Vol. 175, No. 22JOlJRNAL OF BACTERIOLOGY, Nov. 1993, p. 7209-72150021 -9193/93/227209-07$02.00/0Copyright © 1993, American Society for Microbiology

tRNA Modification Activity Is Necessary forTet(M)-Mediated Tetracycline Resistance

VICKERS BURDETT

Department of Microbiology, Duke University Medical Center, Durham, North Carolina 27710

Received 23 June 1993/Accepted 8 September 1993

Tet(M) protein interacts with the protein biosynthetic machinery to render this process resistant to thetetracycline in vivo and in vitro (V. Burdett, J. Biol. Chem. 266:2872-2877, 1991). To understand this processmore completely, a mutant of Escherichia coli which is altered in the ability of Tet(M) to confer resistance hasbeen identified. This mutation maps to miaA and displays phenotypes characteristic of previously isolated miaAmutations. The miaA gene product modifies A37 adjacent to the anticodon of several tRNA species. Both themutant isolated in this work and previously isolated miaA mutants confer tetracycline sensitivity in thepresence of functional Tet(M), both share a slow growth phenotype, and in neither case is a wild-typephenotype restored in trans by F'112 carrying the 89- to 98-min region of the chromosome. These similarphenotypes further substantiate the assignment of the mutation described here to the miaA locus.

Tetracyclines are broad-spectrum antibiotics which haveenjoyed wide clinical application because of their effectivenessagainst both gram-positive and gram-negative pathogens.These antibiotics exert a bacteriostatic effect by inhibitingprotein synthesis, presumably by binding to a high-affinity siteon the 30S ribosomal subunit to prevent stable association ofaminoacyl-tRNA with the A site (18). The increasing occur-rence of clinical resistance to tetracycline has limited the use ofthis class of drugs for treatment of infections. The mostwidespread class of tetracycline resistance element is exempli-fied by the class M resistance determinant, tet(M), which hasbeen identified in a number of gram-positive and -negativebacterial species (26, 37) as a marker on such conjugativetransposons as Tn916 (9). Resistance conferred by this elementhas been attributed to association of the gene product with thebacterial protein synthesis machinery to render this systemresistant to the antibiotic (6, 8).Ribosomes prepared under low-salt conditions from cells

expressing Tet(M) are drug resistant in vitro because of theassociation of the tet(M) gene product. Nevertheless, suchribosomes bind tetracycline to the same extent as thoseisolated from a drug-sensitive strain (8a). Tet(M) has beenpurified to homogeneity from high-salt extracts and fromoverproducing strains (8) and has been shown to be function-ally similar to elongation factor G (EF-G) (8, 8a). This is inaccord with sequence homology demonstrated betweenTet(M), EF-G, and other GTP-binding proteins (7). Neverthe-less, the mechanism by which Tet(M) confers resistance hasremained elusive.To further clarify the mechanism of Tet(M) action, I have

sought bacterial mutants that remain drug sensitive in thepresence of functional Tet(M). The rationale for this approachis based on the reasoning that such mutations would permitidentification of components of the protein biosynthetic sys-tem, the function(s) of which is critical for Tet(M) action. Thisarticle describes one such mutation that maps to the miaAlocus which functions in tRNA modification (14).

MATERIALS AND METHODS

Bacterial strains and plasmids. Genotypes and origins ofbacterial strains and plasmids are shown in Table 1. Rifampin-resistant derivatives of MV3 and EMS71 were selected on

plates containing 100 p.g of drug per ml and are designatedMV3R and EMS71R.Plasmid constructions. Several plasmids carrying the tet(M)

gene from Tn916 were constructed during the course of thiswork (Fig. 1). Plasmid pVBll is a derivative of pACYCI77(10) prepared by insertion of the 4,820-bp Hincd! fragment ofTn916 that encodes Tet(M) (22). A spontaneous deletionwithin the 4,820-bp fragment but external to the tet(M) gene(8b) was identified during subsequent manipulations. Theresulting 3,170-bp fragment was inserted into pACYC177AKto yield clones with tet(M) in two different orientations (Fig. 1).These latter plasmids, pVB201 and pVB202, mediate resis-tance to tetracycline and ampicillin.Recombinant DNA methods were performed as described

by Maniatis et al. (27) and Ausubel et al. (1). Restrictionenzymes and T4 DNA ligase were obtained from U.S. Bio-chemicals (Cleveland, Ohio), Bethesda Research Laborato-ries, and Boehringer Mannheim and used as described in themanufacturers' specifications.

