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The multidrug ABC transporter BmrC/BmrD of Bacillus subtilis is regulated via a ribosome-mediated transcriptional attenuation mechanism.

ABSTRACT: Expression of particular drug transporters in response to antibiotic pressure is a critical element in the development of bacterial multidrug resistance, and represents a serious concern for human health. To obtain a better understanding of underlying regulatory mechanisms, we have dissected the transcriptional activation of the ATP-binding cassette (ABC) transporter BmrC/BmrD of the Gram-positive model bacterium Bacillus subtilis. By using promoter-GFP fusions and live cell array technology, we demonstrate a temporally controlled transcriptional activation of the bmrCD genes in response to antibiotics that target protein synthesis. Intriguingly, bmrCD expression only occurs during the late-exponential and stationary growth stages, irrespective of the timing of the antibiotic challenge. We show that this is due to tight transcriptional control by the transition state regulator AbrB. Moreover, our results show that the bmrCD genes are co-transcribed with bmrB (yheJ), a small open reading frame immediately upstream of bmrC that harbors three alternative stem-loop structures. These stem-loops are apparently crucial for antibiotic-induced bmrCD transcription. Importantly, the antibiotic-induced bmrCD expression requires translation of bmrB, which implies that BmrB serves as a regulatory leader peptide. Altogether, we demonstrate for the first time that a ribosome-mediated transcriptional attenuation mechanism can control the expression of a multidrug ABC transporter.

SUBMITTER: Reilman E

PROVIDER: S-EPMC4191407 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Project description:A complete transcript of the Bacillus subtilis pyr operon contains the following elements in 5' to 3' order: a 151-nucleotide (nt) untranslated leader; pyrR, encoding a 20-kDa protein; a 173-nt intercistronic region; pyrP, encoding a 46-kDa protein; a 145-nt intercistronic region; and eight overlapping cistrons encoding all of the six enzymes for de novo pyrimidine biosynthesis. Transcription is controlled by the availability of pyrimidines via an attenuation mechanism. There are three transcription terminators within the operon, each of which is preceded by another stem-loop structure, the antiterminator, whose formation would prevent formation of the terminator stem-loop. These are located in the leader, the pyrR-pyrP intercistronic region, and the pyrP-pyrB intercistronic region. Northern (RNA) blot analysis has identified transcripts of lengths which coincide with termination at these proposed attenuation sites and whose relative abundances vary in the expected pyrimidine-dependent manner. Each antiterminator contains a 50-base conserved sequence in its promoter-proximal half. Various transcriptional fusions of the pyr promoter and surrounding sequences to promoterless reporter genes support an attenuation mechanism whereby when pyrimidines are abundant, the PyrR protein binds to the conserved sequence in the pyr mRNA and disrupts the antiterminator, permitting terminator hairpin formation and promoting transcription termination. Deletion of pyrR from the chromosome resulted in the constitutive, elevated expression of aspartate transcarbamylase, which is encoded by pyrB, the third gene in the operon. Complementation of an E. coli upp mutant, as well as direct enzymatic assay, has demonstrated that pyrR also confers uracil phosphoribosyltransferase activity. Analysis of pyrR and upp deletion mutants demonstrated that upp, not pyrR, encodes the quantitatively important uracil phosphoribosyltransferase activity. The pyrP gene probably encodes an integral membrane uracil permease.

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The multidrug ABC transporter BmrC/BmrD of Bacillus subtilis is regulated via a ribosome-mediated transcriptional attenuation mechanism.

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