This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tzeng, Y.-L.
Right arrow Articles by Stephens, D. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tzeng, Y.-L.
Right arrow Articles by Stephens, D. S.

 Previous Article  |  Next Article 

Infection and Immunity, February 2008, p. 704-716, Vol. 76, No. 2
0019-9567/08/$08.00+0     doi:10.1128/IAI.01007-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

MisR/MisS Two-Component Regulon in Neisseria meningitidis{triangledown} ,{dagger}

Yih-Ling Tzeng,1* Charlene M. Kahler,3 Xinjian Zhang,1,{ddagger} and David S. Stephens1,2

Department of Medicine, Emory University School of Medicine, Atlanta, Georgia,1 Laboratories of Bacterial Pathogenesis, Department of Veterans Affairs Medical Center, Decatur, Georgia,2 Discipline of Microbiology and Immunology, School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, Perth, Australia3

Received 23 July 2007/ Returned for modification 6 September 2007/ Accepted 20 November 2007


arrow
ABSTRACT
 
Two-component regulatory systems are involved in processes important for bacterial pathogenesis. Inactivation of the misR/misS system in Neisseria meningitidis results in the loss of phosphorylation of the lipooligosaccharide inner core and causes attenuation in a mouse model of meningococcal infection. One hundred seventeen (78 up-regulated and 39 down-regulated) potential regulatory targets of the MisR/MisS (MisR/S) system were identified by transcriptional profiling of the NMBmisR mutant and the parental wild-type meningococcal strain NMB. The regulatory effect was further confirmed in a subset of target genes by quantitative real-time PCR and β-galactosidase transcriptional fusion reporter assays. The MisR regulon includes genes encoding proteins necessary for protein folding in the bacterial cytoplasm and periplasm, transcriptional regulation, metabolism, iron assimilation, and type I protein transport. Mutation in the MisR/S system caused increased sensitivity to oxidative stress and also resulted in decreased susceptibility to complement-mediated killing by normal human serum. To identify the direct targets of MisR regulation, electrophoretic mobility shift assays were carried out using purified MisR-His6 protein. Among 22 genes examined, misR directly interacted with 14 promoter regions. Six promoters were further investigated by DNase I protection assays, and a MisR-binding consensus sequence was proposed. Thus, the direct regulatory targets of MisR and the minimal regulon of the meningococcal MisR/S two-component signal transduction system were characterized. These data indicate that the MisR/S system influences a wide range of biological functions in N. meningitidis either directly or via intermediate regulators.


arrow
INTRODUCTION
 
Two-component regulatory systems are one of the most common bacterial signal transduction mechanisms controlling responses and adaptation to environmental changes (17). Many such systems act as global regulators in coordinating the expression of virulence determinants in bacterial pathogens. Often, these systems are composed of an inner membrane-bound histidine kinase that, upon sensing specific signals, undergoes autophosphorylation at a conserved histidine residue and a cytoplasmic response regulator partner that receives the phosphoryl group at an invariant aspartate residue (16) which consequently modulates DNA-binding activity.

Neisseria meningitidis is an obligate pathogen which inhabits the human nasopharynx but can rapidly disseminate to cause sepsis and meningitis during invasive infection (50, 57). Likely because of the restricted habitat of the meningococcus, the organism has a relatively small genome and few two-component systems, only four predicted pairs (39, 52), compared to other pathogens experiencing more-complex environments (36, 66). The persistence of these few functional two-component regulatory systems implies an important role in regulating meningococcal colonization and virulence. Sequence comparison suggests that one system (NMB0114/NMB0115) shares amino acid sequence similarities to NtrY/NtrX (COG5000/COG2204 domain family), which regulates nitrogen metabolism in Azorhizobium caulinodans (40), while the second pair, NMB1606/NMB1607, shares homology with pilS/pilR, which regulates piliation in Pseudomonas aeruginosa (15). The NMB1249/NMB1250 two-component system exhibits amino acid sequence similarities with NarQ/NarP, and the equivalent gonococcal system has been shown to respond to anaerobic growth (30, 38). We have reported that inactivation of the fourth two-component system, encoded by NMB0595/NMB0594, designated misR/misS, results in the loss of phosphoethanolamine (PEA) substitutions on the lipooligosaccharide (LOS) inner core, with an increased sensitivity to cationic antimicrobial peptides (58). LOS or endotoxin is a major virulence factor of N. meningitidis, and structural changes in LOS are important in meningococcal pathogenesis. In addition, a misR response regulator mutant of a serogroup C meningococcal strain is avirulent in a mouse model of infection (34).