Culture media and growth conditions. Bacterial strains weregrown in E medium (43), LB broth (30), and Terrific Broth(40). Solid medium contained 1.4% agar. Supplements wereadded to E medium as necessary at the following concentra-tions: glucose, 0.25%; thiamine, 2 pLg/ml; L-tryptophan, 0.1mM; and Casamino Acids, 0.4%. Growth of bacterial cultureswas monitored with a Klett-Summerson colorimeter with agreen filter (1 Klett unit equals approximately 5 x 10" cells perml). Antibiotics were generally used at the following concen-trations: ampicillin, 50 p.g/ml; chloramphenicol, 25 p.g/ml;kanamycin, 30 pg/ml; nalidixic acid, 30 ,ug/ml; rifampin, 100p.g/ml; and tetracycline, as indicated.

Genetic methods. Mutagenesis of strain MV3 was by treat-ment with 0.2 M ethyl methanesulfonate to 50% survival (30).This procedure yielded mutations to nalidixic acid resistance ata frequency of 2.5 x 10-'. Plasmid pVBl I was introducedinto the population of ethyl methanesulfonate-treated cells byelectroporation (13) with selection for kanamycin resistance.Tetracycline-sensitive strains were identified by replica platingonto LB agar containing 5 pg of tetracycline per ml. Onemutant identified in this manner and further characterizedhere was EMS71 (and the corresponding mutation rtp7l).

PlvirA transductions were carried out as described by Stern-berg and Maurer (39). Transductants obtained with phage

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TABLE 1. Genotypes and origins of bacterial strains and plasmids

Strain or plasmid Genotype and relevant markers Source

StrainsMV3 trpR trpEl0220 leu-6 thi-I supE44 lacY] C. YanofskyEMS 71 trpR trpE10220 leu-6 thi-l supE44 lacYl miaA71 This studyE5014 A(gpt-lac)5 supE44 re/Al? rpsE2112 malA24 thi-1/F'128 B. BachmannKL729 /euB6 tonA2 lacYl supE44 gal-6 hisGi recAl argG6 rpsLI04 B. Bachmann

malA I xyl- 7 mtl-2 metBlF'1 12W3110::Tn5 collection Wild-type E. coli K-12::Tn5 random pool LaboratoryES4 purA45 B. BachmannMA150 hflA 150 B. BachmannX9205 hflAl Banuett and Herskowitz (2)X9246 hflAl purA::Tn5 Banuett and Herskowitz (2)DEV15 miaA thi re/A spoT lacZ(UGA) miaA Petrullo et al. (33)TX2561 miaA::Kmr M. WinklerNK7510 mutL::Tn5 Kleckner et al. (24)

PlasmidspVBII tet(M) Kmr Laboratory collection

(see Fig. 1)pVB201 tet(M) Apr This study (see Fig. 1)pVB202 tet(M) Apr This study (see Fig. 1)pKT71.3 pUC18:chromosomal EcoRI fragment containing zjf::Tn5 This studypNU127.2 miaA+; Cmr; TnlO00 insertion in tet(C) gene; derivative of This study

pNU127 (11)pNU133.1 miaA::Kmr; Cmr; TnlOOO insertion in tet(C) gene; derivative of This study

pNU133 (11)pTX348 miaA hfq hflX hfiK; Apr M. WinklerpFB501 hfq hflX hflK hflC purA; Apr Banuett and Herskowitz (2)

grown on donor strains containing Tn5 were allowed to expresskanamycin resistance prior to plating: after removal of unad-sorbed phage following centrifugation, cells were incubated for1 h in LB broth containing 10 mM sodium citrate. Resistant

transductants were selected on LB agar containing 10 mMsodium citrate and antibiotic.Tn]000 (y8) insertions in pTX348, pNU127, and pNU133

