Vol. 169, No. 1JOURNAL OF BACTERIOLOGY, Jan. 1987, p. 340-3500021-9193/87/010340-11$02.00/0Copyright C 1987, American Society for Microbiology

Cloning and Analysis of ermG, a NewMacrolide-Lincosamide-Streptogramin B Resistance Element from

Bacillus sphaericusM. MONOD,t S. MOHAN, AND D. DUBNAU*

Department of Microbiology, Public Health Research Institute, New York, New York 10016

Received 12 August 1986/Accepted 20 October 1986

To analyze the regulation of a newly discovered macrolide-lincosamide-streptogramin B resistance element(ermG) found in a soil isolate ofBacillus sphaericus, we cloned this determinant and obtained its DNA sequence.Minicell analysis revealed that ermG specifies a 29,000-dalton protein, the synthesis of which is induced byerythromycin. Si nuclease mapping was used to identify the transcriptional start site. These experimentsdemonstrated the presence on the ermG mRNA of a 197 to 198-base leader. Within the leader are two smallopen reading frames (ORFs) capable of encoding 11- and 19-amino-acid peptides. Each ORF is preceded by asuitably spaced Shine-Dalgarno sequence. The ermG protein is encoded by a large ORF that encodes a244-amino-acid protein, in agreement with the miniceli results. This protein and the 19-amino-acid peptide arehighly homologous to the equivalent products of ermC and ermA. We conclude, on the basis of this homology,that ermG encodes an rRNA transmethylase. The leader of ermG can be folded into a structure that sequestersthe Shine-Dalgarno sequence and start codon for the large ORF (SD3). On the basis of these data and on theobserved greater responsiveness of the ermG system than of the ermC system to low concentrations oferythromycin, we propose a model for the regulation of this gene in which the stalling of a ribosome under theinfluence of an inducer, while reading either peptide, suffices to uncover SD3 and allow translation of the rRNAtransmethylase. The evolution of ermG is discussed.

Naturally occurring resistance to the macrolide-lin-cosamide-streptogramin B (MLS) antibiotics is usually con-ferred by the addition of methyl groups to the N6 position ofa particular adenine residue in 23S rRNA (15, 27). Thismodification results in a decrease in ribosomal affinity forthese antibiotics (26, 34). Several MLS resistance elementshave been studied, and their DNA base sequences have beendetermined (9, 10, 13, 14, 18, 20, 23, 30). The products ofthese genes have been shown to be rRNA methyltransfer-ases. Interest has focused on two aspects. First, MLSresistance is in many cases inducible by the macrolideerythromycin. The mechanism of induction has been estab-lished in the case of ermC, a gene located on the Staphylo-coccus aureus plasmid pE194 (4, 32). The addition of eryth-romycin causes a ribosome to stall while translating a19-amino-acid leader peptide that precedes the major ermCopen reading frame (ORF). Ribosome stalling opens a hair-pin loop, rendering the ribosome-binding site for the ermCgene product accessible for the initiation of translation. Inaddition to this positive mode of regulation, we have re-cently shown that ermC is subject to autoregulation (3). TheermC gene product represses its own translation, presum-ably by interacting with mRNA. Finally, we have observedthat the addition of erythromycin results in a specific anddramatic stabilization of ermC mRNA (26), and the mecha-nism of this effect has been investigated (D. Bechhofer andD. Dubnau, Proc. Natl. Acad. Sci., in press).

Interest in MLS resistance has also focused on the prop-erties of the rRNA methyltransferases. These pose interest-

* Corresponding author.t Present address: Biotechnology Department, Ciba Geigy AG,

Basel, Switzerland.

ing problems in protein-RNA recognition, and in view of thewide distribution and diversity of MLS determinants, theyare an attractive subject for evolutionary investigation (D.Dubnau and M. Monod, in S. Levy and R. P. Novick, ed.,Evolution and Spread of Antibiotic Resistance Genes, inpress).We present here the isolation by molecular cloning of a

new MLS gene from Bacillus sphaericus. The sequence andsome properties of this resistance element (ermG) are pre-sented and compared with those of other MLS determinants.

MATERIALS AND METHODSStrains and plasmids. All B. subtilis strains were deriva-

tives of 168. The strains and plasmids used are listed in Table1. The MLS-resistant soil isolates were kindly provided by J.Pollak.

Isolation of plasmid DNA and transformation. PlasmidDNA was isolated from stationary-phase cultures by thesodium dodecyl sulfate-NaCl method (11), followed by CsCl-ethidium bromide centrifugation as described for B. subtilis(7). The preparation of competent cells and plasmid trans-formation were done as described previously (1).

Restriction endonuclease cleavage, restriction mapping, andligation. Restriction endonucleases and T4 DNA ligase wereobtained from Boehringer Mannheim Biochemicals, NewEngland BioLabs, Inc., and Collaborative Research, Inc.,and used in accordance with the specifications of the suppli-ers. Mapping of restriction sites was done by multipleenzyme cutting and analysis of digestion products by elec-trophoresis on agarose gels with Tris-borate buffer (6).

Southern blotting and hybridization. DNA probes werelabeled by nick translation with [a-32P]dATP and [a-32P]dCTP to specific activities of 1 x 108 to 3 x 101 (22).

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CLONING AND ANALYSIS OF ermG FROM B. SPHAERICUS

TABLE 1. B. subtilis strains and plasmids

Strain Plasmid Characteristics Reference

CU403 thyA thyB metB divIVB1 21BD624 pBD64 trpC2 thr-5 8BD629 pUB110 leu met hisHBD630 leu met hisHBD1117 pBD347 leu met hisH 17BD1146 pBD364 leu met hisHBD1156 pBD370 leu met hisHBD1107 pBD142 thyA thyB metB divIVBJBD1158 pBD370 thyA thyB metB divIVB1

Transfer of DNA from agarose gels to nitrocellulose mem-

branes was done by the method of Southern (28). Mem-branes were prehybridized for 4 h at 650C in 5 x SSC (lxSSC is 0.15 M NaCl plus 0.015 M sodium citrate)-SxDenhardt reagent (lx Denhardt reagent is 0.02% each ofFicoll, polyvinylpyrrolidone, and bovine serum albumin)(2)-200 ,ug of denatured salmon sperm DNA per ml and thenhybridized at 65°C overnight with the denatured probe in 5 xSSC-1.2x Denhardt reagent-100 ,ug of denatured salmonsperm DNA per ml. After hybridization, the filters werewashed under conditions of high stringency (0.1 x SSC-0.1%sodium dodecyl sulfate at 65°C) and exposed to X-ray film.

