JOURNAL OF BACTERIOLOGY, Dec. 1991, p. 7416-7422 Vol. 173, No. 230021-9193/91/237416-07$02.00/0Copyright C 1991, American Society for Microbiology

The Staphylococcus aureus mec Determinant Comprises an UnusualCluster of Direct Repeats and Codes for a Gene ProductSimilar to the Escherichia coli sn-Glycerophosphoryl

Diester PhosphodiesteraseCRISTINA RYFFEL,1* ROBERTO BUCHER,2 FRITZ H. KAYSER,1 AND BRIGITTE BERGER BACHI

Institute of Medical Microbiology, University ofZurich, Gloriastrasse 32, Postfach CH 8028, Zurich,1and Scuola Tecnica Superiore, CH-6952 Canobbio, Switzerland

Received 2 August 1991/Accepted 27 September 1991

The DNA sequence located between mecA, the gene that codes for penicillin-binding protein PBP2', andinsertion sequence-like element IS431mec has been termed hypervariable because of its length polymorphismamong different staphylococcal isolates. We sequenced and characterized the hypervariable region of themethicillin resistance determinant (mec) isolated from Staphylococcus aureus BB270. Within the 2,040-bphypervariable region, we identified an unusual accumulation of long direct repeats. Analysis of the DNAsequence revealed a minimal direct repeat unit (dru) of 40 bp which was repeated 10 times within 500 bp. Thedru sequences are responsible for the length polymorphism of mec. Moreover, we identified an open readingframe that codes for 145 amino acids (0RF145), whose deduced amino acid sequence showed 57% amino acidsequence similarity to the N terminus of the glycerophosphoryl diester phosphodiesterase (UgpQ) ofEscherichia coli.

The methicillin resistance (Mc') determinant (mec) isresponsible for the intrinsic resistance of staphylococci topenicillinase-resistant 3-lactam antibiotics (21, 41, 45). Themec DNA is over 30 kb long and integrates into a specific siteof the SmaI G fragment of the Staphylococcus aureus NCTC8325 chromosome (30). Methicillin-susceptible (Mcs) strainslack an allelic site for mec. The mec determinant carries themecA gene, which codes for the low-affinity penicillin-binding protein PBP2' (or PBP2a) (18, 47, 48), the compo-nent required for expression of Mcr. The mecA gene hasbeen cloned and sequenced by different researchers (5, 27,34, 38, 40, 41). Transformation of Mcs staphylococci with aplasmid bearing mecA renders the recipient strain resistantto methicillin (21, 32, 41, 45).On the mec determinant, Barberis-Maino et al. (3) have

identified an insertion sequence-like element, termed IS431(also reported as IS257), which is associated with mecA (3,25, 36). The intervening DNA sequence between IS431mecand mecA of various mec determinants has been claimed tobe hypervariable, because the IS431mec-mecA region showsDNA restriction length polymorphism (11, 14, 22, 45). Vir-tually no other sequences on mec, except mecA andIS431mec, have been determined.

In staphylococci, many resistance determinants, locatedeither on plasmids or on the chromosome, are flanked bysingle or multiple copies of insertion sequence-like elements,and insertion sequences have also been found to flank the tragenes of conjugative plasmids (5, 10, 13, 15). Insertionsequences or transposon attachment sites on the chromo-some may function as target sites for recombination ofdifferent genetic elements such as, e.g., resistance genes,plasmids, bacteriophages, and transposons. Therefore, thepossible involvement of IS431mec in gene transfer is atempting hypothesis. The region comprising mecA and

* Corresponding author.

IS431mec seems indeed to be active in promoting rearrange-ments of mec-associated DNA segments. Trees and landolo(44) have reported the transient mobilization ofmec from thechromosome onto penicillinase plasmid pI524 (44), whileMatthews and Stewart (28) have shown that specific mec-associated sequences have the potential to amplify when thecells are grown under increasing concentrations of methicil-lin. The amplified DNA region involved in these eventscomprised IS431 and its flanking region. Deletions occurringin the mec region involving mec and associated flankingsequences were observed repeatedly (17, 27, 31, 49). Theelimination of different resistance determinants related tomec were found to be bound to loss of Mc'. The site ofexcision was often located within or near IS431 (22, 27, 31,49). All of these phenomena account for the instability orfluctuation of mec-associated sequences and appear to sharea common site, namely, the DNA comprising IS431 and itsflanking DNA regions. To investigate the nature of thisinteresting DNA region and its associated length polymor-phism, we subcloned and sequenced this DNA region of themec determinant isolated from Mcr S. aureus BB270.

