattern in pools from mouse 1, 2 and 4 (Fig. 3A, bottom curves).However, to determine if the FLA was limited in detecting smaller population changes in T-and CT-tract length, we next analyzed ten random clones from two distinctly different output pools not shifted in the FLA and with the lowest and highest CFU counts: the sLex-negative pool from mouse 2 and the sLex-positive pool from mouse 4 (Fig. 3A).First we determined the frequency of sLex-receptor binding in each population and as expected, all clones from mouse 4 showed binding (10/10 clones) while only 3/10 clones from mouse 2 showed sLex-receptor binding (Fig. 3B).The 59 region of the sabA locus, in four representative clones from each bacterial pool, was sequenced and revealed that all sLex-negative clones were CT 8off, and all sLex-positive clones were CT 7 -On (Fig. 3B).Furthermore, among the sLex-positive clones, different binding activities were observed.The clones with highest sLex-receptor binding activity (4-5 and 4-10) had T 16 -tract in their PsabA region, in contrast to the other clones, which had T 17 (Fig. 3B).FLA-spectra obtained for these clones corroborated the sequencing results (data not shown).Hence we concluded that although FLA gives a view of general population shifts in nucleotide repeat tract length, it is limited in detecting more discrete shifts as was seen in output pools from mouse 2 and 4. Overall, our results confirm the in vitro data obtained from SMI109 and J99, illustrating that small changes in T-tract length have major impact on sLex-receptor binding activity (Fig. 2B and Fig. S3).
To further investigate T-tract length alterations in vivo, we analyzed H. pylori output pools isolated from both antrum and corpus regions of the stomach, from three Swedish patients (here called: 101, 1026 and 1106).These bacterial pools were first analyzed for sLex-receptor binding activity.Bacterial pools from patient 101 showed high sLex-receptor binding, whereas lower binding was observed for the antrum-pools from patients 1026 and 1106.The sLex-receptor binding of the corpus-pools from patients 1026 and 1106 was barely detectable (Fig. 3C, bar diagram).We also isolated genomic DNA to obtain FLA-spectra of the 59 region of the sabA locus from each pool (Fig. 3C, right curves).This revealed distinct curve patterns, in the bacterial pools from each stomach region and from each patient, respectively.FLA analysis of PsabA region alone, using primers excluding the CT-tract, showed similar results (data not shown).To confirm that the observed FLA alterations were a consequence of T-tract length variations, genomic DNA was isolated from two clones of each output pool, and the 59-end of sabA locus was sequenced.The result showed similar T-tract length variations as detected by the FLA (Fig. 3C).Although it was not possible to make quantitative measurements using FLA technique (Fig. S4), it is a powerful tool to estimate variations in repeat tract lengths in heterogeneous populations.
Our data support the idea of individual selection since analysis of output clones, from infected mice as well as from distinct regions of the human stomach, revealed populations of heterogeneous sLex-receptor binding phenotypes and different T-tract lengths, which changed along the course of infection (Fig. 3).Interestingly, in one stomach we found a sLex-negative population (CT-off) that displayed a higher degree of T-tract length heterogeneity (Fig. 3C,  sample 1026C).This suggests that without SabA-mediated adhesion, and corresponding host cell responses, there is no selection pressure directed against clones with certain SabAexpressing phenotypes and thus, all T-tract variants generated by SSM are preserved.It has been suggested that SSM frequencies could be affected by environmental stresses [6], however, how these signals are transduced to modulate switching rates are still unclear.
The T-tract modifies RNA polymerase binding efficiency to the sabA promoter SSRs located in intergenic regions have been reported to affect transcription by different mechanisms.SSRs positioned between the 210 and 235 promoter elements affect the docking of the RNAP sigma factor [42][43][44][45].SSRs positioned upstream of the 2 35 element have been reported to affect binding of trans-acting factors and interaction with the RNAP [46][47][48].SSRs located downstream of transcriptional start sites affect mRNA stability or binding of regulatory proteins [49,50].A recent study of a SSR in H. pylori shows that expression of the chemotaxis receptor tlpB is affected by a variable G-tract located downstream of the 210 element, via small RNA-mediated posttranscriptional regulation [51].Depending of the length of the G-tract and interaction with the sRNA, expression of TlpB is either increased or decreased.Having ascertained that the length of our described T-tract affects sabA mRNA levels, we hypothesized that changes in RNA polymerase (RNAP) interaction with the PsabA DNA could underlie the observed variations in mRNA levels, since the T-tract is positioned adjacent to the 235 element.
The core promoter of PsabA (SMI109, TGGAAT-16 bp-TAAAAT) in strain SMI109 is similar to that of the E. coli housekeeping s 70 consensus binding site (TTGACA-17+/21 bp-TATAAT), and highly homologous between different H. pylori strains (Fig. 1C and Fig. S2).No functional RNAP holoenzyme has yet been purified from H. pylori, however, the E. coli s 70 -RNAP can bind and transcribe H. pylori promoters [52,53].Therefore, we tested binding of the E. coli s 70 -RNAP to PsabA DNA fragments, using electrophoretic mobility shift assay (EMSA), and found it to interact strongly (Fig. S5, picture).No interaction was observed to sabA CDS DNA or when only the core RNAP was used (data not shown).When we analyzed s 70 -RNAP binding to PsabA with varying T-tract lengths by EMSA, we could not detect differences in the amount of shifted DNA as the T-tract length was varied (Fig. S5, bar diagram).We instead decided to use high-resolution surface plasmon resonance (SPR) to obtain sensorgrams of s 70 -RNAP binding to immobilized PsabA fragments with various Ttract lengths (T 5 , T 9 , T 13 , and T 18 ).Now, we could clearly distinguish variations in binding strength to the PsabA fragments (Fig. 4A).As a control, the EMSA-inactive DNA fragment of sabA CDS showed no specific binding in the SPR analysis and was subtracted from each of the sensorgrams in Fig. 4. The results showed that s 70 -RNAP displayed weakest binding to T 9 , but stronger binding to both T 5 and T 18 , as compared to T 13 (wt).The relative binding was comparable to the promoter activity of the various PsabA fragments, as measured by b-galactosidase assays using transcriptional fusions in E. coli (Fig. 4A inlay and Fig. S1C).
In order to investigate if the T-tract acts as a spacer, i.e. changing the distance and position of a binding site, we started by replacing the PsabA nucleotide content of the T-tract, without changing the length.The wt T 13 -tract was exchanged to A 13 or C 13 in the corresponding PsabA::lacZ fusion plasmids.Measurements of the promoter activities in E. coli showed that the PsabA activity, in both A 13 and C 13 , increased relative the T 13 variant (Fig. 4B, inlay).SPR analysis revealed higher binding of s 70 -RNAP to the A 13 then to the T 13 variant, comparable to the PsabA activity (Fig. 4B).Conversely, for the C 13 variant, the binding of s 70 -RNAP was similar to that of the T 13 variant.We also created isogenic A-and C-tract variants in strain SMI109 and found that replacement of T's to A's indeed gave higher SabA expression and sLex-receptor binding, whereas substitution of T's to C's gave slightly lower SabA expression, matching the SPR results (Fig. 4B-C).These results excluded that the T-tract merely acts as a spacer, as there were still variations in SabA expression levels, though the tract length was kept constant.
Thus, our results suggest that the T-tract modulates sabA transcription by changing the efficiency of RNAP binding.Nevertheless, sabA expression in H. pylori and in vitro RNAP binding did not exactly match.This could possibly be explained by alternative display of the RNAP binding site, caused by different organization of genomic DNA in vivo versus the shorter DNA fragments used in the in vitro SPR-analyses, or conceivably by additional unknown factors that impact sabA transcription in H. pylori.Another contributing factor could be the structural differences in the RNAP subunits between E. coli and H. pylori.The band b9-subunits have 45% identity to E. coli counterparts (RpoB and RpoC) but are expressed as a fused polypeptide in H. pylori [54].This has been implied to facilitate the assembly of the holoenzyme [55] and to give a selective advantage for H. pylori fitness in the acidic human stomach [56].The housekeeping sigma factor (s 80 ) from H. pylori has 32% identity to E. coli s 70 .The most divergent region is the N-terminal part of the protein (region 1.1) involved in formation of transcription initiating complex and the spacer region [52,53].In spite of these differences, the E. coli RNAP can bind and transcribe H. pylori promoters both in vivo and in vitro, but not the other way around [52,53].The T-tract modulates the RNAP a-subunits interaction to PsabA Besides docking of the s-factor to the core promoter (235/210 elements), RNAP binding can also include interaction of the Cterminal domain of the a subunits (aCTDs) to UP-elements located upstream of the core promoter [57][58][59].To elucidate if the T-tract affects RNAP binding by influencing s-factor or aCTDs binding, we obtained DNase I footprints of s 70 -RNAP and PsabA DNA fragments with different repeat tract length and composition (T 9 , T 13 , T 18 , A 13 and C 13 ).The results showed strong binding to the core promoter (235 to +20) with all variants, and no direct interaction to the T-, A-or C-repeat tracts (Fig. 5B, data not shown).Previous studies show that UP-elements positioned close to the 235 element have a larger impact on aCTDs binding than UP-elements located further upstream.UP-elements positioned upstream of 260 have not been shown to influence transcription of promoters in E. coli [59].Interestingly, we could observe a clear DNase I protected region, positioned at 295 to 250, upstream of the repetitive tract (Fig. 5B-C, blue line) in some of the variants: T 13 , A 13 and C 13 .
As the RNAP aCTDs often interact with AT-rich DNA sequences, we scrutinized the PsabA nucleotide content and it showed more than 74% A/T-residues.This can be compared to 61% A/T-content in the whole genome of H. pylori strains.Dissecting the PsabA region revealed two regions, upstream of the T-tract, with three to four short repetitive A-boxes in each region (Fig. 5A and S2, red boxes).This suggested that the RNAP aCTDs might interact to more than one site.Corroborating this, we could clearly observe an additional protected region in the A 13 variant, located further upstream of the proximal region, positioned approximately at position 2130 to 2105 (Fig. 5C, green line).The footprint analysis revealed that several of these A-boxes were located within the DNase I protected regions observed in Fig. 5B-C.Two of A-boxes were perfectly phased, i.e. spaced by 10 bp, similar to an E. coli UP-element (NNAAAWWTWTTTTN-NAAANNN).However, the regions containing the A-boxes are located further away from the core promoter and did not display complete sequence consensus to an UP-element.Therefore, we considered the regions containing the A-boxes in PsabA to be UPlike elements (Fig, 5A and S2, blue and green lines).
Based on these findings, we generated PsabA DNA fragments lacking the proximal UP-like element (D 46 between 297 to 249), effectively positioning the distal UP-like element closer to the Ttract and the core promoter (Fig. 5A, green line).We speculated that the tract length/composition would optimize the RNAP aCTDs binding to the distal UP-like element differently in the D 46 fragments, relative to the full-length fragments.As hypothesized, DNase I footprint analysis of the PsabA D 46 fragments showed a stronger and more distinct protected region upstream of the repetitive tract as compared to the wt fragments, respectively (Fig. S6A).This was the case in all variants except for the D 46 T 9 fragment.This further demonstrates the RNAP aCTDs capability to also interact with the more distal region.We also assayed the s 70 -RNAP binding in an additional SPR-system where the same series of PsabA D 46 fragments as above, were analyzed simultaneously as their respective wt fragments.We found that the s 70 -RNAP bound stronger to the PsabA D 46 T 9 DNA when the proximal UP-like element was removed, although we could not detect any interaction with the UP-like element (compare Fig. 5D  and S6A).We also observed that RNAP bound slightly stronger to PsabA D 46 T 18 , corroborating the footprint results (Fig. 5D and  S6A).Furthermore, the RNAP bound weaker to PsabA D 46 T 13 and PsabA D 46 A 13 (Fig. 5D), respectively, although we still could observe an interaction between RNAP to distal UP-like element.
Next we analyzed promoter activity of these fragments lacking the proximal UP-like element as transcriptional lacZ fusions in E. coli and found that the expression patterns to large extent match the SPR results (Fig. 5E).We observed increased promoter activity from PsabA D 46 T 9 and PsabA D 46 T 18 variants, following the increased binding of RNAP, and we also found a concomitant decrease of PsabA D 46 A 13 promoter activity.If the binding of the RNAP aCTDs were restricted to the proximal UP-like element, just upstream of the core promoter, the sensorgrams and promoter activities of all deletion fragments should have converged to identical ones, due to the interaction of s 70 -subunit alone.Instead, the D 46 fragments, with varying tract length/composition, still displayed different RNAP binding efficiencies and promoter activities, suggesting that the RNAP can bind to additional regions upstream of T-tract, corroborating the footprint results.
To confirm interaction of RNAP with the UP-like elements we scrambled the A-boxes by exchanging every second A in the A-box with a C or a G (see Materials and Methods for details).Promoter activity of PsabA DNA with scrambled UP-like elements (A-boxes) was analyzed as transcriptional lacZ fusions in E. coli.First, the four A-boxes in proximal UP-like element (Fig. 5A, blue line) or the three A-boxes in the distal UP-like element (Fig. 5A, green line) were scrambled separately.The results showed a clear effect on promoter activity, as expression from PsabA was increased in both variants by 2-fold (Fig. 5F).Concurrently, when both UP-like elements were changed an even more pronounced up-regulation (3-fold) of PsabA activity was observed.Regulation of transcriptional output from a promoter is a complex multi-step process, involving binding of RNAP, separation of DNA strands, initiation of transcription and promoter escape.Ellinger et al showed that depending on a promoter's rate-limiting step in the pathway to productive transcription, A-tracts/UP-elements can either activate or inhibit promoter activity [60].To determine at what step PsabA activity is limiting, extensive in vitro transcriptional studies are required.
Taken together, our results show that the two UP-like elements are important for PsabA activity and that the RNAP interacts directly with both regions upstream of the core promoter.The Ttract length modulates this interaction, by changing the affinity of the RNAP to the promoter, and thereby affecting PsabA activity.During transcription initiation the DNA is wrapped .300uaround the RNAP [61] and although most of the effect of UP-like elements occurs via interaction with the aCTDs, we cannot exclude the possibility that some of the upstream sequences is also interacting with other surfaces on the RNAP.