(Table 1) were isolated as described by Guyer (19) and Sancar

HincilBamHl

amp

(pACYC177~3940 bp/

Haeal Haell

eI kaI

\tM, 4820b

BamHl

pVBll8760 bp

Haell / Haell

Haell digest Hincil

BamHl amp|pAC1f7lK\ 2510bpJ

Haell A

E -0 Eco I CUm

TetM fragment3170 bp

Sstl

Hindlil T

BamHl pVB201 BamHI5680 bp

Haell ampHincll

BamHl digestligate and transform

Sstl

TtMHindll

BamHl pVB202 BamHl5680 bp

HaellamHincll

FIG. 1. Construction of Tet(M) plasmids. Plasmid pVB1I carries the 4,820-bp Hincl! fragment of Tn916 known to encode Tet(M) inpACYC177, while pVB201 and pVB202 carry a 3,170-bp Tet(M) fragment at the BamHI site of pACYC177AK. Since the presence of thekanamycin resistance gene of pACYC177 complicated its use in the mapping experiments, these sequences were removed by deletion of the1,430-bp HaeII fragment to generate pACYC177AK. BamHI linkers were added to the 3,170-bp tet(M) fragment and cloned into BamHl-cleavedpACYCI77AK. Only restriction sites relevant to the manipulations shown are included for simplicity.

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et al. (36). The donor, E5014, containing F'128, was trans-formed to introduce the plasmid to be mobilized. The donorstrains and appropriate recipient (EMS71RIpVB11 as recipi-ent for pTX348 or DH1 for pNU127 and pNU133) were grown

to 2 x 108 cells per ml and mixed at a ratio of five donors per

recipient cell. Mating mixes were incubated at 37°C for 120min with slow shaking, and samples were plated on LB agar

containing ampicillin plus rifampin (pTX348 transconjugants)or chloramphenicol plus nalidixic acid (pNU127 and pNU133transconjugants). Plasmid DNA was isolated from transconju-gants, and the position and orientation of the TnlOOO insertswere determined by restriction enzyme analysis. PlasmidspNU127.2 and pNU133.1 contain TnlOOO in the tetA(C) gene

of the vector (pBR325).Plasmid curing. Strains were cured of resident plasmid by

growing cultures overnight in LB broth containing doublingconcentrations of nalidixic acid (29). Cells were plated onto LBagar from the culture containing the highest concentration ofnalidixic acid in which dense growth occurred, usually 4 to 8 jig

ml-1. After overnight incubation, colonies were tested byreplica plating onto LB agar containing an appropriate antibi-otic to screen for the presence of plasmid. The absence ofplasmid in strains identified in this manner was confirmed byagarose gel electrophoresis of lysates made from three to fourindependent isolates (3). Curing occurred at frequencies of 1 to20% but was always successful in the case of the pACYC177-based vectors used here.

Antibiotic susceptibilities. Cultures were grown in 75 ,ul ofLB broth in a sterile microtiter plate for 6 to 7 h (or overnight)prior to transferring a droplet of culture with a 48-prongedinoculator onto the surface of LB agar plates containingtwofold serially increasing concentrations of antibiotic (44). Bythis method, 48 colonies could be tested on a single plate. Theresistance level (subinhibitory concentration) was taken as thehighest concentration of drug showing growth comparable tothat observed in the absence of antibiotic. The method de-scribed here proved more reproducible than replica plating fordetermining resistance levels and was much more convenientthan broth dilution tests when large numbers of cultures were

to be examined. Antibiotics tested by this method includedtetracycline, oxytetracycline, and minocycline from U.S. Bio-chemicals, doxycycline and demeclocycline (6-demethyl,7-chlorotetracycline) from Sigma, chelocardin (a gift of AbbottLaboratories), and anhydrotetracycline (a gift of Pfizer Re-search).Assay for Tet(M) activity. Cells used for extract preparation

were grown in Terrific Broth (40) and lysed by sonication, andthe debris was removed by centrifugation as described previ-ously (8). Extracts were treated with 0.56 g of ammoniumsulfate per ml, and the resulting precipitate was dialyzed at 4°Cwith three to four changes (1 h per change) against 10 mMTris-HCl (pH 7.6)-0.1 mM EDTA-0.5 mM dithiothreitol-50jiM phenylmethylsulfonyl fluoride. Tet(M) specific activity inthis fraction was determined by an assay of tetracycline-resistant protein synthesis as described previously (8) by uti-lizing ribosomes and enzyme fractions from Streptococcus hirae9790 (Streptococcus faecalis 9790).

