Regulatory role of the MisR/S two-component system in hemoglobin utilization in Neisseria meningitidis

Abstract

Outer membrane iron receptors are some of the major surface entities that are critical for meningococcal pathogenesis. The gene encoding the meningococcal hemoglobin receptor, HmbR, is both independently transcribed and transcriptionally linked to the upstream gene hemO, which encodes a heme oxygenase. The MisR/S two-component system was previously determined to regulate hmbR transcription, and its hemO and hmbR regulatory mechanisms were characterized further here. The expression of hemO and hmbR was downregulated in misR/S mutants under both iron-replete and iron-restricted conditions, and the downregulation could be reversed by complementation. No significant changes in expression of other iron receptors were detected, suggesting that the MisR/S system specifically regulates hmbR. When hemoglobin was the sole iron source, growth defects were detected in the mutants. Primer extension analysis identified a promoter upstream of the hemO-associated Correia element (CE) and another promoter at the proximal end of CE, and processed transcripts previously identified for other cotranscribed CEs were also detected, suggesting that there may be posttranscriptional regulation. MisR directly interacts with sequences upstream of the CE and upstream of the hmbR Fur binding site and thus independently regulates hemO and hmbR. Analysis of transcriptional reporters of hemO and hmbR further demonstrated the positive role of the MisR/S system and showed that the transcription of hmbR initiated from hemO was significantly reduced. A comparison of the effects of the misS mutation under iron-replete and iron-depleted conditions suggested that activation by the MisR/S system and iron-mediated repression by Fur act independently. Thus, the expression of hemO and hmbR is coordinately controlled by multiple independent regulatory mechanisms, including the MisR/S two-component system.

FIG. 1.

qRT-PCR determination of relative transcriptional changes for hmbR (A), hemO (B), and tbpB and lbpB (C) in the misR/S mutants. Total RNAs were isolated from mid-log-phase cultures that were treated (gray bars) or not treated (black bars) with 100 μM dipyridyl for 45 min. The relative transcriptional differences between the mutants and the wild-type strain were calculated by the method (30), using the transcriptional level of the wild-type strain under iron-rich conditions as the calibrator. Each qRT-PCR was performed in triplicate and was repeated with at least two independent RNA preparations. The asterisks indicate statistically significant differences between the wild-type strain and the mutant as determined by the Student t test with a two-tailed distribution (P < 0.01). WT, wild type; comp., complemented.

FIG. 2.

(A) Organization of the hemO-hmbR locus in meningococcal strain NMB. The hemO gene is transcribed divergently from the pqiA (hmp) gene with a 286-bp intergenic space that includes a full-length Correia element (CE) (6), while hemO is separated from hmbR by a 191-bp intergenic region. The bent arrows represent the promoters of hemO and hmbR. The schematic diagram is not to scale. (B) Sequence of the 5′ region of hemO. The ATG start codons are in bold type and underlined, and the large arrows show the direction of transcription. The sequence of the CE is in gray type, the terminal inverted repeats (TIR) are indicated by dotted arrows, and the IHF binding sequence in the CE is underlined, with the nucleotides matching the consensus sequence nucleotides (7) in bold type. The putative transcript processing sites in the TIRs are indicated by bold type and labeled d and p. The putative Fur binding motif matching the (NATWAT)₃ motif is indicated by an arrow. The sequence protected by MisR in the DNase I protection assay is in bold type and shaded, and the sequences matching the MisR binding motif are indicated by double overlining. The 5′ ends of two hemO::lacZ fusions are indicated by bent arrows, and the hemO-pR1-ERI primer is the corresponding 3′ end. The hemO-pF1-ERI primer used to generate probes for DNase I protection assay and the hemO-PE3 primer used in primer extension are indicated by dashed arrows. The two transcriptional start sites and the two corresponding promoter elements are enclosed in boxes. (C) Nucleotide sequence of the hmbR promoter region. The stop codon of hemO and the start codon of hmbR are in bold type and underlined. The putative −10 and −35 hexamers are in bold type and enclosed in boxes. The sequence protected by MisR is shaded, and the two matching MisR consensus motifs are indicated by double underlining. The Fur-protected sequence reported by Delany et al. (10) is indicated by a dashed line. The primers used for the EMSA and DNase I protection assay, YT173 and hmbR-pR, are indicated by dashed arrows. The dotted bent arrows indicate the 3′ ends of promoter fragments cloned in reporter strains O305, O501, and R1203.

FIG. 3.

