Regulation of iron transport in Streptococcus pneumoniae by RitR, an orphan response regulator

Abstract

RitR (formerly RR489) is an orphan two-component signal transduction response regulator in Streptococcus pneumoniae that has been shown to be required for lung pathogenicity. In the present study, by using the rough strain R800, inactivation of the orphan response regulator gene ritR by allele replacement reduced pathogenicity in a cyclophosphamide-treated mouse lung model but not in a thigh model, suggesting a role for RitR in regulation of tissue-specific virulence factors. Analysis of changes in genome-wide transcript mRNA levels associated with the inactivation of ritR compared to wild-type cells was performed by the use of high-density DNA microarrays. Genes with a change in transcript abundance associated with inactivation of ritR included piuB, encoding an Fe permease subunit, and piuA, encoding an Fe carrier-binding protein. In addition, a dpr ortholog, encoding an H(2)O(2) resistance protein that has been shown to reduce synthesis of reactive oxygen intermediates, was activated in the wild-type (ritR(+)) strain. Microarray experiments suggested that RitR represses Fe uptake in vitro by negatively regulating the Piu hemin-iron transport system. Footprinting experiments confirmed site-specific DNA-binding activity for RitR and identified three binding sites that partly overlap the +1 site for transcription initiation upstream of piuB. Transcripts belonging to other gene categories found to be differentially expressed in our array studies include those associated with (i) H(2)O(2) resistance, (ii) repair of DNA damage, (iii) sugar transport and capsule biosynthesis, and (iv) two-component signal transduction elements. These observations suggest that RitR is an important response regulator whose primary role is to maintain iron homeostasis in S. pneumoniae. The name ritR (repressor of iron transport) for the orphan response regulator gene, rr489, is proposed.

FIG. 1.

Tissue selectivity of S. pneumoniae ΔritR for infectivity. S. pneumoniae RU402 (ΔritR, allele replacement mutant of R800) and R800 (ritR⁺ R6 derivative) were compared with respect to the ability to infect the mouse lung or thigh. RU402 infected the thigh but not the lung (A), whereas R800 infected both the lung and the thigh (B).

FIG. 2.

Timing of ritR mRNA synthesis during the growth of S. pneumoniae. A culture of S. pneumoniae CPU401 containing a promoterless E. coli LacZ reporter inserted into the ritR open reading frame was sampled as indicated for determination of turbidity, measured at 600 nm (A600), and for determination of E. coli LacZ activity, measured fluorimetrically at 480 nm (F480) with methylumbelliferyl galactoside as a LacZ substrate. The normalized specific activity of LacZ is plotted as F480/A600. The ritR promoter activity reached the maximal value during exponential growth and decreased as cells entered the stationary phase. To purify RNA for microarray studies, cells were harvested at an A₆₀₀ of 0.4, as indicated by the arrow, which corresponded to the time at which the ritR mRNA initially reached the maximum level.

FIG. 3.

Scatter plot analysis of the array data. The normalized average fluorescence signal for RU402 on the y axis is plotted versus the normalized average fluorescence signal for R800 on the x axis. Genes that were significantly differentially expressed (PDE, ≥0.5) appear as off-diagonal blue squares, whereas genes that were differentially expressed and had a PDE of <0.5 are represented by red squares. Genes above the diagonal were repressed in the presence of ritR, whereas genes below the diagonal were activated.

FIG. 4.

Northern blot analysis of ritR and neighboring genes. The RNA fraction from S. pneumoniae R800 (ritR⁺) contains a 2.2-kb transcript that hybridizes with either gnd or ritR but not with cbpF probes, indicating that gnd and ritR are cotranscribed independent of the 1-kb cbpF transcript located immediately downstream.

FIG. 5.

