MisR/MisS Two-Component Regulon in Neisseria meningitidis

Bacterial strains, medium, and reagents.The strains and plasmids used in this study are listed in Table 1, while the primers used in this study are listed in Table S3 in the supplemental material. Meningococcal strain NMB (CDC 8201085) is a serogroup B N. meningitidis strain originally isolated from the cerebrospinal fluid of a patient with meningococcal meningitis in Pennsylvania in 1982. Meningococcal strains were grown with 3.5% CO₂ at 37°C unless specified otherwise. Gonococcal (GC) base agar (Difco) supplemented with 0.4% glucose and 0.68 mM Fe(NO₃)₃ or GC broth with the same supplements and 0.043% NaHCO₃ was used. Brain heart infusion medium (37 g/liter brain heart infusion) with 1.25% fetal bovine serum was used when kanamycin selection was required. The antibiotic concentrations (in μg/ml) used for Escherichia coli strains were as follows: ampicillin, 100; kanamycin, 50; and erythromycin, 300. Those for N. meningitidis strains were as follows: kanamycin, 80; and erythromycin, 3. E. coli strain DH5α cultured on Luria-Bertani (LB) medium was used for the cloning and propagation of plasmids. Meningococci were transformed by the procedure of Janik et al. (20). E. coli strains were transformed by electroporation with GenePulser (Bio-Rad) according to the manufacturer's protocol.

Construction of the misRS double mutant and complementation of the misR mutation.A 737-bp PCR product containing the 5′ region of misR and another 684-bp PCR product containing the 3′ region of misS were amplified separately using primer pairs YT174HindIII-YT175SmaI and YT177SmaI-YT178EcoRI. Two PCR products were digested with their respective restriction enzymes and then ligated with HindIII-EcoRI-restricted pUC18, yielding pYT334. The correct plasmid carrying the newly created SmaI site was confirmed by restriction digestion and sequencing analysis. The aphA-3 cassette released from pUC18K by EcoRI-BamHI digestion and Klenow treatment was inserted into the SmaI site of pYT334, yielding pYT336. Removal of the misR and misS internal sequence and the presence of a correctly oriented aphA-3 cassette in the resulting pYT336 plasmid were confirmed by a panel of colony PCRs and sequencing analysis. This construct removed most of the misR and misS sequences and contained the kanamycin resistance gene. To generate the meningococcal misRS mutant, pYT336 was linearized with ScaI and the digestion mixture was used to transform the meningococcal strains. Kanamycin-resistant colonies were selected, and the mutation was confirmed as described above.

The pJKD2581 plasmid (C. M. Kahler and J. Davies, unpublished data), which contains the Hermes-inducible expression cassette (27) with the spectinomycin marker flanked by iga sequences on a low-copy suicide vector with a pSC101 origin, was used to complement the misR mutation. The coding sequence of misR was amplified using primers cpxR5B and cpxR7B containing flanking BamHI sites and cloned into the HincII site of pWSK29 (61) to create pJKD2582. Both pJKD2582 and pJKD2581 were digested with BamHI and ligated, and chloramphenicol-resistant transformants, named pJKD2583, were selected for further analysis. The pJKD2583 plasmid contains the misR gene under the control of the P_trc promoter. MscI-linearized pJKD2583 was used to transform meningococcal strain NMB, and spectinomycin-resistant transformants were isolated. A panel of PCR amplifications was performed on the resulting strain, CMK30, to confirm integration of the Hermes-misR expression cassette into iga: DAP139 and DAP119 for the left flank, DAP227 and DAP120 for the right flank, and DAP486 and DAP276 for the internal part of the cassette. Due to the difficulty of transforming the misR mutant, the misR::aphA-3 mutation was introduced into CMK30 and Sp^r/Km^r transformants were selected. A panel of PCRs was done to ensure the presence of the misR::aphA-3 mutation at the wild-type locus and that the second copy of the intact misR gene remained at the iga locus.