The T-tract length influences local DNA structure
Short phased A-tracts act as major determinants of DNA curvature by forming intrinsically bent DNA and depending on the tract periodicity this alters the DNA structure to different conformations [62][63][64].Our results suggest that the A-boxes in the UP-like elements upstream of the T-tract is important for PsabA activity, therefore we hypothesized that the effect on RNAP binding can be a result of changes in local DNA structure.We made in silico structure predictions of the PsabA DNA (2166 to + 74) of different T-variants (T 9 , T 13 , T 18 and A 13 ), which supported this hypothesis by showing structural changes of the DNA in several orientations (Fig. 6A).This was further supported by PAGE separation of PsabA DNA fragments with varying tract length/composition where we observed altered migration patterns that can be explained by structural differences rather than by the small differences in size (Fig. 6B).The T 9 and T 18 variants have an apparent similar structure (Fig. 6A), although the sabA expression are slightly different, low vs. intermediate (Fig. 2).Possibly, the longer T-tract in T 18 may give a more flexible DNA that allows for some contact between the RNAP aCTDs and the UP-like elements, as our SPR and footprint data from the wt and D 46 PsabA DNA suggested (Fig. 5D and S6A).To further look into this, we made in silico structure predictions of PsabA DNA with sequential nucleotide extensions in T-tract length (T 13 to T 18 ).Evidently, a distinct 3D DNA structure was observed for each variant (Fig. 6C), since the DNA was converted both in the y and in the z orientation by each thymine addition (Fig. 6D).This is in line with the alterations in mRNA levels we detected in the H. pylori T-variants with one deleted (T 12 , 60%) or two added (T 15 , 75%) T's, as compared to the wt (T 13 , 100%, Fig. 2C).This illustrates the influence of small alterations in T-tract length on the final SabA expression and sLex binding activity in H. pylori.This is also visible in the heterogeneous populations isolated post-infection (Fig. 3).Some of the A-boxes, described in the preceding section, have a perfectly phased location (10)(11) nucleotides in between) in the DNA helix (Fig. 5A and S2).We propose that the A-boxes are interaction sites for RNAP aCTDs and also contribute to the intrinsic DNA curvature in the promoter (Fig. 5-6).Such curvature has previously been shown to affect both binding of RNAP (formation of closed complex), of DNA strands (formation of open complex), release from promoters (promoter escape) and binding of trans-acting factors, which argues that upstream static DNA bends can influence promoter activity at several levels [65].Structural predictions of PsabA fragments lacking the proximal UP-like element showed major structural alterations in PsabA DNA as compared to the wt fragments (Fig. S6B).This explains the SPR and promoter activity results where we observe a stronger interaction and increased promoter activity with low-expressing T 9 and T 18 variants as this region is missing (Fig. 5D-E).Probably, the A-boxes in the distal UP-like element is in a more favorably phasing in the D 46 T 9 and D 46 T 18 , promoting DNA curvature and optimal contact to RNAP, than in the D 46 A 13 variant (Fig. 5E and S6B).The overall effect on promoter activity observed in these variants is probably due to a combination of the changed RNAP binding and DNA structure.Structure predictions of the scrambled UP-like elements revealed that it is the A-box located between the T-tract and the proximal UP-like element that has most impact on DNA structure (Fig. S6C).This A-box is missing in the D 46 fragments, probably resulting in observed changes in DNA structure (Fig. S6B) but was kept unchanged in our scrambled UP-like elements (Fig. S6C).In conclusion, our results suggest that the T-tract length drives the A-boxes into different phasing of the DNA, thereby altering the threedimensional architecture of PsabA DNA.Furthermore, this changes the angular orientation between the core promoter and UP-like elements resulting in enhanced or decreased interaction of RNAP with DNA, giving the observed multiphasic expression pattern of SabA protein and sLex-receptor binding activity (Fig. 2).

