Revisiting the mechanism of macrolide-antibioticresistance mediated by ribosomal protein L22Sean D. Moore1 and Robert T. Sauer2

Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139

Contributed by Robert T. Sauer, October 16, 2008 (sent for review September 13, 2008)

Bacterial antibiotic resistance can occur by many mechanisms. Anintriguing class of mutants is resistant to macrolide antibiotics eventhough these drugs still bind to their targets. For example, a3-residue deletion (�MKR) in ribosomal protein L22 distorts a loopthat forms a constriction in the ribosome exit tunnel, apparentlyallowing nascent-chain egress and translation in the presence ofbound macrolides. Here, however, we demonstrate that �MKR andwild-type ribosomes show comparable macrolide sensitivity invitro. In Escherichia coli, we find that this mutation reducesantibiotic occupancy of the target site on ribosomes in a mannerlargely dependent on the AcrAB-TolC efflux system. We propose amodel for antibiotic resistance in which �MKR ribosomes alter thetranslation of specific proteins, possibly via changes in pro-grammed stalling, and modify the cell envelope in a manner thatlowers steady-state macrolide levels.

ermC � erythromycin � ribosome � TolC

Many antibiotics inhibit bacterial protein synthesis. Under-standing how microbes become antibiotic resistant is

important both for developing effective treatment regimens anddesigning new therapeutics. Macrolides consist of a 14- to16-member lactone ring with different appended sugars andcomprise a key group of inhibitors of bacterial translation (1, 2).The inhibitory activity of macrolides, including erythromycin,depends on binding to a site near the polypeptide exit tunnel ofthe large ribosomal subunit (3, 4). Because macrolides do notbind to ribosomes with an occupied exit tunnel and cause thesynthesis of 2–10 residue peptides in translation assays in vitro,it has been proposed that drug binding physically blocks elon-gation of nascent proteins beyond this size (5–7).

Some macrolide-resistance mutations alter the ribosomal targetsite and prevent binding (4, 8). Intriguingly, other mutations conferresistance despite the fact that macrolides still bind the mutantribosome well (8–10). For example, deletion of the M82K83R84

sequence in Escherichia coli ribosomal protein L22 (�MKR) allowsgrowth in the presence of high levels of erythromycin and othermacrolides (11–13). The same mutation makes Haemophilus influ-enzae resistant to numerous macrolides (14); different L22 muta-tions also confer macrolide resistance in other bacterial species (2).When binding has been measured, ribosomes with macrolide-resistant alterations in L22 bind erythromycin with near wild-typeaffinity (8, 10, 12, 15).

In a crystal structure of the E. coli ribosome, the MKR sequenceis part of an extended L22 loop, which together with a similar loopin protein L4 forms a narrow constriction in the exit tunnel (Fig. 1A)(16, 17). Cryo-EM studies initially revealed a widened exit tunnelin E. coli �MKR ribosomes (18). This loop is also displaced tocreate an expanded tunnel in structures of �MKR ribosomes fromThermus thermophilus and Haloarcula marismortui (4, 19). Theseresults explain the altered chemical reactivity in E. coli �MKRribosomes of 23S-RNA bases (13). Together, these results supporta prevailing model in which the L22 �MKR deletion confersantibiotic resistance by allowing nascent proteins to enter theribosome exit tunnel despite bound macrolides (18, 19).

Here, we rule out the accepted model of L22-mediated macrolideresistance by showing that E. coli �MKR ribosomes are inhibited

by erythromycin in vitro and in vivo. Instead, the �MKR mutationappears to reduce the intracellular concentration of macrolides. Wepresent evidence that links changes in the activity of the AcrAB-TolC efflux system to antibiotic resistance in the �MKR strain andsuggest that changes in translation of specific proteins are respon-sible for �MKR-linked macrolide resistance.

