Ribosome Assembly As Antimicrobial Target

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Article Ribosome Assembly as Antimicrobial Target

Rainer Nikolay 1,*,†, Sabine Schmidt 2,†, Renate Schlömer 2, Elke Deuerling 2,* and Knud H. Nierhaus 1 1 Institut für Medizinische Physik und Biophysik, Charité—Universitätsmedizin Berlin, 10117 Berlin, Germany; [email protected] 2 Molecular Microbiology, University of Konstanz, Konstanz 78457, Germany; [email protected] (S.S.); [email protected] (R.S.) * Correspondence: [email protected] (R.N.); [email protected] (E.D.); Tel.: +49-30-450-524165 (R.N.); +49-7531-88-2647 (E.D.) † These authors contributed equally to this work.

Academic Editor: Claudio O. Gualerzi Received: 31 March 2016; Accepted: 16 May 2016; Published: 27 May 2016

Abstract: Many antibiotics target the ribosome and interfere with its translation cycle. Since translation is the source of all cellular proteins including ribosomal proteins, protein synthesis and ribosome assembly are interdependent. As a consequence, the activity of translation inhibitors might indirectly cause defective ribosome assembly. Due to the difﬁculty in distinguishing between direct and indirect effects, and because assembly is probably a target in its own right, concepts are needed to identify small molecules that directly inhibit ribosome assembly. Here, we summarize the basic facts of ribosome targeting antibiotics. Furthermore, we present an in vivo screening strategy that focuses on ribosome assembly by a direct ﬂuorescence based read-out that aims to identify and characterize small molecules acting as primary assembly inhibitors.

Keywords: protein synthesis as preferential target of antibiotics; ribosome as antibiotic target; inhibitors of ribosome assembly; concepts for identifying assembly inhibitors

1. Introduction Most antibiotics are microbial secondary metabolites mainly produced by actinomycetes. The production is usually induced by unfavorable growth conditions, when the producer has turned into the stationary or sporulation phase and its own metabolic activity is sharply reduced. Under these conditions, antibiotics will block the growth of better adapted competitors and thus prevent a further impairment of life conditions (for review, see [1]). Antibiotics therefore improve the survival chance of the producer, which represents the selective force for the natural development of antibiotics. Under these restricted conditions, synthesis of nucleic acids and protein synthesis is strongly reduced in the producer cell, which explains that, in many cases, the producer is not resistant against its own product. Antibiotics are chemically extremely diverse. The synthesis by microorganisms and a molecular weight less than 1000 Da are the only common features [2]. Nowadays, the term antimicrobials (short for antimicrobial substances) is used, which includes both natural products (antibiotics) and semi-synthetic or full-synthetic substances (chemotherapeutics). In addition to the wide chemical spectrum, the target spectrum is also enormous: More than 160 cellular targets have been described [3]. Examples are replication (e.g., nalidixic acid), transcription (e.g., rifamycin), translation (e.g., chloramphenicol) and the synthesis of the cell wall (e.g., penicillin). Most of the antibiotics inhibit the translating ribosome because, due to its complicated structure, it offers many interference points. The Escherichia coli ribosome contains 57 different components, 54 proteins and three ribosomal RNAs [4], and all of them are present in one copy per ribosome except the protein bL12, which is

Antibiotics 2016, 5, 18; doi:10.3390/antibiotics5020018 www.mdpi.com/journal/antibiotics Antibiotics 2016, 5, 18 2 of 13

presentAntibiotics in four 2016 copies., 5, 18 Nevertheless, the binding sites of the antibiotics are concentrated at and2 of around 13 functional hot spots of the small 30S and the large 50S subunit of the bacterial ribosome (Figure1a,b). copies. Nevertheless, the binding sites of the antibiotics are concentrated at and around functional These sites are often dominated or exclusively built by elements of the rRNA (ribosomal RNA), and, hot spots of the small 30S and the large 50S subunit of the bacterial ribosome (Figure 1a,b). These sites importantly,are often dominated the multiplicity or exclusively of rrn operons built by presentelements in of most the rRNA bacterial (ribosomal species RNA), aggravates and, importantly, development of resistancethe multiplicity [5]. Mutations of rrn operons in onlypresent one in genemost bacterial have low specie impacts aggravates and multiple development mutations of resistance are of low probability[5]. Mutations [6]. in only one gene have low impact and multiple mutations are of low probability [6].