Microarray transcription profile comparison has frequently been used to obtain a general picture of a particular transcriptome. However, this strategy does not allow the delineation of direct versus indirect regulatory targets. A microarray study of the serogroup C misR mutant reported previously by Newcombe et al. (35) used RNAs isolated from cells grown on blood agar. A total of 281 genes were identified as being significantly up- or down-regulated in the mutant compared to those in the parent strain. However, these microarray observations were not validated by other biochemical or genetic means, nor were the direct regulatory targets of MisR identified. We have previously shown that the misRS operon is under the control of autoactivation, and this has been the only proven direct target of MisR (60). As phenotypic differences have been noted between the serogroup C misR mutant and the serogroup B misR mutant studied by our group (21, 35, 58, 60), we carried out transcriptional profile analyses of the NMBmisR mutant to independently identify MisR-regulated genes. Real-time reverse transcription (RT)-PCR and reporter assays were performed to confirm the potential regulatory effects of MisR on genes identified by microarray and to define the minimal regulon. Further, direct regulatory targets of MisR were identified by electrophoretic mobility shift assay (EMSA), and the MisR-binding sequences of several target genes were mapped by DNase I protection assays. With curation of the in vitro biochemical data and the bioinformatic analysis of the binding sequences, a MisR-binding motif is proposed.


arrow
MATERIALS AND METHODS
 
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 DH5{alpha} 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.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Strains and plasmids used in this study

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 Ptrc 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 Spr/Kmr 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 0.2x SSC (1x 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 0.2x SSC-0.1% sodium dodecyl sulfate for 5 min at 42°C, followed by consecutive rinses in 0.2x SSC-0.1% sodium dodecyl sulfate for 10 min at room temperature (two times) and 0.1x 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 {gamma}-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 {gamma}-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–{Delta}{Delta}CT 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).


arrow
RESULTS
 
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.


View this table:
[in this window]
[in a new window]

  		TABLE 2. Genes analyzed by qRT-PCR and EMSA

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.


Figure 1
View larger version (11K):
[in this window]
[in a new window]

  		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).


Figure 2
View larger version (83K):
[in this window]
[in a new window]

  		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.


Figure 3
View larger version (150K):
[in this window]
[in a new window]

  		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.


Figure 4
View larger version (38K):
[in this window]
[in a new window]

  		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.


View this table:
[in this window]
[in a new window]

  		TABLE 3. misRS mutation increases sensitivity to oxidative stress

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 Ptrc 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.


Figure 5
View larger version (20K):
[in this window]
[in a new window]

  		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.


arrow
DISCUSSION
 
The limited number of two-component regulatory systems in N. meningitidis, an obligate human pathogen, suggests that they regulate genes important for maintaining the lifestyle of the organism and possibly for virulence. For example, NarQ/NarP has been shown to regulate the denitrification pathway, which is important under oxygen-limited in vivo growth conditions, such as those present inside vacuoles containing gonococci or meningococci in human epithelial cells (30, 38).

Although interesting phenotypes associated with virulence have been observed for the MisR/S two-component system (34, 58), the regulatory targets of this system remain to be defined. Previous microarray studies of a misR mutant created in a meningococcal serogroup C strain grown on blood agar revealed a total of 281 genes that were up- or down-regulated compared to those of the parental strain. However, this microarray observation was not validated by other experimental methods, nor was information on direct targets of regulation provided. As phenotypic differences between the serogroup C and the serogroup B mutants have been noted previously (21, 35, 58, 60), we carried out transcriptional profile analyses of the NMBmisR mutant to independently identify MisR-regulated genes. Our microarray data indicated that 117 genes displayed altered expression in the misR mutant, with 78 up-regulated and 39 down-regulated, respectively. The overlap of MisR regulatory targets between the study of the serogroup C misR mutant (15) and our study of the serogroup B NMBmisR mutant was approximately 30%. The limited overlap between these two studies may be due to the fact that the two strains may have different genotypes and were grown under different culturing conditions. Using quantitative real-time PCR, a β-galactosidase reporter assay, and an EMSA, we further characterized 25 potential MisR targets (Table 2). Fourteen promoters controlling the transcription of 15 genes were defined as direct targets of MisR, including several genes not seen in the other microarray study. Overall, the MisR/S regulon included genes involved in protein folding, chaperones, metabolism, iron assimilation, type I protein transport, and sensitivity to oxidative stress and human serum.

General protein folding machinery has been implicated in the virulence of many pathogens. Although it is likely that the transcriptional changes of some genes involved in protein folding were caused by the growth rate difference between the wild-type parent strain and the mutant, the MisR/S two-component system appears to directly regulate aspects of the cytoplasmic protein folding machinery, including dnaJ, clpB, and fkpA, as shown by EMSAs. In addition, the expression of dsbD, an integral membrane protein necessary for the protein isomerization pathway in the periplasm, also significantly decreased in the misR mutant. Differences in the genetic organization and regulation of chaperones between Neisseria spp. and other bacteria are significant. First, unlike genes in E. coli, which encode chaperones often organized as an operon, meningococcal dnaJ, dnaK, and grpE are independently transcribed at three distant chromosomal loci. Second, although Neisseria belongs to the betaproteobacteria that commonly utilize the HrcA/CIRCE system for chaperone regulation, no HrcA homologue has been found in the neisserial genomes (41). Instead, dnaJ, dnaK, and grpE are under the positive control of the heat shock sigma factor {sigma}32 (RpoH) (14, 29) in gonococci and thus are influenced by the regulatory mechanisms controlling the amount and activity of RpoH itself. Our study indicates that MisR negatively regulates some of these chaperone genes. MisR was shown to bind the dnaJ promoter with low affinity, which appears to correlate with a modest up-regulation, while MisR bound the clpB promoter with high affinity, resulting in a more significant increase in the transcription level when MisR was absent. This observation parallels that seen in Streptococcus pyogenes, where inactivation of the response regulator covR resulted in the derepression of dnaK, dnaJ, grpE, and groEL transcripts (12). In addition to this emerging mechanism of regulation of chaperone expression, dnaK and clpB were shown to be up-regulated in a gonococcal fur null mutant, and this regulation is independent of iron concentration (8). Thus, in addition to the general stress response (RpoH-dependent positive control) (14) and the Fur-mediated negative regulation (8), the MisR/S two-component system is another negative regulatory mechanism of a subset of chaperone proteins in N. meningitidis. The biological significance of these interactions is as yet unclear; however, RpoH, the positive regulator of chaperone expression, was up-regulated, while NG0177, the equivalent of misR in Neisseria gonorrhoeae, was down-regulated when gonococci adhered to human epithelial cells (10), indicating a possible role for MisR in attachment and invasion.

