Autoregulation of the MisR/MisS two-component signal transduction system in Neisseria meningitidis

Abstract

Two-component regulatory systems are involved in processes important for bacterial pathogenesis. The proposed misR/misS (or phoP/phoQ) system is one of four two-component systems of the obligate human pathogen Neisseria meningitidis. Inactivation of this system results in loss of phosphorylation of the lipooligosaccharide inner core and causes attenuation in a mouse model of meningococcal infection. MisR and the cytoplasmic domain of MisS were purified as His6 and maltose binding protein fusion proteins, respectively. The MisS fusion was shown to be autophosphorylated in the presence of ATP, and the phosphoryl group was subsequently transferred to MisR. The phosphotransfer reaction was halted with a MisR/D52A mutation, while a MisS/H246A mutation prevented autophosphorylation. Specific interaction of phosphorylated MisR (MisR approximately P) and MisR with the misR promoter was demonstrated by gel mobility shift assays, where MisR approximately P exhibited higher affinity than did the nonphosphorylated protein. The transcriptional start site of the misRS operon was mapped, and DNase I protection assays revealed that MisR interacted with a 15-bp region upstream of the transcriptional start site that shared no similarity to binding motifs of other two-component systems. Transcriptional reporter studies suggested that MisR phosphorylation is critical for the autoinduction of the misRS operon. Limited Mg2+ concentration failed to induce expression of the misRS operon, which is the only operon now proven to be under the direct control of the MisRS two-component system. Thus, these results indicate that the meningococcal MisRS system constitutes a functional signal transduction circuit and that both components are critical in the autoregulation of their expression.

FIG. 1.

In vitro phosphorylation of MisS and MisR. MisS was autophosphorylated with 0.4 mM ATP for 10 min prior to the addition of MisR, and the reaction was allowed to proceed for an additional 30 min before being quenched by SDS-PAGE loading buffer. The reaction mixtures were resolved on a 12% gel, and the radioactivity was detected by a phosphorimager. The final concentration of each protein is indicated above each panel. (A) Wild-type MisS and wild-type MisR; (B) MisS246HA and wild-type MisR; (C) wild-type MisS and MisR52DA.

FIG. 2.

Primer extension analysis using a primer complementary to misR and the total RNA isolated from the wild-type strain NMB. Lanes G, A, T, and C indicate the dideoxy sequencing reactions. The asterisk indicates the transcriptional start site.

FIG. 3.

Transcriptional activities of various misR::lacZ promoter fusions. The schematic diagram of the misRS operon shows the locations of primers used to generate the promoter fragment. The cloned region with respect to the transcriptional start site (+1) is shown in parentheses. The dashed line indicates the 33-bp deleted region within the promoter in strain YT0324. β-Galactosidase activities of strains grown in GC broth to mid-log phase are expressed as Miller units ± standard deviation corresponding to mean values of triplicate measurements. The activity of a promoterless lacZ construct (strain 263) is 57.4 ± 1.7 Miller units.

FIG. 4.

EMSA of MisR-misR promoter interaction. (A) A 608-bp PCR product (∼5 fmol) of a DNA fragment containing the misR promoter region (−504 to +104) was ³²P-labeled by T4 kinase, mixed with increasing amounts of MisR∼P (left) and MisR (right) for 20 min at 30°C and then subjected to gel electrophoresis. MisR∼P was generated by incubation with 50 mM acetyl phosphate for 30 min at 37°C. The amounts of MisR protein are 0, 34, 68, 136, 204, 272, and 340 pmol. (B) Competition EMSA. ³²P-labeled misR probe was mixed with the indicated amounts of either specific or nonspecific DNA, incubated with MisR∼P (102 pmol), and analyzed as described above.

FIG. 5.

Mapping of the MisR binding site within the misR promoter region by EMSA using overlapping DNA fragments. (A) Schematic presentation of each probe used in this study with the primers used to generate these DNA fragments depicted above. Hatched boxes represent probes that are shifted by MisR, and gray boxes indicate PCR fragments that are not shifted. Numbers adjacent to the boxes indicate the endpoints of the fragment with respect to the start site of transcription. (B) In each panel, the left lane contains DNA probe only, the middle lane contains DNA probe and MisR∼P (340 pmol), and the right lane contains DNA probe and MisR (340 pmol). The binding conditions are as described in the legend to Fig. 4.

FIG. 6.

Transcription of misR (A) and MisR protein production (B) are reduced in the misS mutant. (A) β-Galactosidase activities of misR::lacZ fusions in various genetic backgrounds. The misRS genotypes are indicated below the strains. Strain YT263 is the promoterless negative control. (B) Western blotting and probing with polyclonal anti-MisR antisera were performed on whole-cell lysates of the wild-type strain, the misR mutant, and the misS mutant. Equal cell numbers (1 × 10⁸ cells) were loaded in each sample.

FIG. 7.

DNase I protection of the misR promoters by MisR∼P. The coding (A) or noncoding (B) strand of ³²P-end-labeled misR promoter fragment between −205 to +104 with respect to the misR transcriptional start site was incubated with increasing amounts of MisR (left in panels A and B) or MisR∼P (right in panels A and B) for 20 min at 30°C and then subjected to DNase I digestion. The amounts of MisR used in panel A are 0, 85, 170, 255, and 340 pmol for both MisR and MisR∼P, while those used in panel B are 0, 170, 340, 680, 1,360, and 2,720 pmol for MisR and 0, 170, and 340 pmol for MisR∼P. Dideoxy chain termination sequences corresponding to the probes are shown in the order of G, A, T, and C. Black bars indicate regions protected, while the gray bar in panel B indicates a region of the noncoding strand that was not reproducibly protected in all footprinting experiments. (C) Sequence of the misR promoter region used in the DNase I footprinting experiments. The protected regions for MisR (boxed and shaded) and MisR∼P (shaded) are indicated for both strands. The transcriptional start site and −10 and −35 promoter elements are boxed, while the start codons are underlined. Arrows indicate primers used to generate the probes.

FIG. 8.

The misR promoter activity is not affected by different magnesium concentrations. β-Galactosidase activities of reporter strains grown in peptone broth supplemented with various amounts of magnesium were determined. Black bar, 0 mM; dotted bar, 1 mM; hatched bar, 2 mM; gray bar, 10 mM; white bar, 20 mM. A representative result from two independent experiments is shown where each condition was assayed in four replicates. Error bars indicate standard deviations.