Available online at www.sciencedirect.com ScienceDirect Regulation of antibiotic-resistance by non-coding RNAs in bacteria Daniel Dar and Rotem Sorek Antibiotic resistance genes are commonly regulated by overcome antibiotics through various mechanisms, such sophisticated mechanisms that activate gene expression in as the ejection of antibiotics from the cell via efflux response to antibiotic exposure. Growing evidence suggest pumps, enzymatic deactivation of the antibiotic mole- that cis-acting non-coding RNAs play a major role in regulating cules, and protection of antibiotic cellular targets via the expression of many resistance genes, specifically those chemical modifications (e.g. modifying the ribosome or which counteract the effects of translation-inhibiting the cell wall) [4]. Together, such resistance mechanisms 0 antibiotics. These ncRNAs reside in the 5 UTR of the regulated threaten the continued efficacy of antibiotics in medicine gene, and sense the presence of the antibiotics by recruiting [3]. translating ribosomes onto short upstream open reading frames (uORFs) embedded in the ncRNA. In the presence of translation-inhibiting antibiotics ribosomes arrest over the Although resistance genes provide bacteria with a distinct uORF, altering the RNA structure of the regulator and switching advantage during exposure to antibiotic, they are gener- the expression of the resistance gene to ‘ON’. The specificity of ally thought to carry a fitness burden at times when these riboregulators is tuned to sense-specific classes of antibiotics are not applied [2,5,6]. For example methyl- antibiotics based on the length and composition of the ation of specific residues in the 23S ribosomal RNA respective uORF. Here we review recent work describing new protects bacteria from macrolide antibiotics, but also types of antibiotic-sensing RNA-based regulators and causes cell-wide disruption in protein synthesis, which elucidating the molecular mechanisms by which they function lead to major fitness defects [7 ]. Therefore, to mitigate to control antibiotic resistance in bacteria. the negative effects of antibiotic resistance, bacteria employ regulatory mechanisms that can sense the antibi- otic molecule, and then selectively activate the expres- Address sion of the relevant antibiotic resistance genes only dur- Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel ing exposure to the antibiotic, effectively circumventing the fitness burden and facilitating the long term mainte- Corresponding author: Sorek, Rotem ([email protected]) nance of such genes in the genome [2,5,6,7 ]. Current Opinion in Microbiology 2017, 36:111–117 This review comes from a themed issue on Cell regulation One of the common mechanisms underlying antibiotic- dependent activation of resistance genes is via specific Edited by Petra Dersch and Michael T Laub transcription factors that can sense the presence of the antibiotic [4]. In Gram-negative bacteria such regulation is manifested, for example in the context of the tetracy- http://dx.doi.org/10.1016/j.mib.2017.02.005 cline efflux pump, tetA. In the absence of tetracycline, the transcriptional repressor TetR constitutively binds the 1369-5274/ã 2017 Elsevier Ltd. All rights reserved. tetA promoter and inhibits the expression of the tetA resistance gene [8]. When tetracycline antibiotic is pres- ent in the cell, direct binding of tetracycline to the tetR repressor leads to its dissociation from the DNA and drives tetA expression, leading to antibiotic resistance Introduction [8] (Figure 1a). Transcription regulation has also been detected in the vancomycin resistance operon, vanHAX, The discovery of antibiotic compounds had made a commonly found in Enterococci [9]. In this case, however, profound impact on modern medicine, extending both the presence of vancomycin is detected by a membrane life span and life quality. Presently, antibiotics are perva- sensory kinase (VanS) which, in turn, phosphorylates and sively administered in clinical and in veterinary care, as activates VanR, a transcription regulator that drives the well as in agricultural applications, resulting in a tremen- expression of the vanHAX resistance operon [9] dous flow of antibiotics into the environment [1]. The (Figure 1b). Analogous forms of transcription regulation immense selective pressure caused by exposure to anti- occur in many bacteria, where they play important roles in biotic has driven the rapid spread and evolution of anti- controlling the expression of antibiotic resistance genes biotic-resistance genes among pathogenic and commen- [2,5]. sal bacteria [2,3]. These resistance genes allow bacteria to www.sciencedirect.com Current Opinion in Microbiology 2017, 36:111–117 112 Cell regulation Figure 1 (a) Regulation by the TetR repressor (b) Regulation by the VanS/VanR two-component system TetR No transcription Vancomycin tetA DNA Cell wall tetA promoter VanS Plasma Tetracycline binding releases the TetR repressor membrane TetR VanR P tetracycline P Activated VanR RNA-polymerase RNA-polymerase Active transcription Active transcription P tetA DNA vanJKHAX DNA tetA promoter (c ) Regulation by translational attenuation “OFF”Inhibition of translation initiation “ON” Antibiotics RBS is sequestered RBS is free uORF by RNA structure RBS Translation can inititate ermC mRNA ermC mRNA Riboregulator RBS Ribosome arrests over uORF (d) Regulation by transcriptional attenuation “OFF” Premature transcription termination “ON” Antibiotics uORF Terminator stem-loop formation Terminator is bypassed leading leads to premature termination to transcription readthrough UUUUU UUUUU resistance gene mRNA Riboregulator Ribosome arrests over uORF Current Opinion in Microbiology Control of antibiotic resistance by protein or RNA-based regulators. (a) The TetR DNA-binding repressor targets the promoter of tetA and blocks transcription. Upon binding to tetracycline, the repressor dissociates, promoting tetA transcription initiation. (b) The membrane protein VanS senses vancomycin and then phosphorylates the response regulator VanR, which activates the transcription of the vancomycin resistance gene operon. (c) Schematic representation of translational attenuation. In the absence of antibiotics the riboregulator folds into an RBS sequestering structure, such that translation of the mRNA of the resistance gene cannot be initiated. Subsequent exposure to translation inhibiting antibiotics causes the ribosome to stall over a specific position in the uORF, resulting in structural reshaping of the riboregulator such that the RBS is released, thus enabling translation to initiate. (d) Transcriptional attenuation regulates expression by controlling the formation of a premature transcription terminator—a stem-loop structure immediately followed by a poly uridine tract. In the absence of antibiotics, transcription begins, yet terminates prematurely. Ribosome stalling over the uORF inhibits terminator stem-loop formation and promotes transcription of the resistance gene. RNA-mediated regulation of antibiotic ncRNAs, also known as riboregulators, are structured 0 resistance RNA elements that reside within 5 untranslated regions 0 Growing evidence show that, in addition to classic tran- (5 UTRs) of antibiotic resistance genes, in particular scription-factor mediated gene regulation, bacteria fre- those that provide resistance to ribosome-inhibiting anti- quently employ cis-regulatory non-coding RNAs biotics. In the absence of antibiotics these RNA regula- (ncRNAs) that sense the presence of antibiotics and tors inhibit the expression of the resistance gene in cis by regulate resistance genes accordingly [10–12,13 ]. These either masking the ribosome binding site (RBS) or Current Opinion in Microbiology 2017, 36:111–117 www.sciencedirect.com Regulation of antibiotic-resistance by non-coding RNAs in bacteria Dar and Sorek 113 generating a premature transcriptional terminator within dependent ribosome stalling over the short uORF dis- 0 the 5 UTR [14,15] (Figure 1c,d). Conversely, when the rupts the terminator stem-loop base-pairing, enabling antibiotic is present in the cell, the RNA structure of transcriptional readthrough and synthesis of the bmrCD these regulators is altered, resulting in alternative base- mRNA [28] (Figure 1d). Transcriptional attenuation has pairing patterns that do not attenuate transcription or been implicated in the regulation of several antibiotic translation, and so activate the expression of the regulated resistance genes including ribosome methylating gene (Figure 1c,d). enzymes [15], antibiotic efflux pumps [28,30], and ribo- some rescue factors [31,32]. The majority of antibiotic responsive riboregulators have been found to sense the presence of translation-inhibiting Until recently, most antibiotic-responsive riboregulation antibiotics by directly measuring the activity the ribo- was thought to function via RBS-mediated translational some, that is the target of the antibiotic [11]. This attenuation, with transcriptional attenuation (regulation regulatory mechanism, also known as attenuation, is via the generation of a premature transcriptional termi- 0 enabled by the utilization of short upstream open reading nator in the 5 UTR) considered relatively rare [11,15]. A frames (uORF) that recruit translating ribosomes to the recent study, which employed term-seq, a high-through- 0 regulatory 5 UTR element. In the presence of certain put RNA-sequencing method for detecting premature antibiotics, translating ribosomes