The T-tract length affects PsabA activity without involvement of known DNA binding proteins
Not only AT-rich DNA is known to bend DNA, but also binding of nucleoid-associated proteins (NAPs).SSRs positioned upstream of 235 promoter elements frequently influence the binding of a trans-acting regulatory factor exemplified by; the TAAA tract of the nadA promoter in Neisseria meningitidis, affecting binding of integration host factor (IHF); the GAA tract of pMGA in Mycobacterium gallisepticum, affecting binding of a putative regulator HAP; and the A-tract of PatzDEF in Pseudomonas putida, affecting binding of AztR [48,66,67].Typical for many of the classical trans-acting transcriptional regulators in other species, such as H-NS, cAMP receptor protein CRP, and LysR-type regulators, are their ability to interact with AT-rich DNA [68][69][70].Though, there is no H-NS or IHF homolog present in H. pylori, two other NAPs have been described; the HU homolog Hup [71,72] and the Dps homolog NapA [73,74].HU is one of the NAPs conserved in eubacteria.
In order to explore if these DNA binding proteins affect sabA expression, we constructed hup and napA mutants in strain SMI109 and analyzed changes in expression by RT-qPCR (mRNA levels), Western (protein expression) and RIA (receptor binding activity).However, we could not observe an effect on sabA expression, at any level, in either the hup or the napA mutant (Fig. S7B-C).We also analyzed sabA expression in hup mutants with various T-tract lengths, and again no effect was observed (Fig. S7D-E).We cannot yet exclude that no additional factors are involved in regulating sabA mRNA levels in combination with the T-tract.To our knowledge the only trans-acting factor that affects SabA expression is the acid responsive ArsRS system that represses SabA expression at acidic conditions [33].How this repression operates in molecular terms and if the regulation occurs by direct interaction with PsabA, is not yet known.Nonetheless, our results show that the T-tract length located adjacent to the 235 element of the sabA promoter affects binding of the RNAP and thereby the transcriptional output, without involvement of any known DNA binding proteins.
The recurrent multiphasic SabA expression pattern observed in the T-variants supports the hypothesis that it is the structure of promoter DNA and RNAP interaction, rather than binding of a trans-acting factor, that is important for expression.The multiphasic pattern was much more pronounced in H. pylori (Fig. 2) than when promoter activity was analyzed in E. coli (Fig. S1C).Two of the T-variants, T 18 and C 13 , displayed divergent expression levels in H. pylori as compared to the in vitro data (compare Fig. 2 and 4).Nonetheless, SPR analysis of s 70 -RNAP binding and Figure 5. a-subunits of RNAP bind to A-boxes upstream of the T-tract.A) DNA sequence of the PsabA upstream region showing the predicted UP-like elements and multiple A-boxes (red boxes).Red, blue and green lines mark the interaction sites of s 70 -RNAP found by Footprint analysis, correspondingly, see Fig. 5B-C.B-C) Mapping of the binding site for s 70 -RNAP to PsabA DNA using DNase I footprint assay.10 nM of [c 32 P]ATP-labeled PsabA DNA (2166 to +74) were mixed with increasing concentrations of s 70 -RNAP (0, 6.25, 12.5, 25, or 50 nM).The regions protected from DNase I cleavage are marked by red (core promoter), blue (proximal UP-like element) and green (distal UP-like element) lines.The positions of the T-tract, predicted 235 and 210, and +1 transcriptional start site, are indicated to the left.The stars mark the region of the promoter that was deleted in D 46 variants (297 to 249, see also Fig. S2 and S6A).Nucleotide positions, relative to the transcriptional start site, are shown to the right.D) Binding of s 70 -RNAP (55 nM) to PsabA DNA (2166 to +74), with different repeat tract compositions and promoter mutant variants, analyzed by SPR.The sensorgrams show values normalized to that of the full-length T 13 -variant.Binding to a sabA CDS-fragment, also used in Fig. 4, is shown as a background curve in the top diagram.The bottom diagram is an enlargement of the dotted-lined square in the top diagram.E) Promoter activity of PsabA::lacZ transcriptional fusion plasmids, containing PsabA with proximal UP-like element deleted.The constructs contain different tract lengths and compositions (see Fig. 5B-C and S6A).Black bars represent wt promoters and white bars D 46 variants, respectively.b-galactosidase assays were performed in the E. coli strain AAG1, with cultures grown to OD 600 of 2 and analyzed as described in Materials and Methods.Data is presented as relative values with activity of PsabA T 13 wt set to 1. F) Promoter activity of PsabA::lacZ transcriptional fusion plasmids, containing sabA promoter with scrambled UP-like elements.b-galactosidase assays were performed as described in Fig. 5E and data is presented as relative values with activity of PsabA wt set to 1. doi:10.1371/journal.ppat.1004234.g005 the promoter activities analyzed in E. coli show comparable results (Fig. 4A-B).It is therefore tempting to speculate that the dissimilarities could be due to structural differences of the E. coli and H. pylori RNAPs.Our results indicate that it is the interaction between a-subunit of the RNAP and the UP-like elements that is affected by the T-tract length, through change in DNA structure.Homology predictions has shown that the RNAP aand v-subunits are more divergent between different bacterial species than the remaining subunits [75] and thus, interaction of RNAP to DNA structures or DNA binding trans-acting factors might deviate from E. coli.Borin et al showed that the linker region between the aCTD and aNTD is longer in H. pylori compared to the E. coli a-subunit.The H. pylori aCTDs have an additional amphipathic helix in the Cterminal [76], which could explain why the highest expression in H. pylori is T 13 whereas it is T 18 in E. coli.The H. pylori a-subunit should, due to these structural differences, be able to reach further upstream than the E. coli one, to make contact with the UP-like elements or potential trans-acting factors.
T-or A-tracts adjacent to 235 elements affect transcription in H. pylori SSR motifs located between the 235 and 210 promoter elements affect docking of the RNAP s-factor, motifs located upstream of the 235 element affect binding of regulatory factors [6], and as we show here for sabA, motifs located adjacent to the 2 35 element adjust transcription initiation by affecting local DNA structure.To dissect if this finding is a general phenomenon in H. pylori, we searched the genome of strain 26695 for additional genes with T-or A-tracts (.9 nucleotides) close to the 235 element.Among the predicted promoters of H. pylori [77], we found twentyfive genes with appropriately located T-or A-tracts (Table 2).Interestingly, loci encoding outer membrane proteins were again overrepresented among these genes (15 of 25 genes).
Among the twenty-five loci, nine had a T-or A-tract located between the 235 and 210 elements, two replaced the 235 element, six were located approximately 30, 31, 59, 68 and 86 nt upstream of 235 element, respectively, and two were located downstream of the transcriptional start site.Furthermore, five loci had T-or A-tracts located adjacent (,20 nt) to the 235 element, similar to that of sabA: sabB (HP_0722), hopD (HP_0025), hofA (HP_0209), hopM (HP_0227), and hp_0350 (Table 2).We compared the tract lengths of these five loci in the forty-nine publically available genome sequences (Table S1).Our comparison showed that all T-or A-tracts displayed great length variability, in line with the individual selection and stochastic switching hypotheses discussed in preceding sections (Fig. S8).We also found four intergenic regions with predicted divergent transcriptional start sites, and T-or A-tracts located in between their respective promoters (Table 2).The adjacent genes, hp_0350 (unknown) and pyrG (CTP synthase), have an A-tract located just three nucleotides upstream of the 235 element of hp_0350, and a T-tract positioned 30 nucleotides upstream of the 235 element of pyrG (Fig. 7A).These two loci were found in all sequenced H. pylori genomes, and additionally, in other Helicobacter species such as H. acinonychis isolated from cheetahs and H. cetorum isolated from Atlantic white-sided dolphins (Table S1).In order to test if the length of the T-or A-tract influences the expression of these genes, we created lacZ transcriptional fusion plasmids of hp_0350 and pyrG promoter regions from strain SMI109 (A 14 /T 14 ).Additionally, we constructed the corresponding promoter variants with 5 nucleotides shorter A-or T-tracts (A 9 /T 9 ), i.e. half the distance between min/max expression levels observed for sabA (Fig. 2).The promoter activities were analyzed by b-galactosidase assay in H. pylori, and as hypothesized the hp_0350 promoter showed distinct differences in activity in the A-tract length variants, whereas in contrast, the pyrG promoter activity remained unaffected by the change in T-tract length (Fig. 7A).The experiment was repeated at different growth phases and these lacZ fusions were also analyzed in E. coli, with the same results (data not shown).
In addition, we made in silico DNA curvature predictions of three additional promoter regions (hp_0350, hofA and hopM) and found that the DNA structure was affected as the length of the repetitive tract was decreased by 5 nucleotides (Fig. 7B), just as we observed for sabA (Fig. 6A).Alignment of the hp_0350 promoter sequences also revealed high homology between different strains (Fig. S9) and we could observe conserved A-boxes located upstream of the A-tract, forming potential UP-like elements, where the RNAP a-subunits likely interact.Our findings suggest that poly A-or T-tracts located adjacent to 235 elements finetune promoter activity, and thereby mRNA levels, by changing the DNA structure as the tract length is altered.