Microarray.Bacterial strains grown in supplemented GC broth to mid-exponential phase were collected, and the total RNA was extracted using an RNeasy midi kit (Qiagen) according to the protocol recommended by the manufacturer. The RNA samples were further treated with DNase for 1 h at 37°C to remove contaminating chromosomal DNA and then purified through phenol-chloroform extraction and ethanol precipitated. The final RNA preparations were used as templates in standard PCR amplification of an unrelated chromosomal region to ensure the absence of DNA contamination. Reaction mixtures containing 40 μg total RNA and 0.1 pmol of a mixture of 3′-end-specific primers, which were used in generating the PCR products for printing slides (Eurogentec), were first denatured at 70°C for 10 min, and then the primers were allowed to anneal to RNA on ice. Cy3- and Cy5-labeled cDNAs were generated using a CyScribe first-strand cDNA labeling kit (Amersham Pharmacia Biotech) and then purified by a QIAquick PCR purification kit (Qiagen). The percentage of incorporated label was estimated using the following extinction coefficients: DNA (6,000 M⁻¹ cm⁻¹ at an optical density at 260 nm [OD₂₆₀]), Cy3 (150,000 M⁻¹ cm⁻¹ at OD₅₅₀), and Cy5 (250,000 M⁻¹ cm⁻¹ at OD₆₅₀). The labeled cDNAs were then mixed together prior to hybridization to the array slides. A prehybridization step was performed by incubating the microarray slide in a solution of 2.5 μl of salmon sperm DNA (10 mg/ml) diluted in 47.5 μl of Dig Easy solution (Roche) under a coverslip in a humidified chamber (Corning) for 1 h at 42°C. After prehybridization, the slide was rinsed in 0.2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) buffer, water, and then isopropanol and dried by brief low-speed centrifugation. The probes, resuspended to a total of 10 μl, were mixed with 3 μl of sperm DNA (10 mg/ml), and the solution was denatured at 95°C for 2 min and cooled on ice before the addition of 20 μl Dig Easy solution. The hybridization was performed at 42°C overnight. The slide was washed in 0.2× SSC-0.1% sodium dodecyl sulfate for 5 min at 42°C, followed by consecutive rinses in 0.2× SSC-0.1% sodium dodecyl sulfate for 10 min at room temperature (two times) and 0.1× SSC for 5 min at room temperature (two times). The slide was dried by brief low-speed centrifugation and then scanned with a GMS 428 array scanner (Genetic Microsystems). Data analyses were performed with both Jaguar software (Affymetrix) and ImaGene/Genesight software (Biodiscovery), which allow automatic gridding of the array and an attached database for flagging nonoptimal spots that can be removed from later analyses. Measuring the signal intensities detected for both fluorophores and calculating the signal ratio determine relative amounts of a particular gene transcript in the two samples. Signal ratios of fluorescence were generated from each spot and then analyzed by Genesight (BioDiscovery). The standard twofold cutoff was used as a minimum value for induction and repression. The array glass slide (Eurogentec) contains 2,191 open reading frames (ORFs) of the serogroup A strain Z2491 and 73 specific ORFs of the serogroup B strain MC58 that were printed in duplicate. This represents ∼98% of all ORFs of the serogroup A and B genomes. One array analysis with fluorescence labels swapping between test and reference cDNA was done as a technical control, and three independent sets of wild-type and mutant RNA preparations were studied.

Construction of reporter strains and β-galactosidase reporter assays.The promoter fragments were obtained by PCR amplification and cloned into pCR2.1 by using a TOPO-TA cloning kit (Invitrogen). The inserts were released by EcoRI digestion, gel purified, and then ligated with pYT328, which had been digested with EcoRI and treated with shrimp alkaline phosphatase. Colony PCR using primer YT168, an outward primer at the 5′ end of lacZ, and the forward primer within the promoter identified clones with the correct orientation of the promoter to the lacZ gene. The NcoI-linearized plasmid constructs were used to transform the wild-type meningococcal strain NMB and the NMBmisS mutant. A panel of colony PCRs with a chromosome-specific primer and the lacZ-specific primer confirmed allelic exchanges into the expected locus. The wild-type reporter strains were transformed using the plate transformation method (20), with chromosomal DNA isolated from the NMBmisRS mutant to generate reporter strains with the misRS double mutation. Transformants resistant to both erythromycin and kanamycin were selected on brain heart infusion plates and confirmed by colony PCR. The β-galactosidase activity assays were performed as described previously (60).

Protein purification and Western blots.The purification of the MisR-His₆ protein and the Western blot procedure using polyclonal sera against the MisR-His₆ protein have been described previously (60).