Minicell studies. Plasmids were introduced into theminicell strain CU403 by transformation. Minicells wereisolated, stored, and used to study the incorporation of[35S]methionine as described by Shivakumar et al. (25).Minicell lysates were analyzed by discontinuous sodiumdodecyl sulfate-polyacrylamide gel electrophoresis as de-scribed before (23). Minicell autoradiographs were traced byusing a Shimadzu dual-wavelength chromatogram scanner(model CS-910). Integration of the resulting peaks was

accomplished by cutting them out from the tracing andweighing them.DNA sequence determination. Fragments of pBD370 were

cloned into M13mplO and M13mpll (16), and the resultingDNA preparations were used for sequencing by thedideoxynucleotide method of Sanger et al. (24). Thin (0.35mm) urea-polyacrylamide gels (6%) were used.Computer analysis. The ermG sequence was analyzed by

using a Sun-3 minicomputer running version 4.2 BSD of theUnix operating system. The Sequence Analysis Packagedeveloped by the Biomathematics Computation Laboratory,Department of Biochemistry and Biophysics, University ofCalifornia at San Francisco, was used.

RESULTS

Characterization of MLS-resistant strains. Several bacte-rial strains were isolated by J. Pollak from a swine feedlotsoil sample and shown to be resistant to erythromycin (J.Pollak, personal communication). These organisms, allgram-positive sporulating bacilli, were investigated by fur-ther tests based on the taxonomic key of Gordon et al. (5).Strains 27 and 33 were found to be lysozyme sensitive,Voges-Proskauer negative, and weakly catalase positive.They did not hydrolyze starch, failed to grow anaerobically,did not grow in 5% NaCl, did not clear tyrosine, andproduced no acid from glucose. They deaminated phenylal-anine. On the basis of these tests, the isolates could beunambiguously classified as B. sphaericus.The isolates were tested further for MLS inducibility by

streaking on tylosin-containing media in the presence or

absence of an inducing concentration of erythromycin.Strains 27 and 33 could be induced by either erythromycin orthe closely related macrolide oleandomycin but not bytylosin, clindamycin, or lincomycin. In this respect, theywere similar to strains carrying ermC or ermD.

Cloning of ermnG. Chromosomal DNA isolated from B.sphaericus 33 was cleaved with BclI and ligated to pBD64DNA cut with BglII. The ligation mix was used to transformcompetent BD629, with selection for chloramphenicol anderythromycin resistance. Both antibiotics were used at 5,ug/ml. BD629 carries pUB110 and acts as a helper by virtueof the homology between pBD64 and pUB110 (8). Fiveclones were isolated and shown to exhibit inducible resist-ance to MLS antibiotics by the criteria described above.Plasmid DNA was isolated from these strains and used totransform a plasmid-free recipient (BD630) to chloramphen-icol and erythromycin resistance. This retransformation stepserved to separate the recombinant plasmid carrying the B.sphaericus MLS determinant (ermG) from the pUB110helper plasmid. The ermG plasmid (pBD364) was found tocontain an insert of approximately 3.6 kilobases (kb).

Southern hybridization with ermG. pBD364 DNA was nicktranslated and used as a probe in a Southern blottingexperiment with undigested chromosomal DNA from B.sphaericus 33 and 27 and from a number of plasmids carryingermD, ermC, ermB, and ermA (data not shown). A clearsignal was obtained only with the two samples of B.sphaericus chromosomal DNA; a weak signal was obtainedwith the ermC DNA. Hybridization to material that comi-grated with the chromosomal DNA was observed. Theintensity of hybridization to 2 jig of chromosomal DNA wasequivalent to that observed with 0.005 pug of pBD364 DNA.This plasmid contains about 7.2 kb. On the basis of thesedata, it appears that each genome equivalent of B.sphaericus DNA contains one to two copies of ermG. Itseems likely that ermG is a chromosomal element. In an-other experiment, B. sphaericus 33 DNA was cut with BclI,electrophoresed, blotted to nitrocellulose, and hybridizedwith nick-translated pBD364 DNA. A hybridizing band ofabout 3.6 kb was uniquely present, consistent with theestimate for the size of the cloned fragment (Fig. 1).

Subcloning of ermG. To facilitate the analysis of the ermG

AB C

23130.*9416k65574361 -360023222027

FIG. 1. Southern blotting of pBD364 to B. sphaericus 33 DNA.pBD364 DNA was labeled by nick translation and hybridized to amembrane filter to which a Bcll digest of B. sphaericus 33 chromo-somal DNA had been blotted. Lanes: A, size standards (HindIII-cleaved bacteriophage X DNA); B, ethidium bromide-stainedagarose gel on which the cleaved chromosomal DNA had beenelectrophoresed; C, autoradiograph of the hybridized filter. Num-bers represent bases pairs.