Concluding remarks
In conclusion, we describe a general mechanism where certain simple sequence repeats (SSRs) in H. pylori changes the local DNA structure, which by a rheostat-like mechanism affects interaction of the RNAP, to fine-tune gene expression via slipped strand mispairing (SSM).For SabA, optimal T-tract length (T 13 in strain SMI109) positions the upstream-located A-boxes in a favorable phasing, aligning the UP-like elements and the core promoter, to enhance RNAP interaction, and resulting in higher promoter activity (Fig. 8).Thus, low transcriptional activity occurs when the UP-like elements and the core promoter are skewed relative each other, and the interaction of the RNAP a-subunits to the UP-like elements is disturbed (Fig. 8).Each T-tract length variant displayed a unique 3D DNA structure, contributing to the multiphasic transcriptional output observed from the sabA promoter.The mechanism described in this paper is possibly of significance to other bacterial systems that like H. pylori have a limited repertoire of trans-acting transcription factors and numerous SSRs, e.g.Haemophilus influenzae and N. meningitidis.
SabA is just one, of many outer membrane proteins in H. pylori, which have SSRs in its inter-and intragenic sequences.Intragenic CT-tracts have been reported for loci encoding BabA, BabB, SabB, OipA and HopZ, and we show that a set of them also harbor T-or A-tracts in their promoter regions, respectively.Taken together, all the SSR regions found in a genome create multiple contingency loci of hypermutable DNA that via different mechanisms blindly mediate causal and heritable genotypes, and contribute to stochastic switching.This cost-beneficial approach Table 2. Loci in strain 26695 with T-or A-tracts ($9 bp) located close to 235 promoter elements.meets the need to control gene expression at various levels and can despite the lack of specific trans-acting regulators pilot persistent infections in fluctuating host environments through production of heterogeneous bacterial populations of best-fit phenotypes.

Ethical statement
The animal studies were approved by the Animal Care and Use Committee of Umea ˚University and by the ethical committee of Swedish Board of Agriculture (Decision No. A120-06).Experiments were conducted in accordance with Guidelines for Care and Use of Laboratory Animals.