Hemoglobin utilization assay. Bacteria grown overnight on appropriate selection plates were collected and resuspended at an OD₆₀₀ of 0.1 in GC broth. Aliquots (100 μl) were plated onto GCB agar plates containing 100 μM Desferal. Filter paper disks were placed on the plates and then soaked with 10 μl of a human hemoglobin solution (1, 3, and 5 mg/ml [1 Hb, 3 Hb, and 5 Hb respectively]) or a ferric nitrate solution (5 mg/ml [5 Fe⁺³]) as a control. The zones of growth around disks were measured after 48 h of incubation at 37°C. The results for one representative of six independent experiments are shown. The growth curves for all of the strains tested in standard GC broth are shown on the right. (A) hmbR^off hpuA^off background; (B) hmbR^off hpuB::erm background; (C) hmbR^on hpuB::erm background. NMB, IR3261, and IR3287 are wild-type parental strains. SZT1001, SZT1004, and SZT1009 are misR::aphA-3 mutants. SZT1002, SZT1005, and SZT1010 are misS::aphA-3 mutants. SZT1003 and SZT1006 are misRS::aphA-3 double mutants.

FIG. 4.

(A) Primer extension analysis of hemO using the hemO-PE3 primer (A) and hemO-pR1-ERI primer (B). Lanes G, A, T, and C contained the dideoxy sequencing reaction mixtures. The asterisks indicate the transcriptional start sites, while the mRNA processing sites (d site and p site) as described by Mazzone et al. are indicated by arrows.

FIG. 5.

(A) Competition EMSA experiments with the hmbR promoter and MisR. A 326-bp hmbR fragment (YT173-hmbR-pLR) was end labeled with [γ-³²P]ATP using T4 kinase and incubated with the MisR protein as described previously (52). Lane 1, DNA probe; lanes 2 to 6, DNA probe with MisR protein (77 pmol, 5 μM) (lane 2, no DNA competitor; lanes 3 and 4, probe with 1.5 and 2 μg specific DNA competitor, respectively; lanes 5 and 6, probe with 1.5 and 2 μg nonspecific DNA competitor, respectively). (B) (Left panel) Dose-dependent shift of the hemO promoter with MisR. Lanes 1 and 5, free probe; lanes 2 to 4, 0.6, 2.6, and 3.8 μM MisR protein, respectively. (Right panel) Competition EMSA with the hemO promoter. Lane 6, free probe; lanes 7 to 9, DNA probe with MisR protein (38 pmol, 2.5 μM). One microgram of cold specific DNA was added to lane 8, while 1 μg of nonspecific DNA was added to lane 9. (C and D) DNase I protection assays with the hemO promoter (C) and the hmbR promoter (D). The noncoding strand of the promoter fragment was end labeled with ³²P as described in Materials and Methods, incubated with increasing amounts of MisR for 20 min at 30°C, and then subjected to DNase I digestion in a 20-μl (total volume) reaction mixture. MisR∼P was prepared by incubation with acetyl phosphate (50 mM) at 37°C for 30 min. For hemO, the amounts of MisR tested were 0, 93, 186, 232, and 278 pmol and the total amounts of MisR∼P tested were 0, 93, 139, 232, and 371 pmol. For hmbR, the amounts of MisR tested were 0, 170, 340, 510, and 850 pmol. The results for dideoxy chain termination sequencing ladders corresponding to the probes are shown on the left (lanes A, T, G, and C). The solid line indicates the sequence protected by MisR, while the Fur protected region determined by Delany et al. (10) is indicated by a dotted line.

FIG. 6.

(A) Schematic diagram of the hemO-hmbR locus. The MisR binding sites are indicated by vertical lines with circles at the ends, while the Fur binding sites are indicated by vertical lines with diamonds at the ends. The location of clustered repeats is indicated by a gray vertical line. The BamHI (Bm) and HincII (Hc) sites used for inserting the lacZ-erm cassette to generate the hemO::lacZ (O101) and hmbR::lacZ (R103) fusions, respectively, at the native locus are also indicated. The DNA fragments used to generate transcriptional lacZ reporter fusions at a permissive ectopic genomic location are indicated by black arrows. The schematic diagram is not drawn to scale. (B) Promoter activities of hemO::lacZ (O1101 and O2002) and hmbR::lacZ (GM101 and R1203) reporter strains integrated into a permissive chromosomal locus or the native locus (O101 and R103). Black bars, wild-type background; gray bars, misS background. (C) Comparison of reporter activities with various promoters. Black bars, wild-type background; gray bars, misS background. (D) Differential activation of hemO::lacZ (O1101 and O101) and hmbR::lacZ (R1203 and R103) fusions by iron limitation. Induction ratios were determined by dividing the promoter activity of the iron-depleted culture by the promoter activity of the iron-replete culture. Black bars, wild-type background; gray bars, misS background. (Inset) Alignment of the hemO Fur box and the best hmbR Fur box with the Fur box consensus sequence. Mismatched nucleotides are underlined and in bold type.