EMSA and DNase I protection assay.Procedures for the EMSA and DNase I protection assay have been reported previously (60). Briefly, probes for the EMSA were generated by T4 kinase-mediated γ-³²P end labeling of PCR promoter fragments and purified using the QIAquick PCR purification kit (Qiagen). The binding reactions for mixtures consisting of 5 fmol labeled DNA and various amounts of protein in a binding buffer (20 mM HEPES, pH 7.9, 60 mM KCl, 5 mM MgCl₂, 1 mM EDTA, 1 mM dithiothreitol, 0.3 mg/ml bovine serum albumin, 0.2 mg/ml salmon sperm DNA, and 10% glycerol) were carried out at 30°C for 20 min and analyzed with 6% polyacrylamide gels. The gels were dried under vacuum without prior fixing, and the resulting radioactive electrophoretic patterns were analyzed with a PhosphorImager (Molecular Dynamics). The binding reactions were examined with phosphorylated (50 mM acetyl phosphate for 30 min at 37°C) or nonphosphorylated MisR. Competition with excess specific competitors (unlabeled probes) and nonspecific competitors, a 593-bp internal coding sequence of misR, obtained by PCR amplification using primers YT45 and YT46, was performed to assess the specificity of the interaction.

For the DNase I protection assay, primers were end labeled using T4 polynucleotide kinase and γ-³²P and purified with the QIAquick nucleotide removal kit (Qiagen). PCR fragments of the promoter region were then amplified with the radiolabeled primer and the corresponding reverse primer, and the labeled DNA products were purified with the QIAquick PCR purification kit. Using the binding conditions defined in the EMSA studies, the DNA-protein complex in the binding buffer without salmon sperm DNA was treated with diluted DNase for 1 min at room temperature before being quenched with a phenol-chloroform extraction. Nucleic acids were recovered by ethanol precipitation and analyzed on 6% sequencing gels along with a sequencing ladder obtained with the same labeled primer.

Real-time quantitative RT-PCR assay.Reverse transcriptions were carried out with a GeneAmp kit by using 1 μg of total RNA according to the procedure suggested by the manufacturer (Applied Biosystems), and reactions without the reverse transcriptase were used as a negative control. The double-strand DNA-binding dye Sybr green detection method was employed to quantify the amount of mRNA from the RT reactions, and the assay conditions have been described previously (58). The primers used are listed in Table S3 in the supplemental material. All primer sets have been confirmed to yield similar amplification efficiencies. Melting curve analyses were performed following each RT-PCR experiment to ensure that each reaction mixture contained only a single specific product. rpsE encoding the constitutively and highly expressed meningococcal 30S ribosomal protein was used in each experiment as an endogenous reference gene for normalization. RT negative-control reactions were also analyzed by real-time RT-PCR to confirm that they were free of contaminating chromosomal DNA. The relative changes were calculated using the 2^−ΔΔC_T method (32). Each set of RT-PCRs was examined in triplicate and was repeated with at least two independent RNA preparations. Student's t test with a two-tailed hypothesis was used to determine the significant difference (P < 0.01) between two variables in this study.

Oxidative stress sensitivity assay and serum bactericidal assay.The sensitivities of meningococcal strains to hydrogen peroxide (H₂O₂) and paraquat were determined by disk diffusion growth inhibition assays. Briefly, cells from plate-grown cultures were suspended in GC broth to an OD₆₀₀ of 0.15. Fifty microliters of cell suspension was mixed with 5 ml of GC soft agar (GC broth with 0.5% agar) kept at 45°C, and the mixture was poured over a plate containing 15 ml of solidified GC agar. Disks of 6 mm diameter were placed onto soft agar, and 5-μl aliquots of tested agents were spotted on disks. Plates were incubated at 37°C for 24 h, and zones of growth inhibition were measured. Each condition was tested with three disks, and the assays were repeated three times. The procedure for the serum bactericidal assay has been described previously (24). The expression of Ptrc::misR in the complemented strain was induced by 1 mM IPTG (isopropyl-β-d-thiogalactopyranoside) in broth culture.

Capsule ELISA.Serogroup B capsule-specific immunoglobulin M monoclonal antibody 2-2-B was used in the whole-cell enzyme-linked immunosorbent assay (ELISA) as previously described (59).

