ABSTRACT
MtaN (Multidrug Transporter Activation, N terminus) is a constitutive, transcriptionally active 109-residue truncation mutant, which contains only the N-terminal DNA-binding and dimerization domains of MerR family member Mta. The 2.75 A resolution crystal structure of apo-MtaN reveals a winged helix-turn-helix protein with a protruding 8-turn helix (alpha5) that is involved in dimerization by the formation of an antiparallel coiled-coil. The hydrophobic core and helices alpha1 through alpha4 are structurally homologous to MerR family member BmrR bound to DNA, whereas one wing (Wing 1) is shifted. Differences between the orientation of alpha5 with respect to the core and the revolution of the antiparallel coiled-coil lead to significantly altered conformations of MtaN and BmrR dimers. These shifts result in a conformation of MtaN that appears to be incompatible with the transcription activation mechanism of BmrR and suggest that additional DNA-induced structural changes are necessary.
Subject(s)
DNA-Binding Proteins/chemistry , Trans-Activators/chemistry , Amino Acid Sequence , Bacterial Proteins/chemistry , Biological Transport , Crystallography, X-Ray , DNA/metabolism , DNA-Binding Proteins/metabolism , Dimerization , Drug Resistance, Multiple , Escherichia coli/metabolism , Models, Molecular , Molecular Sequence Data , Promoter Regions, Genetic , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , Trans-Activators/metabolism , Transcription, GeneticABSTRACT
The N-terminal DNA-binding domain of the multidrug transporter activation protein (MtaN) was crystallized by the hanging-drop vapour-diffusion method using lithium chloride as a precipitant. The crystals are orthorhombic and belong to the space group I2(1)2(1)2(1), with unit-cell parameters a = 49.4, b = 67.8, c = 115. 0 A. Diffraction data have been collected at 100 K to 2.75 A resolution at a synchrotron-radiation source.
Subject(s)
Bacillus subtilis/metabolism , Bacterial Proteins/chemistry , DNA/metabolism , Bacterial Proteins/metabolism , Crystallization , Crystallography, X-Ray , Protein ConformationABSTRACT
Little is known about the natural functions of multidrug-efflux transporters expressed by bacteria. Although identified as membrane proteins actively extruding exogenous toxins from the cell, they may actually be involved in the transport of as yet unidentified specific natural substrates. The expression of two highly similar multidrug transporters of Bacillus subtilis, Bmr and Blt, is regulated by specific transcriptional activators, BmrR and BltR, respectively, which respond to different inducer molecules, thus suggesting distinct functions for the two transporters. Here, we describe an alternative mechanism of regulation, which involves a global transcriptional activator, Mta, a member of the MerR family of bacterial regulatory proteins. The individually expressed N-terminal DNA-binding domain of Mta interacts directly with the promoters of bmr and blt and induces transcription of these genes. Additionally, this domain stimulates the expression of the mta gene itself and at least one more gene, ydfK, which encodes a hypothetical membrane protein. These results and the similarity of Mta to the thiostrepton-induced protein TipA of Streptomyces lividans strongly suggest that Mta is an autogenously controlled global transcriptional regulator, whose activity is stimulated by an as yet unidentified inducer. This stimulation is mimicked by the removal of the C-terminal inducer-binding domain. The fact that both Bmr and Blt are controlled by this regulator demonstrates that some of their functions are either identical or, at least, related. Further analysis of Mta-mediated regulation may reveal the natural function of the system of multidrug transporters in B. subtilis and serve as a paradigm for similar systems in other bacteria.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Acetyltransferases , Bacillus subtilis/genetics , Bacterial Proteins/genetics , DNA-Binding Proteins/genetics , Gene Expression Regulation, Bacterial , Genes, Regulator , Membrane Transport Proteins , Trans-Activators/genetics , Amino Acid Sequence , Anti-Bacterial Agents/pharmacology , Blotting, Northern , Carrier Proteins/genetics , Chromosome Mapping , DNA Footprinting , DNA Mutational Analysis , Genotype , Molecular Sequence Data , Multigene Family/genetics , Promoter Regions, Genetic , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid , Thiostrepton/pharmacology , Transcription, Genetic , Transduction, GeneticABSTRACT
A Streptococcus pneumoniae strain selected for resistance to ethidium bromide demonstrated enhanced energy-dependent efflux of this toxic dye. Both the ethidium resistance and the ethidium efflux could be inhibited by the plant alkaloid reserpine. The ethidium-selected cells demonstrated cross-resistance to the fluoroquinolones norfloxacin and ciprofloxacin; this resistance could also be completely reversed by reserpine. Furthermore, reserpine potentiated the susceptibility of wild-type S. pneumoniae to fluoroquinolones and ethidium. The most plausible explanation for these results is that S. pneumoniae, like some other gram-positive bacteria, expresses a reserpine-sensitive multidrug transporter, which may play an important role in both intrinsic and acquired resistances of this pathogen to fluoroquinolone therapy.