FigureFigure 1. Antibiotic1. Antibiotic target target sites sites of of bacterial bacterial ribosomes.ribosomes. (a (a) )a afew few antibiotic antibiotic target target sites sites on onthe the30S 30S subunitsubunit mentioned mentioned in thein the text. text. Tet, Tet, tetracycline; tetracycline; Neo,Neo, neomycin, Str, Str, streptomycin; streptomycin; (b) ( bsome) some antibiotic antibiotic target sites on the 50S subunit. Cam, chloramphenicol; Lin, lincomycin. (from [7], modified, courtesy target sites on the 50S subunit. Cam, chloramphenicol; Lin, lincomycin. (from [7], modified, courtesy of Daniel N. Wilson, Munich Gene Center). of Daniel N. Wilson, Munich Gene Center). Most of the natural antibiotics inhibit the peptidyltransferase center (PTC) on the 50S subunit (FigureMost of 1a; the [8-10]). natural Reasons antibiotics for the inhibitprevalence the of peptidyltransferase the PTC as antibiotic center target (PTC)are (i) the on high the 50S number subunit (Figureof crevices1a; [ 8–10 allowing]). Reasons binding for theof small prevalence molecules of the with PTC high as affinity; antibiotic (ii) targetthe fact are that (i) the PTC high needs number a of creviceshigh allowingamount of binding structural of small flexibility molecules and any with interference high affinity; by drug (ii) the binding fact that or mutations the PTC needs causing a high amountresistance of structural might hamper flexibility the andspeed any and/or interference accuracy byof translation, drug binding which ormutations leads to the causing final outcome resistance mightthat hamper any rescuing the speed mutations and/or within accuracy the PTC of are translation, associated whichwith high leads fitness to the costs final [10]. outcome that any The most common target site of the 30S subunit is the decoding center of the A site, where the rescuing mutations within the PTC are associated with high fitness costs [10]. large group of aminoglycosides interferes with the fidelity of aminoacyl-tRNA(aa-tRNA)·EF-Tu·GTP The most common target site of the 30S subunit is the decoding center of the A site, where the selection (Figure 1b). Examples of binding sites on the 30S part of the A site outside the decoding center large group of aminoglycosides interferes with the fidelity of aminoacyl-tRNA(aa-tRNA)¨ EF-Tu¨ GTP are the tetracyclines, which block stable binding of aa-tRNAs to the A site. The sketch of the ribosomal selectionfunctions (Figure in Figure1b). Examples 2 summarizes of binding the interference sites on points the 30S of partthe various of the Agroups site outside of antibiotics the decoding with centerribosomal are the functions. tetracyclines, Some antibiotics which block act directly stable on binding translational of aa-tRNAs G-proteins. to theOne Aexample site. The is kirromycin, sketch of the ribosomalwhich functionsbinds to the in elongation Figure2 summarizes factor EF-Tu·GTP the interference and blocks points its switch of the into various the GDP groups conformer of antibiotics after withdelivery ribosomal of the functions. aa-tRNA Someto the antibioticsA site. The result act directly is that onEF-Tu translational remains on G-proteins. the ribosome One and examplethus is kirromycin,blocks protein which synthesis. binds Another to the elongation example is fusidic factor acid EF-Tu (FA)¨ GTP acting and on blocks the second its switch elongation into factor the GDP conformerG (EF-G). after EF-G delivery is responsible of the aa-tRNA for the translocation to the A site. of Theribosomes result isto thatthe next EF-Tu codon remains of the on mRNA the ribosome after anda thus decoding blocks step. protein FA also synthesis. blocks the Another conformational example isswitch fusidic into acid the (FA)GDP actingconformer on the of secondEF-G, which elongation is factornecessary G (EF-G). for EF-Gthe dissociation is responsible of this for factor the translocation after it has fulfilled of ribosomes its ribosomal to the function. next codon of the mRNA after a decoding step. FA also blocks the conformational switch into the GDP conformer of EF-G, which is necessary for the dissociation of this factor after it has fulfilled its ribosomal function. Antibiotics 2016, 5, 18 3 of 13

Antibiotics 2016, 5, 18 3 of 13

Kir, Str Tet Ksg Ede Cam Pmn

70S-scanning 50S initiation Ery, Tel

FA Neo HygB Cam Ths Pmn Vio

Figure 2. Overview of some antibiotics interfering with ribosomal functions. Kir, kirromycin; Str, Figure 2. Overview of some antibiotics interfering with ribosomal functions. Kir, kirromycin; Str, streptomycin, tet, tetracycline; Cam, chloramphenicol; Pmn, puromycin; FA, fusidic acid; Neo, streptomycin, tet, tetracycline; Cam, chloramphenicol; Pmn, puromycin; FA, fusidic acid; Neo, neomycin, HygB, hygromycin B; Ths, thiostrepton; Vio, viomycin; Ery, erythromycin, Tel, neomycin, HygB, hygromycin B; Ths, thiostrepton; Vio, viomycin; Ery, erythromycin, Tel, telithromycin. telithromycin. 70S-scanning initiation is a newly detected initiation mode in bacteria, which can 70S-scanning initiation is a newly detected initiation mode in bacteria, which can replace the RRF/EF-G replace the RRF/EF-G dependent recycling phase. Modified, from [11]. dependent recycling phase. Modified, from [11]. Antibiotics are estimated to have originated between 2 billion. and 40 million years ago [12]. Consequently,Antibiotics areresistance estimated mechanis to havems originated have been between evolving 2 for billion. the same and 40large million amount years of agotime. [12 ]. Consequently,Recently, 30,000-year-old resistance mechanisms bacterial isolates have been of Canadian evolving forpermafrost the same soil large confirmed amount ofthat time. resistance Recently, 30,000-year-oldpredated the bacterialindustrial isolates use of ofantibiotics Canadian [12]. permafrost Five different soil confirmed types of resistance that resistance mechanisms predated are the industrialknown: use of antibiotics [12]. Five different types of resistance mechanisms are known: (1)(1)Mutations Mutations of of membrane membrane components components affectaffect the pe permeabilityrmeability barrier barrier and, and, alternatively, alternatively, transport transport proteinsproteins are are affected, affected, shifting shifting the the import:export import:export ratio towards the the export. export. (2)(2)The The antibiotic antibiotic target targetis isaltered altered byby blockingblocking binding or modifying modifying the the binding binding site, site, causing causing −6 −8 insensitivityinsensitivity to to the the drug. drug. MutationMutation rates rates for for types types 1 1and and 2 are 2 are in inthe the range range of 10 of 10to´ 106 to, i.e. 10,´ one8, i.e. , bacterium out of 106 to 108 is resistant to the respective drug. one bacterium out of 106 to 108 is resistant to the respective drug. (3) Plasmids coding for enzymes that modify (acetylation, phosphorylation of adenylation) or (3) Plasmids coding for enzymes that modify (acetylation, phosphorylation of adenylation) or degrade the antibiotic [3]. degrade the antibiotic [3]. (4) Special factors remove the antibiotic from the target. For example, the EF-G derivative Tet(O) (4) Specialprotein factors chases remove bound tetracycline the antibiotic off the from ribosome the target. [13,14]. For Interestingly, example, the resistance-types EF-G derivative 1–4 Tet(O) are proteinknown chases for the bound tetracycline tetracycline group off[15]. the ribosome [13,14]. Interestingly, resistance-types 1–4 are (5)known Rare mechanisms for the tetracycline are dilution group (overproduction) [15]. of the target molecule or activation of alternative (5) Rarepathways. mechanisms Both are are known dilution for (overproduction) trimethoprim that of inhibits the target dihydrofolate molecule or reductase activation [16]. of alternative pathways. Both are known for trimethoprim that inhibits dihydrofolate reductase [16]. Wasteful use of antibiotics in animal fattening and clinical applications have strengthened the spreadWasteful of resistant use of antibiotics and often inmulti-resistant animal fattening germs and via clinical horizontal applications gene transfer have strengthened[17], which is the spreadconsidered of resistant one of and the often major multi-resistant threads of medical germs ther viaapies. horizontal It is therefore gene transfer obvious [17 ],that which new isdrugs considered and onepossibly of the major new targets threads are of needed medical [5,18]. therapies. It is therefore obvious that new drugs and possibly new targets areFacing needed the enormous [5,18]. accumulation of knowledge about all aspects of antibiotics, it is astonishing thatFacing the highly the enormous complicated accumulation process ofof assembly knowledge of the about bacterial all aspects ribosomes of antibiotics, does not itseem is astonishing to be a thatmajor the highly target complicatedfor antibiotics process [19]. One of assembly possible ofreason the bacterial is the lack ribosomes or scarcity does of not suitable seem to screening be a major techniques to identify antibiotics specifically interfering with the ribosomal biogenesis. In this target for antibiotics [19]. One possible reason is the lack or scarcity of suitable screening techniques respect, some promising progress has been achieved in recent years, which we will consider in the to identify antibiotics specifically interfering with the ribosomal biogenesis. In this respect, some following section. promising progress has been achieved in recent years, which we will consider in the following section. Antibiotics 2016, 5, 18 4 of 13

