regulation of transcription of the mph(a) gene for …(11). thus, the medical use of macrolide...

JOURNAL OF BACTERIOLOGY,0021-9193/00/$04.0010

Sept. 2000, p. 5052–5058 Vol. 182, No. 18

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Regulation of Transcription of the mph(A) Gene for Macrolide29-Phosphotransferase I in Escherichia coli: Characterization

of the Regulatory Gene mphR(A)NORIHISA NOGUCHI,* KATSUTOSHI TAKADA, JIN KATAYAMA, AYAKO EMURA,

AND MASANORI SASATSU

Department of Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Science,1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan

Received 7 February 2000/Accepted 5 July 2000

The synthesis of macrolide 2*-phosphotransferase I [Mph(A)], which inactivates erythromycin, is inducibleby erythromycin. The expression of high-level resistance to erythromycin requires the mph(A) and mrx genes,which encode Mph(A) and an unidentified protein, respectively. We have studied the mphR(A) gene, whichregulates the inducible expression of mph(A). An analysis of the synthesis of Mph(A) in minicells and resultsof a complementation test indicated that mphR(A) is located downstream from mrx and that its product,MphR(A), represses the production of Mph(A). DNA sequencing indicated that the mph(A), mrx, and mphR(A)genes exist as a cluster that begins with mph(A) and that the deduced amino acid sequence of MphR(A) canadopt an a-helix–turn–a-helix structure. To study the regulation of gene expression by MphR(A), we per-formed Northern blotting and primer extension. A transcript of 2.9 kb that corresponded to the transcript ofmph(A) through mphR(A) was detected, and its level was elevated upon exposure of cells to erythromycin. Gelmobility shift assays and DNase I footprinting indicated that MphR(A) binds specifically to the promoterregion of mph(A), and the amount of DNA shifted as a results of the binding of MphR(A) decreased as theconcentration of erythromycin was increased. These results indicate that transcription of the mph(A)-mrx-mphR(A) operon is negatively regulated by the binding of a repressor protein, MphR(A), to the promoter of themph(A) gene and is activated upon inhibition of binding of MphR(A) to the promoter in the presence oferythromycin.

Macrolide antibiotics are active mainly against gram-positivebacteria, but erythromycin (EM) and clarithromycin are activeagainst Helicobacter, Legionella, and Mycoplasma spp. (6, 31).Furthermore, it is becoming clear that some of these antibiot-ics have various other pharmacological activities, for example,as anti-inflammatory agents in addition to antibacterial agents(11). Thus, the medical use of macrolide antibiotics has in-creased significantly in recent years.

Resistance to macrolide antibiotics is usually due to modi-fication of the target site (12, 29), active efflux of the antibiotic(24), or inactivation of the antibiotic (13). However, macrolide-inactivating enzymes, namely, EM esterases (1, 20) and macro-lide 29-phosphotransferases (10, 19), that mediate such resis-tance have been found in clinical isolates of Escherichia coli,which is naturally resistant to macrolides. Furthermore, almostall macrolide-inactivating enzymes are constitutively producedin E. coli (1, 2, 10, 20). However, the production of macrolide29-phosphotransferase I [Mph(A); formerly MPH(29)I] (22),which is a strong inactivator of 14-member ring macrolides,such as EM and oleandomycin (OL), is induced by EM in theoriginal strain E. coli Tf481A (19). Furthermore, although themph(A) gene confers low-level resistance to EM, cells thatcarry the mph(A) and mrx genes come to exhibit high-levelresistance to EM. The deduced product of the mrx gene, Mrx,

is a hydrophobic protein, but its function remains to be deter-mined.

As an inducible macrolide resistance gene, the ermC gene,which encodes an rRNA methylase, is been well known (29).Inducible expression of ermC is regulated at the posttranscrip-tional level by attenuation of translation (30). However, notonly the mechanism of the macrolide resistance conferred byermC but also the structure of the ermC gene is completelydifferent from that of the mph(A) gene.