Both Fur and the MisR/S system are also involved in the control of the hemoglobin receptor hmbR. Fur has been shown to bind directly to the promoter region of hmbR and act as a repressor under iron-replete conditions (46). The array and real-time experiments reported here were performed with RNAs isolated from iron-replete cultures. Thus, the expression of hmbR was further decreased in the misR mutant, indicating that MisR functions as an activator in the presence of Fur repression. Whether MisR acts alone as an activator or functions through antagonizing the repressor effect of Fur remains to be clarified. On the other hand, the expression of the bfrA-bfrB operon is clearly regulated by iron availability (5), but no direct interaction of Fur with the bfrA promoter has been reported. We show that MisR interacts directly with the bfrA promoter. This represents the first example of a two-component regulatory system mediating the expression of iron storage proteins important in maintaining iron homeostasis. Similarly, as the expression of tdfH is not regulated by iron (56), the direct control by the MisR/S system might be the only regulatory mechanism modulating the expression of tdfH.

The EMSA data indicate that MisR indirectly regulates dsbD, the integral membrane component of the protein disulfide bond isomerization pathway in the periplasm. DsbD participates in correcting misformed disulfide bonds of periplasmic proteins and is critical in providing the reducing power for cytochrome c assembly in the periplasm (26). Among all Dsb proteins, only DsbA has been shown to be a member of the Cpx two-component system regulon of E. coli (43). The regulatory mechanisms controlling other Dsb proteins have not been characterized. In addition to its involvement in the refolding of periplasmic proteins and cytochrome c assembly, DsbD can reoxidize the methionine sulfoxide reductase (MsrA/B) domains of PilB in N. gonorrhoeae, which repairs methionine damaged by reactive oxygen species (3), an important arsenal elicited by host defense. Further, the methionine sulfoxide reductase activity of PilB also contributes to the maintenance of adhesins in Streptococcus pneumoniae, N. gonorrhoeae, and E. coli (62). In correlation with the role of DsbD in maintaining the function of PilB and the down-regulation of azurin, which is involved in defense against hydrogen peroxide (65), we found that the misRS and misR mutants exhibited increased sensitivities to hydrogen peroxide and paraquat (Table 3).

One of the original phenotypes of the misR mutant in strain NMB was the disappearance of O-6-linked PEA attached to the HepII of the LOS inner core (58). However, the expression of lpt6, encoding the O-6 PEA transferase (23, 64), was unchanged in the misR mutant when examined by microarray and real-time PCR (data not shown). Without the transcriptional down-regulation of lpt6 expression, two models are being examined. In one model, Lpt6 may be misfolded or degraded in the periplasm, due to the impairment of the protein folding pathway dependent upon DsbD. Alternatively, Lpt6 activity may remain intact in the NMBmisR mutant, with the up-regulation of a putative PEA hydrolase removing all the HepII PEA substituents during the biosynthesis and assembly of the LOS. While plausible, such PEA hydrolase activity has yet to be observed in meningococci. Clearly, more studies are needed to address the mechanism leading to the loss of O-6 PEA phosphorylation. Another possible indirect target of MisR is a putative hemolysin encoded by NMB1900. This gene has been shown to be up-regulated when meningococci attach to endothelial, but not epithelial, cells and is down-regulated when exposed to human serum (9, 28). However, its role in meningococcal pathogenesis has not been defined. Other exotoxin family proteins expressed by meningococci with unknown pathogenic functions are the RTX toxins (FrpC). High levels of antibodies recognizing a recombinant FrpC protein were detected in convalescent-phase sera of patients recovering from invasive meningococcal disease and were absent in the sera at hospital admission, indicating the expression of FrpC-like proteins during disease (37). A type I secretion machinery homologous to HlyB/HlyD/TolC of E. coli is responsible for the secretion of FrpC RTX toxin family proteins, and this secretion system is shown here to be directly and negatively controlled by the MisRS two-component system.