Protein synthesis in toluenized cells. Protein synthesis intoluene-treated cells was assayed by a modification of theprocedure of Halegoua et al. (20). Cells were grown to a

concentration of 5 x 108 CFU ml- I in LB broth, harvested at4°C by centrifugation, and resuspended in 1/100 volume of 50mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonicacid; pH 7.6)-15 mM Mg(CH3COO)2-60 mM NH4CI-2 mMATP-1 mM dithiothreitol. Toluene (1% [vol/vol]) was added,and the suspension was shaken gently for 10 min in an ice bath.

At time zero, the suspension was diluted fivefold into theappropriate buffer prewarmed to 37°C for assay of proteinsynthesis. Final reaction mixtures contained 50 mM HEPES(pH 7.6), 15 mM Mg(CH3COO)2, 60 mM NH4Cl, a mixture of19 amino acids (all amino acids minus phenylalanine, 0.1 mMeach), 0.5 mM CTP, 0.5 mM GTP, 0.5 mM UTP, 10 mM ATP,1 mM dithiothreitol, 4 jIM [3H]phenylalanine (4 Ci/mmol;DuPont New England Nuclear), and tetracycline as indicatedin a final volume of 0.25 ml. Reactions were stopped after 15min by adding 0.05-ml samples to 5 ml of 10% trichloroaceticacid. Samples were heated to 90°C for 10 min and cooled to23'C, and precipitates were collected on GF/C filters whichwere washed prior to counting. Incorporation was linear for atleast 20 min after the addition of label. Toluene-treated cells,washed once with buffer, were stable for at least 1 month at- 200C.Preparation of probe DNA and hybridization. Labeled

pKT71.3 probe DNA was prepared by utilizing the random-primed DNA labeling method (U.S. Biochemicals) in thepresence of [oc-32P]dATP. Labeled probe DNA was separatedfrom unincorporated nucleotide by chromatography throughSephacryl S-300 equilibrated with 10 mM Tris hydrochloride(pH 7.6)-i mM EDTA. This DNA was then used to probe theEscherichia coli Gene Mapping Membrane (Takara Biochemi-cals, Inc., Berkeley, Calif.) (31), consisting of the ordered Xlibrary of the E. coli W3110 chromosome constructed byKohara et al. (25).

RESULTS

Isolation of tetracycline-sensitive mutants. We have previ-ously shown that the Tet(M) protein interacts with the bacte-rial translation machinery to render protein synthesis resistantto tetracycline (8). To identify host components required forTet(M) action, we have sought chromosomal mutations thatalter the ability of Tet(M) to confer tetracycline resistance.These experiments utilized an E. coli host since tet(M) conferstetracycline resistance in this host (22) and E. coli provides awell-characterized genetic system compared with other bacte-rial species in which tet(M) has been identified.

E. coli MV3 was mutagenized with ethyl methanesulfonate,and an unmutagenized plasmid, pVB11, carrying Tet(M) and akanamycin resistance marker (Fig. 1), was then introduced intothe mutagenized culture with selection for kanamycin resis-tance (see Materials and Methods). The resulting colonies(>50,000) were screened for tetracycline sensitivity by beingreplica plated onto media containing 5 jig of tetracycline perml. Eleven presumptive tetracycline-sensitive mutants wereidentified from screens of two batches of independently mu-tagenized cells.To ensure that the mutations were not due to alterations of

the resident plasmid, plasmid DNAs isolated from the pre-sumptive mutants were screened for physical or genetic alter-ations. Of the 11 selected strains, two lacked detectableplasmid and two contained plasmid with obvious alterations insize and restriction map. Of the plasmid DNAs from theremaining seven presumptive mutant isolates, which appearedto be unaltered on the basis of the above physical criteria, sixwere able to express tetracycline resistance normally whenintroduced into the wild-type parent strain, MV3. Further-more, the plasmid copy number appeared to be normal inthese six strains.The tetracycline resistance level expressed in each of these

six isolates was examined in detail by drug dilution tests. Fourof the isolates were found to be only slightly more tetracyclinesensitive than the wild type containing pVB11. The remaining

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TABLE 2. Tetracycline resistance levels expressed by wild-type andmutant isolates