In the present study of the inducible expression of mph(A),we identified the gene that regulates its expression and char-acterized the gene product. We also developed a model thatexplains the inducible expression of mph(A).

MATERIALS AND METHODS

Bacterial strains, plasmids, and antibiotics. The bacterial strains and plasmidsused in this study are listed in Table 1. Expression of the mph(A) gene wasinduced by treatment with a subinhibitory concentration of EM (25 or 50 mg/ml)(16, 19).

Analysis of protein expression in minicells. Minicells were prepared from E.coli TH1219 that carried the indicated plasmid, and plasmid-encoded proteinswere analyzed as described previously (17). Standard proteins for determinationof molecular weight were purchased from Bio-Rad Laboratories.

Enzymatic inactivation of antibiotics. A crude preparation of enzymes wasused as the source of Mph(A). The preparation of enzymes and the enzymaticinactivation of macrolide antibiotics were performed as described previously(18).

DNA manipulation and sequencing. All DNA manipulations were performedby standard methods (26). DNA amplification by PCR was performed usingGene Taq polymerase (Wako Pure Chemical Industries Ltd., Osaka, Japan).Nucleotide sequences were determined with an automated DNA sequencer(PRIZM 377) and a dye terminator cycle sequencing kit (both from PE AppliedBiosystems, Foster City, Calif.).

Northern blotting and primer extension analysis. RNA was prepared from E.coli as described previously (18). A 508-bp fragment that had been amplified by

* Corresponding author. Mailing address: Department of Microbi-ology, School of Pharmacy, Tokyo University of Pharmacy and LifeScience, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan. Phone:81-426-76-5619. Fax: 81-426-76-5647. E-mail: [email protected].

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PCR with primers mphA1835 (59-GCTCGACTATAGGATCGTGATCGC)and mphA21343 (59-CGTAGAGATCGCCATGCACCAC) was used as theprobe for mph(A) transcripts. The probe was labeled with an AlkPhos Directlabeling kit (Amersham Pharmacia Biotech Inc.) and allowed to hybridize withRNA in accordance with the instructions from the manufacturer of the labelingkit. Primer extension analysis was performed as described previously (18), usingprimer mphA2795 (59-CCATGTCGGGCTGCAAGTGCGTACAGTTGGG),which was end labeled with [g-32P]ATP and T4 polynucleotide kinase.

Construction of a plasmid carrying a fusion gene for MphR(A)-GST andpurification of MphR(A). A DNA fragment containing the mphR(A) structuralgene was amplified by PCR with two primers that included EcoRI and SalIrestriction sites (underlined), namely, mphR11 (59-AAGGTGAGAATTCATGCCCCGCCCCAAGCT) and mphR21 (59-GGACTCTGTCGACCTCCGTTTACGCATGTG), respectively. Plasmid pGEX-mphR(A) was constructed by sub-cloning an EcoRI-SalI mphR(A) fragment from pTZ3509 between the EcoRIand SalI sites of pGEXT4-1. The synthesis of the glutathione S-transferase(GST)-MphR(A) fusion protein and purification of MphR(A) were performedas described by Smith and Johnson (27). The concentration of protein wasestimated by Bradford’s method (3) with bovine serum albumin as the standard.