Growth conditions and strains
Bacterial strains used in this study are listed in Table 1.H. pylori strains were routinely grown on Brucella agar (Difco) supplemented with 10% citrated bovine blood (Svenska Labfab), 1% IsoVitox (Becton Dickinson, US) and an antibiotic mix (4 mg/L amphotericin B, 5 mg/L trimethoprim and 10 mg/L vancomycin).When needed, H. pylori strains were grown in culture medium containing Brucella Broth (Difco), 1% Isovitox and 10% fetal calf serum (Gibco).Plates or broth were, when required, supplemented with chloramphenicol (20 mg/L) and/or kanamycin (25 mg/L).Bacteria were grown at 37uC under microaerophilic conditions (5% O 2 , 10% CO 2 , and 85% N 2 ).For the analysis of sabA mRNA levels, protein expression and sLex-receptor binding, equal amounts of each strain were re-plated onto Brucella blood agar plates, and the bacteria were collected after 16 h of growth, in order to have all strains in the same growth phase.For the Dhup strain the plates were left for 40 h due to the delayed onset of growth (Fig. S7A).E. coli strains were cultured in Luria broth (LB) agar at 37uC, supplemented with carbenicillin (100 mg/L) and/or kanamycin (25 mg/L).Growth was measured by OD at 600 nm using the spectrophotometer Ultrospec2100 PRO (GE healthcare).
SMI109 DsabA was created by transformation of a plasmid containing the DsabA::cam construct [13].Deletion of the sabA gene, loss of sLex-receptor binding, and absence of SabA expression was verified by PCR, RadioImmunoAssay (RIA), and immunoblot assays, respectively.We also determined, by diagnostic PCR, as previously described [18], that the sabA homolog sabB is absent in strain SMI109.SMI109 Dhup was created by transformation of a Dhup::kan PCR fragment generated by hup-1 and hup-5 primers, and pAAG178 as template.SMI109 DnapA was created by transformation of a DnapA::kan PCR fragment generated by napA1F and napA1R primers, and pBlue-DnapA::kan [78] as template.Deletion of the hup and napA genes was verified by PCR using hup-2/hup-in and napA2F/napA2R primers, respectively.Plasmids used are shown in Table 1 and primers in Table S2.
J99 StrR was constructed by transformation of plasmid pEG21 (a kind gift from Prof Rainer Haas, Ludwig Maximilians University, Munich, Germany) into J99.The bacteria were plated on plates containing 500 mg/L streptomycin to obtain single colonies and a sabA T 17 and CT 8 -off clone was selected and used for animal studies.
SMI109 pyrG::lacZ and hp_0350::lacZ strains were constructed by transformation of pAAG202-205 plasmids into SMI109.Correct incorporation in the chromosome was verified by PCR.

Genetic techniques
Basic molecular genetic manipulations were performed essentially as described previously [79].Genomic DNA was isolated as previously described [80] from bacteria grown on plate.Polymerase chain reactions (PCR) were carried out according to the manufacturer's instruction, using GoTaq polymerase (Promega) or Phusion Hot start DNA polymerase (Thermo Scientific), on a MJ PTC-200 thermal cycler (MJ Research).For isolation of plasmid DNA, the E.Z.N.A Mini and Midi column plasmid purification kits were used and purification of PCR products were done with the E.Z.N.A Cycle Pure or Gel Extraction kits (OMEGA bio-tek, USA).Plasmids and/or PCR products were sequenced at Eurofins MWG Biotech (Germany).

Construction of lacZ transcriptional fusion plasmids
The sabA transcriptional lacZ fusion plasmids were obtained by cloning a PCR-amplified fragment (sabA-1 and sabA-3) spanning 310 bp of the sabA promoter region and 8 bp of the CDS (2244 to +74) between the EcoRI-BamHI sites in pRZ5202 creating a transcriptional fusion (Fig. S1A).As template, genomic DNA from different H. pylori strains (26695, J99, G27, 17875/sLex and SMI109) were used.Site-directed mutagenesis, using primers spanning ,20 bp on either side of the T-tract (see example sabA-Tf/sabA-Tr in Table S2), were used to change the length of the Ttract in the sabA::lacZ promoter fusions.
The D 46 promoter fragments were constructed with overlapping PCR using primers P163-165 and P167 (different variants), and 162 (Table S2).As template PsabA DNA from SMI109 was used.Mutations were verified by sequencing and a PCR-amplified fragment (sabA-1 and sabA-3) was cloned between EcoRI-BamHI sites in pRZ5202, creating lacZ transcriptional fusions.For SPR and footprint analysis, PCR fragments generated with primers sabA-5 and sabA-8 were used.
Scrambling of the A-boxes in UP-like elements of PsabA was generated by site-directed mutagenesis, using primers spanning the proximal (Amut1) or/and distal (Amut2) elements (Table S2).As template PsabA DNA from SMI109 cloned in pUC19 was used.Mutations were verified by sequencing and a PCR-amplified fragment (sabA-1 and sabA-3) was cloned between EcoRI-BamHI sites in pRZ5202, creating lacZ transcriptional fusions.
The hp_0350 and pyrG promoter lacZ fusion plasmids were obtained by cloning the PCR-amplified fragments (hyp F/hyp R or pyrGp F/pyrGp R) spanning the hp_0350/pyrG promoter region between SalI-BglII sites in pBW.As template, genomic DNA from strain SMI109 was used.Stitch PCR using primers spanning ,20 bp on either side of the T-or A-tract (pyrG 9Tf/pyrG 9Tr), were used to change the length of the T-or A-tract in the pyrG::lacZ and hp_0350::lacZ promoter fusions.

Construction of T-tract mutants in H. pylori
Isogenic sabA repeat tract variants were constructed by contraselection in strain SMI109, as previously described [81].In short, the sabA promoter region was removed and replaced by an antibiotic resistance cassette, generating the SMI109Dsa-bA::rpsLCAT strain, using primers LA-F, LA-R, RA-F, RA-R, rpsLCAT-F, rpsLCAT-R.PCR fragments harboring the sabA promoter region, with different lengths or composition of the repeat tract, were generated by stitch PCR using (P93, Tf, Tr, P96) and transformed into the SMI109DsabA::rpsLCAT strain, to replace the antibiotic cassette.Tf and Tr refer to the complementary primers spanning the region determining the tract length or composition (see Table S2).Clones were verified by PCR and sequencing using P93 and P96 primers.The protocol was optimized to fit the SMI109 strain by first determining the frequency of false positive clones (as contraselection is somewhat leaky).The amount of bacteria that corresponded to ,10 false positives (in mock transformation) was used for transformation, which greatly facilitated the yield of mutants.

Construction of hup deletion/insertion fragment
A deletion/insertion fragment of the hup gene from SMI109 was generated by removal of the hup CDS and replacing that with a kanamycin resistance cassette.DNA regions upstream and downstream of the hup gene (HP_0835) were amplified by PCR using SMI109 genomic DNA as template, hup-1/hup-3Km and hup-4Km/hup-5 primers generating PCR fragments with flanking regions homologous to a kanamycin cassette.The kanamycin cassette from pKD4 was amplified using Km-up/Km-dn primers.The three PCR fragments were stitched together using equimolar amounts of each PCR fragment and hup-1/hup-5 primers.The hup deletion/insertion fragment was cloned in SmaI cut pUC19, generating the plasmid pAAG178, and analyzed by PCR and sequencing.

RadioImmunoAssay (RIA)
Measurement of binding to soluble 125 I-sLex-receptor conjugate was performed as previously described [82].Samples were assayed in duplicates and minimum two independent sets of each experiment, plotted with standard deviations.