Results�MKR Ribosomes Are Inhibited by Erythromycin in Vitro. The E. coliS10 operon, encoding L22 and additional ribosomal proteins, canbe deleted without substantially altering the growth rate if the cellscontain a plasmid-borne operon encoding L22 or L22 fused to thetitin-I27 domain (20). The L22-titin fusions are longer than un-modified L22 and this feature allowed us to establish unambigu-ously that mutant forms of L22 were the only versions present in cell(20). Using this system, we constructed a strain expressing onlyL22-titin with the �MKR mutation. We purified ribosomes fromstrains expressing just L22-titin or just �MKR-L22-titin; SDS-PAGE of these ribosomes revealed that the �MKR L22 variant hada slightly faster electrophoretic mobility (Fig. 1B). In coupledtranscription/translation reactions, both types of ribosomes wereequally active in the synthesis of 14C-labeled test proteins (Fig. 1CLeft) and had activities comparable to ribosomes containing un-fused L22 (data not shown). Surprisingly, however, increasingconcentrations of erythromycin inhibited the �MKR-L22 ribo-somes and the ‘‘wild-type’’ ribosomes (Fig. 1 C and D). Fitting thesedata gave apparent inhibition constants (Ki) of 41 � 9 nM for theL22-titin ribosomes and 26 � 5 nM for the �MKR-L22-titinribosomes (Fig. 1D). The Ki values were within the range of KDvalues (�10–100 nM) measured for erythromycin binding to ribo-somes containing wild-type L22 or �MKR-L22 (21–24). At thehighest concentrations of erythromycin tested, no translation by�MKR ribosomes was detected (Fig. 1C Bottom). Erythromycinalso completely inhibited translation of another mRNA by L22-titinand �MKR-L22-titin ribosomes (data not shown). Thus, theseexperiments are inconsistent with models in which the �MKRdeletion in L22 allows the ribosome to function when erythromycinis bound.

Erythromycin Sensitivity in Vivo. As anticipated from previousstudies, in liquid cultures of strains expressing only L22-titin or�MKR-L22-titin, the mutation increased the erythromycin mini-mal inhibitory concentration (MIC) from �125 to �850 �� (Fig.2A, dashed lines), doubled the spiramycin MIC (�500 to �1000�g/ml; data not shown), and quadrupled the tylosin MIC (�500 �Mto �2 mM; data not shown). E. coli strains containing plasmid-borne L22 variants lacking the titin-fusion domain behaved simi-

Author contributions: S.D.M. designed research; S.D.M. performed research; S.D.M. con-tributed new reagents/analytic tools; S.D.M. analyzed data; and S.D.M. and R.T.S. wrote thepaper.

The authors declare no conflict of interest.

1To whom correspondence may be addressed at the present address: Department of MolecularBiology and Microbiology, Burnett School of Biomedical Sciences, University of Central Flor-ida, Orlando, FL 32816. E-mail: semoore@mail.ucf.edu.

2To whom correspondence may be addressed. E-mail: bobsauer@mit.edu.

© 2008 by The National Academy of Sciences of the USA

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larly (data not shown) as did strains with chromosomal alleles ofwild-type L22 or �MKR L22 (2, 11–13). Although sufficiently highconcentrations of macrolides inhibited growth of both the L22-titinand �MKR-L22-titin strains, as expected from our results in vitro,the differential macrolide sensitivity in vivo was not recapitulatedin the in vitro assays.

Efflux Pumps and Macrolide Resistance. Efflux pumps can dramat-ically alter the sensitivity of bacteria to antibiotics (2). For example,phenylanine-arginine-�-napthylamide (PA�N) inhibits multidrugefflux pumps and markedly lowers the MIC of erythromycin andother antibiotics (25, 26). In strains expressing only L22-titin or only�MKR-L22-titin, 30 �g/ml PA�N lowered the MIC for erythro-mycin �20-fold (Fig. 2A, solid lines) and the MICs for tylosin andspiramycin �10-fold (data not shown). Therefore, drug efflux playsa significant role in determining the macrolide resistance of bothstrains. In liquid culture, some growth of the L22-titin strain wasobserved 2 h after addition of 200 �M erythromycin (Fig. 2B). Bycontrast, growth ceased almost immediately with 30 �g/ml PA�Nand 200 �M erythromycin (Fig. 2B), suggesting that active eryth-romycin efflux normally delays growth inhibition. For the �MKR-L22-titin strain, 200 �M erythromycin had little effect on growth inthe absence of the efflux inhibitor but caused rapid cessation ofgrowth in its presence (Fig. 2C).

