OmpR-Mediated Transcriptional Regulation and Function of Two Heme Receptor Proteins of Yersinia enterocolitica Bio-Serotype 2/O:9

Abstract

We show that Yersinia enterocolitica strain Ye9 (bio-serotype 2/O:9) utilizes heme-containing molecules as an iron source. The Ye9 genome contains two multigenic clusters, hemPRSTUV-1 and hemPRST-2, encoding putative heme receptors HemR1 and HemR2, that share 62% amino acid identity. Expression of these proteins in an Escherichia coli mutant defective in heme biosynthesis allowed this strain to use hemin and hemoglobin as a source of porphyrin. The hemPRSTUV-1 and hemPRST-2 clusters are organized as operons, expressed from the p_hem-1 and weaker p_hem-2 promoters, respectively. Expression of both operons is negatively regulated by iron and the iron-responsive transcriptional repressor Fur. In addition, OmpR, the response regulator of two component system (TCSs) EnvZ/OmpR, represses transcription of both operons through interaction with binding sequences overlapping the -35 region of their promoters. Western blot analysis of the level of HemR1 in ompR, fur, and ompRfur mutants, showed an additive effect of these mutations, indicating that OmpR may regulate HemR expression independently of Fur. However, the effect of OmpR on the activity of the p_hem-1 promoter and on HemR1 production was observed in both iron-depleted and iron-replete conditions, i.e., when Fur represses the iron-regulated promoter. In addition, a hairpin RNA thermometer, composed of four uracil residues (FourU) that pair with the ribosome-binding site in the 5'-untranslated region (5'-UTR) of hemR1 was predicted by in silico analysis. However, thermoregulated expression of HemR1 could not be demonstrated. Taken together, these data suggest that Fur and OmpR control iron/heme acquisition via a complex mechanism based on negative regulation of hemR1 and hemR2 at the transcriptional level. This interplay could fine-tune the level of heme receptor proteins to allow Y. enterocolitica to fulfill its iron/heme requirements without over-accumulation, which might be important for pathogenic growth within human hosts.

Keywords: Fur; HemR1; HemR2; OmpR; Yersinia enterocolitica.

Figure 1

Y. enterocolitica Ye9 siderophore production and heme-containing molecule utilization. (A) Siderophore activity was assessed using CAS agar plates. No color change is seen on the plate carrying growth of Ye9. In contrast, a change in color from blue to orange is observed when iron is removed from the iron-CAS complex by the siderophore yersiniabactin produced by JB580v cells. (B) The growth yield of Ye9 determined by measuring the OD₆₀₀ after 28-h incubation at 26 or 37°C in LB, LB with 10 μM FeCl₃, LB with 100, 150, 200, or 300 μM 2,2′-dipyridyl (DPD). (C) The growth yield of Ye9 in LBD medium (150 μM DPD) containing hemin (He, 10 μM) or hemoglobin (Hb, 2.5 μM). The graphs present the results of one of two separate experiments performed in triplicate.

Figure 2

Organization of two heme uptake loci of Y. enterocolitica Ye9 and comparison of the encoded HemR homologs. (A) Genetic maps of heme transport loci 1 and 2. (B) Alignment of HemR1 and HemR2. The conserved histidine residues are in bold, and the TonB box and region V characteristic of all TonB-dependent OM proteins are boxed. Conserved amino acids are indicated by asterisks.