Microarray identification of MisR-regulated genes.To assess the scope of MisR regulation, the transcriptome of the mutant was analyzed by a whole-genome microarray. Commercially available meningococcal genomic microarrays (Eurogentec, Belgium) based on the serogroup A genome (39) were used. The serogroup A genome is 91.2% similar to the serogroup B MC58 genome (52). ORFs unique to the MC58 genome that are found within the three major putative islands of horizontally transferred DNA were also included in the array. One of the islands contains the genes encoding the serogroup B capsule biosynthesis and transport proteins, and the other two contain genes encoding mostly hypothetical proteins (52). The total RNA of the wild-type parent strain NMB and the NMBmisR mutant was isolated from cultures grown in standard GC broth. cDNAs were generated using the CyScribe first-strand cDNA labeling kit with a mixture of 3′-end-specific primers. The individually labeled cDNAs were purified, combined, and hybridized with the array slides as described in Materials and Methods. Three independent RNA preparations of both the wild type and the NMBmisR mutant were examined, including one dye swap comparison. Genes that exhibited >2-fold change in two of three microarray experiments were designated as potential regulatory targets of the MisR response regulator (see Tables S1 and S2 in the supplemental material). A total of 78 genes were up-regulated and 39 genes were down-regulated in the NMBmisR mutant. As the mutants displayed increased doubling time compared to that of the wild-type parent strain, which can be corrected when complemented with a second copy of misR (data not shown), transcriptional changes due to the growth difference between the mutant and the wild-type strain were likely to be present in the microarray comparison. However, this pleiotropic phenotype is a likely indication that the regulatory targets of the MisR/S two-component system are involved in important physiological functions of N. meningitidis.

As the microarray results did not differentiate between direct and indirect regulatory roles of MisR, a panel of 25 MisR-regulated genes was selected for further detailed biochemical characterization and could be classified into five important functional categories: protein folding/stress response, regulation, metabolism, iron assimilation, and transport/secretion (Table 2). While 12 genes were down-regulated in the NMBmisR null mutant, indicative of an activator role for MisR, the expression levels of 20 genes were increased. This observation indicated that either MisR has a dual function as a repressor or alteration in the expression of several putative transcriptional regulators results in indirect MisR regulation. However, indirect transcriptional changes mediated by intermediate regulators under the control of misR and those caused by growth rate differences cannot be distinguished.

Characteristics of potential MisR regulatory targets.As shown in Table 2, the group of protein folding/stress response genes included dnaJ, fkpA, hslO, grpE, htpX, clpB, and dsbD. The significant changes observed in the transcription of dsbD and several genes involved in the general stress response may explain the pleiotropic effect of the misR mutation on growth. DsbD is an inner-membrane thiol:disulfide interchange protein that maintains the periplasmic disulfide isomerase DsbC in its active form by using the reducing power from cytosolic thioredoxin (22); thus, the decreased expression of DsbD is expected to result in the accumulation of misfolded proteins in this compartment. ClpB is part of the Clp protease complex, which operates in concert with DnaJ/DnaK/GrpE chaperones to mediate the refolding and degradation of misfolded proteins in the cytoplasmic compartment of the cell. FkpA has homology to the C-terminal domain of the periplasmic FK506 binding protein-type peptidyl-prolyl cis-trans isomerase in E. coli and is transcribed divergently from hprA encoding a glycerate dehydrogenase, also up-regulated in the misR mutant. A gene cluster consisting of fkpA located upstream of eight pilS cassettes was shown to up-regulate (see Table S1 in the supplemental material) in the array analysis. HslO, or Hsp33, belongs to a family of redox-sensing cytoplasmic heat shock proteins (Hsp33) (1, 19) and works with DnaJ/DnaK/GrpE to support the refolding of the substrate proteins (18). HtpX is a membrane-bound zinc metallopeptidase with a cytosolic active site (47).

The next group of genes identified as potential MisR regulons includes three up-regulated putative transcriptional regulators and the misRS operon (60). NMA0738 contains domains that are found in a family of repressor proteins that includes the phage CI repressor and LexA. Interestingly, NMA0738 is near dnaK (NMA0736) and is divergently transcribed from a conserved hypothetical protein, NMA0737, which was also up-regulated. NMA1593 has one Rrf2-type DNA-binding domain and may encode a putative IscR, an iron-sulfur binding transcription factor of the iron-sulfur cluster assembly system, which is located immediately downstream (NMA1594 to NMA1598). However, no changes were detected in the transcription of the iron-sulfur cluster assembly genes. Finally, NMA1751 contain domains that are well conserved in the GntR regulator family. The identification of transcriptional regulators under the control of MisR suggested that MisR can indirectly regulate the expression of additional genes.