Tetracycline resistance level (Lg/ml)"Strain

No plasmid pVBI pVB2(1 pVB202

MV3 0.8 6.25 25 25EMS7 0.8EMS71 0.8 0.8 3.12 3.12

" Tetracycline resistance levels were determined as described in Materials andMethods. EMS7 is the original isolate, which contains pVBI 1. EMS71 has beencured of pVBII and a number of plasmids have been introduced by transfor-mation.

two mutant isolates (EMS2 and EMS7) were as sensitive totetracycline as the wild-type parent in the absence of plasmid(Table 2). One of these, EMS7, was chosen for more completeanalysis.To confirm that the alteration in the tetracycline resistance

phenotype in the mutant was in fact due to a chromosomalmutation, a plasmid-free derivative (EMS71) of the originalisolate was obtained by the curing procedure described inMaterials and Methods. Tetracycline resistance levels of theoriginal and cured isolates were accurately determined (Table2). The plasmid-free mutant strain was as susceptible totetracycline as the parental strain, MV3. This suggests thatprotein synthesis in the plasmid-free mutant isolate does notexhibit enhanced tetracycline sensitivity relative to the parentalstrain. When the plasmid used in the initial selection, pVBI 1,was reintroduced into the mutant (EMS71/pVB11), the origi-nal phenotype was reproduced; tetracycline susceptibilitieswere identical to those obtained with the original isolate(EMS7) which contained pVB11.During the course of this work, a deletion of sequences

upstream of tet(M) was identified. This deletion results inenhanced resistance in the wild-type strain although the ori-entation and cloning site in the pACYC177 vector have littleeffect on expression (not shown). When plasmids pVB201 andpVB202, containing the deletion fragment, were introducedinto EMS71, the level of resistance increased to 3 p.g/ml (Table2). However, in each case, tetracycline resistance in the mutantisolates was only 12.5% of that observed in the wild-typeparent. Thus, the data presented in Table 2 illustrate severalpoints. First, a tet(M) plasmid can be introduced into a mutantstrain cured of the original plasmid, and the original phenotypeis maintained. Second, the plasmid-free mutant isolate is nomore sensitive to tetracycline than the wild-type parent. Third,the level of tetracycline sensitivity observed is plasmid depen-dent whether in the mutant or parental strain.

I have previously shown (9) and others have confirmed (32)that tet(M) mediates resistance to tetracycline and minocyclinebut not to chelocardin. I demonstrate here that, in addition tothese tetracyclines, tet(M) also mediates resistance to doxycy-cline, demeclocycline, and oxytetracycline. In this context, Ihave compared resistance to these antibiotics in the wild typeand the EMS71 mutant strain in the presence and absence oftet(M) (Table 3). In every instance, the mutant strain withtet(M) is much more sensitive to the tetracycline being testedthan the wild-type strain with tet(M). None of the strains testedhere exhibited resistance to chelocardin or anhydrotetracycline(data not shown), consistent with previous observations (9, 32).The sensitivity of E. coli to anhydrotetracycline and chelocar-din is not mediated at the level of protein synthesis (34).Mutant isolates contain wild-type levels of Tet(M) activity.

Although the analyses described above indicate that the defect

TABLE 3. Resistances to tetracyclines conferred by tet(M) inmutant and wild-type strains

Resistance level (pug/ml)"Antibiotic

MV3 MV3/pVB202 EMS71 EMS71/pVB202

Tetracycline 0.8 25 0.8 3.12Minocycline 0.4 25 0.4 3.12Oxytetracycline 0.2 12.5 0.2 1.6Doxycycline t).8 25 0.8 3.12Demeclocycline 0.2 6.25 0.2 0.4

' Resistance levels were determined as described in Materials and Methods.

in EMS71 is due to alteration of a chromosomally encodedproduct, the defect could be at the level of tet(M) geneexpression. To test this possibility, ribosome-free extracts ofMV3/pVB1I and EMS71/pVB11 were prepared and assayedfor the level of Tet(M) protein by using the in vitro proteinsynthesis system described previously (8). Extracts from MV3/pVB II and EMS71/pVB11 contained 1.5 x 103 and 1.6 x 103U mg of protein', respectively. Since the levels of Tet(M)activity are identical in the mutant and parental strains, theinability of Tet(M) to confer drug resistance in the mutantbackground is presumably due to the alteration of a hostcomponent that is involved in Tet(M) action.