Figure 3

sqRT-PCR analysis of the hemPRSTUV-1 and hemPRST-2 gene clusters of Y. enterocolitica Ye9. (A,C) Scheme showing primers used in sqRT-PCR analysis of hem clusters 1 and 2. The sequences of the following primers are listed in Table S2. 1, RThPR1F; 2, RThPR1R; 3, RThRS1F; 4, RThRS1R; 5, RThPV1R; 6, RThPR2F; 7, RThPR2R; 8, RThRS2F; 9, RThRS2R; 10, RThPT2R. (B,D) Agarose gel electrophoresis of sqRT-PCR products from hem clusters 1 and 2. Total RNA isolated from strain Ye9 grown in iron-depleted LBD medium (LB with the iron chelator 2,2′-dipyridyl) at 37°C was DNase I treated and then reverse transcribed with random hexamers. The obtained cDNA was used as a template in PCR reaction with pairs of primers shown in (A) and (C). RNA was used as the template in negative control reactions. M, GeneRuler 1 kb DNA Ladder.

Figure 4

Growth promotion of E. coli ΔhemA strain SASX77 expressing receptors HemR1 or HemR2 in the presence of hemin and hemoglobin as porphyrin sources. (A) Ability of the E. coli ΔhemA strain expressing HemR1 or HemR2 to utilize hemin and hemoglobin. Diluted overnight cultures of SASX77/pHEM1 and SASX77/pHEM2 were mixed with agarose and overlaid on LB or LBD agar plates. Sterile paper discs wetted with 10 μl of hemin (10 mM) or hemoglobin (0.1 mM) were placed on these plates and zones of growth around these discs were analyzed after 1–4 days of incubation at 37°C. (B) Immunodetection of HemR1 in Y. enterocolitica Ye9N (wt) and E. coli SASX77 carrying plasmids pHEM1 (HemR⁺), pHEM2 (HemR2⁺), or pACYC184. Cell extracts from overnight cultures were analyzed by Western blotting with a polyclonal antibody directed against HemR1 (upper) and by TGX Stain free (lower, loading control). MW, 3-Color Prestained Protein Marker; kDa. This result is representative of at least three independent experiments.

Figure 5

OmpR-dependent regulation of hem-1 and hem-2 operon expression. (A) Activity of the p_hem−1 and p_hem−2 promoters in E. coli BW25113 harboring the p_hem−1::lacZ (pCM1) or p_hem−2::lacZ (pCM2) fusion plasmids, compared with the same strain carrying parent vector pCM132. Strains were grown overnight in LB+FeCl₃ or LBD medium at 37°C. (B,C) OmpR-dependent regulation of hem-1 and hem-2 expression in Y. enterocolitica strains grown to stationary phase in LB+FeCl₃ or LBD medium at 26 or 37°C. (B) The wild-type Ye9N (wt), wt/pBOmpR, AR4 (ompR), and ompR/pBOmpR, harboring fusion p_hem−1::lacZ (pCM1). (C) The wild-type Ye9N (wt) and wt/pBOmpR harboring fusion p_hem−2::lacZ (pCM2). Plasmid pBOmpR carries the wild-type ompR allele. β-galactosidase activity was determined for each culture and expressed in Miller units. The data represent mean activity values with standard deviation from two independent experiments, each performed using at least triplicate cultures of each strain. Significance was calculated using one-way ANOVA [ns (non-significant) P > 0.05, ^*P < 0.05, ^****P < 0.00001].

Figure 6

Effect of iron, Fur, and OmpR on HemR1 production. The level of HemR1 in cell extracts of strains grown overnight in LB+FeCl₃ or LBD at 37°C analyzed by immunoblotting with a polyclonal HemR1 antibody. (A,B) The HemR1 level in the wild-type strain (wt), wt/pBBR1 (empty vector), wt/pBOmR (overexpression), the ompR mutant AR4, ompR/pBOmpR (complementation), and ompR/pBBR1 (empty vector) shown on Western blots (upper) with the stained gels as a loading control (bottom). (C) The HemR1 level in the fur mutant background, i.e., fur mutant, furompR double mutant, and furompR/pBOmpR. The percentage values indicating the HemR1 band intensities in the tested strains relative to the wt (B) and fur mutant (C), were determined using Amersham Imager 600 Analysis Software V1.0.0 (GE Healthcare). Stained gels shown as a loading control: (A,C) TGX Stain-Free gels (BioRad), (B) Coomassie blue stained gel. MW, 3-Color Prestained Protein Marker, kDa. The size of the HemR1 band is approximately 75 kDa on the Western blots. This result is representative of at least three independent experiments.