MATERIALS AND METHODS

Bacterial strains and plasmids. Mc' S. aureus BB270 (7)was the DNA source for all of the experiments in this study.Mcr S. aureus BB565 was BB270 carrying penicillinaseplasmid pI524 (33), and Mcs S. aureus BB308 is a femAmutant of BB270 (6). The Escherichia coli strains used wereJM109 (50) for the M13-based sequencing strategy and DH5a(Gibco BRL) for pTZ18R and pGEM11 cloning. Plasmid-encoded proteins were expressed in the maxicell system ofE. coli CSR603 (35). E. coli Brz495 (ugpQ plsB plsX), whichdoes not grow on glycerophosphorylcholine as the phospho-lipid source except when complemented by ugpQ+ (9), wasused for complementation analysis with plasmid pBBB56.Plasmid pBBB56 carries a 3.5-kb BglIl insert which starts at

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the end of the mecA gene and spans the hypervariable region(see Fig. 1A) and ends within IS431mec. Plasmid pBBB30carries a 3.6-kb XbaI fragment; this insert starts within themecA gene and spans the hypervariable region up toIS431mec. The cloning vector for pBBB56 and pBBB30waspTZ18R (USB, Cleveland, Ohio). Plasmid pBBB83 was

pGEM11 (Promega, Madison, Wis.) carrying the MaeIII-Hindlll fragment spanning nucleotides (nt) 1000 to 1477 (seeFig. 2A).Growth conditions. All bacterial strains were grown in LB

broth (26) at 37°C. Ampicillin (50 jig/ml) was added whereneeded. For complementation analysis in the E. coli ugpQmutant, minimal medium A (29) containing 0.2% glucose and1 mM glycerophosphorylcholine was used (9).DNA manipulations. All DNA standard manipulations

were done as described by Maniatis et al. (26) or Ausubel etal. (1). Nucleotide sequences were determined by dideoxy-chain termination (36) by using the Sequenase sequencing kit

(USB). Transformation of S. aureus with recombinant plas-mid DNA was done by electroporation by following the

protocol described previously (8).RNA manipulations. For extraction of staphylococcal total

RNA, 8-ml aliquots of exponentially growing cell cultureswere pelleted at 103 rpm for 3 min in a Sorvall SS34 rotor at

4°C. All following manipulations were done rapidly and at

0°C to prevent RNA degradation. The cell pellet was sus-

pended by vortexing in 700 ,ulof RNA extraction buffer (7.2M urea, 1.2% sodium dodecyl sulfate, 35 mM NaCl, 15 mM

EDTA, 10 mM Tris-HCl [pH 7.5] per ml), transferred to a

microcentrifuge tube containing 300 RI of 25:24:1 phenol-chloroform-isoamyl alcohol (1), and quickly frozen in a dryice-ethanol bath. The aliquots were then thawed at 65°C for

5 min, vortexed, and centrifuged at 4°C for 5 min in an SS34

rotor at 18,000 rpm. DNase I digestion and further purifica-tion steps were done as described by Ausubel et al. (1). Gel

electrophoresis, transfer, and hybridization were done as

described by Gilmann and Chamberlin (16), Staeheli et al.

(39), and Church and Gilbert (12), respectively.Computer analysis. The DNA sequences were assembled

with the DNA Inspector Ile program (Textco, Inc., West

Lebanon, N.H.). All further analyses were performed with

the GCG program (University of Wisconsin Genetics Com-

puter Group, Madison). The NBRF, Swissprot, EMBL, and

MIPX protein data banks were searched for amino acid

homologies with ORF145. For the first search, we used the

Wordsearch program.Nucleotide sequence accession number. The nucleotide

sequence of the hypervariable region of S. aureus BB270 has

been submitted to the EMBL/GenBank data base under

accession no. X52594.

RESULTS

Genetic organization of the hypervariable region of S.

aureus. The genetic organization and a restriction map of the

mec-associated DNA containing the hypervariable region of

Mcr S. aureus BB270 is presented in Fig. 1A. To illustrate

the variable section of the DNA, we show a Southern blot

hybridization of different selected DNAs extracted from Mcr

S. aureus clinical isolates collected at the University Hospi-tal of Zurich during a period of over 20 years (Fig. 1B). To

target the sequence of interest, the DNA was digested with

TaqI and hybridized with the 1,475-bp XbaI-HindIII frag-ment (termed the dru probe in Fig. 1A), which covers the