Gel mobility shift assay. Labeled DNA fragments were prepared by PCR usingprimers that had been end labeled with [g-32P]ATP. The 66-bp fragment desig-nated DNA-1, containing the promoter region of mph(A), was generated withprimers mphA1638 (59-CTGCCTCATCGCTAACTTTG) and mphA2703 (59-CCTAAATGTAACAGTCA). The 77- and 60-bp fragments designated DNA-2and DNA-3 were generated with primers mphA1591 (59-GGTAAGCAGAGTTTTTGAAATGTAAGGCCT) and mphA2667 (59-GGCACTGTTGCAAAGTTA) and primers mphA1696 (59-CATTTAGGTGGCTAAACCC) andmphA2755 (59-CGGTCGTGACTACGGTCATGA), respectively. The 32P-la-beled DNA fragments (approximately 5 3 103 cpm/reaction) were incubatedwith MphR(A) in DNA binding buffer that contained 5 mg of bovine serumalbumin, 1 mg of poly(dI-dC), and 10% glycerol in a total volume 30 ml (7). Aftera 30-min incubation at room temperature, the reaction mixtures were analyzedby 8% polyacrylamide gel electrophoresis in Tris-borate buffer.

DNase I footprinting. DNase I footprinting of the promoter region of mph(A)was performed using a 165-bp DNA fragment that had been amplified by PCRwith primer mphA2755, which had been end labeled with [g-32P]ATP, andprimer mphA1591 (4). Reaction mixtures for analysis of binding containedapproximately 1 pmol of labeled DNA and 10, 25, or 50 pmol of purifiedMphR(A) in 50 ml of a reaction buffer composed of 50 mM Tris-HCl (pH 7.4),

50 mM KCl, 1 mM EDTA, 1 mM 2-mercaptoethanol, 5 mg of bovine serumalbumin, 0.2 mg of poly(dI-dC), and 10% glycerol. Each reaction mixture wasincubated for 30 min at 37°C. Then, 0.5 U of DNase I (Promega) was added tothe reaction mixture and the mixture was incubated for 1 min at room temper-ature. The DNA fragments were analyzed by 8% polyacrylamide gel electro-phoresis sequencing with a G1A sequencing ladder (15).

Nucleotide sequence accession number. The nucleotide sequence reportedhere has been deposited in the DDBJ, EMBL, and GenBank databases underaccession number AB038042.

FIG. 1. Autoradiogram after sodium dodecyl sulfate-polyacrylamide gel elec-trophoresis showing plasmid-encoded proteins labeled with [14C]leucine thatwere produced in minicells. The autoradiograms show radioactive proteins syn-thesized in the absence of EM (2 lanes) and in the presence of EM (1 lanes).The mobilities and molecular weights (in thousands) of standard proteins areindicated on the left. CAT, chloramphenicol acetyltransferase; Mph(A), macro-lide 29-phosphotransferase I.

TABLE 1. Bacterial strains and plasmids used in this study

Strain or plasmid Description [genotype of plasmid insert(s)] Reference or source

E. coli strainsTf481A Emr; clinical isolate 19MV1189 Strain used for complementation and sequencing experiments 26DH5a Strain used for general cloning experiments 26TH1219 Minicell-producing strain 25BL21(DE3) Host strain for expression of pGEX 5

Bacillus subtilis ATCC 6633 Indicator strain used in microbioassaysPlasmids

pUC119 Apr; cloning vector 26pSTV28 Cmr; cloning vector with origin of pACYC184 that is compatible with pUC119 Takaraa

pHSG399 Cmr; pUC19-derived cloning vector in which bla was replaced with cat Takara 28pTZ3509 Apr Emr [mph(A) mrx mphR(A)]; pUC119 with 4.1-kb PstI fragment 16pTZ3519 Apr Emr [mph(A) mrx mphR(A)]; pUC119 with 3.3-kb BamHI-PstI fragment 16pTZ3519ORF3TGA Apr [mrx]; derivative of pTZ3519 carrying mph(A) with nonsense mutation 16pTZ3519ORF4TGA Apr Emr [mph(A)]; derivative of pTZ3519 carrying mrx with nonsense mutation 16pTZ3519D318 Apr Emr [mph(A)]; derivative of pTZ3519 without mrx 16pSTV3519D318 Cmr Emr [mph(A)]; pSTV29 with 2.2-kb PstI-BamHI fragment of pTZ3519D318 This studypTZ3509D361 Apr [mph(A)]; derivative of pTZ3509 without mph(A) and mrx 16pSTV3509D361 Cmr [mphR(A)]; pSTV29 with 1.5-kb PstI-EcoRI fragment of pTZ3509D361 This studypTZ3510 Cmr Emr [mph(A) mrx mphR(A)]; pHSG399 with 4.1-kb PstI fragment of

pTZ3509This study

pTZ3513 Cmr [mrx mphR(A)]; pHSG399 with 4.1-kb pstI fragment of pTZ3509 carryingmph(A) with nonsense mutation