Tetracycline sensitivity of protein synthesis in toluenizedcells. The results described above suggest that EMS71 containsa mutation that alters the ability of Tet(M) to circumvent theinhibitory action of tetracycline. This possibility was confirmedby testing the sensitivity of protein synthesis to the antibiotic intoluene-permeabilized cells.

Protein synthesis in non-plasmid-containing strains, eitherwild type or mutant, is very sensitive to inhibition by addedtetracycline (Fig. 2); confirming previous observations to thiseffect (28), synthesis was inhibited 50% at 10 ,uM tetracyclineand 85% at 250 ,uM antibiotic. In contrast, protein synthesis in

100

90

80-

70-

>N 60-

50s- X20

0 40- \

Cd 20 - [10 fX f f__10 -

10 O_I0 5 25 50 100 250

Tetracycline, IIMFIG. 2. Sensitivity of protein synthesis in toluene-permeabilized

cells to tetracycline. Cells were labeled with [3H]phenylalanine in thepresence of different amounts of tetracycline as indicated. Afterlabeling, the protein was precipitated by hot trichloroacetic acid, andthe radioactivity was counted in a scintillation counter. The data areexpressed as percentage of remaining activity compared with an assaywithout tetracycline. Toluenized cells were prepared from MV3 (0),MV3/pVB202 (0), EMS71 (O), and EMS71/pVB202 (-).

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HOST FACTOR FOR Tet(M) FUNCTION 7213

wild-type cells expressing Tet(M) is essentially completelytetracycline resistant (20% inhibition) at the highest level oftetracycline tested (250 FM). When the mutant cell lineEMS71/pVB202 was tested, protein synthesis displayed inter-mediate sensitivity, with 50% inhibition at 250 F.M antibiotic.These results are consistent with the intermediate tetracyclineresistance levels observed in vivo for EMS71/pVB202 (3.12p.g/ml) compared with the low resistance levels of sensitiveplasmid-free strains (0.8 pLg/ml) and the high levels of fullyresistant strains (25 K.g/ml for MV3/pVB202; Table 2). Proteinsynthesis in S30 extracts paralleled the results obtained herewith permeabilized cells (not shown).Mapping of the loci responsible for tetracycline sensitivity

in the presence of Tet(M). The mutation in EMS71 has beendesignated tp or required for Tet(M) protection; I will refer tortp71 as the mutation present in this strain. The rtp71 mutationwas mapped by utilizing a transposon tagging strategy (24)since strategies based on matings with Hfr donors did not givereliable results. EMS71/pVB202 was transduced with a PlvirAlysate grown on W3110::Tn5 with random transposon inser-tions. Several Rtp+ Kanr transductants were isolated. Subse-quent analysis of these strains revealed that, in one isolate, thekanamycin resistance marker cotransduced with rtp+ with afrequency of 83%. When the wild-type allele of rtp7l wasintroduced into EMS71/pVB202, the resistance level was re-stored to that of the wild-type parental strain MV3/pVB202.Furthermore, the mutant allele could be transduced into awild-type strain with similar efficiency by Plvir grown on astrain in which rtp7l was linked to Tn5.To determine the chromosomal location of Tn5, the chro-

mosomal EcoRI fragment containing the entire Tn5 elementwas cloned since the transposon does not contain any EcoRIsites (19). Although the resulting clone (pKT71.3) containsTn5 and neighboring chromosomal sequences, it did notcomplement rtp7l. When pKT71.3 was used as a probe, stronghybridization to phages corresponding to 95 min on the E. colilinkage map (phages 653 and 654) was observed. This Tn5insertion was designated zjf-71::Tn5.