The genes prpB, prpC, and acnA, which are clustered and whose products participate in the 2-methylcitric acid cycle, were significantly induced in the misR mutant. Whether these genes constitute an operon was not clear since the expression of NMA2053, located between prpC and acnA, was not altered. The up-regulation of the prp genes whose products are involved in catabolism of the short fatty acid, propionic acid, in the misR mutant suggested that MisR influenced the expression of this alternative metabolic pathway.

Microarray analyses indicated that the expression levels of the iron-related genes, hmbR, bfrAB, and tdfH, were reduced in the misR mutant compared to those in the parent wild-type strain NMB (Table 2). BfrA and BfrB most likely form a transcriptional unit and are homologous bacterioferritin proteins that manage the intracellular storage of iron and protect the cell from iron-mediated oxidative stress (5). HmbR encodes a meningococcus-specific hemoglobin receptor (51). TdfH contains a TonB box and is annotated to be a TonB-dependent outer-membrane iron-siderophore receptor; however, the specific substrate(s) has not been identified (52, 56).

The last group of genes identified encodes putative cell surface-localized proteins that include several transporters (e.g., the capsular polysaccharide transport protein CtrD) and a putative hemolysin. In particular, the HlyB and HlyD proteins together with TolC, an outer-membrane channel protein that is encoded in the same putative operon as hlyD, constitute the type I secretion machinery responsible for the secretion of several iron-inducible repeat-in-toxin (RTX) toxin homologues (53-55, 63).

Validation of microarray by quantitative real-time PCR and β-galactosidase reporter assays.Quantitative RT-PCR and lacZ transcriptional reporter fusion were employed as independent methods to assess the differential regulation of genes selected from the microarray experiments and to validate the microarray-based observations. Quantitative real-time RT-PCRs were performed using three independent sets of wild-type and mutant RNAs, of which two were also examined by the microarray experiments. The transcription of all genes examined by quantitative RT-PCR correlated with the microarray data (Table 2). Using primer pairs annealed to the 5′ coding sequence upstream of the cassette insertion site, the expression of misR itself was one of the most significant reductions in the NMBmisR mutant, confirming that MisR functions as an autoactivator of the misR promoter (60).

To further confirm the importance of the MisR/MisS (MisR/S) two-component system in regulating the transcription of genes identified by the array study, transcriptional fusions of a β-galactosidase reporter were generated as a single copy on a permissive chromosomal locus. As the phosphorylation of MisR is critical in the activation of its regulatory function, the reporter was also inserted in the misS::aphA-3 mutant strain (60) to access the regulatory roles of MisR phosphorylation by MisS kinase. Additionally, reporter fusions of mtr, bfrA, hlyD, and misR were created in the misRS::aphA-3 double mutant background, and the resulting reporter activities were compared to those of the corresponding misS mutants to test whether nonphosphorylated MisR exhibited additional regulation. The double mutant with deletion-insertion mutations in both misR and misS was constructed by replacing an ∼1.6-kb coding sequence of misR and misS with an aphA-3 cassette as described in Materials and Methods. All mutations have been confirmed by colony PCRs, Southern blot analyses, and Western blot analyses (data not shown). As shown in Fig. 1, changes in the reporter activities of each gene examined were consistent with those obtained by arrays and quantitative RT-PCRs, although differences in the magnitude of changes were observed between the three methods. Comparable reporter activities were obtained in both the misS and the misRS mutants, indicating that the phosphorylation of MisR was critical in its regulatory activity, at least for the four genes studied.

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FIG. 1.

β-Galactosidase reporter activities of MisR-regulated genes. The reporter fusion was integrated into a permissive chromosomal locus as a single copy. β-Galactosidase activities of the wild type and misS and misRS mutants grown in GC broth to mid-log phase are expressed as Miller units and correspond to mean values for at least three independent experiments, each with triplicate measurements. Black bars, reporter strains in a wild-type background; gray bars, reporter strains with a misS::aphA-3 mutation; hatched bars, reporter strains with a misRS::aphA-3 double mutation.