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342 MONOD ET AL.

determinant, we subcloned it into pBD347, a derivative ofpIM13 in which the MLS gene has been replaced by thechloramphenicol resistance gene from pC194 (17). Thisvector was cut at its unique MboI site and ligated to pBD364DNA that had been partially cut with MboI. The 3.6-kb B.sphaericus insert in pBD364 consisted of three MboI frag-ments. The ligation mix was used to transform a plasmid-freecompetent culture of BD630, with selection for chloram-phenicol and erythromycin resistance. Twelve transform-ants were tested and found to express inducible MLSresistance. One of these transformants was selected forfurther study and found to contain a plasmid (pBD370) thatconsisted of pBD347 plus two additional MboI fragments.These corresponded to two of the fragments from pBD364and together comprised about 1.9 kb. It is noteworthy thatstrains carrying either pBD364 or pBD370 were unable togrow on tylosin-containing media in the absence of inducer.The copy numbers of these two plasmids were about 30 and150 to 200, respectively. ermC-containing plasmids withcopy numbers of about 30 or higher confer the ability to growon media containing tylosin because of their basal synthesisof transmethylase (33; R. Villafane and D. Dubnau, unpub-lished data). The failure of strains carrying pBD364 orpBD370 to grow on tylosin-containing media indicates thatermG expresses a low basal level of resistance, in contrast toermC.

Minicell analysis. The gene product of ermG and itsresponse to erythromycin induction were studied inminicells. Figure 2 shows the results of an experiment inwhich the induction of the ermG and ermC products wasexamined as a function of erythromycin concentration. Theelectrophoretic mobilities of the two proteins were similar.The ermG product appeared to be an erythromycin-inducibleprotein with an apparent Mr of 29,000. The response of thesetwo determinants to various concentrations of inducer wasdifferent. The interpretation of this difference will be dis-cussed below.DNA sequence of ermG. Figure 3 shows the strategy used

to determine the DNA sequence of ermG. Sequencing wascarried out by the dideoxynucleotide method after cloninginto suitable M13 derivatives. The entire insert in pBD370was sequenced on both strands, and all cloning sites werecrossed. To facilitate the crossing of an EcoRI site, weprepared and used a synthetic oligonucleotide primer com-plementary to an upstream sequence (Fig. 3). The DNAsequence of the insert is shown in Fig. 4. The sequenceincludes a large ORF capable of encoding a 244-amino-acidprotein with a predicted molecular weight of 28,562, in closeagreement with the minicell results. The sequence of thisprotein shows striking homology to that of the other MLSresistance proteins for which data are available and, inparticular, to the sequences of the ermC and ermA geneproducts (see below). Thus, we can confidently concludethat this ORF encodes an rRNA transmethylase. Precedingthe rRNA transmethylase-coding region is a ribosome-binding sequence (SD3).

Si nuclease mapping. To determine the start site of ermGtranscription, we carried out S1 nuclease mapping. Thesmall EcoRI fragment from the pBD370 insert (Fig. 3 and 4)was isolated and 5' terminally labeled with polynucleotidekinase. The labeled fragment was cut with AluI, and thelarger AluI-EcoRI fragment was isolated and annealed toRNA prepared from a B. subtilis strain carrying pBD370.Figure 5 shows the results of this experiment. A 167- to168-base-pair fragment was protected from Si nucleasedigestion by being annealed to the RNA sample. A second

A1 2 3 4 5 6

.012

i0

U)

'E

0-

.00

L-

aC

c @0

B1 2345 6

......1..i9

.02

Erythromycin ( pg/ ml ).04

FIG. 2. Minicell analysis of ermC and ermG induction. Minicellscarrying pBD142 (A) or pBD370 (B) were induced with the followingconcentrations of erythromycin (micrograms per milliliter: none)(lanes 1), 0.001 (lanes 2), 0.005 (lanes 3), 0.01 (lanes 4), 0.02 (lanes5), and 0.05 (lanes 6). After 10 min of incubation, [3H]leucine wasadded, and the incubation was continued for an additional 30 min.pBD142 is a derivative of pE194 that carries the cop-6 mutation aswell as a chloramphenicol resistance determinant from pC194. (C)Synthesis of ermC (0) and ermG (0) transmethylases, determinedfrom tracings of the gels shown in panels A and B. Results arepresented in arbitrary units proportional to absorbances on thefluorographic film.

fragment corresponding in size to the complete AluI-EcoRIfragment also appeared to be protected. These results sug-gest that ermG transcription begins at positions 474 and 475and that a second promoter upstream of the protectedfragment may also be used. To test the latter possibility, wecleaved pBD370 with EcoRI and self-ligated it to remove thesmall EcoRI fragment that contains the start site at positions474 to 475. This sample was used to transform BD630, withselection for chloramphenicol resistance. Several transform-

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CLONING AND ANALYSIS OF ermG FROM B. SPHAERICUS

HindIIIAluI MbcI

1000

AluIEcoRI

EcoRI AluI AluII I

ermG

I------------->

I.-------------_>1 .-------------->

0 >

<----.----

----- I<______________-I<.----------I<-----

FIG. 3. Sequencing strategy. The restriction fragments indicated by the arrows were cloned into M13 derivatives. Sequencing by thedideoxynucleotide procedure was performed in the directions shown by the arrows. A synthetic primer was used to sequence one region (0).

ants were selected and found to be erythromycin sensitive,and all were shown to lack the small EcoRI fragment. Itshould be noted that the Eco1K1 site at position 641 is about19 bases upstream from SD3 (Fig 4). If an upstream pro-.

moter had been present, therefore, the deleted plasrnidswould have expressed erythromycin resistance. These re-

sults suggest that the start site for ermG identified by S1nuclease mapping at positions 474 to 475 is probably themajor one utilized in vivo and that no significant in vivotranscription initiates upstreamh from the EcoRI site locatedat position 412. The larger radioactive band seen in Fig. 5,lane B, is therefore probably undigested probe remainingbecause of incomplete digestion by Si nuclease.

DISCUSSION

We have cloned and sequenced ermG, a new MLS resist-ance determinant from the soil bacterium B. sphaericus.This determinant is contained within two MboI fragments, ispresumably located on the chromosome of B. sphaericus,and includes the genetic information necessary to specify theinducible synthesis of an rRNA transmethylase.