Antibiotics 2016, 5, 18 4 of 13 2. Ribosome Assembly as Attractive Target for New Antimicrobials 2. Ribosome Assembly as Attractive Target for New Antimicrobials The bacterial ribosome is one of the most intricate structures in the bacterial cell, and its assembly The bacterial ribosome is one of the most intricate structures in the bacterial cell, and its assembly is a highly complex process. The fact that the small and large ribosomal subunits can be reconstituted is a highly complex process. The fact that the small and large ribosomal subunits can be reconstituted in vitro [20,21] indicates that the complete information for the assembly process is intrinsically present in vitro [20,21] indicates that the complete information for the assembly process is intrinsically present in thein the sequences sequences of of ribosomal ribosomal proteins proteins and and rRNAsrRNAs [20] [20].. With With the the help help of ofthe the reconstitution reconstitution technique, technique, importantimportant mechanistic mechanistic assembly assembly steps could could be be unraveled, unraveled, and and examples examples are identification are identification of the two of the twoassembly assembly initiator initiator proteins proteins [22] [22 and] and the the five five early early assembly assembly proteins proteins necessary necessary and and sufficient sufficient for one for one of theof the most most dramatic dramatic conformational conformational changes changes known, which which occurs occurs during during the the assembly assembly of precursor of precursor intermediatesintermediates (a 33S(a 33S particle particle becomes becomes a highlya highly compact compact 41S 41S particle; particle; [23 [23]).]). Two Two ribosomal ribosomal proteins, proteins, L24 andL24 L20, and could L20, becould mere be assemblymere assembly proteins proteins without without important important functions functions in in the the mature mature ribosome ribosome [ 24]. The[24]. analyses The analyses cumulated cumulated in assembly in assembly maps formaps both for theboth small the small and large and large subunit subunit (Figure (Figure3a,b; 3a,b; [ 25, 26]; for[25,26]; review, for see review, [27]). see [27]).

5' 3' (a) 16S RNA S20 S4 S18 S17 S4 S20 S15 S17 S16 S15 S16 S8 S6:S18 S8 S6 S12 S11 S12 S5 S11 S7 S7 S5 S10 S19 S9 S13 S19 S9 S13 S10 S14 S14 S2 S3 S21 S3 S2 S21

5' 3' (b) 23S RNA L24 L22 L24 L20 L21 L20 L21 L22 L4 L15 L17 L23 L1 L3 L3 L1 L13 L23 L13 L4 L15 L17 L5 L18 L34 L29 L5 L18 L29 L34 L11 L33 L19 L32 L14 L2 5S L19 L32 L14 L2 L11 L6 L28 L9 L28 L33 L9 L6 L36 L35 L30 L10 L16 L25 L30 L27 L7/12 L27 L25 L10 L7 L16 L35 L36 L31 L31 FigureFigure 3. Assembly 3. Assembly maps. maps. (a )( mapa) map of theof the small small 30S 30S subunit; subunit; (b )(b map) map of theof the large large 50S 50S subunit. subunit. The The maps of Nomuramaps of Nomura (a) and Nierhaus (a) and Nierhaus (b) are color(b) are coded color fromcoded blue from to blue red to according red according to their to their appearance appearance during theduring assembly the processassemblyin process vivo, modiﬁed in vivo, modified from Reference from Reference [28]. The [28]. assembly The assembly maps showing maps showing the assembly the dependenciesassembly dependencies of the ribosomal of the proteins ribosomal during proteins the during total reconstitution the total reconstitutionin vitro demonstrate in vitro demonstrate an excellent agreementan excellent with agreement the in vivo withdata. the Please in vivocheck data. Please if there check is copyright if there is issue. copyright issue.