Erythromycin Inhibits L22-�MKR Ribosomes in Vivo. Is erythromycinsensitivity of the �MKR-L22-titin strain a consequence of drugbinding to the mutant ribosome or a different cellular target? Toanswer this question, we used ErmC, which methylates A2058 in23S rRNA and blocks erythromycin binding (27, 28). Constitutiveexpression of ErmC allowed L22-titin or �MKR-L22-titin strains togrow in the presence of 200 �M erythromycin and PA�N (Fig. 2D).The growth rates were approximately half those without erythro-mycin, which could be caused by incomplete ErmC methylation.Indeed, consistent with a previous report (28), a primer-extensionassay, using reverse transcriptase showed that A2058 was methyl-ated in only �60% of the ribosomes (Fig. 2E). Because ErmC-mediated resistance to erythromycin correlated approximately withthe extent of A2058 methylation, we conclude that erythromycininhibits growth of the �MKR-L22-titin strain by occupying itsnormal binding site in the exit tunnel of �MKR ribosomes.

Erythromycin Binding to Ribosomes in Vivo. If �MKR cells accumu-lated or retained less erythromycin than wild-type cells, then�MKR ribosomes would appear to be more erthyromycin resistantin vivo. Because erythromycin protects A2058 and A2059 in the 23SrRNA of ribosomes from dimethyl sulfate (DMS) modification invitro (22, 29), we used DMS reactivity in vivo to monitor erythro-mycin binding. L22-titin or �MKR-L22-titin cells were grownwithout drug, with 200 �M erythromycin, or with 200 �M eryth-

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Fig. 1. Ribosomal protein L22 and erythromycin inhibition of translation in vitro. (A) Crystal structure of L22 protein (green; M82K83R84 is shown in red), L4 protein(blue), and 23S and 5S RNA (orange) from the 50S subunit of the E. coli ribosome (PDB entry 2AWB). The exit tunnel is visible through the center of the subunit. (B)SDS-PAGE of proteins from purified ribosomes with wild-type L22, an L22-titin fusion, or a �MKR L22-titin fusion. Wild-type L22 (12 kDa) is not resolved from otherribosomal protein in the Left lane. The L22 fusion proteins are visible as separate bands (Center and Right lanes). (C) Autoradiograms of translation reactions containing100 nM wild-type or �MKR ribosomes and increasing amounts of erythromycin. (D) Integrated band intensities from the autoradiogram shown in the Top and Middleof C are plotted as a percentage of the intensity of the band from the reaction without erythromycin. These data were fit to a quadratic binding equation to obtainthe inhibition constant (Ki).

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romycin and PA�N, DMS was added, and total RNA was purifiedand used for primer-extension reactions. The bands correspondingto A2058/A2059 exhibited similar intensities in both strains grownwithout erythromycin and showed reduced but comparable inten-sities in L22-titin or �MKR-L22-titin cells treated with erythromy-cin and PA�N (Fig. 3A). By contrast, A2058/A2059 were modifiedto a greater extent in the �MKR-L22-titin strain than in theL22-titin strain after growth in erthyromycin alone (Fig. 3A). Thereactivity of A2062 did not change substantially with or withouterythromycin, allowing normalization of the A2058/A2059 inten-sities for quantitative comparisons (Fig. 3B). Irrespective of the L22allele, A2058/A2059 reactivities were high in the absence of eryth-romycin and low in the presence of very high intracellular concen-trations of erythromycin (mediated by PA�N inhibition of efflux).Importantly, A2058/A2059 reactivities were approximately twice ashigh in �MKR-L22-titin ribosomes than in L22-titin ribosomesduring growth in erthyromycin alone, supporting a model in whichthe intracellular concentration of erythromycin is lower in the�MKR cells.