This study

pTZ3514 Cmr Emr [mph(A) mphR(A)]; pHSG399 with 4.1-kb PstI fragment of pTZ3509carrying mrx with nonsense mutation

This study

pTZ3517 Cmr Emr [mph(A) mrx]; pHSG399 with 3.3-kb BamHI-PstI fragment ofpTZ3519

This study

pGEX4T-1 Apr; cloning vector for expression of GST fusion protein Pharmaciab

pGEX4T-mphR(A) Apr [mphR(A)]; pGEX4T-1 with 0.6-kb EcoRI-SalI fragment carrying mphR(A) This study

a Takara Shuzo Co.b Amersham Pharmacia Biotech Inc.

VOL. 182, 2000 TRANSCRIPTIONAL REGULATION OF mph(A) IN E. COLI 5053


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RESULTS AND DISCUSSIONIdentification and nucleotide sequence of the mphR(A) gene.

To confirm the production of Mph(A) directly and to identifythe regulatory gene [mphR(A)] that controls the inducible ex-pression of the mph(A) gene for Mph(A) in minicells, werecloned the inserts in pUC119 into pHSG399, in which theampicillin resistance gene of pUC19 had been replaced with a

chloramphenicol resistance gene, since the electrophoreticmobility of b-lactamase (32 kDa), which was generated fromthe ampicillin resistance gene on the pUC119 vector, was sim-ilar to that of Mph(A) (16). We introduced various derivativesof pHSG399 into minicell-producing E. coli and analyzed theradiolabeled products (Fig. 1). In the case of plasmid pTZ3517,which contained a 3.3-kb PstI-BamHI fragment that included

FIG. 2. Structures of the mph(A), mrx, and mphR(A) genes. The nucleotide sequence from nt 521 to nt 4070 is shown. The 235 and 210 sequences that definethe promoter and ribosome-binding site (RBS), as well as various restriction sites, are underlined. The arrows indicate the positions and directions of primers used inthis study. The initiation transcription site and IS174 are indicated by hooked arrows. The binding site of MphR(A) is shown by a dotted line. The putative a-helicesand turn in MphR(A) are boxed and underlined, respectively.

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the mph(A) and mrx genes, high-level production of Mph(A)was recognized when cells were cultured in the presence and inthe absence of EM (Fig. 1). However, in case of plasmidpTZ3510, which contained a 4.1-kb PstI fragment that con-sisted of 0.8 kb of the BamHI-PstI fragment that included thedownstream region of the mrx gene and the above-mentioned3.3-kb PstI-BamHI fragment, the level of Mph(A) produced inthe absence of EM was lower than that produced in the pres-ence of EM. This result indicated that the regulatory genemphR(A) is located downstream from the mrx gene and thatthe expression of mph(A) is negatively regulated by mphR(A).However, no specific products encoded by mrx and mphR(A),respectively, were detected after autoradiography of the pro-teins produced in minicells. Furthermore, although the prod-uct of the mrx gene is required for expression of the high-levelresistance to EM mediated by Mph(A), the production ofMph(A) in the minicells carrying pTZ3514, which included mrxwith a nonsense mutation, was as enhanced in the presence ofEM as that of the minicells carrying pTZ3510. This resultindicated that mrx is not required for the inducible expressionof mph(A).