The increased resistance of the misRS mutants to killing by normal human serum bactericidal activity was striking (Fig. 5A). Multiple mechanisms are utilized by meningococci to avoid killing by human complement (11, 24, 44). Two main contributors to resistance are capsular polysaccharide and LOS. Strain NMB in broth culture expresses a mixture of LOS structures, with all inner cores carrying O-6 PEA groups and 70% carrying O-3-linked glucose. The misR mutant expresses a homogenous population of LOS structures, all of which lack the O-6 PEA of the inner-core HepII residue while being completely substituted with O-3-linked glucose (58). This effect has been shown to be the result of derepression of the O-3-linked glucosyltransferase gene, lgtG, in the NMBmisR mutant. Ram et al. (43a) have previously demonstrated that the O-6-linked PEA of the LOS inner core is a preferred target for binding complement component C4b, thus resulting in increased susceptibility to complement-mediated killing in serum bactericidal assays. However, an lpt6 mutant in strain NMB did not display an increase in serum resistance (data not shown), indicating that the loss of O-6-linked PEA alone from the LOS inner core in the misRS mutant could not explain this phenotype. An examination of the total capsule by whole-cell capsule ELISA indicated that the misRS mutants expressed ~30 to 50% more capsule than the wild-type parent strain (Fig. 5B). This phenotype could potentially arise from the modest increase (~1.5-fold) in the transcription of the capsule biosynthesis and capsule transport genes in the misR and misS mutants. We have previously shown that small modulations in capsule expression of approximately 25% can result in detectable changes in serum resistance (25), indicating that these relatively small changes have important biological effects.

In summary, we have defined a minimal regulon of the MisR/S two-component system through microarray, quantitative real-time PCR, reporter fusion, EMSAs, and DNase I protection assays. The MisR-binding motif was clearly different from those of the PhoP and CpxR response regulators, which are potential functional homologues of MisR (13, 42). The MisR/S system directly or indirectly regulates genes involved in a diverse array of functional categories, many with implicated roles in meningococcal pathogenesis. Additional data are needed to refine the MisR-binding motif and to identify critical nucleotides within the motif. The results reported here should enable a better understanding of the interesting regulatory circuitry of the important MisR/S two-component system in N. meningitidis.


arrow
ACKNOWLEDGMENTS
 
This work was supported by grants R01 AI061031 (Y.-L.T.), R01 AI033517 (D.S.S. and C.M.K.), and R01 AI40247 (D.S.S.) from the National Institutes of Health. C.M.K. was supported by National Health and Medical Research Council Project grant ID124465.

We thank Larry Martin, Shaojia Bao, and Soma Sannagrahi for excellent technical assistance and Lane Pucko for administrative assistance. We are grateful to John Davies for sharing the complementation vector.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Woodruff Memorial Research Building, Room 2101, 1639 Pierce Drive, Atlanta, GA 30322. Phone: (404) 727-8393. Fax: (404) 712-2278. E-mail: ytzeng{at}emory.edu Back

{triangledown} Published ahead of print on 3 December 2007. Back

{dagger} Supplemental material for this article may be found at http://iai.asm.org/. Back

Editor: V. J. DiRita

{ddagger} Present address: Department of Chemistry, Georgia State University, Atlanta, GA. Back