Iron concentration is not the inducing signal of the MisRS two-component system.Iron availability is known to regulate proteins involved in iron metabolism through the function of the ferric uptake regulator, Fur (2, 7), and both hmbR (4, 51) and bfrAB (5) have been shown to be iron regulated. To determine whether iron availability is the signal activating the MisR regulon, the expression levels of the autoregulated misRS operon were compared under iron-replete and -deplete growth conditions. The wild-type strain NMB was grown to mid-log phase in the presence of 25 μM deferoxamine (Desferal) to deplete free iron, and total RNA was isolated and analyzed by real-time PCR. As controls, the expression levels of hmbR, tdfH, and bfrA were also examined. Consistent with published results (4, 5, 51), iron limitation caused an increase in hmbR expression and a down-regulation of bfrA, confirming that the cultures were iron starved (see Fig. S1 in the supplemental material). tdfH is not iron regulated (56), and tdfH expression was not altered with iron starvation (see Fig. S1 in the supplemental material). No changes were seen in the expression of misR (see Fig. S1 in the supplemental material), indicating that iron concentration was not involved in the control of the autoregulated misRS operon. This result indicated that the decrease in the expression of hmbR, bfrA, and tdfH observed in the NMBmisR mutant was not directly associated with changes in iron availability but was the result of an unknown inducing signal transmitted by MisS. Thus, an alternative environmental sensing mechanism involving the MisR/S two-component regulatory system may regulate a subset of iron assimilation processes. Interestingly, among the TonB-dependent receptors, only hmbR and tdfH, but not the other iron receptors for transferrin, lactoferrin, or siderophores, were affected by the misR mutation, suggesting that the biological function(s) regulated by the misRS system may involve specific pathways of iron metabolism.

MisR specifically binds to a subset of regulated genes identified by microarray analysis.Response regulators typically affect gene expression by directly binding to specific sequences in the promoters of genes that they control; however, since altered expression of additional transcriptional regulators was identified, expression changes in the NMBmisR mutant may be indirectly related to the loss of MisR. To better delineate the direct regulatory effects of MisR, EMSAs were conducted on a subset of 22 genes, and the results indicated that MisR directly interacted with 14 promoter regions of these genes (Table 2 and Fig. 2). The remaining genes without detectable direct interaction with MisR may be indirectly regulated by MisR or, alternatively, were detected by microarray analysis due to growth differences. A relatively large probe size (∼500 bp) was chosen to ensure the coverage of distant regulatory elements. However, it remains possible that MisR-binding sites were not included in the designed probes. Competition EMSAs were performed to demonstrate that the interaction of MisR with these promoter fragments was specific (see Fig. 2 for results of bfrA, tdfH, and B0556 competition experiments; also data not shown). MisR directly interacted with the promoters of clpB, dnaJ, fkpA, bfrA, tdfH, hmbR, and three genes, NMA0738, NMA1593, and NMA1751, encoding the transcriptional regulators. In addition, MisR directly controlled the expression of hlyB and hlyD (Fig. 2). The autoregulatory mechanism of the misRS operon by MisR has been reported previously (60). The titration experiments whose results are shown in Fig. 2 indicated that generally phosphorylated MisR (MisR∼P) exhibited higher affinity than nonphosphorylated MisR and that differences in binding affinity among the various promoters were present. A single protein complex species was observed for both MisR and MisR∼P. As many response regulators interact with their target DNA as oligomers, the single shifted band of the MisR-DNA complex implied a potentially high cooperativity of MisR∼P self-self interaction upon DNA binding. Although the expression of dsbD was significantly altered in the NMBmisR mutant (Table 2), MisR appears not to directly regulate the transcription of this gene since an interaction between MisR and the dsbD promoter was not demonstrated by EMSA (Fig. 2).

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FIG. 2.