Larger Deletions of the MKR Loop Do Not Confer ErythromycinResistance. Ribosomes containing L22 mutants with larger dele-tions in the MKR loop appear to be functional in merodiploidstrains, because they are found with wild-type ribosomes in poly-

somes (30). To test whether these larger deletions allowed ribosomefunction in the presence of erythromycin, we constructed strainsexpressing just L22-titin variants missing residues 85–95 (�loop1) orresidues 82–100 (�loop2) (Fig. 4A). In growth-rate studies, thedoubling times were 34 min (L22-titin), 53 min (�MKR-L22-titin),57 min (�loop1-L22-titin), and 62 min (�loop2-L22-titin) (data notshown). Thus, all of the deletions allow bacterial growth but withsignificant growth defects. The �loop1 and �loop2 strains did notgrow on plates with 200 �M erythromycin (Fig. 4B) and haderythromycin MICs in liquid culture of �125 �M and �150 �M,respectively (data not shown). Thus, partial or complete removal ofthe MKR loop from the narrow constriction in the exit tunnel is notsufficient to confer significant increases in erythromycin resistance.Because strains with the extended loop mutants grew more slowlythan the �MKR mutant, these results also show that L22-mediatedslowing of the growth rate is not sufficient to confer enhancedmacrolide resistance. We also constructed a strain in whichM82K83R84 of L22 was replaced with A82A83A84. This strain grew aswell as wild type in the absence of erythromycin and was equallysensitive to the antibiotic (data not shown). Thus, the chemicalidentity of the side chains at these residue positions is unrelated tothe resistance phenotype caused by the �MKR deletion.

acrAB-tolC System and �MKR Macrolide Resistance. In E. coli, theAcrAB-TolC efflux system is primarily responsible for resistance to

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Fig. 2. Erythromycinsensitivity increaseswhendrugefflux isblocked. (A)Erythromycinresistanceof strainscontainingwild-typeL22-titin (triangles)or�MKRL22-titin(circles). The turbidity (600 nm) of 16 h cultures grown at 37 °C in the presence of erythromycin (dashed lines) or erythromycin and 30 �g/ml PA�N (solid lines) is plottedas a percentage of the value of a culture grown without drug. (B) Growth at 37 °C of cultures containing the wild-type L22-titin fusion in the absence of erythromycin(opencircles), afteradditionof200 �Merythromycin (filledcircles),orafteradditionof200 �Merythromycinand30 �g/mlPA�N(filledtriangles). (C)Growthofculturescontaining the �MKR mutation in L22 with the same symbols as in B. (D) Strains containing wild-type (circles) or �MKR L22 (filled circles) and an induced ermC genewere grown in medium with 30 �g/ml PA�N to early log phase and erythromycin was added to 200 �M at the time indicated by the arrows. (E) ErmC methylation ofA2058 in 23S RNA assayed by primer extension. An oligonucleotide complementary to 23S rRNA bases 2066–2102 was used to prime a reverse-transcription reactioncontaining dideoxy-ATP and RNA purified from strains containing wild-type or �MKR L22-titin fusions grown with or without induction of ermC. Reaction productswere electrophoresed on a urea-acrylamide gel. In the presence of template RNA, reverse transcription proceeded until blocked by ErmC-mediated methylation ofA2058 or until incorporation of dideoxy-ATP at the position corresponding to U2041.

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low levels of erythromycin and a variety of other toxic compounds(31–32). To determine whether this system was responsible for theincreased resistance conferred by the �MKR mutation, we deletedeither the AcrA/AcrB or TolC ORFs and assayed resistance toerythromycin. Deletion of either tolC or acrAB abolished much ofthe difference in erythromycin resistance between strains express-ing L22-titin or �MKR-L22-titin and substantially increased thesensitivity of both strains (Fig. 5 A and B). Therefore, increaseddrug efflux mediated by AcrAB-TolC appears to be largely respon-sible for the increased erythromycin resistance conferred by the�MKR mutation. The tolC deletion reduced the difference inerythromycin sensitivity between the wild-type and �MKR strainsto a greater extent than the acrAB deletion. This result can berationalized because several TolC-dependent efflux pumps in ad-dition to ArcAB-TolC have also been shown to plays roles inerythromycin resistance (32, 34).

DiscussionThe discovery that bacterial resistance to erythromycin can becaused by mutations in ribosomal proteins was first reported in1967, and the �MKR deletion in protein L22 was subsequentlyshown to cause this phenotype (8, 11, 12). Since these initialobservations, consensus molecular mechanisms for macrolide in-hibition of translation and for the effects of the �MKR mutationhave emerged from biochemical and structural studies (3, 4, 7, 18,19). According to the exit-tunnel blockade/bypass model, macro-lides sterically block peptide elongation during translation by bind-

ing in the exit tunnel of the large ribosomal subunit, with the �MKRdeletion serving to relieve this blockade by providing additionalspace for the nascent chain to bypass the drug and enter the exittunnel.

