A Single Nucleotide Change Affects Fur-Dependent Regulation of sodB in H. pylori

Helicobacter pylori is a significant human pathogen that has adapted to survive the many stresses found within the gastric environment. Superoxide Dismutase (SodB) is an important factor that helps H. pylori combat oxidative stress. sodB was previously shown to be repressed by the Ferric Uptake Regulator (Fur) in the absence of iron (apo-Fur regulation) [1]. Herein, we show that apo regulation is not fully conserved among all strains of H. pylori. apo-Fur dependent changes in sodB expression are not observed under iron deplete conditions in H. pylori strains G27, HPAG1, or J99. However, Fur regulation of pfr and amiE occurs as expected. Comparative analysis of the Fur coding sequence between G27 and 26695 revealed a single amino acid difference, which was not responsible for the altered sodB regulation. Comparison of the sodB promoters from G27 and 26695 also revealed a single nucleotide difference within the predicted Fur binding site. Alteration of this nucleotide in G27 to that of 26695 restored apo-Fur dependent sodB regulation, indicating that a single base difference is at least partially responsible for the difference in sodB regulation observed among these H. pylori strains. Fur binding studies revealed that alteration of this single nucleotide in G27 increased the affinity of Fur for the sodB promoter. Additionally, the single base change in G27 enabled the sodB promoter to bind to apo-Fur with affinities similar to the 26695 sodB promoter. Taken together these data indicate that this nucleotide residue is important for direct apo-Fur binding to the sodB promoter

Flow Cytometry analysis of <i>sodB</i> and <i>pfr</i> GFP reporters.

<p>Strains bearing <i>sodB::gfpmut3</i> or <i>pfr::gfpmut3</i> promoter fusions were grown overnight in either iron replete or iron depleted media. Changes in fluorescence were analyzed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005369#s2" target="_blank">Materials and Methods</a>. Results for the <i>sodB</i> promoter fusions are displayed in Panel 1A, and results for the <i>pfr</i> promoter fusions are displayed in Panel 1B. For both A and B, solid lines indicate the plasmid in WT <i>H. pylori</i> G27 grown in iron replete conditions, dotted lines indicate the plasmid in WT bacteria grown in iron deplete conditions, and dashed lines indicate the plasmid in Δ<i>fur</i> bacteria grown in iron replete conditions. Fluorescence is measured in relative units, and the data are representative of multiple independent flow analyses.</p

Role of the −5 bp in <i>sodB</i> regulation.

<p>WT G27, WT 26695, and the “−5 bp swap” strain were grown as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005369#s2" target="_blank">Materials and Methods</a>, and RNA was isolated under iron replete and iron-depletion shock conditions. RPAs were performed on RNA isolated from 4 biologically independent experiments using <i>sodB</i> and <i>pfr</i> riboprobes. Data from <i>sodB</i> RPAs are presented in Panel A, and data from <i>pfr</i> RPAs are presented in Panel B. Each square, diamond, triangle, and circle represent the average fold decrease calculated from three technical repeats with each independent set of RNA for each strain and growth condition combination. Median fold decrease is represented as a bar for each combination, and the dotted-dashed line represents the 2-fold significance cut-off. ^*p-value of 0.0001. ^#p-value of 0.006.</p

Direct Comparison of <i>sodB</i> Regulation in <i>H. pylori</i> Strains G27 and 26695.

<p>WT and Δ<i>fur</i> strains of G27 and 26695 were grown to exponential (A) and stationary (B) phase in iron replete and iron-limited (growth) media (60 µM dpp). After growth overnight, one-half of the exponential phase, iron replete culture was removed for RNA isolation. 200 µM dpp (final concentration) was added to create an iron-depletion shock condition to the remaining half of the iron replete cultures, and those cultures were grown for an additional hour prior to RNA isolation. The same procedure was applied the following day to the iron replete, stationary phase culture. After overnight growth, one-half of the iron-limited growth culture was removed for RNA isolation in exponential phase while the remaining half was allowed to grow into stationary phase, and RNA was isolated the following day. RNase Protection Assays (RPAs) were performed on RNA isolated from these strains using <i>sodB</i>, <i>pfr</i>, and <i>amiE</i> riboprobes. Data for Exponential phase cultures are shown in Panel A, and data for Stationary phase cultures are shown in Panel B. Fold-changes are indicated below each pair and were calculated by comparing either the relative amount of protected riboprobe in the iron-depletion shock environment (S) or the relative amount of protected riboprobe in the iron limited growth environment (G) to the iron replete lane (N). These data are representative of multiple independent experiments.</p

Fur binding to the <i>sodB</i> promoters.