arrow
REFERENCES
 
    1
  1. Aslund, F., and J. Beckwith. 1999. Bridge over troubled waters: sensing stress by disulfide bond formation. Cell 96:751-753.[CrossRef][Medline]
    2. 2
  2. Braun, V. 2003. Iron uptake by Escherichia coli. Front. Biosci. 8:s1409-s1421.[Medline]
    3. 3
  3. Brot, N., J. F. Collet, L. C. Johnson, T. J. Jonsson, H. Weissbach, and W. T. Lowther. 2006. The thioredoxin domain of Neisseria gonorrhoeae PilB can use electrons from DsbD to reduce downstream methionine sulfoxide reductases. J. Biol. Chem. 281:32668-32675.[Abstract/Free Full Text]
    4. 4
  4. Chen, C. J., P. F. Sparling, L. A. Lewis, D. W. Dyer, and C. Elkins. 1996. Identification and purification of a hemoglobin-binding outer membrane protein from Neisseria gonorrhoeae. Infect. Immun. 64:5008-5014.[Abstract]
    5. 5
  5. Chen, C. Y., and S. A. Morse. 1999. Neisseria gonorrhoeae bacterioferritin: structural heterogeneity, involvement in iron storage and protection against oxidative stress. Microbiology 145:2967-2975.[Abstract/Free Full Text]
    6. 6
  6. Crooks, G. E., G. Hon, J. M. Chandonia, and S. E. Brenner. 2004. WebLogo: a sequence logo generator. Genome Res. 14:1188-1190.[Abstract/Free Full Text]
    7. 7
  7. Crosa, J. H. 1997. Signal transduction and transcriptional and posttranscriptional control of iron-regulated genes in bacteria. Microbiol. Mol. Biol. Rev. 61:319-336.[Abstract]
    8. 8
  8. Delany, I., R. Grifantini, E. Bartolini, R. Rappuoli, and V. Scarlato. 2006. Effect of Neisseria meningitidis fur mutations on global control of gene transcription. J. Bacteriol. 188:2483-2492.[Abstract/Free Full Text]
    9. 9
  9. Dietrich, G., S. Kurz, C. Hubner, C. Aepinus, S. Theiss, M. Guckenberger, U. Panzner, J. Weber, and M. Frosch. 2003. Transcriptome analysis of Neisseria meningitidis during infection. J. Bacteriol. 185:155-164.[Abstract/Free Full Text]
    10. 10
  10. Du, Y., J. Lenz, and C. G. Arvidson. 2005. Global gene expression and the role of sigma factors in Neisseria gonorrhoeae in interactions with epithelial cells. Infect. Immun. 73:4834-4845.[Abstract/Free Full Text]
    11. 11
  11. Geoffroy, M. C., S. Floquet, A. Metais, X. Nassif, and V. Pelicic. 2003. Large-scale analysis of the meningococcus genome by gene disruption: resistance to complement-mediated lysis. Genome Res. 13:391-398.[Abstract/Free Full Text]
    12. 12
  12. Graham, M. R., L. M. Smoot, C. A. Migliaccio, K. Virtaneva, D. E. Sturdevant, S. F. Porcella, M. J. Federle, G. J. Adams, J. R. Scott, and J. M. Musser. 2002. Virulence control in group A Streptococcus by a two-component gene regulatory system: global expression profiling and in vivo infection modeling. Proc. Natl. Acad. Sci. USA 99:13855-13860.[Abstract/Free Full Text]
    13. 13
  13. Groisman, E. A. 2001. The pleiotropic two-component regulatory system PhoP-PhoQ. J. Bacteriol. 183:1835-1842.[Free Full Text]
    14. 14
  14. Gunesekere, I. C., C. M. Kahler, D. R. Powell, L. A. Snyder, N. J. Saunders, J. I. Rood, and J. K. Davies. 2006. Comparison of the RpoH-dependent regulon and general stress response in Neisseria gonorrhoeae. J. Bacteriol. 188:4769-4776.[Abstract/Free Full Text]
    15. 15
  15. Hobbs, M., E. S. Collie, P. D. Free, S. P. Livingston, and J. S. Mattick. 1993. PilS and PilR, a two-component transcriptional regulatory system controlling expression of type 4 fimbriae in Pseudomonas aeruginosa. Mol. Microbiol. 7:669-682.[Medline]
    16. 16
  16. Hoch, J. A. 2000. Two-component and phosphorelay signal transduction. Curr. Opin. Microbiol. 3:165-170.[CrossRef][Medline]
    17. 17
  17. Hoch, J. A., and T. J. Silhavy. 1995. Two-component signal transduction. American Society for Microbiology, Washington, DC.
    18. 18
  18. Hoffmann, J. H., K. Linke, P. C. Graf, H. Lilie, and U. Jakob. 2004. Identification of a redox-regulated chaperone network. EMBO J. 23:160-168.[CrossRef][Medline]
    19. 19
  19. Jakob, U., W. Muse, M. Eser, and J. C. Bardwell. 1999. Chaperone activity with a redox switch. Cell 96:341-352.[CrossRef][Medline]
    20. 20
  20. Janik, A., E. Juni, and G. A. Heym. 1976. Genetic transformation as a tool for detection of Neisseria gonorrhoeae. J. Clin. Microbiol. 4:71-81.[Abstract/Free Full Text]
    21. 21
  21. Johnson, C. R., J. Newcombe, S. Thorne, H. A. Borde, L. J. Eales-Reynolds, A. R. Gorringe, S. G. Funnell, and J. J. McFadden. 2001. Generation and characterization of a PhoP homologue mutant of Neisseria meningitidis. Mol. Microbiol. 39:1345-1355.[CrossRef][Medline]
    22. 22