What are the advantages of ribosome assembly as a target for antimicrobials? The inhibition of ribosomeWhat are assembly the advantages precedes ofthe ribosome interference assembly with functions as a target of the for operating antimicrobials? ribosome. The Inhibition inhibition of of ribosome assembly precedes the interference with functions of the operating ribosome. Inhibition of Antibiotics 2016, 5, 18 5 of 13 Antibiotics 2016, 5, 18 5 of 13 early assembly events result in premature precursors, so called assembly dead ends, which, due to early assembly events result in premature precursors, so called assembly dead ends, which, due to their loose structures, are cleared by proteases and RNases [29]. While such a scenario is irreversible, their loose structures, are cleared by proteases and RNases [29]. While such a scenario is irreversible, inhibition of translation is not. As soon as the inhibitor is gone, translation can recommence [30]. In inhibition of translation is not. As soon as the inhibitor is gone, translation can recommence [30]. addition, assembly differs significantly between bacterial and mitochondrial ribosomes [31]. This is In addition, assembly differs signiﬁcantly between bacterial and mitochondrial ribosomes [31]. This is an important notion because the latter ones are frequent targets of adverse reactions as has been an important notion because the latter ones are frequent targets of adverse reactions as has been shown shown for chloramphenicol [32], linezolid [33] and aminoglycosides [34]. for chloramphenicol [32], linezolid [33] and aminoglycosides [34].

3.3. Ribosome Ribosome Assembly Assembly and and Translation Translation Are Are Coupled Coupled in in Bacteria Bacteria FacingFacing both both the the highly highly complicated complicated process process of of ri ribosomebosome assembly assembly and and the the fact fact that that the the majority majority ofof antibiotics antibiotics inhibit inhibit functions functions of of the the bacterial bacterial ribosomes, ribosomes, it it is is surprising that that hitherto no no antibiotics havehave been been detected, detected, which which primarily primarily and and specifically speciﬁcally block block ribosomal ribosomal biogenesis. biogenesis. It is Itexpected is expected that translationthat translation inhibitors inhibitors will willaffect affect also also the thesynthesis synthesis of ofribosomal ribosomal proteins proteins and and thus thus the the assembly assembly processprocess (Figure (Figure 44a).a). Consequently, Consequently, assembly assembly and and tr translationanslation are are interdependent, interdependent, which which results results in in a chicken-and-egga chicken-and-egg problem problem [19]. [19 ].

(a)

(b)

(c)

FigureFigure 4. SchemeScheme illustrating interconnectivity interconnectivity of of ribosome assembly and translation. ( (aa)) ribosome ribosome assemblyassembly is is the the prerequisite prerequisite for for translation, translation, which which in inturn turn is required is required for forribosome ribosome assembly; assembly; (b) inhibition(b) inhibition of translation of translation by a bytranslation a translation inhibitor inhibitor is the iscausative the causative mechanistic mechanistic element. element. As a consequence,As a consequence this ,results this results in inhibition in inhibition (or at (or least at least reduction) reduction) of ofthe the assembly assembly process, process, which, which, in in turn, turn, inhibitsinhibits translation translation completing completing the the negative negative feedba feedbackck loop. loop. Both Both processes processes are are clearly clearly interconnected interconnected inin vivo vivo and underlie complex regulation; ( (cc)) inhibition inhibition of of ribosomal ribosomal subunit subunit assembly assembly by by a a putative putative primaryprimary assembly assembly inhibitor inhibitor acting acting on on one one of of the the tw twoo subunits subunits (cause) (cause) will will result result in in inhibition inhibition of of translationtranslation (consequence), (consequence), which which addi additionallytionally inhibits inhibits assembly. assembly. This This also also creates creates a a negative negative feedback feedback loop.loop. Legend: Legend: BlackBlack lineslines with with arrows: arrows: positive positive effects; effects; redred lines:lines: direct direct (causative) (causative) inhibitory inhibitory effects; effects; orangeorange lines:lines: resulting resulting (consequent) (consequent) inhibitory inhibitory effects; effects; asterisks: asterisks: site site of of inhibition; inhibition; darkdark gray gray circle:circle: largelarge ribosomal ribosomal subunit; subunit; lightlight gray gray circle:circle: small small ribosomal ribosomal subunit; subunit; dark gray line:line: mRNA.

ForFor this reason, it is difﬁcultdifficult toto distinguishdistinguish betweenbetween causecause andand consequence.consequence. Inhibition Inhibition of of translationtranslation (cause) (cause) would would result result in in a decrease a decrease in translation in translation (consequence) (consequence) (Figure (Figure 4b).4 However,b). However, this isthis difficult is difﬁcult to distinguish to distinguish from from a scenario a scenario where where inhibition inhibition of assembly of assembly would would be bethe the cause cause and and a decreased translation the consequence (Figure 4c). This interdependence was observed by Nomura Antibiotics 2016, 5, 18 6 of 13