Since rtp7J could reside either clockwise or counterclock-wise of zjf-71::Tn5 on the E. coli linkage map, PlvirA trans-duction was utilized to orient the position of rtp71 relative tozjf-71::TnS and to other markers in this region. The 90%cotransduction of rtp7l with mutL::Tn5 and 91% cotransduc-tion with purA::TnS places rtp between these markers. Al-though rtp7l could be readily mapped to this position by P1transduction, rtp71 could not be complemented in trans byF'112, which carries the chromosomal region from 89 to 98min of the genome (see below).There is 9,763 bp of continuous sequence information

available for this region (12, 23, 45; for information regardingan unpublished sequence from GenBank release 75, see refer-ence 2) with the clockwise gene order mutL-miaA-hfq-hflA(hflX-hflK-hfiC)-purA. To test whether mutations in any ofthese genes alter the tetracycline resistance phenotype nor-mally conferred by Tet(M), a plasmid carrying tet(M) wasintroduced into strains defective in hflA (2) and miaA (11, 12).Two different hflA strains, MA150 and X9246, transformedwith a Tet(M) plasmid, pVB202, were tetracycline resistant.On the other hand, the miaA strain TX2561 (miaA::Km)remained tetracycline sensitive (3.1 ,ug/ml) after establishmentof the same plasmid. The parental strain of TX2561, W3110,was tetracycline resistant (25 p.g/ml) after transformation withpVB202.

Several recombinant plasmids carrying miaA+ and nearbymarkers were tested for their ability to complement rtp7l byusing vectors compatible with the pACYC177-derived tet(M)

plasmids (Fig. 3). pFB501, which contains an 18-kb BamHIchromosomal fragment which complements both purA andhflA, fails to restore a tetracycline-resistant phenotype toEMS71/pVB11. Tetracycline resistance was restored bypNU127.2 but not by the derivative plasmid pNU133.1, inwhich the miaA gene is interrupted by a kanamycin cassette inthe SmaI site (11). Further, TnlOOO mutagenesis of pTX348was also consistent with miaA encoding the product in ques-tion. TnlOOO insertions in mutL, hflX, and hflC were withouteffect on the ability of this plasmid to complement rtp71;insertions in miaA abolished complementation. AlthoughTn1000 insertions in hfq were not recovered, it seems unlikelythat hfq is involved. Plasmid pNU127.2, which complementsrtp71, contains only 75% of the hfq gene. It could be arguedthat this is sufficient for biological function and that thekanamycin insertion present in pNU133.1 could be polar onthis expression. However, this seems highly unlikely sincemiaA::Km insertions present on pNU133.1 and in the chromo-some of TX2561 are upstream of two major promoters for hfqand have little if any effect on hfq expression (41, 42) and hfqshould be fully expressed from pFB501 (these data are sum-marized in Fig. 3). Thus, the proper name of our mutationshould be miaA71.

Despite these findings that miaA could be complemented bythe wild-type gene on a multicopy plasmid, F'112, which isknown to span the miaA-purA region since it spans the 89- to98-min region, failed to complement rtp71 to restore thetetracycline resistance phenotype or to restore the miaA+phenotype to DEV15 miaA. This is not surprising since theoriginal miaA (trpX) mutation has been shown previously to bedominant in trans (46) with respect to F' 112. Our finding thattetracycline sensitivity conferred by rtp7l and the miaA phe-notype is also dominant in trans further substantiates theconclusion that these two mutations reside in the same gene.

DISCUSSION

The Tet(M) protein interacts with the bacterial translationmachinery to render protein synthesis resistant to the presenceof tetracycline. Although Tet(M) protein has been purified tohomogeneity (8)? the biochemical basis for the resistance is stillnot understood. The protein is known to associate with ribo-somes in a salt-labile manner (8), and ribosomes from resistantand sensitive cells have been found to bind equivalent amountsof tetracycline (8a). This suggests that association of Tet(M)protein with the ribosome may prevent tetracycline frominhibiting protein synthesis even in the presence of bounddrug. The amino acid sequences and biochemical activities ofTet(M) are similar to those of bacterial EF-G (7, 8). However,reactions known to be mediated by EF-G (translocation of thepeptidyl-tRNA from the A site to the P site and ribosome-dependent hydrolysis of GTP) are not inhibited by tetracycline(21, 35).