Our results disprove the prevailing �MKR bypass model (18, 19),which predicts that ribosomes harboring the L22 �MKR deletionshould be active in the presence of bound macrolides. By contrast,we find that erythromycin inhibits translation by �MKR ribosomesin vitro, with the mutant ribosomes being as sensitive to inhibitionas wild-type ribosomes. In the cell, we find that �MKR ribosomesare also sensitive to macrolides added at sufficiently high concen-trations, in agreement with previous studies (12). In E. coli express-ing �MKR-L22-titin, the MIC for erythromycin is 850 �M, but thisvalue drops to �10 �M when PA�N, an inhibitor of efflux pumpsis also present. Similarly, mutations that inactivate the AcrAB-TolCefflux system reduce the MIC for erythromycin to 5–10 �M for�MKR strains. These inhibitory effects are caused by macrolidebinding to the site near the exit tunnel of �MKR ribosomes,because ErmC methylation of a base within this site alleviatesinhibition both in vitro (data not shown) and in vivo. These resultsdo not support models that rely on macrolide-bound �MKRribosomes functioning better than macrolide-bound wild-typeribosomes.

Macrolide concentrations appear to be lower in �MKR strainsthan in wild-type strains. For example, when �MKR or wild-typecells are grown in 200 �M erythromycin, footprinting experimentsshow lower occupancy of the macrolide-binding site in �MKRribosomes, but similar occupancy in the presence of an effluxinhibitor. This result is unlikely to arise from differences in affinity,because we find that both types of ribosomes show similar sensitivityto erythromycin inhibition in vitro and previous studies show thaterythromycin binds �MKR ribosomes similarly to wild-type ribo-somes (8, 12). Moreover, small differences in the affinity oferythromycin for wild-type and �MKR ribosomes in the cell shouldhave no appreciable effect on occupancy, because near stoichio-metric binding would be expected in both cases because the

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Fig. 3. Reduced erythromycin binding to ribosomes with �MKR L22 in vivo.Cultures were treated with dimethyl sulfate (DMS), reactions were quenched,and total RNA was purified and used in primer-extension assays to detect basemodifications near the erythromycin-binding site in 23S rRNA. (A) Gel ofprimer-extension products from the following RNA templates: (Lane 1) Non-DMS-treated ErmC-modified template (product terminates at the positioncorresponding to A2058). (Lanes 2 and 3) Templates from cultures grownwithout erythromycin. (Lanes 4 and 5) Templates from cultures grown with200 �M erythromycin. (Lanes 6 and 7) Templates from cultures grown with 200�M erythromycin and 30 �g/ml PA�N. (B) Plots of the ratio of band intensitiescorresponding to A2058 (filled bars) or A2059 (hatched bars) in A divided bythe intensity of A2062.

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Fig. 4. Extended L22 loop deletions. (A) Ribbon drawings of E. coli L22 with theM82-K83-R84 residues marked in red (PDB 2AWB). The �loop1 and �loop2 dele-tions remove residues 85–95 and 82–100, respectively. (B) Growth of strains on aplate containing 200 �M erythromycin.

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intracellular concentration of ribosomes exceeds the KD for eryth-romycin binding by 100-fold or more (35). Two mechanisms couldpotentially give rise to reduced drug concentration in the �MKRstrain: (i) a factor sequesters or destroys the antibiotic; or (ii)cellular uptake of the drug is reduced or efflux is increased.Although either of these mechanisms are feasible, we favor thesecond possibility, which is consistent with our findings that block-ing efflux with PA�N makes cells more sensitive to erythromycinand deleting either the AcrA/AcrB or TolC components of theAcrAB-TolC efflux pump reduces the erythromycin resistance ofa strain with �MKR-L22-titin ribosomes compared with a strainwith L22-titin ribosomes.