  22. Kadokura, H., F. Katzen, and J. Beckwith. 2003. Protein disulfide bond formation in prokaryotes. Annu. Rev. Biochem. 72:111-135.[CrossRef][Medline]
    23. 23
  23. Kahler, C. M., A. Datta, Y. L. Tzeng, R. W. Carlson, and D. S. Stephens. 2005. Inner core assembly and structure of the lipooligosaccharide of Neisseria meningitidis: capacity of strain NMB to express all known immunotype epitopes. Glycobiology 15:409-419.[Abstract/Free Full Text]
    24. 24
  24. Kahler, C. M., L. E. Martin, G. C. Shih, M. M. Rahman, R. W. Carlson, and D. S. Stephens. 1998. The ({alpha}2->8)-linked polysialic acid capsule and lipooligosaccharide structure both contribute to the ability of serogroup B Neisseria meningitidis to resist the bactericidal activity of normal human serum. Infect. Immun. 66:5939-5947.[Abstract/Free Full Text]
    25. 25
  25. Kahler, C. M., L. E. Martin, Y.-L. Tzeng, Y. K. Miller, K. Sharkey, D. S. Stephens, and J. K. Davies. 2001. Polymorphisms in pilin glycosylation locus of Neisseria meningitidis expressing class II pili. Infect. Immun. 69:3597-3604.[Abstract/Free Full Text]
    26. 26
  26. Kranz, R. G., C. S. Beckett, and B. S. Goldman. 2002. Genomic analyses of bacterial respiratory and cytochrome c assembly systems: Bordetella as a model for the system II cytochrome c biogenesis pathway. Res. Microbiol. 153:1-6.[Medline]
    27. 27
  27. Kupsch, E. M., D. Aubel, C. P. Gibbs, A. F. Kahrs, T. Rudel, and T. F. Meyer. 1996. Construction of Hermes shuttle vectors: a versatile system useful for genetic complementation of transformable and non-transformable Neisseria mutants. Mol. Gen. Genet. 250:558-569.[Medline]
    28. 28
  28. Kurz, S., C. Hubner, C. Aepinus, S. Theiss, M. Guckenberger, U. Panzner, J. Weber, M. Frosch, and G. Dietrich. 2003. Transcriptome-based antigen identification for Neisseria meningitidis. Vaccine 21:768-775.[CrossRef][Medline]
    29. 29
  29. Laskos, L., C. S. Ryan, J. A. Fyfe, and J. K. Davies. 2004. The RpoH-mediated stress response in Neisseria gonorrhoeae is regulated at the level of activity. J. Bacteriol. 186:8443-8452.[Abstract/Free Full Text]
    30. 30
  30. Lissenden, S., S. Mohan, T. Overton, T. Regan, H. Crooke, J. A. Cardinale, T. C. Householder, P. Adams, C. D. O'Conner, V. L. Clark, H. Smith, and J. A. Cole. 2000. Identification of transcription activators that regulate gonococcal adaptation from aerobic to anaerobic or oxygen-limited growth. Mol. Microbiol. 37:839-855.[CrossRef][Medline]
    31. 31
  31. Liu, X., D. L. Brutlag, and J. S. Liu. 2001. BioProspector: discovering conserved DNA motifs in upstream regulatory regions of co-expressed genes. Pac. Symp. Biocomput. 6:127-138.
    32. 32
  32. Livak, K. J., and T. D. Schmittgen. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25:402-408.[CrossRef][Medline]
    33. 33
  33. Ménard, R., P. J. Sansonetti, and C. Parsot. 1993. Nonpolar mutagenesis of the ipa genes defines IpaB, IpaC, and IpaD as effectors of Shigella flexneri entry into epithelial cells. J. Bacteriol. 175:5899-5906.[Abstract/Free Full Text]
    34. 34
  34. Newcombe, J., L. J. Eales-Reynolds, L. Wootton, A. R. Gorringe, S. G. Funnell, S. C. Taylor, and J. J. McFadden. 2004. Infection with an avirulent phoP mutant of Neisseria meningitidis confers broad cross-reactive immunity. Infect. Immun. 72:338-344.[Abstract/Free Full Text]
    35. 35
  35. Newcombe, J., J. C. Jeynes, E. Mendoza, J. Hinds, G. L. Marsden, R. A. Stabler, M. Marti, and J. J. McFadden. 2005. Phenotypic and transcriptional characterization of the meningococcal PhoPQ system, a magnesium-sensing two-component regulatory system that controls genes involved in remodeling the meningococcal cell surface. J. Bacteriol. 187:4967-4975.[Abstract/Free Full Text]
    36. 36
  36. Oshima, T., H. Aiba, Y. Masuda, S. Kanaya, M. Sugiura, B. L. Wanner, H. Mori, and T. Mizuno. 2002. Transcriptome analysis of all two-component regulatory system mutants of Escherichia coli K-12. Mol. Microbiol. 46:281-291.[CrossRef][Medline]
    37. 37
  37. Osicka, R., J. Kalmusova, P. Krizova, and P. Sebo. 2001. Neisseria meningitidis RTX protein FrpC induces high levels of serum antibodies during invasive disease: polymorphism of frpC alleles and purification of recombinant FrpC. Infect. Immun. 69:5509-5519.[Abstract/Free Full Text]
    38. 38
  38. Overton, T. W., R. Whitehead, Y. Li, L. A. Snyder, N. J. Saunders, H. Smith, and J. A. Cole. 2006. Coordinated regulation of the Neisseria gonorrhoeae-truncated denitrification pathway by the nitric oxide-sensitive repressor, NsrR, and nitrite-insensitive NarQ-NarP. J. Biol. Chem. 281:33115-33126.[Abstract/Free Full Text]