Regulation of ermG, The induction specificity in B.sphaericus with a single copy per genome is indistinguish-able from that in B. subtilis with a multicopy plasmid.Furthermore, this specificity, induction by erythromycin andoleandomycin but not by other MLS antibiotics, is identicalto that of ermC. The induction of ermC by erythromycin hasbeen intensively studied (4, 32). It has been shown that ermCrmRNA exists in a stable conformation in which SD2 issequestered by base pairing and thus rendered inaccessiblefor ribosome binding. A ribosome bound to an erythromycinmolecule stalls while translating the leader peptide. Thisstalling event opens the structure, exposing SD2 and permit-ting translation of the ermC methylase to occur. ermA has a

regulatory region similar to that of ermC but with a secondsmallet leader peptide encoded upstream from the 19-amino-acid peptide (18). A cascade model for ermA induction hasbeen proposed (18) in which stalling of a ribosome within thefirst peptide under the influence of erythromycin frees SD2.An erythromycin-free ribosome initiates translation of thesecond peptide and then stalls in the presence of erythromy-cim. SD3 is then freed, permitting the synthesis of methylaseprotein by erythromycin-free or methylated ribosomes.The erOnG transcriptional start site at positions 474 to 4t5

and the translational initiation site at positidn 672 indicate

that ermG mRNA is synthesized with a 5' leader of 197 to198 bases. Within this leader are two short ORFs sufficient toencode peptides of 11 and 19 amino acids. Each ORF ispreceded by a suitably spaced Shine-Dalgarno (SD) se-

quence (Fig. 4).We have detertnined the predicted minimal-energy folded

structure of the promoter-proximal 281 base pairs of ermGmRNA by using a folding program based on that of Zuckerand Stiegler (35) (Fig. 6A). SD3 is partially base paired, as isits associated AUG initiation codon. The configuration nearthis ribosome-binding site is very similar to that in ermC. Wehave shown that the ermC ribosome-binding site is inacces-sible to ribosomes in the absence of erythromycin (19).Consequently, it appears that the predicted equilibriumstructure shown in Fig. 6A will be inactive for the synthesisof methylase. In this structure, the sequences flanking SD2are also base paired, and the associated AUG initiationcodon is partly base paired, although the GGAGG sequencethat forms part of SD2 is unpaired. SD1 is also partly basepaired, and its associated AUG initiation codon is free. Thepairing of the 5'-terminal AAU triplet shown in the comput-er-predicted structure is very doubtful, and SD1 is likely toexist in a largely unpaired configuration. These featuressuggest that a cascade model similar to the one proposed forermA (18) might be applicable to ermG, if we assume thatpairing in the SD2 region is sufficient to prevent ribosomeinitiation. According to this model, a ribosome would initiateat SD1 and commence the traaslation of peptide 1. In thepresence of erythromycin the ribosome would stall in an

appropriate position to unpair the sequence containing SD2.A ribosome in an initiation complex protects 25 to 40 basepairs from nuclease action (29). If a stalled ribosome pro-tected an equivalent segment, the stall would have to occuranywhere in the C-terminal half of peptide 1 to free thesecond ribosome-binding site. Translation would then beginat this binding site, and?-a second stalling event would serveto free SD3, permitting nmethylase synthesis to occur. Inview of the sequence similarity of the peptide 2-codingregion of ermG to that of the peptide 2-coding region ofermC, the second stalling event would be similar for the twogenes.Murphy (18) has suggested that a cascade-type induction

mechanism such as those proposed for ermA and ermGwould be characterized by a weak response to low concen-trations of inducer. This follows from the requirement fortwo stalling events for induction to occur. At low concen-

1917

MboI AluI

I-------------->

MboI

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344 MONOD ET AL. J. BACTERIOL.

MboIGATCATAAAAATTCACCAGAGAAATTTTATGATAATATAGAGCATCATTATGAATTTATTGGAGATAAACT

.100GATAATAGGCAAGTTCTGCGCAATAGCAGAGGGTGTGAAGTTTATTATGAATGGTGCAAATCATAGAATGG

.200ATGGAATCACAACCTATCCCTTTAATATCTTTGGCTGTGGATGGGAAAAGGTTACTCCTACAATAGAACAA

BstNlCTTCCTTTTAAGGGAGATACTGTGATTGGTAATGATGTCTGGATTGGTCAAAATGTAACCATTATGCCAGG

.300TGTTATAATTGGTGATGGTGCGATTATTGCCGCTAATTCAACAGTAGTGAAAAGTGTTGAACCCTATTCAA

.400 . EcoRITATATAGTGGGAATCCAGCTAAGTTTATCAAAAAACGCTTTAGTGACGAAAAGATTGAATTCTTACTAAAG

CTAGAGTGGTGGAACTGGAGCGGAGAGGAAATATTTGATAATCTTGAAATTCTAACATCCGAAGCAGGGTT-35 -10

.500 Met Asn Lys Tyr Ser Lys Arg Asp Ala Ile Asn ENDAGAGGAATTA ATG AAT AAA TAT TCG AAA AGA GAT GCA ATA AAT TAAGGAGGTTTTCTA

SD1 SD2.600

Met Gly Leu Tyr Ser Ile Phe Val Ile Glu Thr Val His Tyr Gln Pro AsnATG GGT TTA TAC TCA ATT TTT GTA ATA GAA ACA GTT CAT TAT CAA CCA AAT

Glu Lys END . . . EcoRIGAA AAA TAAAAGGTTATAATGAATTGTTGATATGAATTCATTATAACCTTTAAGGAGAGGTTATA

SD3.700

Met Asn Lys Val Asn Ile Lys Asp Ser Gln Asn Phe Ile Thr Ser Lys Tyr HisATG AAC AAA GTA AAT ATA AAA GAT AGT CAA AAT TTT ATT ACT TCA AAA TAT CAC

Ile Glu Lys Ile Met Asn Cys Ile Ser Leu Asp Glu Lys Asp Asn Ile Phe GluATA GAA AAA ATA ATG AAT TGC ATA AGT TTA GAT GAA AAA GAT AAC ATC TTT GAA