The �MKR mutation is known to affect translation of certainmRNAs by reducing programmed ribosome stalling (36, 37). Howmight �MKR-linked translational changes affect macrolide uptakeor efflux? It is possible that AcrAB and/or TolC activities areincreased through direct effects on translation of these proteins orindirect effects on translation of a regulator. However, preliminaryexperiments, using Western blot analysis with anti-AcrA and anti-TolC antibodies did not reveal up-regulation of these proteins in the�MKR strain (data not shown). Moreover, our �MKR strain didnot show enhanced resistance to SDS or ethidium bromide (datanot shown), which are also pumped from the cell by the AcrAB-TolC system (38, 39), although this result could be complicated by

the regulatory mechanisms by which these compounds are sensedby the cell (40). Because erythromycin must enter and cross theinner membrane to gain access to ribosomes, a delay in this stepcould result in the efflux pumps being more effective at sheddingthe drug in the �MKR strain, which would not necessarily requireincreased pump levels.

Although the mechanism by which the �MKR mutation in-creases macrolide resistance remains to be determined, it seemsplausible that L22-mediated ribosome stalling plays a role indetermining the balance of cell envelope components, which, inturn, affects macrolide resistance by altering the efficiency of effluxpumps. Moreover, given that the antibiotic resistance of wild-typeand �MKR strains often changed in parallel when efflux systemswere perturbed, it seems likely that the �MKR mutation simplyenhances a ribosome-mediated defense mechanism against antibi-otics that also occurs in wild-type cells.

We note that L22 mutations can cause macrolide resistance inGram-positive and Gram-negative bacteria (2). Although Gram-positive strains lack TolC-mediated efflux systems, they do containnumerous membrane efflux pumps capable of transporting a widevariety of compounds including macrolides (2). Moreover, syner-gistic relationships between L22-mediated antibiotic resistance andefflux systems have been reported in strains of Haemophilus andCampylobacter (41, 42). Thus, the connection between L22 functionand drug efflux seems to be highly conserved. Our experimentsshow that �MKR ribosomes are not inherently resistant to mac-rolides and support a new model in which changes in translationalter bacterial physiology and reduce macrolide accumulation.Because wild-type and �MKR strains are efficiently inhibited bylow concentrations of macrolides when drug efflux is blocked,combination therapies, using macrolides and efflux inhibitors maybe an appealing antibacterial strategy that precludes many normalroutes to drug resistance.

Experimental ProceduresStrains and Plasmids. L22 fusions containing an N-terminal His6 tag and aC-terminal titin-I27-ssrA domain were encoded on plasmid pS10 and maintainedin clpX�, clpA�, rna� cells with a deletion of the chromosomal S10 operon (20).Weconfirmedthat themacrolideresistancemediatedbytheL22�MKRmutationwas not altered in clpX�, clpA�, rna� strains or when the C-terminal I27-ssrAfusion was omitted (data not shown). The tolC� strains were constructed by P1transducing tolC::kan from strain JW5503–1 of the Keio collection (43) into strainSM1090 (X90, clpX�, clpA�, rna�), removing the kan marker with FLP recombi-nase (44), transforming with either pS10-H6-L22WT-I27-ssrA or pS10-H6-L22�MKR-I27-ssrA, and then deleting the chromosomal S10 operon (20). The acrAB� strainswere constructed by replacing the acrAB ORF with a PCR product containing thecat gene, using the recombination plasmid pSIM-5 (45), and then transducing thecat gene by selection on chloramphenicol into strains SM1145 (wild-type L22) orSM1211 (L22 �MKR) (20). All constructs were verified using diagnostic PCR andthe wild-type, �MKR, �loop1, and �loop2 L22 fusions constructs were verified asthe only copies of L22 genes in the cell by Southern blot analysis with a probecomplementary to the antisense strand of the rplV gene (20). A plasmid contain-ing only the ORF of the ermC gene from Bacillus subtilis under control of anarabinose-inducible promoter (pBAD24-ermC) was a gift from C. Hayes (Univer-sity of California, Santa Barbara, CA) and was introduced into wild-type and�MKR strains and grown in the presence of 0.2% arabinose for induction.