Immunoblot analysis
Immunoblot analysis was performed as previously described [17].Antibodies against SabA (AK278) and AlpB (AK262) [83] was used in combination with secondary a-rabbit IgG-HRP (DAKO A/S, Denmark).Blots were developed with SuperSignal (Pierce, Rockford, IL) ECL and detected on High Performance Chemiluminescence film (GE Healthcare).SabA protein densities were measured by ImageJ software (NIH) and normalized to the corresponding PAGE Blue stained SDS-PAGE gel (for Fig. 1A) or AlpB protein density (Fig. 2A, 4C, S6C and S6E) to calculate relative protein expression levels (Fig. S10).
b-galactosidase assay b-galactosidase activity measurements were performed as described by Miller [84].Data shown are mean values of duplicate determinations of at least three independent experiments, plotted with standard deviations.

RNA isolation
Total RNA was extracted using the SDS/hot phenol method, as previously described [85].Contaminating DNA was removed by 10 U DNase I (Ambion 2 U/ml) treatment for 10 min at 37uC, followed by phenol/chloroform extraction.Quality and concentration of the total RNA was examined in a 1.2% agarose gel and by measurement on a micro-spectrophotometer (Nanodrop, ND-1000).The total RNA was stored at 280uC until used.

59-Rapid Amplification of cDNA Ends (59-RACE) and primer extension
The 59-RACE analysis was made using FirstChoice RLM-RACE kit (Ambion) according to the manufacturer's protocol.In brief, ,1 mg of total RNA isolated from different H. pylori strains was used in each ligation and cDNA synthesis reaction.For the first PCR reaction RACE-outer and AB35 primers were used, and for the second PCR reaction RACE-inner and J99-8 primers were used.The PCR fragment of expected size was cloned in SmaI cut pUC19 and sequenced using universal M13F/R primers.
Primer extension analysis was done as previously described [86] using total RNA samples from H. pylori and E. coli (20 mg), and [c-32 P]-ATP kinase-labeled sabA-8 primer.

cDNA synthesis and quantitative real-time PCR (qPCR) analysis
The cDNA synthesis and RT-qPCR analysis was performed in accordance to the MIQUE guidelines [87].Total RNA was isolated from H. pylori strains grown on Brucella blood agar plates (as described above).Before cDNA synthesis the total RNA (250 mg/ml) was treated an extra time with Turbo DNase I (Ambion) to remove any residual DNA.cDNA synthesis was performed in 20 ml reactions using 500 ng Turbo DNase treated total RNA, Transcriptor First Strand cDNA Synthesis kit (Roche Applied Science) and random hexamers (60 mM) provided with the kit, according to the manufacturer's protocol.cDNA synthesis was performed at 25uC for 10 min and at 55uC for 30 min.The enzyme was inactivated at 85uC for 5 min.The cDNA was diluted with 80 ml DEPC MQ before it was used as template in qPCR reactions.
Quantitative real-time reverse transcriptase PCR (RT-qPCR) was used to determine the mRNA levels of sabA in different H. pylori strains, and the primers used are listed in Table S2.As reference genes, expression of gyrA, ppk and rrnA were simultaneously analyzed.RT-qPCR analysis was done in 20 ml reactions in 96-well plates using cDNA from 10 or 100 ng RNA as template, 26 FastStart Essential Green Master (Roche Applied Science), gene specific primers (5 mM each) and LightCycler 96 instrument (Roche Applied Science).Cycling conditions were; 10 min at 95uC and 40 cycles of; 20 s at 95uC, 20 s at 58uC and 20 s at 72uC.Fluorescence was detected at the end of each extension step, and the Cq values and relative ratios were calculated using the LightCycler 96 software (Roche Applied Science).After each run a melt curve analysis was performed and the size of the PCR products were analyzed by gel electrophoresis.In each run, a nontemplate control for each cDNA sample (NTC, RNA only), and a PCR negative control was included for each primer pair.At least duplicate samples were analyzed in each experiment and expression ratios were determined from at least two biological replicates.The PCR efficiencies were determined using standard curve analysis and cDNA from SMI109 as template, and were as follows: sabA-1 1.9860.09,sabA-2 1.9960.06,ppk-2 1.9960.05,gyrA-1 1.9660.03and rrnA-2 1.9060.03.
Semi-quantitive RT-PCR analysis was performed using the same cDNA and primers as in RT-qPCR analysis.20 ml reactions, using cDNA from 100 ng of RNA as template, Phusion Hot Start DNA polymerase (Thermo Scientific) and gene specific primers (5 mM each), were run on MJ PTC-200 thermal cycler (MJ Research).Cycling conditions were; 30 s 98uC followed by 10 (rrnA amplicon) or 20 (sabA and ppk amplicons) cycles of 15 s at 98uC, 30 s at 55uC and 30 s at 72uC. 5 ml of each PCR reaction was separated in a 1.5% agarose TAE gel, stained with GelRed (Biotium) and scanned using the Kodak Image station 2000R.
Animal experiments and collection of bacterial sweeps FVB/N male mice (6-8 weeks of age), transgenic for the human a-1,3/4-fucosyltransferase gene, resulting in the expression of ABO Lewis blood group antigen in the epithelial lining of the stomach, were used in this study [40].Transgenicity was confirmed as described previously [40].Mice were bred and kept in separate cages during the study and kept on a 12-h light-dark cycle.Water and standard pellet diet was provided ad libitum.Animals were infected with 2610 8 H. pylori (J99 StrR sabA T 17 and CT 8 -off), twice a week during two weeks, via oro-gastrical gavage.To evaluate the H. pylori infection load, mice were sacrificed 4 weeks post-infection and stomach tissue samples were collected and quantitatively cultured on plates supplemented with 500 mg/ L streptomycin.
Bacterial sweeps prepared from antrum and corpus biopsies during the same gastroscopy session [88] were thawed for reculturing of H. pylori under standard conditions.Genomic DNA isolation and receptor binding analysis of these bacterial pools was performed as described in preceding sections.

Fragment length analysis (FLA)
All fragment length analysis was performed at MWG Eurofins Medigenomix GmbH (Germany).FAM-labeled primers (see Table S2) were designed and optimized by using the Lasergene software (DNASTAR) and synthesized on site at MWG. Repetitive PCR-amplifications, with three different DNA polymerases (AmpliTaq Gold, Takara Taq, Bioline MyTaq), gave identical peak distribution at different dilutions, although with varying amplification strength (Fig. S4).For all FLA assays at least two dilutions with equal amounts of genomic DNA was used with comparable results.

Electric mobility shift assay (EMSA)
Linear DNA containing the sabA promoter region (spanning 2 166 to +74) was generated by high-fidelity PCR using genomic DNA from SMI109 as template and primers sabA-5 and sabA-8.Radio-labeled DNA fragments were generated by first pre-labeling the sabA-5 primer using [c 32 P]ATP (.3000 Ci/mmol; Perkin Elmer) and T4 kinase (Thermo Scientific).The binding reactions with 10 nM DNA and increasing concentrations of E. coli s 70 -RNAP (Holoenzyme, Epicentre) were done as previously described [89].The samples were separated on 4.5% Tris-Glycine (pH 8.5) polyarcylamide gel.The bands were visualized using Phosphor screen cassette, Typhoon scanner 9400 (GE Healthcare) and ImageJ software (NIH).