To determine whether inducible expression of the mph(A)gene requires the product of mphR(A), we performed acomplementation analysis and assessed the induction of mph(A)by monitoring the inactivation of OL. When pSTV3509D361carrying the mphR(A) gene was introduced into cells that har-bored pTZ3519D318 carrying the mph(A) gene, an extractfrom the cells that had been cultured in the presence of EMinactivated OL more strongly than that from cells cultured inthe absence of EM (data not shown). The complementationassay indicated that inducible expression of the mph(A) generequires the product, MphR(A), of the mphR(A) gene.

We determined the nucleotide sequence of the region (a0.9-kb BamHI-PstI fragment) downstream of the mrx gene,and the sequence, including that of the mph(A) gene, is shownin Fig. 2. The region downstream of the mrx gene contains asingle open reading frame that starts with the ATG initiationcodon at nucleotide (nt) 2877. The open reading frame en-codes a putative protein of 194 amino acids with a molecularweight of 21,627. The amino-terminal region of the putativeprotein includes an a-helix–turn–a-helix structure of the type

conserved in DNA-binding proteins (21). The ATG initiationcodon of the mrx gene overlaps the termination codon ofmph(A) (Fig. 2). Similarly, the initiation codon of mphR(A)overlaps the termination codon of the mrx gene. Therefore, itis clear that mph(A), mrx, and mphR(A) are arranged in closeproximity and form a gene cluster that begins with mph(A).

Repression of transcription of mph(A) by the MphR(A) pro-tein. To determine whether the expression of the mph(A) geneis regulated at the transcriptional level, we analyzed the tran-scription of mph(A) by Northern blotting (Fig. 3). Althoughthere was no difference in the transcript of mph(A) frompTZ3519 in the presence and in the absence of EM, as ob-served in our minicell experiment, the level of the transcriptsfrom pTZ3509 was raised when cells were cultured with EM.Additionally, we found an mRNA of 2.9 kb among transcriptsin cells that harbored pTZ3509 but not in those that harboredpTZ3519. The level of the 2.9-kb mRNA was markedly ele-vated in the presence of EM. Therefore, the expression ofmph(A) was clearly regulated at the transcriptional level.

In order to identify the 59 end of the transcript of mph(A),we performed primer extension analysis (Fig. 4). Total RNAwas isolated from cells that harbored pTZ3509 and had beengrown in the presence of EM. The resulting autoradiogramshowed a single transcription initiation site that corresponds toG, at the RNA level, and is located 31 bp upstream from thetranslation initiation site of mph(A). The result was differentfrom that obtained for the mph(K) gene, which is almost iden-tical to the mph(A) gene (9, 22). Therefore, it appeared thatthe 2.9-kb mRNA was transcribed at least from the initiationsite of the mph(A) gene to the 39 terminus of the mphR(A)gene. Parts of the promoter sequence deduced from the primerextension experiment resembled the consensus sequences forthe so-called 235 and 210 boxes, TTGAat and TAcAtT, re-spectively (where uppercase letters denote identity to the con-sensus sequences) (23). The distance between the putative 210and 235 sequences was 17 nt. The results indicated that mrxand mphR(A) were transcribed from the promoter of themph(A) gene by readthrough transcription. In the Northernblotting experiment with the cells that harbored pTZ3509, we

FIG. 3. Northern blotting analysis of transcripts from E. coli cells harboringpTZ3519 and pTZ3509. Cells were grown in the absence of EM (2 lanes) and inthe presence of EM (1 lanes). The mobilities and sizes (kilobases) of standardsare indicated on the left. Specific transcripts of interest are indicated on the right,along with their lengths in kilobases.

FIG. 4. Mapping of the transcription initiation site of mph(A). Size markers(lanes G, A, T, and C) were generated in sequencing reactions performed withpTZ3519 DNA. The arrow on the right indicates the product of the extensionreaction (PE).