Figure 7

OmpR binding to fragments of the hem-1 and hem-2 promoter regions. (A,B) OmpR- and Fur-binding sites identified in promoter regions of the two heme uptake loci. The −35 and −10 promoter elements are underlined and putative regulator-binding sites are boxed. The ATG start codon of the first ORF of each operon is shown in bold. FBS, Fur-binding site; OBS, OmpR-binding site. (C) Schematic representation of fragments used in EMSA experiments presented in (D) and (E). The numbered arrows represent the primers used to amplify the DNA fragments used in EMSAs: 1, hem1F; 2, hem1R, 3, hem1-aF; 4, hem1-aR; 5, hem1-bF; 6, hem2F; 7, hem2R. (D) EMSA with fragment F2 (326 bp) containing OBS-1 and neighboring fragments F1 (415 bp, upstream) and F3 (107 bp, downstream). A 16S rDNA fragment (211 bp) was used as a negative control. The reaction mixtures contained 0.05 pmol of each fragment and increasing amounts of purified OmpR-His₆ were added: lane 1, no protein; lanes 2–5, 0.65, 1.31, 2.61, and 3.92 μM of OmpR-His₆, respectively. (E) Binding of purified OmpR-His₆ to fragments containing OBS-1 (F2, lanes 1–4) and OBS-2 (F4, lanes 5–8). The 16S rDNA fragment was used as a negative control. The reaction mixtures contained 0.05 pmol of each fragment and increasing amounts of purified OmpR-His₆ were added: lanes 1 and 5, no protein; lanes 2 and 6, 0.65 μM; lanes 3 and 7, 1.31 μM; lanes 4 and 8, 3.92 μM.

Figure 8

Influence of temperature on hemR1 expression. (A) Potential FourU thermometer RNA secondary structures in the 5′-UTRs of Y. enterocolitica Ye9 hemR1 and S. dysenteriae shuA, composed of a stretch of FourU and the ribosomal binding site (RBS), revealed by Mfold analysis (http://mfold.rna.albany.edu). Boxes indicate the location of the FourU motif (shaded) and putative RBS, the translation start codon is marked red, and unpaired nucleotides within the predicted secondary structure are in green. (B) Temperature-dependent HemR1 expression in Y. enterocolitica examined by immunoblotting. The analyzed samples were cell lysates of the fur mutant grown at 26 or 37°C in LB medium. The top panel shows the Western blot probed with a polyclonal antibody against HemR1, the bottom panel shows the Coomassie blue-stained gel as a loading control. The percentage values on the blot, indicating the HemR1 band intensities relative to that of cells grown at 26°C, were determined using Amersham Imager 600 Analysis Software V1.0.0 (GE Healthcare). (C) Expression of a hemR1′-′gfp translational fusion at different temperatures, monitored by fluorescence intensity. E. coli BW25113 harboring the reporter plasmid pFX-P_lac-hemR1 or control plasmids pFX-0 and pFX-1 were grown in LB medium at 26 or 37°C. The GFP fluorescence intensity (RFU) of overnight cultures was determined. The data represent the averages ± SD from at least three experiments with duplicate cultures. Significance was calculated using one-way ANOVA [P > 0.05, ^*P < 0.05, ^***P < 0.001]. (D) GFP abundance examined by immunoblotting. The analyzed samples were cell lysates of E. coli BW25113 harboring the plasmids pFX-0, pFX-1 or pFX-P_lac-hemR1. The top panel shows the immunoblot probed with antibody against GFP, the bottom panel shows the TGX Stain-Free gel as a loading control. MW, 3-Color Prestained Protein Marker; kDa. These results are representative of at least three independent experiments.