Antibiotics 2016, 5, 18 6 of 13 aand decreased coworkers, translation and they the concluded consequence that (Figure any inhibiti4c). Thison of interdependence translation forces was assembly observed defects by Nomura due to andthe arising coworkers, imbalance and they between concluded rRNA that and any r-protein inhibition production of translation [35]. forces assembly defects due to the arisingIndeed, imbalance macrolides between such as rRNA erythromycin and r-protein have production been demonstrated [35]. to block the assembly of the largeIndeed, 50S ribosomal macrolides subunit such [36]. as erythromycinChloramphenicol have and been erythromycin demonstrated have to been block shown the assembly to decrease of thethe largeproduction 50S ribosomal of some subunitproteins [36 (among]. Chloramphenicol others r-proteins). and erythromycinPrecursor particles have beenisolated shown upon to decreaseantibiotic the treatment production were of found some proteinsto have (amongreducedothers levels r-proteins).of exactly those Precursor proteins particles that were isolated reduced upon antibioticin production treatment [37,38]. were Similarly, found to it have hasreduced been described levels of exactlythat the those macrolide proteins erythromycin that were reduced and the in productionketolide telithromycin [37,38]. Similarly, do not itblock has beenthe passage described of nascent that the polypeptide macrolide erythromycin chains through and the the exit ketolide tunnel telithromycincompletely but do notrather block in the a passagecase-dependent of nascent manne polypeptider. Biosynthesis chains through of a number the exit tunnel of polypeptides completely butcontaining rather in a anewly case-dependent identified N manner.-terminal Biosynthesis signal sequence of a number did occur of and polypeptides was interpreted containing as a strategy a newly identifiedto deregulateN-terminal cellular signal pathways sequence [39]. did In occur that and sense, was interpretedribosome assembly as a strategy defects to deregulate and metabolic cellular pathwaysdysregulation [39]. would In that sense,be indirect ribosome effects assembly caused defectsby translation and metabolic inhibitors. dysregulation would be indirect effectsLikewise, caused by assembly translation of inhibitors.the small 30S subunit is inhibited by aminoglycosides such as streptomycinLikewise, or assembly neomycin of the[40]. small However, 30S subunit it is likely is inhibited that the byprimary aminoglycosides target of the such drugs as streptomycinare functions orof neomycinthe mature [40 ribosome]. However, [37]. it is likely that the primary target of the drugs are functions of the mature ribosomeTaken [37 together,]. it seems that identification of selective inhibitors of assembly requires the ability to distinguishTaken together, between it seems cause that and identification consequence. of A selective possible inhibitors strategy to of face assembly that problem requires is the to ability focus todirectly distinguish on subunit between assembly. cause and consequence. A possible strategy to face that problem is to focus directly on subunit assembly. 4. Specific Readouts for Assembly Are Needed 4. Specific Readouts for Assembly Are Needed A first step towards a specific readout for subunit assembly has been described recently. Two A first step towards a specific readout for subunit assembly has been described recently. reporter strains have been generated possessing one r-protein of the large and the small subunit fused Two reporter strains have been generated possessing one r-protein of the large and the small subunit with green or red fluorescent proteins, respectively. While the reporter strain termed MCrg [41] fused with green or red fluorescent proteins, respectively. While the reporter strain termed MCrg [41] harbors ribosomes with two labeled late assembly proteins (Supplementary Figure S1), in a second harbors ribosomes with two labeled late assembly proteins (Supplementary Figure S1), in a second reporter strain MCrg* [42], two early assembly proteins were labeled (Supplementary Figure S2). reporter strain MCrg* [42], two early assembly proteins were labeled (Supplementary Figure S2). Consequently, in MCrg, only fully assembled subunits are fluorescently marked, which allows for Consequently, in MCrg, only fully assembled subunits are fluorescently marked, which allows for using that strain for identification of assembly inhibitors in small molecule screenings by comparing using that strain for identification of assembly inhibitors in small molecule screenings by comparing the the fluorescence ratio (green/red) of untreated with treated reporter cells. In contrast, in MCrg*, both fluorescence ratio (green/red) of untreated with treated reporter cells. In contrast, in MCrg*, both fully fully assembled and incomplete subunits are fluorescently marked, allowing for monitoring of assembled and incomplete subunits are fluorescently marked, allowing for monitoring of assembly assembly landscapes when ribosome profiles are analyzed fluorometrically [42]. Given the necessity landscapes when ribosome profiles are analyzed fluorometrically [42]. Given the necessity to not only to not only detect but also quantify subunit specific assembly defects, two new strains were created, detect but also quantify subunit specific assembly defects, two new strains were created, which are which are the logical consequence of the two predecessors MCrg and MCrg*. The new strains possess the logical consequence of the two predecessors MCrg and MCrg*. The new strains possess either either large (MCrgL) or small subunits (MCrgS) labeled with fluorescent proteins. In both strains, one large (MCrgL) or small subunits (MCrgS) labeled with fluorescent proteins. In both strains, one early early assembly protein (uL1 or uS15) is labeled with mCherry and one late assembly protein (bL19 or assembly protein (uL1 or uS15) is labeled with mCherry and one late assembly protein (bL19 or uS2) uS2) with mAzami. Both phenotypic and biochemical characterization of MCrgL and MCrgS, with mAzami. Both phenotypic and biochemical characterization of MCrgL and MCrgS, respectively, respectively, revealed that the strains show no defects at 37 or 42 °C and only a mild growth defect revealed that the strains show no defects at 37 or 42 ˝C and only a mild growth defect at very low at very low temperature. In summary, we conclude that their growth properties and translation temperature. In summary, we conclude that their growth properties and translation apparatuses are apparatuses are wild type-like (Figure 5). wild type-like (Figure5).

(a)A) 20 °C 30 °C 37 °C 42°C MC4100

MCrgL MCrgS

FigureFigure 5.5. Cont. Antibiotics 2016, 5, 18 7 of 13 Antibiotics 2016, 5, 18 7 of 13

lysate ribosomes lysate ribosomes (b)B) C)(c) 0 0 0 0 0 L S 0 L S 0 L S 0 L S 1 g g 1 g g 1 g g 1 g g 4 r r 4 r r 4 r r 4 r r 20°C 37°C 42°C C C C C C C C C C C C C M M M M M M M M M M M M M 20°C 37°C 42°C 180 1.0 130 100 75 0.8 uS2-mAzami 63 uL1-mCherry 0.6 bL19-mAzami 48 0.4 uS15-mCherry * 0.2 35 uS2 uL1 normalized growth rate 0 28 0 0 L 0 L 0 S 0 S 0 L S g g g g g g 1 r r 1 r r r r 4 4 41 C C C C C C C C C degradation M M M M M M M M M products 17 bL19 uS15 10 SDS-PAGE WB: αL1, αL19, αS2, αS15