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detected two major mRNA bands of 1.2 and 2.2 kb, whichcorrespond to transcripts of mph(A) and mph(A)-mrx, respec-tively, in addition to an mRNA band of 2.9 kb. We observedthat levels of Mrx and MphR(A) were low in minicells, and nopotential promoter with any homology to the consensus se-quences of promoters in E. coli was found upstream of the mrxand mphR(A) genes. These observations suggest that the 1.2-and 2.2-kb mRNAs were generated during degradation of the2.9-kb transcript in the cells that harbored pTZ3509. Fromthese results, we concluded that this inducible macrolide resis-tance determinant consists of a gene cluster, mph(A)-mrx-mphR(A), designated the mphA operon.

Specific binding of MphR(A) to the promoter region ofmph(A). An analysis of the putative product of the mphR(A)gene revealed that the first 64 amino acids of MphR(A) exhibitstrong homology to proteins in the TetR/AcrR family. Theproteins in this family bind in the vicinity of the promoters oftheir respective target genes (8, 14). To examine whetherMphR(A) is the repressor protein that controls expression ofmph(A), we constructed an mphR(A) expression system andpurified MphR(A). We performed gel mobility shift assaysusing the purified protein and a 66-bp fragment (DNA-1) thatincludes the promoter sequence of mph(A). As shown in Fig.5, the mobility of the DNA-1 fragment was shifted in thepresence of MphR(A). To analyze the binding specificity ofMphR(A) in further detail, we performed a competition ex-periment. The shifted DNA band was almost completely abol-ished when the unlabeled DNA-1 fragment was added as acompetitor to the reaction mixture before addition of the la-beled DNA. By contrast, an excess of individual nonspecificcompetitors that included regions either upstream (DNA-2) ordownstream (DNA-3) from the promoter of mph(A), as well asan excess of poly(dI-dC), had no effect on the binding ofMphR(A) to radiolabeled DNA-1. These results indicated thatMphR(A) bound specifically to the promoter region of mph(A).

Identification of the MphR(A) binding site. DNA-bindingproteins often bind sequences displaying dyad symmetry. How-ever, no repeated sequence was present in the region of the

promoter of the mph(A) gene. We identified the exact locationof the binding site of MphR(A) in a DNase I footprintingexperiment with purified MphR(A) and an end-labeled 165-bpDNA fragment that included the promoter region of mph(A).Relative to the transcription initiation site of mph(A), theregion protected by MphR(A) extended from nt 232 (674 nt)to nt 23 (703 nt) on the coding strand and from nt 237 (669nt) to nt 29 (698 nt) on the noncoding strand (Fig. 6). Thus,the protected region (from nt 237 to nt 23) on the two strandscorresponds exactly to the promoter region of the mph(A)gene. These results indicated that MphR(A) represses the ini-tiation of transcription of the mphA operon by blocking thebinding of RNA polymerase to the promoter of the mph(A)gene.

Effects of macrolides on binding of MphR(A) to the pro-moter. In the presence of a subinhibitory concentration of EM,namely, 25 mg/ml (19), the rate of transcription of mph(A) iselevated. To determine whether macrolides can directly inhibitthe binding of MphR(A) to the promoter of mph(A) and toidentify the kinds of macrolide that can act as inducers, weperformed gel mobility shift assays using a variety of macro-lides at various concentrations. Included were EM and OL asrepresentative 14-member ring macrolides and kitasamycinand josamycin as representative 16-member ring macrolides.Each macrolide in the reaction mixture abolished the bindingof purified MphR(A) to the promoter fragment as the concen-tration of the macrolide was increased. However, the concen-tration at which 14-member ring macrolides inhibited the bind-ing of MphR(A) to the DNA was 100-fold lower than at which16-member ring macrolides inhibited such binding (Fig. 7).