    39. 39
  39. Parkhill, J., M. Achtman, K. D. James, S. D. Bentley, C. Churcher, S. R. Klee, G. Morelli, D. Basham, D. Brown, T. Chillingworth, R. M. Davies, P. Davis, K. Devlin, T. Feltwell, N. Hamlin, S. Holroyd, K. Jagels, S. Leather, S. Moule, K. Mungall, M. A. Quail, M. A. Rajandream, K. M. Rutherford, M. Simmonds, J. Skelton, S. Whitehead, B. G. Spratt, and B. G. Barrell. 2000. Complete DNA sequence of a serogroup A strain of Neisseria meningitidis Z2491. Nature 404:502-506.[CrossRef][Medline]
    40. 40
  40. Pawlowski, K., U. Klosse, and F. J. de Bruijn. 1991. Characterization of a novel Azorhizobium caulinodans ORS571 two-component regulatory system, NtrY/NtrX, involved in nitrogen fixation and metabolism. Mol. Gen. Genet. 231:124-138.[CrossRef][Medline]
    41. 41
  41. Permina, E. A., and M. S. Gelfand. 2003. Heat shock (sigma32 and HrcA/CIRCE) regulons in beta-, gamma- and epsilon-proteobacteria. J. Mol. Microbiol. Biotechnol. 6:174-181.[CrossRef][Medline]
    42. 42
  42. Pogliano, J., A. S. Lynch, D. Belin, E. C. Lin, and J. Beckwith. 1997. Regulation of Escherichia coli cell envelope proteins involved in protein folding and degradation by the Cpx two-component system. Genes Dev. 11:1169-1182.[Abstract/Free Full Text]
    43. 43
  43. Raivio, T. L. 2005. Envelope stress responses and Gram-negative bacterial pathogenesis. Mol. Microbiol. 56:1119-1128.[CrossRef][Medline]
    44. 43a
  44. Ram, S., A. D. Cox, J. C. Wright, U. Vogel, S. Getzlaff, R. Boden, J. Li, J. S. Plested, S. Meri, S. Gulati, D. C. Stein, J. C. Richards, E. R. Moxon, and P. A. Rice. 2003. Neisserial lipooligosaccharide is a target for complement component C4b; inner core phosphoethanolamine residues define C4b linkage specificity. J. Biol. Chem. 278:50853-50862.[Abstract/Free Full Text]
    45. 44
  45. Schneider, M. C., R. M. Exley, S. Ram, R. B. Sim, and C. M. Tang. 2007. Interactions between Neisseria meningitidis and the complement system. Trends Microbiol. 15:233-240.[CrossRef][Medline]
    46. 45
  46. Schneider, T. D., and R. M. Stephens. 1990. Sequence logos: a new way to display consensus sequences. Nucleic Acids Res. 18:6097-6100.[Abstract/Free Full Text]
    47. 46
  47. Sebastian, S., S. Agarwal, J. R. Murphy, and C. A. Genco. 2002. The gonococcal fur regulon: identification of additional genes involved in major catabolic, recombination, and secretory pathways. J. Bacteriol. 184:3965-3974.[Abstract/Free Full Text]
    48. 47
  48. Shimohata, N., S. Chiba, N. Saikawa, K. Ito, and Y. Akiyama. 2002. The Cpx stress response system of Escherichia coli senses plasma membrane proteins and controls HtpX, a membrane protease with a cytosolic active site. Genes Cells 7:653-662.[Abstract]
    49. 48
  49. Skaar, E. P., D. M. Tobiason, J. Quick, R. C. Judd, H. Weissbach, F. Etienne, N. Brot, and H. S. Seifert. 2002. The outer membrane localization of the Neisseria gonorrhoeae MsrA/B is involved in survival against reactive oxygen species. Proc. Natl. Acad. Sci. USA 99:10108-10113.[Abstract/Free Full Text]
    50. 49
  50. Stephens, D. S., J. S. Swartley, S. Kathariou, and S. A. Morse. 1991. Insertion of Tn916 in Neisseria meningitidis resulting in loss of group B capsular polysaccharide. Infect. Immun. 59:4097-4102.[Abstract/Free Full Text]
    51. 50
  51. Stephens, D. S., and S. M. Zimmer. 2002. Pathogenesis, therapy, and prevention of meningococcal sepsis. Curr. Infect. Dis. Rep. 4:377-386.[Medline]
    52. 51
  52. Stojiljkovic, I., J. Larson, V. Hwa, S. Anic, and M. So. 1996. HmbR outer membrane receptors of pathogenic Neisseria spp.: iron-regulated, hemoglobin-binding proteins with a high level of primary structure conservation. J. Bacteriol. 178:4670-4678.[Abstract/Free Full Text]
    53. 52
  53. Tettelin, H., N. J. Saunders, J. Heidelberg, A. C. Jeffries, K. E. Nelson, J. A. Eisen, K. A. Ketchum, D. W. Hood, J. F. Peden, R. J. Dodson, W. C. Nelson, M. L. Gwinn, R. DeBoy, J. D. Peterson, E. K. Hickey, D. H. Haft, S. L. Salzberg, O. White, R. D. Fleischmann, B. A. Dougherty, T. Mason, A. Ciecko, D. S. Parksey, E. Blair, H. Cittone, E. B. Clark, M. D. Cotton, T. R. Utterback, H. Khouri, H. Qin, J. Vamathevan, J. Gill, V. Scarlato, V. Masignani, M. Pizza, G. Grandi, L. Sun, H. O. Smith, C. M. Fraser, E. R. Moxon, R. Rappuoli, and J. C. Venter. 2000. Complete genome sequence of Neisseria meningitidis serogroup B strain MC58. Science 287:1809-1815.[Abstract/Free Full Text]
    54. 53