Study of purified ribosomes and ribosomal subunits hasindicated that tetracycline binds to a high-affinity site locatedon the 30S ribosomal subunit and reduces the affinity of the Asite for aminoacyl-tRNA (15, 17, 18). More recently, tetracy-cline has been shown to also inhibit tRNA binding to the P sitein an allosteric manner (15, 16). Nevertheless, attempts toidentify chromosomal mutations that render protein synthesistetracycline resistant have yielded negative results. This sug-gests that the target(s) of tetracycline action may be morecomplex than initially thought, and clarification of the mecha-nism of Tet(M) action may provide further insight into themechanism of tetracycline bacteriostasis.The E. coli mutant described here expresses drug resistance

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Em0

U)

A)0. = 0 0

mutL | miaA llhfI hflX hfiK hfIC Il purA

-i pFB501

B) I-km

I-

- pNU127.2

A pNU133.1

C) Tn 1000 insertionsin pTX348

D) phenoWpertp

miaA

I 119 613 10

+ +

+ +

FIG. 3. Structure of the 95-min region of the E. coli chromosome. (A) Partial restriction map of the mutL-purA segment of the chromosome.The thick line represents the region of continuous sequence information (9,763 bp) compiled from published sources and GenBank entries (12,23, 45; for information regarding an unpublished sequence from GenBank release 75, see reference 2). The positions of the genes are representedby rectangles, and the direction of transcription is from left to right. (B) Clones tested for their ability to complement rtp7l. The 18-kb BamHIinsert in pFB501 extends from the BamHI site in miaA to the right, while pNU127.2 and pNU133.1 inserts extend from the KpnI site 6.5 kb tothe left as indicated. The miaA gene of pNU133.1 is interrupted by a kanamycin resistance fragment. pTX348 contains a 6.5-kb Sall insert as shown.(C) Locations of TnJOOO insertions in plasmid pTX348 determined by restriction enzyme digestion. The numbers are isolate designations. (D)Phenotypes pTX348::TnJOOO derivatives. The phenotypes are shown as either plus for recovery of wild type or minus for failure to become wild typeafter transformation of the plasmids into the rtp strain EMS71/pVB11 and scoring for tetracycline resistance or into DEV15 miaA and scoring forcomplementation of miaA by determining whether colonies were red (mia+) or white (mia) on MacConkey-lactose agar as described previously (33).

at markedly reduced levels in the presence of functionalTet(M) protein, and the mutation maps to the miaA locus. Themutant also displays phenotypes characteristic of previouslyisolated miaA mutants, namely, slightly reduced growth rate(12) and failure to be complemented in trans by F'112 (46).Moreover, previously isolated miaA mutants fail to supportdrug resistance mediated by Tet(M). The identity of pheno-types, despite the mode of selection, suggests that each of thesemutations is due to loss of function of the miaA product.The miaA gene encodes an activity which modifies position

A37 immediately 3' of the anticodon of tRNAs which readcodons beginning with U, including tRNAs for phenylalanine,tryptophan, tyrosine, cysteine, leucine, and serine (see refer-ence 38). Undermodification of these tRNAs results in aber-rant translation and altered expression of a number of charac-terized genes (4, 5, 33, 46, 47). One example points out theimportance of tRNA modification that is necessary to maintaintranslational efficiency. tRNATrP is an important regulator ofthe trp operon. Unmodified tRNATrP found in miaA (trpX)mutants exhibits reduced codon-anticodon interactions result-ing in a decreased frequency of transcription termination in theattenuator region (46). Thus, modification of tRNA plays animportant part in the efficiency and fidelity of translation.As outlined above, the consequence of tetracycline binding

to the ribosome is to reduce the affinity of tRNA for the A siteof the ribosome and to act against the P site allosterically toreduce tRNA binding (15, 16). The finding that the mutationidentified here maps to miaA suggests that tRNA modificationmay have a role in the mechanism of tetracycline inhibition ofprotein synthesis. Alternatively, appropriate modification oftRNA may be involved in the mechanism by which Tet(M)circumvents tetracycline inhibition. Since part of the basis of

Tet(M) action may be to stabilize the ribosome-tRNA inter-action in the presence of antibiotic, the combination of un-modified tRNA, due to mutation in miaA, and the presence oftetracycline may create a situation in which Tet(M) is unable tostabilize the tRNA interactions sufficiently to allow proteinsynthesis to proceed normally.

ACKNOWLEDGMENTS

I thank Kristin Garrett for excellent technical assistance in theisolation and characterization of these mutants, Carol Caughey andMeg Monahan for assistance with the TnS experiments, and PaulModrich for many useful discussions.

This research was supported by Public Health Service grant Al15619 from the National Institutes of Health.

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