.800Ile Gly Ala Gly Lys Gly His Phe Thr Ala Glu Leu Val Lys Arg Cys Asn PheATA GGT GCA GGG AAA GGT CAT TTT ACT GCT GAA TTG GTA AAG AGA TGT AAT TTT

Val Thr Ala Ile Glu Ile Asp Ser Lys Leu Cys Glu Val Thr Arg Asn Lys LeuGTT ACG GCG ATA GAA ATT GAT TCT AAA TTA TGT GAG GTA ACT CGT AAT AAG CTC

.900Leu Asn Tyr Pro Asn Tyr Gln Ile Val Asn Asp Asp Ile Leu Lys Phe Thr PheTTA AAT TAT CCT AAC TAT CAA ATA GTA AAT GAT GAT ATA CTG AAA TTT ACA TTT

Pro Ser His Asn Pro Tyr Lys Ile Phe Gly Ser Ile Pro Tyr Asn Ile Ser ThrCCT AGC CAC AAT CCA TAT AAA ATA TTT GGC AGC ATA CCT TAC AAC ATA AGC ACA

.1000Asn Ile Ile Arg Lys Ile Val Phe Glu Ser Ser Ala Thr Ile Ser Tyr Leu IleAAT ATA ATT CGA AAA ATT GTT TTT GAA AGT TCA GCC ACA ATA AGT TAT TTA ATA

.MboI .1100Val Glu Tyr Gly Phe Ala Lys Arg Leu Leu Asp Thr Asn Arg Ser Leu Ala LeuGTG GAA TAT GGT TTT GCT AAA AGG TTA TTA GAT ACA AAC AGA TCA CTA GCA TTG

Leu Leu Met Ala Glu Val Asp Ile Ser Ile Leu Ala Lys Ile Pro Arg Tyr TyrCTG TTA ATG GCA GAG GTA GAT ATT TCT ATA TTA GCA AAA ATT CCT AGG TAT TAT

.1200Phe His Pro Lys Pro Lys Val Asp Ser Ala Leu Ile Val Leu Lys Arg Lys ProTTC CAT CCA AAA CCT AAA GTG GAT AGC GCA TTA ATT GTA TTA AAA AGA AAG CCA

Ala Lys Met Ala Phe Lys Glu Arg Lys Lys Tyr Glu Thr Phe Val Met Lys TrpGCA AAA ATG GCA TTT AAA GAG AGA AAA AAA TAT GAA ACT TTT GTA ATG AAA TGGHpaI . .1300 . HindIIIVal Asn Lys Glu Tyr Glu Lys Leu Phe Thr Lys Asn Gln Phe Asn Lys Ala LeuGTT AAC AAA GAG TAT MAA AAA CTG TTT ACA AAA AAT CAA TTT AAT AAA GCT TTA

Lys His Ala Arg Ile Tyr Asp Ile Asn Asn Ile Ser Ph1 Glu Gln Phe Val SerAAA CAT GCG AGA ATA TAT GAT ATA AAC AAT ATT AGT TTC GAA CAA TTT GTA TCG

.1400Leu Phe Asn Sec Tyr Lys Ile Phe Asn Gly END . HaeIII.CTA TTT AAT AGT TAT AAA ATA TTT AAC GGC TAAAACAATAGGCCACATGCAACTGTAAATG

.1500TTTAGTTATAGGTAGGGTAGCATAAGTTAAAATGCTATTCTGCCTTTTAAAAGTATGGTATACATTCCAGA

GAGAGATTGGATATGTTCCCGTATACCGATAGTGCCAAAAGCGAAGTGGATATGTTTCCGAGAACGGACGT

.1600ACTCAAATGACATTCATTCTGTCCTCTCAAGCCATGTTGAGTGTGTGGCATTGCTGGAAAMTGATAAATC

.1700GCAGAAAGCTCGTATAAATCGTTACTGATTTAGCGAGTTTTTTGTTTTTATTCAACTTCCCACAGTCCTTT

TCCACAATGAATCCTCTCAATTCCTTGTTCACAAACCCCATATGAMCGCCATGAACATGTTTCACAMATAG.1800

ATAAAATATCCTTGGGAACGACATATTTTCGGACACGTGTTTCAATATCTCCCCATTTCACAACATCGCCA

.1900 . MboIATTTTAAGTCCCATTTTCTCTAAGGATAGCAGCATCTCTTTCGTAMATATCTTGATC

FIG. 4. Sequence of the pBD370 insert. The sequences of the two MboI fragments of the pBD370 insert are shown, along with thelocations of several restriction sites mentioned in the text. Translations of the two leader peptides and of the ermG transmethylase are shown.The possible positions of the transcriptional start site are indicated by asterisks. The probable locations of the -10 and -35 promotersequences and of the three SD sequences are also marked.

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CLONING AND ANALYSIS OF ermG FROM B. SPHAERICUS

A B C D E298rx

~ 154

-75

FIG. 5. S1 nuclease mapping of the ermG transcript. The EcoRI-AluI DNA fragment described in the text was labeled at the EcoRIterminus and annealed to RNA isolated from a strain carryingpBD370. Lanes A, untreated probe; B, probe annealed to RNA butnot treated with S1 nuclease; C, probe treated with Si nuclease butnot annealed to RNA; D, probe annealed to E. coli tRNA andtreated with S1 nuclease; E, probe annealed to RNA and treatedwith S1 nuclease. The positions of size standards (kilobases; end-labeled Hinfl digest of pBR32; DNA) are indicated.

trations of erythromycin, the probability of both occurringwould equal the product of the individual probabilities, andermG induction would display a quadratic dependence onthe concentration of erythromycin. Figure 2 shows a minicellexperiment in which the induction responses of ermC andermG to various concentrations of erythromycin were deter-mined. Clearly, the response of ermG to low concentrationsof erythromycin was not less than that of ermC, and thequadratic dependence predicted by the cascade model wasnot observed. In fact, ermG seemed to be more responsiveto low concentrations of erythromycin (Fig. 2). It is neces-

sary, therefore, to search for an alternative model for ermGinduction.