FIG. 5. Gel mobility shift assay with the promoter region of the mph(A)gene. A 66-bp fragment (DNA-1) including the promoter of the mph(A) genewas amplified with end-labeled primers mphA1638 and mphA2667 (see Mate-rials and Methods) and used as the probe. Reaction mixtures contained noMphR(A) protein (lane 1); no competitor (lane 2); unlabeled competitor DNA-1(lane 3); the DNA-2 fragment, amplified with primers mphA1591 andmphA2667 (lane 4); and the DNA-3 fragment, amplified with primersmphA1638 and mphA2703 (lane 5).

FIG. 6. DNase I footprinting analysis of protection by MphR(A) of thepromoter region of mph(A). (A) Footprinting analysis of the coding strand. (B)Footprinting analysis of the noncoding strand. G1A indicates patterns ofMaxam-Gilbert chemical cleavage reactions. Lanes 0 through 50 indicate diges-tion with DNase I of DNA in reaction mixtures that contained increasing con-centrations (in micrograms per milliliter) of DNase I. The thick vertical linesdelineate regions protected by MphR(A). The transcription initiation site andthe promoter region (210 and 235) are also indicated. The values to the rightare lengths in kilobases.



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This result is supported by the profiles of inactivation of mac-rolides by Mph(A) and the pattern of susceptibility to macro-lides of E. coli that carried the mphA operon. Although theinhibition of the binding of MphR(A) to the DNA by EM wasslightly stronger than that of OL, we found no difference in theability to induce mph(A) in the presence of EM and OL bymonitoring the inactivation of OL (data not shown). Theseresults indicated that 14-member ring macrolides are the in-ducers of the mphA operon and directly inhibited the bindingof MphR(A) to the promoter of the mph(A) gene.

A model for regulation of expression of the mphA operon.Based on our results, a model of the inducible expression ofthe macrolide resistance determinant mphA is presented inFig. 8. Thus, the mechanism of inducible expression of themphA determinant is entirely different from that of the typicalmacrolide resistance determinant ermC.

This conclusion predicts that the production of the Mrx andMphR(A) proteins in E. coli with an mphA operon should beenhanced in the presence of EM. Since we failed to detectproteins with molecular weights of approximately 41,000 and22,000 that might have been encoded by mrx and mphR(A),respectively, in minicell experiments (Fig. 1), it was not clearwhether levels of Mrx and MphR(A) might be enhanced in thepresence of EM. However, the increase in the level of the2.9-kb transcript upon addition of EM to the culture mediumindicated that the transcription of the mph(A)-mrx-mphR(A)cluster was regulated by MphR(A). Therefore, it appears thatMphR(A) negatively autoregulates the transcription of its owngene.

ACKNOWLEDGMENTS

We thank T. Horii, Y. Fujii, M. Suzuki, and A. Sato for their skilledtechnical assistance.

This work was supported by a grant for private universities from theMinistry of Education, Science, Sports and Culture, Japan, and by agrant from the Promotion and Mutual Aid Corporation for PrivateSchools of Japan.

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FIG. 7. Gel mobility shift assays showing the binding of MphR(A) to thepromoter region of mph(A) in the presence of various macrolides. Lanes 0through 100 contained increasing concentrations (in micrograms per milliliter) ofmacrolides. LM, kitasamycin; JM, josamycin.

FIG. 8. A model for regulation of the expression of the mph(A) operon in E.coli. P represents the promoter of the mph(A) gene. Induced and uninducedexpression is indicated by dotted and solid lines, respectively. The mph(A) geneencodes the repressor protein MphR(A) and the macrolide resistance determi-nant consisted of the mphA operon, which is made up of the mph(A), mrx, andmph(A) genes. The expression of the mphA operon is negatively regulated at thetranscriptional level by the binding of MphR(A) to the promoter of the mph(A)gene. In the presence of a subinhibitory concentration of a 14-member ringmacrolide, transcription of the mphA operon is activated by blockage of thebinding of MphR(A) to the mph(A) promoter.



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