Insights Into the Stress Response Triggered by Kasugamycin in Escherichia Coli

antibiotics Article Insights into the Stress Response Triggered by Kasugamycin in Escherichia coli Christian Müller, Lena Sokol, Oliver Vesper, Martina Sauert and Isabella Moll * Max F. Perutz Laboratories, Center for Molecular Biology, Department of Microbiology, Immunobiology and Genetics, University of Vienna, Vienna Biocenter (VBC), Dr. Bohr-Gasse 9/4, A-1030 Vienna, Austria; [email protected] (C.M.); [email protected] (L.S.); [email protected] (O.V.); [email protected] (M.S.) * Correspondence: [email protected]; Tel.: +43-1-427-754-606 Academic Editor: Claudio O. Gualerzi Received: 1 April 2016; Accepted: 23 May 2016; Published: 1 June 2016 Abstract: The bacteriostatic aminoglycoside antibiotic kasugamycin inhibits protein synthesis at an initial step without affecting translation elongation. It binds to the mRNA track of the ribosome and prevents formation of the translation initiation complex on canonical mRNAs. In contrast, translation of leaderless mRNAs continues in the presence of the drug in vivo. Previously, we have shown that kasugamycin treatment in E. coli stimulates the formation of protein-depleted ribosomes that are selective for leaderless mRNAs. Here, we provide evidence that prolonged kasugamycin treatment leads to selective synthesis of specific proteins. Our studies indicate that leaderless and short-leadered mRNAs are generated by different molecular mechanisms including alternative transcription and RNA processing. Moreover, we provide evidence for ribosome heterogeneity in response to kasugamycin treatment by alteration of the modification status of the stalk proteins bL7/L12. Keywords: kasugamycin; translation initiation; leaderless mRNA; Escherichia coli 1. Introduction The bactericidal aminoglycoside antibiotic kasugamycin (Ksg) inhibits protein synthesis in eukaryotic and prokaryotic cells at an initial step [1] without affecting translational elongation [2]. Several experiments have suggested that it prevents the formation of the pre-initiation complex by interfering with the binding of the initiator fMet-tRNAfMet to the ribosomal P-site in E. coli ribosomes [3,4]. Structural analyses revealed that the antibiotic binds to the mRNA track of the ribosome, thereby preventing formation of the translation initiation complex [5,6]. Resistance to Ksg is caused by the lack of dimethylation of two adjacent adenosines 1518/1519 in helix 45 at the 31-end of the 16S rRNA [7]. Intriguingly, this post-transcriptional modification is the only universally conserved modification common to the rRNA of prokaryotes and eukaryotes [8], suggesting an important function that still remains to be elucidated. Nevertheless, structural analyses indicate that the modification alters the conformation and flexibility of the ultimate stem-loop in the 16S rRNA [9] and, thus, the dimethylated adenosine residues might assist in correct folding of the 31-terminus of the 16S rRNA to ensure the availability of the anti-Shine-Dalgarno (aSD) sequence required for translation initiation on canonical transcripts [10]. At variance with the characterized effect of the antibiotic Ksg on the initiation of protein synthesis on canonical transcripts, translation of leaderless mRNAs (lmRNAs) continues in the presence of the drug in vivo [1,11]. Despite the fact that the conserved nucleotides A1518 and A1519 of 16S rRNA, the methylation of which causes sensitivity to the drug, lie in close proximity to the aSD sequence, Ksg does not interfere with formation of the SD-aSD helix during ternary complex formation [12]. Antibiotics 2016, 5, 19; doi:10.3390/antibiotics5020019 www.mdpi.com/journal/antibiotics Antibiotics 2016, 5, 19 2 of 13 In addition, the activity of initiation factor 3 (IF3) was not affected by Ksg [12], even though IF3 interacts with the ribosome close to the Ksg binding site [13]. However, Ksg differentially affects the formation of 30S and 70S initiation complexes [3,12] underpinning the 70S initiation pathway for lmRNAs [14]. Surprisingly, ribosome profile analyses revealed the appearance of a ribosomal subpopulation after Ksg treatment in E. coli, which sediments at 61S [15]. These ribosomes contain a normal 50S subunit but a 30S species with a reduced protein complement: at least six proteins are lacking from the 30S subunit, among which are the functionally important proteins bS1 and uS2, both of which are essential for translation of canonical mRNAs [16–19], but are dispensable for translation of lmRNAs [15,17–19]. The characterization of the 61S particles revealed that they form initiation complexes on lmRNA comparable to 70S ribosomes. However, in contrast to 70S ribosomes these 61S particles are competent in translation of lmRNA in the presence of Ksg in vivo and in vitro [15]. Treatment of E. coli with antibiotics that inhibit transcription or translation triggers the activation of the mazEF module, a so-called type 2 toxin-antitoxin (TA) system [20,21]. In general, type 2 TA systems comprise two transcriptionally and translationally coupled genes that encode for a labile antitoxin and a stable toxin. As the labile antitoxin interacts with the toxin, the toxic activity is inhibited. However, during stress conditions, which inhibit the continuous expression of the TA genes, degradation of the antitoxin releases the active toxin that interferes with diverse activities in the cell like replication, transcription, or translation [22]. The toxin MazF is an endoribonuclease that degrades the majority of bulk mRNA by cleaving at single-stranded ACA sites [23]. In addition, its activation results in the processing of distinct transcripts within their 51-UTR, which enables their selective synthesis by specialized ribosomes that are likewise modified by the MazF activity [24]. Here, the MazF toxin cleaves the 16S rRNA in the context of mature and active ribosomes close to the 31-terminus, thereby removing the last 43 nucleotides comprising helix 45 and the aSD sequence (from here on referred to as RNA43). Taken together, these particular processing events performed by the activation of a single endoribonuclease result in a fast and energy-saving reprogramming of the translatome in response to diverse stress conditions [25,26]. Given that the addition of the aminoglycoside Ksg mimics the effect of the toxin MazF in rendering the translational machinery specific for lmRNAs, our observation that several proteins are selectively synthesized in the presence of the antibiotic tempted us to speculate that Ksg treatment triggers the mazEF system. We suspected that the MazF toxin is involved in the formation of 51-terminally processed transcripts that are consequently translated by the 61S ribosomes in the presence of Ksg. Thus, we studied the consequences of Ksg treatment on the transcriptome and translatome in E. coli. Our results show that besides formation of protein depleted 61S ribosomes, extended Ksg treatment leads to selective protein synthesis that can be attributed to the formation of leaderless and short-leadered transcripts. Surprisingly, Ksg does not lead to a strong activation of the mazEF TA system but our results suggest that the majority of transcripts with short leaders are rather engendered by alternative transcription. In addition, we observed ribosome heterogeneity in response to kasugamycin treatment that is based on the modification status of the stalk proteins bL7/L12. 2. Results 2.1. Selective Protein Synthesis after Prolonged Kasugamycin Treatment As shown before, the addition of Ksg results in the cessation of E. coli growth (Figure1a) and the inhibition of protein synthesis (Figure1b, lanes 2–3). However, pulse labeling experiments employing E. coli strain MG1655 harboring plasmid pRB381-1 [14], encoding the leaderless cI-lacZ fusion gene, revealed that translation of a leaderless reporter continued in the presence of 0.5 mg/mL Ksg (Figure1b, lanes 2–6; [15]). Intriguingly, 90–120 min after addition of the antibiotic, we observed restored de novo translation of particular proteins (Figure1b, lanes 4–6), which was resistant to Ksg even after retreatment of the cells with the antibiotic at time point 120 min (Figure1b, lane 6). Considering the selective synthesis of proteins encoded by lmRNAs after induction of mazF together with the fact Antibiotics 2016, 5, 19 3 of 13 Antibiotics 2016, 5, 19 3 of 13 that antibiotic treatment can trigger the mazEF system [20,21] we hypothesized that Ksg treatment likewisetreatment results likewise in activation results in activation of MazF. This of MazF. would This in turn would lead in to turn the lead formation to the formation of lmRNAs, of whichlmRNAs, are selectivelywhich are selectively translated translated in the presence in the ofpresence Ksg. To of testKsg. for To this test hypothesisfor this hypothesis we repeated we repeated the growth the analysisgrowth analysis using the using isogenic the isogenicmazF deletion mazF deletion mutant mutant and performed and performed pulse labeling pulse labeling upon addition upon addition of Ksg (Figureof Ksg 1(Figurec,d). Surprisingly, 1c,d). Surprisingly, the experiment the experiment revealed revealed that despite that thedespite lack ofthe MazF lack theof MazF same the proteins same areproteins selectively are selectively synthesized synthesized after Ksg after treatment Ksg trea (Figuretment1d, (Figure lanes 4–51d, andlanes 9–10). 4–5 and This 9–10). result This indicates result thatindicates MazF that is not MazF required is not forrequired the specific for the protein

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