1887

Abstract

The multidrug efflux operon of is regulated by the NfxB repressor. Two forms of NfxB have been reported [Shiba (1995). , 5872) although mutagenesis studies here confirm that the larger protein (199 amino acids, 22.4 kDa) is the functional repressor. NfxB binds upstream of the transcription initiation site to a region containing two inverted repeats, both of which are required for binding. Two-hybrid assays confirmed that NfxB is a multimer, with the C-terminal two-thirds of the repressor required for multimerization. Random mutagenesis identified several mutations within the C-terminal region of NfxB required for multimerization, all of which mapped to a three-helix subdomain of the C-terminal region in a structural model of the repressor, which may thus represent the multimerization domain. These mutations compromised NfxB binding to its target DNA in electromobility shift assays, and their introduction into the chromosome of enhanced expression and promoted multidrug resistance, consistent with the functional NfxB repressor being a multimer. Site-directed and spontaneous mutants showing increased expression and multidrug resistance were also recovered, with mutations mapping to the three-helix subdomain again impacting multimerization and DNA binding. Mutations mapping to the N-terminal helix–turn–helix motif implicated in DNA binding did not impact multimerization although they did render the repressor insoluble and unsuitable for mobility shift assays. Size exclusion column chromatography demonstrated that wild-type NfxB forms tetramers in solution, although a mutant form of the repressor carrying a G192D substitution near the C terminus of the protein and compromised for DNA binding and repressor activity forms dimers. These results suggest that NfxB operates as a tetramer (dimer of dimers) and that the C terminus of the protein serves as a tetramerization domain.

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2013-10-01
2020-10-22
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