Autoregulation of the MisR/MisS Two-Component Signal Transduction System inNeisseria meningitidis

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Abstract
Two-component regulatory systems are involved in processes important for bacterial pathogenesis. The proposedmisR/misS(orphoP/phoQ) system is one of four two-component systems of the obligate human pathogenNeisseria 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 His6and 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∼P) and MisR with themisRpromoter was demonstrated by gel mobility shift assays, where MisR∼P exhibited higher affinity than did the nonphosphorylated protein. The transcriptional start site of themisRSoperon 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 themisRSoperon. Limited Mg2+concentration failed to induce expression of themisRSoperon, 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.