entire putative hypervariable region (Fig. 1A). Restriction

enzyme TaqI cuts six times within the XbaI-HindIII frag-

ment of Mcr S. aureus BB270, giving rise to internal TaqIfragments of 52, 107, 135, 386, and 617 bp. Two additionalfragments of 762 bp and 1.6 kb, located outside the druprobe, are designated as flanking fragments (Fig. 1A). Theflanking 762-bp fragment overlapping the dru probe by only19 bp and the internal 52-bp fragment escaped detectionbecause of experimental limitations. From previous hybrid-ization experiments, we knew that the flanking 1.6-kb frag-ment covering the 3' end of mecA and the flanking sequencesstarting from the XbaI site reaching into the IS431mecsequence (Fig. 1A) were not responsible for the observedlength polymorphism of this DNA region. The Southernblotanalysis shown in Fig. 1B narrowed down the variability toa DNA fragment ranging between 0.8 and 0.54 kb. Theflanking fragments of 386, 135, and 107 bp were the same

sizes in all of the strains analyzed (data not shown). One Mcrstrain apparently had no variable DNA fragment and a bandsmaller than 386 bp (Fig. 1B, lane 7).

Nucleotide sequence. We determined the nucleotide se-

quence of the hypervariable DNA region isolated from McrS. aureus BB270 (Fig. 2A). The 2,040-bp DNA fragmentspanned from the XbaI restriction site located at the 5' endof the published nucleotide sequence of IS431mec in S.aureus (4) up to the published 3' end of the mecA gene (34,38).A self-homology matrix of the S. aureus hypervariable

region (obtained with DNA Inspector Ile; data not shown)revealed several partially overlapping long direct repeats inthe DNA sequence ranging from nt 330 to 820 (Fig. 2A).Beside this particular cluster, we found an open readingframe on the DNA sequence which could code for a proteinof 145 amino acids (ORF145).

Analysis of dru in Mc' S. aureus BB270. The DNA fragmentcontaining the long direct repeat cluster was restrictedwithin 400 bp, ranging from nt 401 to 800 (Fig. 2). Theidentification of the minimal best repeated sequence was

made by the correlation between the 401- to 800-bp segmentand the complete 2,040-bp long DNA sequence. Significantvalues could be seen by shifting a multiple of 40 nt (data not

shown). We concluded that the minimal repeated sequencewas 40 bp long and was directly repeated at least nine timeswithin the hypervariable region. We termed the 40-bp se-

quences the direct repeat unit (dru) and the DNA fragmentcarrying these sequences the dru element. A plot of thecross-correlation for each dru within the 401- to 800-bpsegment was made to identify the best possible dru consen-

sus sequence. By considering homology values greater than60%, we propose for dru the following consensus sequence:CTAAGTAAAATTGCAGATAAGAGGTAAGTTAAAAGCAGTT, whereby we did not define (either theoretically or

experimentally) the precise 5' and 3' ends of the dru consen-

sus sequence, and it should therefore be considered as a

circular string of probable positions for the given nucleotidewithin dru (Fig. 3). The nucleotides of the consensus se-

quence marked in boldface were conserved among all of thedru's studied in this work. Furthermore, we scanned theEMBL data bank for nucleotide sequence similarities to theconsensus sequence of either dru or the dru element itself.However, no similarity to mobile genetic element structuresor to other DNA sequences entries were found.

Analysis of ORF145. From nt 1040 to 1477, we found an

open reading frame that codes for a protein of 145 aminoacids (ORF145). ORF145 is located on the opposite DNAstrand with respect to the transcription direction of the mecAgene and of the putative IS431mec-encoded transposase(Fig. 1A). A potential -10 region and a potential ribosomal

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FIG. 1. (A) Genetic organization of the hypervariable region of Mcr S. aureus BB270. The relative position of the hypervariable region onthe mec determinant of BB270 is presented schematically. The blowup shows the restriction map for HindIlI, TaqI, and XbaI of the region.The dru element is shown by a broken line, and the directions of transcription of mecA, the putative transposase (tnp) encoded by IS431mec,and ORF145 are indicated by arrows. The dru and ORF145 probes used for hybridization are indicated by bars. (B) Identification of dru bySouthern blot. Genomic DNAs of Mcr S. aureus clinical isolates, collected at the University Hospital of Zurich over a period of over 20 years.The DNA was digested with TaqI and hybridized with the 1.5-kb XbaI-HindIII fragment shown in panel A. Control strains: lane 1, Mc' strainBB270; lane 2, its Mcs parent, BB255; lane 3, BB308, an McsfemA mutant derived from BB270. Mcr S. aureus strains were isolated in thefollowing years: lane 4, 1966; lane 5, 1970; lane 6, 1972; lane 7, 1973; lane 8, 1974; lane 9, 1976; lane 10, 1977; lane 11, 1978; lane 12, 1979;lane 13, 1980; lane 14, 1980; lane 15, 1981; lane 16, 1981; lane 17, 1983; lane 18, 1984. The stronger intensity of the 0.54- to 0.8-kb bands isdue to the presence of the dru element. The presence of several bands in lane 10 is due to partial digestion of the DNA. Because the internal52-, 107-, and 135-bp TaqI fragments are not variable (data not shown), they were not included in this figure. The variation of the bandintensities is due to the slightly different DNA contents loaded on the gel.

binding site were identified in the 5' region of ORF145 (Fig.2A), supporting our assumption that ORF145 is expressed inS. aureus.