EMSA results of MisR-promoter interaction. ³²P-labeled promoter fragments (∼5 fmol) were generated by T4 kinase phosphorylation, mixed with increasing amounts of MisR∼P (lanes 2 to 4) and MisR (lanes 5 to 7) for 20 min at 30°C, and then subjected to gel electrophoresis. Free probe is shown in lane 1. MisR∼P was generated by incubation with 50 mM acetyl phosphate for 30 min at 37°C. The amounts of MisR protein are 68 pmol (lanes 2 and 5), 136 pmol (lanes 3 and 6), and 272 pmol (lanes 4 and 7). The last three panels with six samples show the results of representative competition experiments with bfrA, tdfH, and B0556. Lane 1, probe alone; lane 2, probe and MisR; lanes 3 and 5, 2 μg specific and nonspecific competitor DNA, respectively; lanes 4 and 6, 4 μg specific and nonspecific competitor DNA, respectively.

The expression of lgtG, the glucosyltransferase responsible for the glucose substitution of the HepII O-3 position of the LOS inner core, has been shown to increase in the NMBmisR mutant (58); however, a shift could not be detected with an lgtG probe that encompasses an ∼500-bp sequence upstream of lgtG (data not shown). Subsequently, a lacZ reporter strain constructed with the same fragment yielded minimal activity, indicating that there is no functional promoter immediately upstream of lgtG in strain NMB. Interestingly, the mtr gene encoding an amino acid transporter is transcribed in the same direction directly upstream of lgtG and was shown to be directly and negatively regulated by MisR (Fig. 1 and 2). Sequencing analysis of the intergenic region between mtr and lgtG in strain NMB indicated that an 89-bp deletion, in comparison to the MC58 genome, removes half of an inverted repeat motif located downstream of the mtr coding sequence and disrupts a potential stem-loop transcriptional terminator. Thus, the increase in lgtG expression likely resulted from a transcriptional coupling of mtr and lgtG expression in strain NMB and MisR influences lgtG expression indirectly.

Identification of the MisR-binding motif by DNase I protection assays.To understand how MisR regulates its targets, several MisR-regulated promoters have been studied by DNase I protection assays to map the MisR-binding sites. The promoters of hmbR, hlyB, and hylD (data not shown) and fkpA and mtr (Fig. 3) were examined. The sequence length protected by MisR∼P was generally longer than those protected by nonphosphorylated MisR (see reference 60 for misR footprinting data; also data not shown), and this correlated with the affinity differences observed by EMSAs. Together with the previously reported footprinting data for the misR promoter (60), 12 binding sequences have been compiled and a conserved MisR-binding 9-bp core consensus motif, (A/T)(A/T)TGTAA(A/G/C)G, was derived (Fig. 4) using the BioProspector program (31). The importance of this motif correlated with a previous study of the misR promoter region (60). lacZ reporter assays have showed that deleting three bases at the 5′ end of this core motif significantly reduced the promoter activity to a level similar to that of a strain with only the −35 and −10 promoter elements, thus eliminating the activation by MisR. In addition, single base changes at either the first base (A/T→G) or the third base (T→A) of this core sequence (corresponding to bases 3 and 5 of the WebLogo plot in Fig. 4B) yielded a twofold reduction in promoter activity (60). Sequence searches indicated that either single- or multiple-core motifs with a maximum of two mismatches can be identified in the upstream sequence of MisR direct targets; however, the accuracy of MisR-binding sequence prediction will require confirmation with additional DNase I protection assays.

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FIG. 3.

Results of DNase I protection assays of the coding (A) and noncoding (B) strands of the fkpA promoter and the noncoding strand of the mtr promoter (C) by MisR∼P. ³²P-end-labeled promoter fragments were incubated with increasing amounts of MisR∼P for 20 min at 30°C and then subjected to DNase I digestion. MisR∼P was prepared freshly by reaction with 150 mM acetyl phosphate at 37°C for 30 min. The amounts of MisR∼P used in panels A and B were 0, 85, 170, 340, and 510 pmol and in panel C were 0, 42.5, 85, 170, 340, 680, and 1,360 pmol. Dideoxy-chain termination sequences corresponding to the probes are shown in the order of G, A, T, and C. The vertical bars indicate protected regions.

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FIG. 4.

(A) Consensus alignment of MisR-binding sites. Regions protected from DNase I from misR, hmbR, mtr, hlyB, hlyD, and fkpA promoters are aligned by the BioProspector program (31). The bold letters indicate sequences identical to the 15-bp consensus motif. K, G or T; W, A or T; R, A or G; H, any bases except G; N, any bases. (B) Graphic representation of the consensus MisR-binding motif generated by the WebLogo program (6, 45). The overall height of the stack indicates the sequence conservation at that position, while the height of bases within the stack indicates the relative frequency of each base at that position.