Northern blot analysis of S. pneumoniae mRNA. (A) mRNA levels for ritR (lanes 1 and 2), piuB (lanes 3 and 4), piuA (lanes 5 and 6), and dpr (lanes 7 and 8). ritR mRNA levels in S. pneumoniae R800 ritR⁺ (+) and RU402 ΔritR (Δ) were compared. ritR mRNA is absent in ΔritR strain RU402 (lane 2). Repression of puiB and piuA transcription in R800 and the reciprocal relationship between piuA and piuB with respect to dpr are shown. (B) mRNA levels were measured as described above for panel A, except that RNA samples from S. pneumoniae RU403 (lower-activity tet promoter-driven ritR) and RU404 (higher-activity tet promoter-driven ritR), as a function of added anhydrotetracycline, were used. Note the dependence of ritR mRNA abundance and the corresponding repression of piuA on anhydrotetracycline. VncS, whose function is unknown, was also tested and appeared to be strongly dependent on ritR expression compared to the wild-type (wt) control (R800) with no anhydrotetracycline. Lanes 1, 2, and 3 contained RNA extracted from RU403 cells treated with 0, 100, and 300 ng of anhydrotetracycline per ml. Lanes 4, 5, and 6 contained RNA extracted from RU404 cells treated with 0, 100, and 300 ng of anhydrotetracycline per ml. (C) Northern blot analysis of RNA from S. pneumoniae R800 (ritR⁺) and RU402 (ΔritR) with probes specific for piuB and piuA. Probes for piuB and piuA were obtained by PCR with primer pairs 8F-8R and 9F-9R, respectively (Table 2). Inactivation of ritR led to overexpression of the piuBCDA operon, as shown in lanes 2 and 3. The 4-kb fragment is the fragment expected for the full-length piuBCDA mRNA. Faster-moving fragments are presumed to represent degradation products of the piuBCDA message. Except for a 300-bp fragment (lane 1), no hybridization was detected in the RNA obtained from R800 cells, indicating the extent of repression of piuBCDA mRNA by ritR. The 300-bp RNA may be a regulatory RNA, and its origin is unknown. kD, kilodaltons.

FIG. 6.

Susceptibility to streptonigrin and H₂O₂ treatment: ritR⁺ versus ΔritR. (A) S. pneumoniae R800 (ritR⁺) cells were preincubated in the presence or absence of 50 μM iron, followed by addition of 2.5 μg of streptonigrin/ml. The number of viable cells varied little in response to iron in any of the four forms tested (Fe²⁺, Fe³⁺, hemin, and hemoglobin). The data indicate the log percentage of surviving cells compared to the no-iron (No addn) control. Hgb, hemoglobin. (B) The conditions used were the same as those described above for panel A, except that S. pneumoniae RU402 (ΔritR) cells were used. Losses of viability were seen for addition of Fe²⁺ (5 orders of magnitude) and for addition of hemin (3.5 orders of magnitude), whereas Fe³⁺ or hemoglobin had no detectable effect. (C) Effect of hydrogen peroxide on survival of S. pneumoniae. The number of surviving CFU in the culture was determined for S. pneumoniae R800 (ritR⁺) or RU402 (ΔritR) challenged with 40 mM H₂O₂ for various times.

FIG. 7.

Characterization of the piu promoter transcription start site, gel retardation analysis, and RitR footprint. (A) The transcription start site for the piuBCDA message was determined by reverse transcriptase (RT) mapping by using total RNA extracted from S. pneumoniae RU402 (ΔritR). The results indicate that the piuB message starts with G. (B) Gel retardation analysis of the piuB promoter. A 217-nucleotide DNA sequence containing the piu promoter region in ³²P-labeled form was incubated with different concentrations of GST-RitR, and the resultant complexes were fractionated by PAGE. Lanes 1 to 10 contained 0, 0.030, 0.060, 0.125, 0.25, 0.50, 1.0, 2.0, 4.0, and 8.0 μM GST-RitR, respectively. The first appearance of retardation occurred at 0.060 μM GST-RitR (lane 3). P, probe; C, complex. (C) Footprint analysis of RitR bound to the piuB promoter. Two preparations of the piuB promoter region were obtained by PCR, each labeled with ³²P at one 5′ end. GST-RitR was bound to each of the two PCR products. Following incubation with DNase I and PAGE fractionation of the resultant fragments, the footprints were visualized by autoradiography. +P, RitR preincubated with 50 mM acetyl phosphate; −P, no acetyl phosphate added; G/A, G/A ladder. Three distinct binding sites for RitR are labeled. The relationship of the RitR DNA-binding regions to the piu promoter DNA sequence is shown in Fig. 8A.

FIG. 8.

Annotated DNA sequence of the piuB promoter region. (A) DNA sequence of the piuB promoter region with the transcriptional regulatory elements annotated as follows: −35, −10, +1 (start of transcription), RBS (ribosome-binding site for translation initiation), and the first 14 amino acids of PiuB. Three binding sites for RitR and their relationship to the piu promoter are underlined and labeled #1, #2, and #3. (B) Alignment of the three binding site sequences, showing the resultant consensus that was used as a search key to locate additional similar sequences in the S. pneumoniae genome.