Surface plasmon resonance analysis
Binding experiments shown in Fig. 4A and 4B were done with a Biacore3000 (GE Healthcare) at 25uC and analyzed with Scrubber 2 software (BioLogic Software).CM5 sensor chips where precoated with streptavidin (50 mg/ml) by injecting at 5 ml/min until approximately 5000 RU were obtained, using an amine coupling kit according to GE Healthcare protocol.This was followed by ethanolamine blockage and subsequent immobilization of 59 biotin-labeled DNA fragments with running buffer [25 mM HEPES pH 7.5, 150 mM KCl, 10 mM MgCl 2 , 0.1 mM EDTA and 0.005% non-ionic surfactant polyoxyethylenesorbitan (P2O) (GE Healthcare)], by injecting at 5 ml/min to obtain approximately equal RU (,70) for every chip.All results were normalized to a T 13 (wt) DNA-fragment used in all runs.DNA fragments were obtained by PCR amplification of genomic DNA from the A-, C-, or T-tract variants, using high fidelity DNA polymerase and 59-Biotin-labeled primer paired with an unlabeled primer (Biotin-sabA-5 & sabA-8).20 nM of the E. coli s 70 -RNAP (Holoenzyme, Epicentre) was injected for 2 min at the rate of 100 ml/min.Between injections the s 70 -RNAP was regenerated from the chip with a quick injection of 30 ml of 500 mM MgCl 2 .Binding to a DNA fragment of the sabA CDS was used as negative control, and subtracted from all data in Fig. 4. Experiments were performed in at least duplicates.
Binding experiments shown in Fig. 5D were done with the ProteOn system (Bio-Rad) at 25uC and analyzed with ProteOn manager 3.1 software (Bio-Rad).ProteOn GLC Sensor Chips were coated with streptavidin and blocked with ethanolamine, essentially as described in the preceding section.Biotinylated DNA fragments of the sabA promoter were immobilized by injection at 30 ml/min, to obtain approximately 120 RU for the two chips used.The E. coli s 70 -RNAP (Holoenzyme, Epicentre) was injected in 5 different concentrations, 1.25, 2.5, 5, 10 and 20 ng/ml.The results were normalized to that of the T 13 (wt) DNA-fragment.

DNase I footprint assay
Binary complexes were formed by incubating 10 nM [c 32 P]ATP-labeled DNA fragments and increasing concentrations of E. coli s 70 -RNAP (Holoenzyme, Epicentre).Binding reactions were done in buffer B (25 mM HEPES pH 7.5, 0.1 mM EDTA, 5 mM DTT and 10% glycerol), 50 mM KCl and 0.5 mg/ml bovine serum albumin), for 30 min at 30uC.The reactions were subjected to DNase I digestion (0.3 U Recombinant DNase I, Ambion) and treated as previously described [90].The samples were analyzed on a 6% denaturing polyacrylamide-8.3Murea gel.A+G Maxam Gilbert sequencing reactions of the same DNA fragments were loaded alongside the samples.The bands were visualized using Phosphor screen cassette and Typhoon scanner 9400 (GE Healthcare).

Statistical analysis
We used the non-parametric two-tailed Mann-Whitney test for the statistical analyzes.Differences were considered significant when the p value was below 0.05.Significance levels are marked with *,0.05, **,0.01 and ***,0.005.

Supporting Information
Figure S1 The T-tract length affects sabA promoter activity.A) Schematic illustration of the promoter DNA (2244 to +74) cloned in pRZ5202 to create the PsabA::lacZ transcriptional fusion plasmids assayed in Fig. S1B-C.See Materials and Methods for details.B) Promoter activity of PsabA::lacZ transcriptional fusion plasmids, containing sabA promoter from different H. pylori strains (see Fig. 1A-B and Table 1).b-galactosidase assays were performed in the E. coli strain AAG1, with cultures grown to OD 600 of 2 and analyzed as described in Materials and Methods.C) Promoter activity of PsabA::lacZ transcriptional fusion plasmids, containing sabA promoter with different T-tract lengths (see Table 1).Samples were taken and b-galactosidase assays were performed as described in Figure1.Interstrain variation of sabA mRNA levels, SabA protein expression and functional sLex-receptor binding.A) Analysis of SabA expression and sLex-receptor binding activity in a set of five H. pylori strains.Image shows one representative immunoblot analysis with a-SabA antibodies and the numbers above represents SabA expression quantification, with expression in strain SMI109 set to 1. Equal amounts of crude protein extracts were loaded in each lane (Fig.S10A).The graph shows binding to soluble 125 I-sLex-receptor conjugate of the same strains as analyzed in the immunoblot.Bacteria were grown on plate as described in Materials and Methods prior to the analysis.Average and standard deviations are calculated from at least two independent experiments and duplicate samples/analysis of each strain.B) RT-qPCR analysis of sabA mRNA levels in the same set of strains as in Fig.1A.The sabA mRNA levels were normalized to a set of reference genes and data is presented as relative, with the levels in strain SMI109 set to 1. Images show one representative semi-quantitative PCR analysis, using the same primers as in the RT-qPCR analysis; sabA-2 and ppk-2.C) Sequence comparison of the PsabA region (271 to +158, relative to the transcriptional start site) between different H. pylori strains.The +1 transcriptional start sites, as determined by primer extension and 59-RACE, and the predicted 210 and 235 promoter elements, are underlined.Differences in nucleotide sequences are shown in grey color.The regions containing the T-tract and CT-repeats are boxed.The stop codon (TGA) that results in a truncated SabA protein in the CT 6 -off strain 26695 is also underlined.A more extensive comparison, of 44 PsabA sequences, is shown in Fig. S2.D) Distribution of T-tract lengths in the sabA promoter (PsabA) of 51 sequenced H. pylori strains.Black represents number of analyzed genome-sequenced strains, whereas white represents the number of strains where the sequence of the sabA locus was obtained after conventional PCR amplification.doi:10.1371/journal.ppat.1004234.g001

Figure 2 .
Figure 2. The T-tract length alters sLex-receptor binding activity by affecting sabA mRNA levels in H. pylori.A) SabA protein expression analysis in variants of SMI109 harboring different T-tract lengths.Image shows one representative immunoblot with a-SabA antibodies used for the quantification.Expression levels were normalized to expression of the AlpB protein before comparison (Fig. S10B).Data are presented in the bar diagram, as described in Fig. 1A, with the expression in the T 13 (wt) variant set to 1. Stars indicate significant differences from T 13 -variant, * p,0.05, ** p,0.01, ns = non significant.B) Binding to soluble 125 I-sLex-receptor conjugates of the same set of T-variants as in Fig.2A.The data are presented as in Fig.1A, with the binding of the T 13 -variant set to 1. Stars mark significant differences from T 13 -variant, see Fig.2A.C) RT-qPCR analysis of sabA mRNA levels in T-variants of SMI109.Data are presented as in Fig.1B, with the mRNA levels in the T 13 -variant (wt) set to 1.The upper images show result from one semi-quantitative PCR analysis using primers for sabA-1 and rrnA-2.D) Binding of FITC-labeled SMI109 T-variants (T 9 , T 13 and T 18 ) to human gastric tissue sections.SMI109 DsabA mutant was included as a negative control.Images were taken with 1006magnification.For all analyses in Fig.2, bacteria were grown on plate prior to the experiment, as described in Materials and Methods.doi:10.1371/journal.ppat.1004234.g002