It is possible that pairing in the SD2 region is not sufficientto sequester this binding site and th4t peptide 1 is irrelevantto ermG induction. However, the fact that ermA has asimilar organization but no apparent bpase sequence homol-ogy in the peptide 1 region hints at a role for the latter in bothgenes. This fact, plus the apparently greater responsivenessof ermG to low concentrations of erythromycin, suggeststhat the function of peptide 1 translation may be to increasethe sensitivity of the system to inducer by providing analternative device for detecting the presence of antibiotic.Thus, the response of ermG may be determined by the sumrather than the product of the stalling probabilities in pep-tides 1 and 2. As noted above, base pairing in the SD2 regionmay be insufficient to prevent the initiation of translation ofpeptide 2. If a ribosome could initiate at SD2, stalling inpeptide 2 would occur independently of events taking placein peptide 1. Also, a ribosome could sometimes initiate atSD1 and stall in peptide 1 in the presence of erythromycin,freeing the distal half of the peptide 1-coding sequence,permitting a structure to form in which SD3 is exposed, andallowing synthesis of the rRNA transmethylase to proceed(Fig. 6C). Thus, stalling in either peptide 1 or peptide 2would suffice for induction, explaining the apparently greaterresponsiveness of ermG than of ermC.

This hypothesis depends on the assumed ability of aribosome to initiate independently at SD1 and SD2. As notedabove, base pairing of these ribosome-binding sites is notimpressive, and this assumption seems plausible. We haveused the RNA folding program to study the predictedstructures of nascent ermG mRNA. With lengths of about250 bases or less, structures similar to the one shown in Fig.6B are formed. In these structures, SD2 and the associatedAUG initiation codon are completely unpaired, and SD2 isexposed in a loop. Thus, kinetically trapped structures suchas the one shown in Fig. 6B may contribute to the "inde-pendence" of SD2 in the model presented above. Theapparent enhanced responsiveness of ermG compared withthat of ermC may thus be due to the ability of ribosomes toinitiate independently at SD1 and SD2 in mature RNA, innascent RNA, or in both, together with the ability ofribosomes stalled in either peptide 1 or peptide 2 to alter themRNA structure so as to expose SD3.

Basal level of expression. We have suggested elsewherethat the basal level of the ermC methylase is determined bythe existence of a predicted metastable, kinetically trappedform of nascent mR4A that contains an unpaired SD2sequence (12). In fact, the basal level of ermC expression israther high, amounting to approximately 5% of the fullyinduced level. When this basal level is elevated even three-to fourfold, either by an increase in the copy number or bythe introduction of a regulatory mutation, the resulting strainis rendered capable of growth on media containing thenoninducing MLS antibiotic tylosin (33; Villafane andDubnau, unpublished data). We have observed, however,that when ermG is cloned into a derivative of pUB110 (copynumber, about 30) or pIM13 (copy number, 150 to 200), theresulting strain is unable to grow on tylosin-containingmedia. This result suggests that the basal level of ermGexpression is much lower than that of ermC. We haveinvestigated the possible absence of important kineticallytrapped nascent mRNA structures by running the foldingprogram on overlapping RNA segments of increasing length,all beginning with the known initiation site for ermG tran-scription. Although the predicted equilibrium structuresvary with the length ofRNA, no significant structure with anunpaired SD3 is predicted. In other words, the ribosome-binding site for methylase is predicted to pair as soon as it istranscribed. This prediction is in accord with the apparentlylow basal level of ermG expression. Also in accord with thisprediction is the reduced response of ermG to erythromycinconcentrations in excess of 0.02 ,ug/ml, compared with thatof ermC (Fig. 2). This response may be explained by thepresence of premethylated ribosomes in ermC-containingminicells, permitting translation of the rRNA transmethylaseto occur even when most unmethylated ribosomes havebound to erythromycin. Recent in vitro induction experi-ments with ermC and mixtures of methylated and unmethyl-ated ribosomes support this interpretation (C. Narayananand D. Dubnau, submitted for publication).

It appears then, that ermC and ermG may representalternative survival strategies in the face of challenge byinducing MLS antibiotics. In the case of ermC, the highbasal level of expression ensures that some premethylatedribosomes will be present. These will enable induction tooccur even in the presence of a high intracellular concentra-tion of inducer. Ribosomes will be saturated with erythro-mycin and will therefore stall in the leader peptide with a

high probability. However, a small number of methylated(resistant) ribosomes will nevertheless be available to carryout translation from SD2. In the case of ermG, as noted

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346 MONOD FT AL.

G A U

U AU U

G GUAUAAUAUGCUA

AU

AdUA

AUUAUA%GCGCAUAU

UAAUA *** AA AA GA G

A AG GIU AlAAA SD3

CGCGAUAU

C IUAAUUAUA

AUCGUAUA A

AAACAGC A U GAUAGG AAGUA AUAAAA U

A UUCAU-UAUUUU C

UAAUGC A4AAUAUAUAUA

AUACUC

AUUAAUU

UA

UA

GC

AIGCIUA

IAAUUAcoUAUAUAUA

SD2 I GG AIAUIGGA*A

*AU*UAUAAU

Ga AUAAUAUCGGCUArAU

:CMIUACAG aAqmuw -3

A

G U

U A~~u u

UAUAAUAU

AA GCU A UAA U AUA U* AUC A* UAG A* AUU GI UAA GI SD2 UAG Al GCA GI GC

G GI AUA,U . ........AU

AU UAAU

AU A *** AAU A AGC A G

C A GU A AlU G GIAU U AISD3UA- AAAAU I CGIGA CGIGG, AU

AU AUAU C|AU UAUA AUAU UA| A UAI

SD1 C UAGA AUIAU A A CGI

-G- GC A A UACA --G A -IUA U C UA

G GG- GUUAGA AUUAA AU-UUUIG A GCA CC CAAUCU- UAA UA AAAAC AAG UA 5' U UA0

IUACGAU

3' UAAA

U

UAAUUAUAUAUA.

