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.
Highlights
Since the clinical introduction of antibiotics, bacteria have evolved efficient mechanisms to counteract their effects.This has resulted in the emergence of multidrug resistant (MDR) bacterial pathogens, which is a serious threat to public health as the treatment of infections caused by these pathogens is becoming increasingly difficult [1]
To determine the baseline gfp expression in B. subtilis 168 BSBII5 bmrC-gfp, this strain was cultured in Lysogeny broth (LB) without antibiotics
We have employed promoter-green fluorescent protein (GFP) fusions to monitor the expression dynamics of the genes encoding the MDR-ATP-binding cassette (ABC) transporter BmrCD upon exposure to antibiotics. This real-time approach revealed that the antibiotic-induced expression profile of bmrCD is constrained to the transition and stationary growth phases due to a tight control by the transition state regulator AbrB
Summary
Since the clinical introduction of antibiotics, bacteria have evolved efficient mechanisms to counteract their effects This has resulted in the emergence of multidrug resistant (MDR) bacterial pathogens, which is a serious threat to public health as the treatment of infections caused by these pathogens is becoming increasingly difficult [1]. The model bacterium Bacillus subtilis, a close relative of notoriously drug-resistant Gram-positive bacteria, such as Staphylococcus aureus, is equipped with an extensive repertoire of genes that encode for potential drug efflux transporters [3]. These include a significant number of ABC transporters [4]. Among these eight is the heterodimeric ABC transporter YheI/YheH, which was renamed BmrC/BmrD (in short BmrCD) due to its homology to the LmrC/LmrD ABC transporter of Lactococcus lactis [5]
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