  54. Thompson, S. A., and P. F. Sparling. 1993. The RTX cytotoxin-related FrpA protein of Neisseria meningitidis is secreted extracellularly by meningococci and by HlyBD+ Escherichia coli. Infect. Immun. 61:2906-2911.[Abstract/Free Full Text]
    55. 54
  55. Thompson, S. A., L. L. Wang, and P. F. Sparling. 1993. Cloning and nucleotide sequence of frpC, a second gene from Neisseria meningitidis encoding a protein similar to RTX cytotoxins. Mol. Microbiol. 9:85-96.[CrossRef][Medline]
    56. 55
  56. Thompson, S. A., L. L. Wang, A. West, and P. F. Sparling. 1993. Neisseria meningitidis produces iron-regulated proteins related to the RTX family of exoproteins. J. Bacteriol. 175:811-818.[Abstract/Free Full Text]
    57. 56
  57. Turner, P. C., C. E. Thomas, I. Stojiljkovic, C. Elkins, G. Kizel, D. A. Ala'Aldeen, and P. F. Sparling. 2001. Neisserial TonB-dependent outer-membrane proteins: detection, regulation and distribution of three putative candidates identified from the genome sequences. Microbiology 147:1277-1290.[Abstract/Free Full Text]
    58. 57
  58. Tzeng, Y.-L., and D. S. Stephens. 2000. Epidemiology and pathogenesis of Neisseria meningitidis. Microbes Infect. 6:687-700.[CrossRef]
    59. 58
  59. Tzeng, Y.-L., A. Datta, K. D. Ambrose, J. K. Davies, R. W. Carlson, D. S. Stephens, and C. M. Kahler. 2004. The MisR/MisS two-component regulatory system influences inner core structure and immunotype of lipooligosaccharide in Neisseria meningitidis. J. Biol. Chem. 279:35053-35062.[Abstract/Free Full Text]
    60. 59
  60. Tzeng, Y.-L., J. S. Swartley, Y. K. Miller, R. E. Nisbet, L. J. Liu, J. H. Ahn, and D. S. Stephens. 2001. Transcriptional regulation of divergent capsule biosynthesis and transport operon promoters in serogroup B Neisseria meningitidis. Infect. Immun. 69:2502-2511.[Abstract/Free Full Text]
    61. 60
  61. Tzeng, Y.-L., X. Zhou, S. Bao, S. Zhao, C. Noble, and D. S. Stephens. 2006. Autoregulation of the MisR/MisS two-component signal transduction system in Neisseria meningitidis. J. Bacteriol. 188:5055-5065.[Abstract/Free Full Text]
    62. 61
  62. Wang, R. F., and S. R. Kushner. 1991. Construction of versatile low-copy-number vectors for cloning, sequencing and gene expression in Escherichia coli. Gene 100:195-199.[CrossRef][Medline]
    63. 62
  63. Wizemann, T. M., J. Moskovitz, B. J. Pearce, D. Cundell, C. G. Arvidson, M. So, H. Weissbach, N. Brot, and H. R. Masure. 1996. Peptide methionine sulfoxide reductase contributes to the maintenance of adhesins in three major pathogens. Proc. Natl. Acad. Sci. USA 93:7985-7990.[Abstract/Free Full Text]
    64. 63
  64. Wooldridge, K. G., M. Kizil, D. B. Wells, and D. A. Ala'Aldeen. 2005. Unusual genetic organization of a functional type I protein secretion system in Neisseria meningitidis. Infect. Immun. 73:5554-5567.[Abstract/Free Full Text]
    65. 64
  65. Wright, J. C., D. W. Hood, G. A. Randle, K. Makepeace, A. D. Cox, J. Li, R. Chalmers, J. C. Richards, and E. R. Moxon. 2004. lpt6, a gene required for addition of phosphoethanolamine to inner-core lipopolysaccharide of Neisseria meningitidis and Haemophilus influenzae. J. Bacteriol. 186:6970-6982.[Abstract/Free Full Text]
    66. 65
  66. Wu, H. J., K. L. Seib, J. L. Edwards, M. A. Apicella, A. G. McEwan, and M. P. Jennings. 2005. Azurin of pathogenic Neisseria spp. is involved in defense against hydrogen peroxide and survival within cervical epithelial cells. Infect. Immun. 73:8444-8448.[Abstract/Free Full Text]
    67. 66
  67. Yamamoto, K., K. Hirao, T. Oshima, H. Aiba, R. Utsumi, and A. Ishihama. 2005. Functional characterization in vitro of all two-component signal transduction systems from Escherichia coli. J. Biol. Chem. 280:1448-1456.[Abstract/Free Full Text]

Infection and Immunity, February 2008, p. 704-716, Vol. 76, No. 2
0019-9567/08/$08.00+0     doi:10.1128/IAI.01007-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Jamet, A., Rousseau, C., Monfort, J.-B., Frapy, E., Nassif, X., Martin, P. (2009). A two-component system is required for colonization of host cells by meningococcus. Microbiology 155: 2288-2295 [Abstract] [Full Text]  
  • Sannigrahi, S., Zhang, X., Tzeng, Y.-L. (2009). Regulation of the type I protein secretion system by the MisR/MisS two-component system in Neisseria meningitidis. Microbiology 155: 1588-1601 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tzeng, Y.-L.
Right arrow Articles by Stephens, D. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tzeng, Y.-L.
Right arrow Articles by Stephens, D. S.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»