A GA AA UA AU GU

FIG. 6. Predicted secondary structures of the promoter-proximal portions of the ermG transcript. The structures were developed by usingthe program of Zucker ansd Stiegler (35). (A) Predicted minimal-energy structure for the first 281 bases; the predicted value of AG is -89 kcal(ca. 372 kJ). (B) Predicted structure for the first 250 bases; AG = -75 kcal (ca. 314 kJ). (C) Predicted structure if the C terminus of the peptide1-coding sequence is cqnsilered to be unavailable for base pairing because of a ribosome (represented by the large oval) being stalled ir thepresence of erythromycin. Shine-Dalgarno and start codons are indicated by lines; stop codons are indicated by asterisks.

J. BACTERIOL.

A

AGA CG ACG

U ICCAUAUVACGUAIGISD1

AlIUAAUAU

5' AAUGAAUAAAUAUUC

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VOL. 169, 1987 CLONING AND ANALYSIS OF ermG FROM B. SPHAERICUS 347

CAA GAU

C AUGC

AU|AUAUUA

UACUUAUAIcUAUAUA

A IAUCGUA

AG CA C AG A UCG ACG UA

UAA | UACG GAU GAU IGUA UACG IAU*UAI A

G SD1 UGl

UA UA*AU UAAU UA U

5' AAUGAAUAAAUAUUCGAAAAGAGAUGCAAUAAAUUAAGGAGG GGUUAUAAUGAAUU-GU G---L\*-- CCAAUAUUACUUAA UA A

SD2 U G U~~~~~~~~UUAAGGAGAGGUUAUAAUGAA-----3'

SD3

FIG. 6.-Continued

above, the basal level of expression appears to be low. Thus, may permit induction by low erythromycin concentrations,challenge with elevated concentrations of erythromycin is allowing some methylation of rRNA to take place beforepotentially dangerous. Although stalling in the leader pep- erythromycin concentrations rise to a dangerous level. Thetides would occur efficiently, ribosomes would not be avail- effect of enhanced responsiveness would thus be equivalentable to initiate translation from SD3. However, the enhanced to that of an elevated basal level of expression.responsiveness of this system to low levels of erythromycin Comparison of amino acid sequences of erinG and other

Amino acid identities 33023232233333330220232202020203322333233333302333222233333S.aureus ermA MNQKNPKDTQNFITSKKHVKEILNHTNISKQDNVIEIGSGKGHFTKGLVKMSRSVTAIES.aureus ermC MNEKNIKHSQNFITSKHNIDKIMTNIRLNEHDNIFEIGSGKGHFTLELVKRCNFVTAIEB.sphaericus ernmG MNKVNIKDSQNFITSKYHIEKIMNCISLDEKDNIFEIGAGKGHFTAELVKRCNFVTAIE

Ident. 3302330230222000030220203332303322003332323333333232223232303002333332333333ermA IDGGLCQVTKEAVNPSENIKVIQTDILKFsFPKHINyKIyGNIpyNISTDIVKRITFESQAKYSYLIVEKGFAKRLermC IDHKLCKTTENKLVDHDNFQVLNKDILQFKFPKNQSYKIYGNIPYNISTDIIRKIVFDSIANEIYLIVEYGFAKRLermG IDSKLCEVTRNKLLNYPNYQIVNDDILKFTFPSHNPYKIFGSIPYNISTNIIRKIVFESSATISYLIVEYGFAKRL

Ident. 20203232333323233223022320323333232303323032000220320233003323333233022333333ermA QNLQRALELLLMVEMDIKMLKKVPPLYEHPKPSVDSVLIVLERHQPLISKKDYKKYRSFVYKWVNREYRVLFTKNQFermC LNTKRSLALLLMAEVDISILSMVPREYFHPKPKVNSSLIRLSRKKSRISHKDKQKYNYFVMKWVNKEYKKIFTKNQFermG LDTNRSLALLLMAEVDISILAKIPRYYFHPKPKVDSALIVLKRKPAKMAFKERKKYETFVMKWVNKEYEKLFTKNQF

Ident. 20233330202232232333232333332320ermA RQALKHANVTNINKLSKEQFLSIFNSYKLFHermC NNSLKHAGIDDLNNISFEQFLSLFNSYKLFNKermG NKALKHARIYDINNISFEQFVSLFNSYKIFNG

FIG. 7. Comparison of the amino acid sequences of the ermA, ermC, and ermG transmethylases. The predicted amino acid sequences ofthe ermA (18), ermC (9, 14), and ermG transmethylases are aligned, and the number of amino acid identities is noted at each position.

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348 MONOD ET AL.

ermGSD1 Met Asn Lys Tyr Ser Lys Arg Asp Ala

AATTCTAACATCCGAAGCAGGGTTAGAGGAATTA ATG AAT AAA TAT TCG AAA AGA GAT GCA 534

SD1 Met Cys Thr Ser Ile Ala Val Val Glu Ile Thr Leu Ser His Ser ***srmA ACATAAGGAGGTTTCAATT ATG TGC ACC AGT ATC GCA GTA' GTA GAA ATT ACT TTA TCT CAT TCA TAATGAAA 72

Ile Aen ***SD2 Met Gly Leu Tyr Ser Ile Phe Val Ile Glu Thr Val His Tyr Gln ProermG ATA AAT TAAGGAGGTTTTCTA ATG GGT TTA TAC TCA ATT TTT GTA ATA GAA ACA GTT CAT TAT CAA CCA