Figure 3 .
Figure 3.The T-tract length is variable, both in a mouse model and in the human stomach.A) Analysis of bacterial output pools isolated two months post-infection from FVB/N mice.Binding to soluble 125 I-sLex-receptor conjugates is shown in the bar diagram.Values above the bars show the infectious load in each mouse (colony forming units, CFU).Bottom curves show the corresponding FLA-spectra after PCR-amplification of the PsabA region, using genomic DNA isolated from the different output pools as template, including the input strain.Dotted lines serve as length reference for comparison.The arrows mark the FLA peak observed to decrease in output pools of mouse 3 and 5, relative to input strain.B) Binding to 125 I-sLex-receptor conjugates of ten independent clones isolated from the bacterial output-pools of mouse 2 and 4, respectively.The T-and CT-tract lengths of a representative set of clones, as determined by sequencing, are shown above the bars.CT 7 -On = SabA CDS in-frame, CT 8 -off = SabA CDS out of frame.C) Analysis of bacterial output pools, isolated from the antrum (A) and the corpus (C) regions of the stomach, of three Swedish patients.Binding to soluble 125 I-sLex-receptor conjugates of the output pools is shown in the bar diagram, and the corresponding FLA-spectra are shown to the right.The T-tract lengths, of two clones from each bacterial pool, are shown next to the FLA-spectra.doi:10.1371/journal.ppat.1004234.g003

Figure 4 .
Figure 4. Binding of RNAP to PsabA DNA with varying tract length and nucleotide composition.A) Analysis of E. coli s 70 -RNAP binding to PsabA DNA by Surface Plasmon Resonance (SPR).Sensorgrams show injection of the s 70 -RNAP (20 nM) over chips with pre-bound biotinylated-PsabA (2166 to +74) DNA fragments, with different T-tract lengths (T 5 , T 9 , T 13 or T 18 ).Inlay shows promoter activity of the corresponding T-tract variants, assayed in E. coli using transcriptional PsabA::lacZ fusions as described in Fig. S1.B) SPR sensorgrams analyzed as described in 4A but with PsabA DNA fragments containing A 13 -or C 13 -tracts.Inlay shows promoter activity of the corresponding variants, assayed in E. coli using transcriptional PsabA::lacZ fusions, as in Fig. S1.C) Analysis of SabA expression and sLex-receptor binding activity of variants of SMI109 harboring A 13 -or C 13 -tracts in PsabA.The image shows one representative immunoblot with a-SabA antibodies, where numbers above represent relative expression with expression in the T 13 -variant set to 1. Bar diagram show binding to soluble 125 I-sLex-receptor conjugate of the same set of variants as in the immunoblot.Samples were prepared as described in Fig. 1A.Statistical tests showed significant differences to the T 13 (wt) variant (* = p,0.05).doi:10.1371/journal.ppat.1004234.g004

Figure 6 .
Figure 6.The T-tract length affects the local DNA structure of the sabA promoter.A) In silico DNA structure predictions of the PsabA (2166 to +74) harboring different repeat tract lengths and nucleotide compositions, using the AA Wedge model (http://www.lfd.uci.edu/,gohlke/dnacurve/).The analyzed DNA fragments contain T 9 -, T 13 -, T 18 -or A 13 -tracts.The structures shown represent the 3D DNA helix backbone, displayed in three dimensions.B) Gel migration of DNA fragments containing the PsabA with different repeat tract lengths and compositions.The DNA samples, same set as in Fig. 6A, were run at 4uC in a Tris-Glycine 4.5% polyacrylamide gel that was stained with GelRed.The DNA size marker (bp) is shown to the left.C) Alignment of PsabA DNA fragments analyzed as pdb structures in the Protean 3D software (Lasergene, DNASTAR).The T-tract was extended by 1 thymine (T) at a time (from 13 to 18), and predictions were made as in Fig. 6A.The image shows one view from a selected angle, with the T-tract marked in black and by an arrow.The different T-variants are labeled in shades of gray, see Fig. 6D.D) A 1D plot of the shape of the PsabA DNA helix, visualized in the y orientation (left diagram) and in the z orientation (right diagram).The coordinates were generated from the predictions in Fig. 6C.The black arrows mark the position of the T-tract in the DNA helix.doi:10.1371/journal.ppat.1004234.g006 Position abbreviation; 235/210: between elements, ,235: #20 nucleotides upstream of 235, %235: .20 nucleotides upstream of 235, 235: overlapping with 235, 210.: downstream of 210.doi:10.1371/journal.ppat.1004234.t002

Figure 7 .
Figure 7. hp_0350 promoter activity is affected by the A-tract located adjacent to the 235 promoter element.A) Effects on hp_0350 and pyrG promoter activities by the length of the repeat tract located in their divergent promoter regions (A 14 /T 14 [wt] vs.A 9 /T 9 [D 5 ]).Strains were grown in Brucella broth at 37uC in 24-well plates under microaerophilic conditions Expression from the hp_0350::lacZ and pyrG::lacZ reporters in strain SMI109 shown are from samples collected in logarithmic growth phase (OD 600 of 0.2, Fig. S7A).Illustration shows the position of the repeat tract, relative to the 235 elements, of each gene.DNA sequence alignment of the hp_0350/pyrG promoter regions from 45 different strains is shown in Fig. S9.B) In silico DNA structure predictions of the hp_0350, hofA (HP_0209) and hopM (HP_0227) promoter regions based on sequences from strain 26695.The analysis were performed as in Fig. 6A.Images in the left panel show DNA structures with wt tract lengths, and in the right panel, the structures of promoter DNA with 5 nucleotide shorter repeat tracts.doi:10.1371/journal.ppat.1004234.g007

Figure 8 .
Figure 8. T-or A-tracts, adjacent to 235 elements, regulate gene expression by a rheostat-like mechanism.Schematic overview of the T-tract rheostat using the sabA promoter as a model.The predicted interaction of the RNA polymerase with sabA promoter, harboring different Ttract lengths and thereby different local DNA structure, is depicted in the model.The illustration shows the high-expressing T 13 -variant and the lowexpressing T 18 -variant.The region containing the A-boxes, i.e. the proximal UP-like element, is marked in purple (290 to 250), T-tract in blue, and the core promoter (235 to +20) in yellow.Bent arrows indicate the change in local DNA structure that occurs in two orientations as the T-tract length is altered.This is a variable process as the T-tract length can both be lengthened and shortened, as a result of slipped strand mispairing during replication.doi:10.1371/journal.ppat.1004234.g008 Figure S1The T-tract length affects sabA promoter activity.A) Schematic illustration of the promoter DNA (2244 to +74) cloned in pRZ5202 to create the PsabA::lacZ transcriptional fusion plasmids assayed in Fig.S1B-C.See Materials and Methods for details.B) Promoter activity of PsabA::lacZ transcriptional fusion plasmids, containing sabA promoter from different H. pylori strains (see Fig.1A-Band Table1).b-galactosidase assays were performed in the E. coli strain AAG1, with cultures grown to OD 600 of 2 and analyzed as described in Materials and Methods.C) Promoter activity of PsabA::lacZ transcriptional fusion plasmids, containing sabA promoter with different T-tract lengths (see Table1).Samples were taken and b-galactosidase assays were performed as described in Fig. S1B.(TIF) Figure S2 Highly conserved regions among sabA promoter sequences.Alignment of 44 sabA promoter sequences

Table 1 .
Strains and plasmids used in this study.
Table S1 Length variations in repeat tracts of six relevant loci in different Helicobacter genomes.(PDF) Table S2 Oligonucleotides used in this study.(PDF)