<p>EMSAs were performed by incubating various concentrations of purified Fur with radiolabeled fragments of the WT G27, “−5 bp swap,” and WT 26695 <i>sodB</i> promoters as well as the negative control promoter, <i>rpoB</i>, as detailed in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005369#s2" target="_blank">Materials and Methods</a>. In the first four lanes, the Fur concentrations are indicated by the triangle from highest to lowest and range from 1.07 µg/mL to 0.026 µg/mL. A no protein control for each promoter is found in the fifth lanes. The last lane shows the 100× cold (unlabeled) competition control for each promoter fragment, which were each performed with the highest concentration of Fur (1.07 µg/mL). Fur exhibits specific interaction with each of the <i>sodB</i> promoters, and no interaction with the <i>rpoB</i> promoter except for very little non-specific binding at the highest Fur concentration. These data are representative of multiple independent EMSA experiments.</p

Alignments of Fur and of the <i>sodB</i> promoters.

<p>Panel A contains the alignment of the predicted Fur amino acid sequences of G27 and 26695. As indicated by an arrow, amino acid 150 is different between the two strains. Panel B contains the <i>sodB</i> promoter alignment from G27, 26695, J99, and HPAG1 with essential promoter elements indicated. The predicted Fur Box ranges from bases −5 to −47 and is indicated by the dashed box <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005369#pone.0005369-Ernst1" target="_blank">[1]</a>. The −5 bp difference between the strains is indicated with an arrow in Panel B. Alignments for both panels were constructed using MultAlin software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005369#pone.0005369-Corpet1" target="_blank">[37]</a>.</p

Primers used in this study.

a<p>Restriction endonuclease sites are underlined, and linker bases are in bold type.</p>b<p>Important restriction sites are included in parentheses.</p

Strain specific differences in <i>sodB</i> regulation.

<p>Various <i>H. pylori</i> strains were grown to exponential phase as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005369#s2" target="_blank">Materials and Methods</a>, and RNA was isolated from iron replete and iron-depleted shock conditions. RPAs were performed using <i>sodB</i>, <i>pfr</i>, and <i>fur</i> riboprobes and results are displayed in Panels A, B, and D, respectively. Basal levels of <i>fur</i> expression relative to the level of expression in 26695 are depicted in Panel C. Fold decrease in expression for <i>sodB</i> and <i>pfr</i>, fold increase for <i>fur</i>, and relative levels of basal <i>fur</i> expression are plotted as single points for each strain with squares, diamonds, triangles, and circles. Each shape represents a biologically independent set of RNA. Median fold change is represented as a bar for each strain. The dotted-dashed line represents the 2-fold significance cut-off in Panels A, B, and D. In Panel A only, the triangles represent the average of two technical repeats on that independent set of RNA.</p

Competitive Binding Studies.

<p>To assess the relative affinity of Fur for each of the <i>sodB</i> promoter fragments (WT G27, “−5 bp swap,” and WT 26695), Fur was incubated with each radiolabeled promoter and 5×, 10×, or 25× the amount of homologous or heterologous unlabeled <i>sodB</i> promoter fragments as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005369#s2" target="_blank">Materials and Methods</a>. For each labeled promoter, lane one contains a no competition control. Lanes two to four, five to seven, and eight to ten contain the competition EMSAs with unlabeled WT G27, “−5 bp swap,” and WT 26695 <i>sodB</i> fragments, respectively. The percent of labeled promoter that is outcompeted and remains unbound in each lane is given below each image. These data are representative of multiple independent experiments.</p