SD2 Met Gly Thr Phe Ser Ile Phe Val Ile Asn Lys Val Arg Tyr Gln ProermA AAAATGGA-AAGGAGATAAAAGT ATG GGT ACT TTT TCT ATA TTT GTT ATT AAT AAA GTT CGT TAT CAA CCA

SD1 Met Gly Ile Phe Ser Ile Phe Val Ile Ser Thr Val Hia Tyr Gln ProermC AATTTTA-TAAGGAGGAAAAAAT ATG GGC ATT TTT AGT ATT TTT GTA ATC AGC ACA GTT CAT TAT CAA CCA

603

142

265

Asn Glu Lys*rmG AMT GAA AAA

SD3 Met AsnTAAAAGGTTATAATGAATTGTTGATAT ---GAATTCATTATAACCTTTAAGGAG-AGGTTATA

A3n Gln Asn ***ermA AAT CAA AAT TAM

SD3

ATG AAC

Met AsnATG AAC

Asn Lys Lys *** SD2 Met AsnermC AAC AMA AAA TAAGTGGTTATAATGAATCGTTAATGCAAAATTCA-TATAACCAAATTAAAGAGGGTTATA ATG AAC

677

217

336

Lys Vai Asn Ile Lys Asp Ser Gln AsncraG AAA GTA AAT ATA AAA GAT AGT CAA AAT 704

Gln Lys Asn Pro Lys Asp Thr Gln AsnermA CAG AAA AAC CCT AAA GAC ACG CAA AAT 244

Glu Lys Asn Ile Lys His Ser Gln Asn*emC GAG AM AAT ATA AM CAC AGT CAA AAC 369

FIG. 8. Comparison of the ermA, ermC, and ermG regulatory regions. The sequences of the ermA (18), ermC (9, 14), and ermG leadersare aligned. Translations of the leader peptides and starts of the transmethylase translations are shown. The positions of the SD sequencesare shown. Stop codons are indicated by asterisks. The base numbers are from references 9 and 18.

MLS determinants. The amino acid sequence of the ermGgene product is similar to those of the other known MLSdeterminants. Comparisons with the ermC and ermA pro-teins are shown in Fig. 7. These three enzymes are clearlyclosely related. A comparison of the ermG product witheight MLS transmethylases and with the Escherichia coliksgA transmethylase (31) has been carried out (Dubnau andMonod, in press). Similarities are evident with all of theseproteins, and several amino acids are completely conserved.Also striking (Fig. 8) is the relatedness of the 19-amino-acidpeptide specified by the ermG leader region to the 19-amino-acid leader peptides of ermC (15 identities) and ermA (13identities). The close similarity of the ermC peptide topeptide 2 of ermA (13 identities) has been noted previouslyby Murphy (18). We have suggested that the location ofribosome stalling during induction by erythromycin may bedetermined at least in part by the amino acid side chains

TABLE 2. Homology of erm genes

Fraction of identical amino acids"Gene

ermG ermA ermC

ermG 1.00 0.60 0.75ermA 0.73 1.00 0.62ermC 0.70 0.72 1.00

I Numbers printed in boldface type represent results for the trans-methylase-coding sequences ofthe three genes. Numbers printed in italic typerepresent results for the regulatory regions from SD1 (ermC) or SD2 (ermGand ermA) to the starts of the coding regions.

present in the nascent peptides (9). The retention of se-quence similarity in these peptides may reflect the require-ments for precise positioning of the stalling event duringinduction.Comparison of base sequence of ermG and other MLS

determinants. Comparisons of the leaders and a portion ofthe coding sequences of ermG, ermC, and ermA are show inFig. 8. Beginning near the positions of the ermG and ermASD3 sequences (positions 541 and 81, respectively) and theermC SD2 sequence (position 203) and continuing down-stream through the coding regions, the three genes showconsiderable base sequence homology. Further upstream thesequences diverge, and no homology is evident (Fig. 8).Table 2 summarizes these homology relationships in both theleader and coding regions of these three genes. Thereappears to be a tendency toward greater conservation ofhomology in the regulatory regions than in the trans-methylase-coding regions, possibly reflecting restraints be-cause of requirements for a secondary structure togetherwith the need to encode peptides.

Origin of ermG. The ermG determinant has a base com-position of 27% guanine plus cytosine, while that of the B.sphaericus host chromosome is 47%. The latter was deter-mined, with the help of L. Day, from the bouyant density ofa chromosomal DNA sample in CsCl by using a Beckmanmodel E ultracentrifuge. We have argued elsewhere (Dub-nau and Monod, in press) that a marked discrepancy of thisnature can reasonably be interpreted as indicating that ermGis a recent immigrant in B. sphaericus. Its homology to ermCand ermA and the residence of the latter determinants in the

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***

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CLONING AND ANALYSIS OF ermG FROM B. SPHAERICUS

low guanine-plus-cytosine-containing organism S. aureus

support the notion that ermG has migrated horizontally intoB. sphaericus, possibly from S. aureus, and has evolvedfrom a common ancestor of ermA and ermC. Presumably,peptide 2 and its associated regulatory structure were pre-

sent in this ancestral gene. Peptide 1 presents more of a

problem, since it is not obviously similar in ermG and ermA.Either this part of the regulatory sequence has evolved twiceor it was present in the ancestal gene and has diverged.

ACKNOWLEDGMENTS

We thank E. Murphy and C. Denoya for useful discussions and A.Howard for expert secretarial assistance. We gratefully acknowl-edge the assistance of L. Day in determining the buoyant density ofthe B. sphaericus DNA and J. Pollak for providing strains 33 and 27.The computer facilities used in this work were supported by

National Science Foundation equipment grant PCM8313516 andNational Institutes of Health equipment grant RR02990 awarded tothe Public Health Research Institute. This work was supported byPublic Health Service grants A117472 and GM37137, from theNational Institutes of Health. M.M. was supported by a grant fromthe Swiss National Foundation. S.M. is a graduate student in theDepartment of Microbiology at the New York University School ofMedicine and was supported by a National Institutes of Healthtraining grant.

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