Figure 5.5.Phenotypic Phenotypic and and biochemical biochemical characterization characterization of strains of strains MCrgL MCrgL and MCrgS. and MCrgS. (a) Serial ( dilutionsa) Serial ofdilutions MC4100, of MCrgLMC4100, and MCrgL MCrgS and were MCrgS spotted were on LB-agar spotted plates on LB-agar and incubated plates atand temperatures incubated asat indicated; (b) LB cultures of the indicated cells were inoculated to a start OD of 0.05 and cultured temperatures as indicated; (b) LB cultures of the indicated cells were inoculated600 to a start OD600 of at 20, 37 and 42 ˝C for 7 h in triplicates. Growth rates were calculated and normalized; (c) cell 0.05 and cultured at 20, 37 and 42 °C for 7 h in triplicates. Growth rates were calculated and lysates and puriﬁed ribosomes obtained from the indicated strains were subjected to SDS-PAGE normalized; (c) cell lysates and purified ribosomes obtained from the indicated strains were subjected (left panel) or SDS-PAGE with subsequent immunoblotting (right panel). The SDS-PAGE was stained to SDS-PAGE (left panel) or SDS-PAGE with subsequent immunoblotting (right panel). The SDS- with Coomassie-brilliant blue and the Western blot membrane was probed with the indicated primary PAGE was stained with Coomassie-brilliant blue and the Western blot membrane was probed with antibodies in combination with appropriated secondary antibodies. Proteins of interest are labeled. the indicated primary antibodies in combination with appropriated secondary antibodies. Proteins of WB, Western blot; αL1, antibody against ribosomal protein uL1. interest are labeled. WB, Western blot; αL1, antibody against ribosomal protein uL1.

To testtest theirtheir capabilitycapability toto analyzeanalyze subunitsubunit specificspecific assemblyassembly defects,defects, in bothboth strains,strains, conditional knock outsouts ofof ribosomalribosomal protein protein genes genes were were introduced. introduced. The The absence absence of of the the L-protein L-protein uL3 uL3 (encoded (encoded by rplCby rplC) or) theor the S-protein S-protein uS17 uS17 (encoded (encoded by byrpsQ rpsQ) would) would render render either either large large or or smallsmall subunitsubunit assemblyassembly defective, as described previously [[41,43,44].41,43,44]. WhileWhile in MCrgL large subunit,subunit, mono-mono- andand polysomespolysomes contain red and green fluorescencefluorescence (Figure6 6c),c), ininMCrgS MCrgSsmall smallsubunit, subunit, mono-mono- andand polysomespolysomes showshow red andand greengreen fluorescencefluorescence (Figure(Figure6 6d).d). InIn MCrgL,MCrgL, thethe absenceabsence ofof uL3uL3 causescauses defectsdefects inin thethe largelarge subunit as becomes evident by a decrease of the green signal and an additional red fluorescence fluorescence peak (Figure(Figure6 6e).e). DefectiveDefective smallsmall subunitsubunit assembly,assembly, inin thethe absenceabsence ofof uS17,uS17, cannotcannot directlydirectly bebe detecteddetected fluorometrically.fluorometrically. However,However, directlydirectly causedcaused defectsdefects inin smallsmall subunitsubunit assemblyassembly seem to result in slight but detectable assembly defects of the large subunit as indicated by asterisks (Figure6 6g).g). MCrgS,MCrgS, onon the otherother hand,hand, allowsallows detectiondetection of of small small subunit subunit assembly assembly defects, defects, which which were were caused caused by by depletion depletion of uS17,of uS17, directly directly (Figure (Figure6f). 6f). While While the the green green fluorescence fluorescence signal signal within within the 30Sthe 30S region, region, which which reflects reflects the intactthe intact portion portion of the of subunit, the subunit, is decreased, is decreased, the red the fluorescence red fluorescence signal is signal broader is broader and of higher and of intensity, higher clearlyintensity, indicating clearly aindicating small subunit a small assembly subunit defect. assembly In the defect. absence In of the uL3 absence (Figure of6h), uL3 defective (Figure large 6h), subunitdefective assembly large subunit cannot assembly be detected cannot fluorometrically. be detected fluorometrically. However, again However, the subunit again not the affected subunit by genenot affected depletion, by gene in this depletion, case the small in this subunit, case the shows small asubunit, decrease shows in green a decrease fluorescence in green and fluorescence a broadened redand fluorescencea broadened peak red withfluorescence a left-sided peak shoulder. with a left-s Quantitationided shoulder. of the fluorescenceQuantitation profiles of the fluorescence confirms the observedprofiles confirms tendencies the (Figureobserved6i–j). tendencies (Figure 6i–j). Taken together, together, fluorescence fluorescence analyses analyses of sucros of sucrosee density density gradient gradient fractions fractions derived derived from MCrgL from MCrgLand MCrgS and enable MCrgS detection enable detection and quantitation and quantitation of assembly of assembly defects within defects the within large theor small large orsubunit, small subunit,respectively. respectively. Antibiotics 2016, 5, 18 8 of 13 Antibiotics 2016, 5, 18 8 of 13