Inactivation of misRS results in sensitivity to oxidative stress.Gene products that are known to be involved in protection against oxidative stress, including bfrA/bfrB (5) and laz (65), were down-regulated in the misR mutant. In addition, DsbD, which provides reducing power to the peptide methionine sulfoxide reductase MsrA/B protein for repairing oxidative damage of periplasmic proteins (3, 48), was also significantly down-regulated in the misR mutant. These observations suggested that meningococcal misRS mutants might be more sensitive to the reactive oxygen species. The in vitro sensitivities of the mutants to hydrogen peroxide and paraquat were measured using disk diffusion assays, as shown in Table 3. All three mutants exhibited enhanced sensitivities to both oxidative agents.

Inactivation of misRS results in resistance to the bactericidal activity of NHS and increased levels of capsular polysaccharide production.To better correlate the avirulent phenotype reported by Newcombe et al. (34) using a mouse model of infection, the ability of the serogroup B misRS mutants to resist complement-mediated bactericidal activity was examined using serum bactericidal assays with pooled normal human serum (NHS) (24). Surprisingly, the misR and misRS mutants showed enhanced serum resistance at 10% and 25% NHS, whereas the misS mutant showed intermediate resistance at 25% NHS compared to the parental wild-type strain (Fig. 5A). An unencapsulated mutant was completely killed at 5% NHS. Thus, the misRS mutation increased resistance to complement-mediated bactericidal activity of NHS. To ensure that the serum-resistant phenotype is caused by the misR mutation, the mutation was complemented with a second copy of misR under the control of the P_trc promoter and integrated into the iga locus. As shown in Fig. 5A, with induced expression of misR by IPTG (confirmed by Western blot analyses; data not shown), the complemented strain is more sensitive to 25% NHS than the mutant and behaved similarly to the wild-type parent strain, confirming that the misR mutation resulted in increased serum resistance.

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FIG. 5.

(A) Results of serum bactericidal assays. NMB (wild type, filled diamond), M7 mutant (unencapsulated, filled square), NMBmisR mutant (misR::aphA-3, filled triangle), YT0310 mutant (misS::aphA-3, open circle), YT0336 mutant (misRS::aphA-3, asterisk), and C1001 mutant (misR::aphA-3/Ptrc::misR, filled circle with dotted line) strains grown to log phase and resuspended in HEPES-minimal essential medium buffer were exposed to 5, 10, and 25% NHS. The viable cell counts (n = 4) were determined after 30 min of incubation, and the percentages of survival were calculated by comparing these values to the viable counts at time zero. *25% indicates treatment with 25% heat-inactivated serum for 30 min. Averages and standard deviations for at least three independent experiments are shown. Student's t test with a two-tailed hypothesis indicates significant differences between the wild-type strain and three mutants at 10 and 25% serum levels (P ≤ 0.05). (B) Results of capsule-specific whole-cell ELISA. The OD₄₀₅ reading of the serogroup B wild-type strain NMB was used as 100% for normalizing the OD₄₀₅ readings of the YT0310 and the NMBmisR mutants. The nonencapsulated mutant M7 (synA::Tn916) was included as a negative control (n = 3).

Multiple mechanisms are utilized by meningococci to avoid killing by human complement (11, 24, 44). One of the major determinants to serum resistance is capsular polysaccharide. To test whether the level of capsule expression contributes to the increase in resistance, the total capsule expression of the misS and misR mutants was quantified by whole-cell ELISA and compared to that of the parent strain (Fig. 5B). The results indicated that the misRS mutants expressed ∼40% more capsule than the wild-type parent strain. The capsule transport gene ctrD was detected as up-regulated in the misR mutant by microarray analysis (see Table S1 in the supplemental material). To test whether the expression of capsule assembly genes was affected, we assayed the transcriptional levels of several capsule biosynthesis and transport genes, synA, ctrA, ctrD, and ctrE(lipA), by real-time RT-PCR. Modest increases (∼1.5-fold) were detected in the misR and misS mutants and could potentially result in an increase in capsule expression and lead to the serum resistance phenotype.