Antibiotics, Culture Growth, and MIC Determination. Antibiotics and PA�N werepurchased from SIGMA-Aldrich. Stocks of erythromycin were prepared in etha-nol, and the concentration was determined in 10 mM bis-Tris (pH 6.5), using anextinction coefficient at 298 nm of 25.7 M�1�cm�1. MICs for antibiotics weredetermined in Luria–Bertani broth supplemented with 1�‘‘Mops mixture’’ (pH7.2, Teknova), using 150 �L of cultures in 96-well plates containing 1,000 log-phase colony-forming units per well and grown with agitation for 16 h at 37 °C.Culture densities were determined by measuring the turbidity at 600 nm.

Biochemical Assays. Purification of ribosomes and rRNA from log-phase culturesand reverse-transcriptase extension of a 5-TCAATGTTCAGTGTCAAGCTATAGTA-AAGGTTCACG-3 primer complementary to residues 2066–2101 of E. coli 23SrRNA were performed as described in ref. 20. Coupled transcription/translationassays were performed as described in ref. 20 by adding purified ribosomes to

Fig. 5. Contribution of AcrAB-TolC to erythromycin resistance. The TolC orAcrA/AcrB components of the AcrAB-TolC efflux pump were deleted from strainswith wild-type or �MKR ribosomes. (A) Erythromycin resistance of cells lackingtolC with wild-type (triangles) or �MKR (circles) ribosomes. (B) Erythromycinresistance of cells lacking acrA/acrB with wild-type (triangles) or �MKR (circles)ribosomes. Inhibition plots of tolC� acrAB� cells are shown for comparison(dashed lines).

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complete a ribosome-free translation extract. Erythromycin was then added todesired concentrations, the mixtures were incubated at room temperature for 15min, and reactions were started by adding a plasmid encoding the SspB and�-lactamase proteins under control of a T7 promoter. After 1 h of transcription/translation at 37 °C, reactions were stopped and 14C-labeled proteins were visu-alizedusingSDS-PAGEandautoradiography.Someexperimentswereperformedusing a transcription template encoding a variant of the E. coli rbsK gene. Thesetranslation assays produced 14C-labeled proteins in a linear fashion for �120 min,and thus the intensity of the product bands at 60 min is a reasonable estimate ofthe translation rate. Inhibition constants (Ki) were obtained by fitting the eryth-romycin (E) dependence of ribosome (R) inhibition to the equation activity

max�(1 � ((([Etotal] � [Rtotal] � Ki) � SQRT(([Etotal] � [Rtotal] � Ki)2) �

4�[Etotal]�[Rtotal]))/(2�[Rtotal]))).DMS modification of ribosomes in vivo was performed similarly to a published

protocol (46). Cultures of wild-type and �MKR cells were grown in LB broth withshaking at 37 °C until early log phase and erythromycin was then added to 1aliquot of each culture. Growth was continued for 2 h and cells were chilled on

ice, harvested, and resuspended at equal cell densities (�1.2 � 109 cfu permilliliter) in fresh medium with or without erythromycin. Aliquots of each culturewere then placed in clean tubes in a 37 °C water bath and PA�N was added to 30�g/ml in 1 sample of each strain containing erythromycin. After 10 min ofpreincubation, DMS solution (freshly diluted 1/6 in ethanol) was added (�50 mMfinal) to each culture, and the cultures were incubated with shaking for anadditional 15 min. Reactions were stopped by diluting an aliquot of each culture20-fold into ice-cold stop solution (100 mM Tris�Cl (pH 7.5), 0.5 M 2-mercapto-ethanol, 50 mM NaCl, 2 mM MgOAc) and mixing rapidly. Cells were then har-vested at 4 °C, washed twice with ice-cold buffer (50 mM Tris�Cl (pH 7.5), 50 mMNaCl, 2 mM MgOAc), and resuspended in lysis buffer (B-Per II (Pierce) supple-mented with 2 mM MgOAc, 0.5 mM CaCl2, 10 units/ml DNase I (Roche), and 0.1mg/ml lysozyme). After lysis, total RNA was purified using acidic phenol/chloroform extraction and alcohol precipitation (20).

ACKNOWLEDGMENTS. We thank K. Griffith, A. Grossman, and R. Britton forhelpful discussions and C. Hayes for materials and discussions. This work wassupported by National Institutes of Health Grants AI-15706 and AI-16892.

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