Figure 6. Ribosome profilesprofiles after sucrose density gradient centrifugation and fluorescence fluorescence detection. ((a,,b)) schematicschematic drawingdrawing ofof MCrgLMCrgL andand MCrgS*,MCrgS*, respectively.respectively. Depicted are the positions of thethe fluorescentfluorescent proteins proteins and and their their coding coding sequences. sequences. DarkDark gray gray: large: large ribosomal ribosomal subunit; subunit; light graylight: small gray: smallribosomal ribosomal subunit; subunit; green greenbarrel:barrel: EGFP; EGFP; red barrel: red barrel: mCherry; mCherry; gray line:gray mRNA;line: mRNA; curved curved blackblack line: line:bacterial bacterial chromosome chromosome with with red redand and green green strips strips symbolizing symbolizing coding coding sequences sequences of of mCherry mCherry and mAzami, respectively; (c–h) Ribosome profilesprofiles derived from MCrgL ((c),), MCrgLMCrgL∆ΔlC ((d),), MCrgLMCrgL∆ΔsQ (e), MCrgS (f), MCrgS∆sQ (g) and MCrgS∆lC (h); A profiles are given in black lines, mCherry and (e), MCrgS (f), MCrgSΔsQ (g) and MCrgSΔlC (h); A254254 profiles are given in black lines, mCherry and mAzami specificspecific fluorescencefluorescence intensities are given as red oror greengreen bars,bars, respectively.respectively. FluorescenceFluorescence intensities were normalized to the firstfirst polysomepolysome peak.peak. Red andand greengreen fluorescencefluorescence intensitiesintensities werewere i j calculated andand compared compared to to each each other, other, ( , ). ( Totali,j). Total mCherry mCherry and mAzami and mAzami specific fluorescencespecific fluorescence emission of each strain’s profile is given in bar charts. mCherry signals were set to 100% and mAzami signals emission of each strain’s profile is given in bar charts. mCherry signals were set to 100% and mAzami are given as relative values. Error bars show S.D. of two independent experiments. One asterisk signals are given as relative values. Error bars show S.D. of two independent experiments. One indicates additional peak or shoulder within the subunit with the primary defect, two asterisks indicate asterisk indicates additional peak or shoulder within the subunit with the primary defect, two additional peak or shoulder within the subunit was not targeted for assembly defect. asterisks indicate additional peak or shoulder within the subunit was not targeted for assembly defect.

Antibiotics 2016, 5, 18 9 of 13

5. Materials and Methods

5.1. Media, Buffers, Antibodies and Antibiotics LB-Medium (0.5% (w/v) yeast extract, 1% (w/v) tryptone, 86 mM NaCl; for growth on solid media additionally 1.5% (w/v) bacto agar); 5ˆ M9 salts (63 mM Na2HPO4¨ 7H2O, 110 mM KH2PO4, 43 mM NaCl, 94 mM NH4Cl); M9 medium (1ˆ M9 salts, 2 mM MgSO4, 0.1 mM CaCl2, 0.4% (w/v) glucose); PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4). Horseradish peroxidase (HRP)-conjugated rabbit anti-sheep (CodeNo: 313-035-003; LotNo: 106383) and donkey anti-rabbit (CodeNo: 711-035-152; LotNo: 103871) and donkey anti-rabbit (CodeNo: 711-035-152; LotNo: 103871) secondary antibodies were from Jackson ImmunoResearch (West Grove, Pennsylvania, USA). HRP substrate for detection: 1 mL solution A + 100 µl solution B + 1 µL solution C (solution A: 0.1 mM TRIS (pH 8.6), 25 mg Luminol, 100 mL distilled H2O; solution B: 11 mg p-hydroxycoumaric acid in 10 mL DMSO; solution C: 30% H2O2 (w/v)). Antibiotics were used in concentrations as indicated: Ampicillin 100 µg/mL (Applichem-A0839, Darmstadt, Germany), kanamycin 50 µg/mL (Carl Roth-T832.4, Karlsruhe, Germany) and chloramphenicol 7 µg/mL (Sigma-C0378, St. Louis, MO, USA).

5.2. Plasmids and Bacterial Strains rpsQ and rplC were amplified from genomic E. coli DNA, brought into DH5α-Z1 and isolated as described earlier [41]. MC4100 (F-[araD139]B/r ∆(argF-lac)169 lambda-e14-flhD5301 ∆(fruK-yeiR)725 (fruA25) relA1 rpsL150(strR) rbsR22 ∆(fimB-fimE)632(::IS1) deoC1); DY330 (W3110 ∆ lacU169 gal490 λcI857 ∆ (cro-bioA)) [45], DH5α-Z1 (F endA1 hsdR17(rk mk+) supE44 thi-1 recA1 gyrA relA1 ∆ (lacZYAargF) U169 deoR Φ80 lacZ∆ M15 LacR TetR and Spr).

5.3. λ-Red Recombineering Fusion proteins of the ribosomal proteins with the FPs mAzami green and mCherry were generated by λ red recombineering and brought into strains of interest using P1-phage transduction as described previously. Genomic integration was conﬁrmed by colony-PCR and DNA-sequencing. Gene deletions of rpsQ and rplC were also accomplished as described earlier [41].

5.4. Cell Growth Analyses For growth on solid media, E. coli cells were grown at 37 ˝C until stationary phase in LB medium and diluted to a cell density of OD600 = 0.025. Serial dilutions with a dilution factor of 0.2 were prepared and transferred onto LB agar plates using a plating stamp. Plates were incubated at 20 ˝C, 30 ˝C, 37 ˝C and 42 ˝C until single colonies were visible. For growth in liquid media, stationary E. coli cells were grown until stationary phase and diluted ˝ ˝ ˝ to an initial OD600 = 0.025 for incubation at 42 C and to OD600 = 0.05 for incubation at 37 C and 20 C. They were incubated in bafﬂed ﬂasks in a water bath incubator with a shaking frequency of 200 rpm until stationary or exponential phase was reached. Cell density was measured using a photometer (Ultrospec 3000, GE Healthcare, Little Chalfont, UK) and growth rates were calculated for periods of exponential growth. Growth analyses in liquid media were conducted in biological triplicates.

5.5. Puriﬁcation of Ribosomes by Sucrose Cushion Centrifugation E. coli cells were cultured at 37 ˝C in LB medium until stationary phase was reached. Pre-cultures were ˝ diluted to an OD600 = 0.5 and incubated at 37 C until an OD600 = 0.8. 250 µg/mL chloramphenicol was added to stop translation 5 min before harvesting. For this purpose, cultures were incubated on ice for 10 min and sedimented for 10 min at 4400 rpm and 4 ˝C. Pellets were resuspended in lysis buffer I (100 mM TRIS, 10 mM MgCl2, 100 mM NaCl, 15% (w/v) sucrose, 100 µg/mL chloramphenicol, pH 7.5), snap frozen in liquid nitrogen and stored at ´80 ˝C. For further preparation, the frozen Antibiotics 2016, 5, 18 10 of 13

cell pellets were thawed on ice and resuspended in 3ˆ volumes of lysis buffer II (10 mM MgCl2, 100 mM NaCl). Cells were then lysed using FastPrep®-24 (MP Biomedicals, Eschwede, Germany). Protein concentrations of the cleared lysates were determined by Bradford assay. In addition, 300 µL of cleared lysates were loaded on 700 µL of 20% sucrose cushion (20 mM TRIS, 10 mM MgCl2, 100 mM KCl, 5 mM β-mercaptoethanol, 20% sucrose (w/v), pH 7.5) and sedimented for 1 h 20 min at 65,000 rpm at 4 ˝C in an S140-AT rotor (Thermoﬁscher Scientiﬁc, Waltham, MA, USA). Pellets harboring the ribosomes were resuspended in buffer III (10 mM TRIS, 12 mM MgCl2, 30 mM NaCl, 4 mM β-mercaptoethanol, pH 7.5). A260 values were determined using a spectrophotometer (NanoVueTM Plus UV/Visible Spectrophotometer (GE Healthcare).

5.6. Sucrose Gradient Centrifugation E. coli cells of the different strains were cultured at 37 ˝C until stationary phase. The pre-cultures were washed 3 times and diluted in M9 minimal medium to OD600 = 0.05 and cultured to OD600 of about 0.3. Five minutes before harvesting, chloramphenicol (250 µg/mL) was added. The pellets were sedimented, snap frozen in liquid nitrogen and stored at ´80 ˝C. For further preparation, pellets were resuspended in lysis buffer (10 mM TRIS, 10 mM MgCl2, 100 mM NH4Cl, 250 µg/mL chloramphenicol, 0.5 mM DTT, 1 mM PMSF, 1x TM CompleteTM (05056489001, (Roche-05056489001, Basel, Swiss) pH7.5) and lyzed using FastPrep®-24. A260 values of the cleared lysates were determined, the concentrations were adjusted to A260 = 20. In addition, 100 µL were then loaded on 10%–40% sucrose gradients and centrifuged at 4 ˝C and 41,000 rpm for 2 h 40 min using a Sorvall TH-641 rotor (Thermoﬁscher Scientiﬁc, Waltham, MA, USA).

5.7. Polysome Analysis and Fluorometric Analysis of the Sucrose Fractions Separated ribosomal populations were analyzed using a Teledyne Isco gradient reader (Teledyne ISCOr, Lincoln, Nebraska, USA). A254 profiles were recorded and the obtained fractions were collected in 96-well plates (5 drops per well) for the following fluorometric analyses. mAzami and mCherry specific fluorescence was determined using Infinite F500 (Tecan, Männedorf, Swiss) fluorescence microplate reader (filter combinations 485/535 nm and 535/612 nm, respectively). The fluorescence intensities were normalized to the first polysome peak.

6. Conclusions The intricate assembly of the macromolecular complex “ribosome” offers many distinct interference points. Even though the structure of ribosomes is highly conserved across domains, a number of speciﬁc features of the biogenesis of the bacterial ribosomes exist. Only recently suitable methods have been developed to trace assembly inhibitors. We can expect that the near future will reveal inhibitors speciﬁcally blocking ribosome assembly in bacteria, which might pave the way for development of urgently needed new therapeutics against bacterial infections.

Supplementary Materials: The following are available online at www.mdpi.com/2079-6382/5/2/18/s1. Figure S1: Ribosome profile analysis using MCrg, Figure S2: Ribosome profile analysis using MCrg*. Acknowledgments: We thank Daniel N. Wilson for help and discussion (Gene Center, University of Munich, Germany). This work was supported by a Human Frontier Science Program Organization (HFSP-Ref. RGP0008/2014) to K.H.N. and a grant from the Deutsche Forschungsgemeinschaft DFG (A01, SFB969) to E.D. Author Contributions: R.N., S.S. and E.D. conceived and designed the experiments; S.S., R.S. and R.N. performed the experiments; R.N., S.S., E.D. and K.H.N. analyzed the data; R.N., S.S., E.D. and K.H.N. wrote the paper. Conflicts of Interest: Engineered bacterial strains described in this study are part of a patent application (application No: 13175775.9-1410) that is pending. Antibiotics 2016, 5, 18 11 of 13

Abbreviations The following abbreviations are used in this manuscript: Da molecular mass in Dalton EGFP Enhanced green fluorescent protein LB Lysogeny broth (medium) LB agar LB and agar containing breeding grounds rRNA ribosomal RNA PTC peptidyl transferase center tRNA transfer RNA TRIS Tris(hydroxymethyl)-aminomethan aa-tRNA aminoacyl tRNA GTP guanosin triphosphate GDP guanosin diphosphate EF-Tu elongation factor thermo-unstable EF-G elongation factor G FA fusidic acid mAzami Monomeric green fluorescent protein mCherry Monomeric red fluorescent protein OD600 Optical density at 600 nm wavelength S.D. standard deviation SDS PAGE Sodium dodecyl sulfate poly acrylamide gel electrophoresis

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