Total RNAs of different Mcr S. aureus strains wereanalyzed by Northern (RNA) blotting for expression of theORF145 gene. Here we show the result of the analysis oftwoMcr S. aureus strains. The Northern blots were probed byusing a 477-bp MaeIII-HindIII DNA fragment, the ORF145probe (Fig. 1A), which covered ORF145. This experimentshowed that ORF145 codes for a small mRNA of approxi-mately 600 nt (Fig. 4), which would span the length of theopen reading frame. With the ORF145 probe, we alsodetected a second transcript of 1,000 nt. This second bandwas not constantly apparent in the Mcr S. aureus analyzed.

Expression of 0RF145 in E. coli maxicells. Recombinantplasmid pBBB30, carrying the entire hypervariable region,was used to transform E. coli CSR603 cells to analyze theplasmid-encoded proteins (Fig. 5). We detected a proteinwith an apparent molecular mass of 16.5 kDa that corre-sponded well to the predicted size of 16.3 kDa for theORF145 protein.The product of 0RF145 is related to E. coli UgpQ glycero-

phosphoryl diester phosphodiesterase. The deduced aminoacid sequence of ORF145 was used for a homology search ofthe protein data banks. The amino acid sequence alignmentin Fig. 2B showed a significant similarity of 57% (total aminoacid identity of 36%) to UgpQ, a glycerophosphoryl diesterphosphodiesterase, the product of the fifth gene (ugpQ) ofthe inducible sn-glycerol phosphate uptake regulon in E. coli(9, 24, 43). The similarities were located in the N-terminalsequence and covered the putative catalytic domain ofUgpQ(42).

Furthermore, plasmid pBBB56 was able to complementugpQ E. coli mutant Brz495, allowing growth within 48 h onminimal medium when supplemented with glycerophospho-rylcholine as the phospholipid source, whereas the vectorpTZ18R alone did not, suggesting that ORF145 can act as aglycerophosphoryl diester phosphodiesterase in the gram-negative host.

DISCUSSION

In the present work, we characterized the mec-associatedhypervariable region of Mcr S. aureus BB270. We showed

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HYPERVARIABLE REGION ON mec IN S. AUREUS 7419

TCTAGAGAGCAATTATCAGTCGAAGAATACGAACATTCTTAACAGCATTTGATAATCAAGAATTTGATTTCGAACGTGAATTGACACAAGATCCATAT

TCAAAAGTATACTTATACAGTATAGAAGACCATATCAGAACATATAAGATAGAGAAATAAACTAGTGGCCGATTGTGCTTGATGAGCTTGGGACATAAATI R . IR IR

CCTAACTCGAAATAAATAAGCATATCAC!TAAACTGATTTTTTAAAGTTTACGT-GATATG----------TATCTTACeTTTGxACGTGCATGCTTG

-TAGGGGTATOGCTCGAGCCATTAGTCTCTCGCACATACTATTCCCTCAGGCGTCCAGACTTACAAAATCGGTTGTAAT TCATTTTTATACGCATTCT. dru441 . . dru481

TACTGAGATTATACTAATAAGAGGAATAGTAAAAGCAATTCTAAGTAAAATTGCAGATAAGAGGTTTGTTAAAAGCAGTSTCTAAGTAAAATTGCAGATA7A. druS22 . . . . dru562

GAGGTTTGTTAAAAGCAGTCCAGTAAAATTACAGGATAAGAGGTAGCTTAAAAATT=AAGTTTAAAAGCAG* . L*. . dru642 .dru62

TCAGATAAC AATTGCAGATACGAGGTACGTTAAACATTCATGCAAAATTGCTGATAAGGGGTAAGTTAAAAGCAGATCTCAGTAAAATTGCAGA0A.dru722 .. .dru763.

_A__GTAGTAAAGAtTAGGAATGAGAAGAGGTGATCGTTAA-AAG-Ct~-AGAATCTTAGGAGTAACA. dru803.

irCCAAGTAAAATTGCAGATAAGGGGTACAGAAAACTAGACTTGATTACAAAATGGAGCTTGGGAGTCATAAATGATTTTTTAAAAATGAGATGAGACG. . . . . . . . . ~~~~~~~~~~~~~-10

TAGATTAACTCCATAATcAATAcGAATcTATcGAcTTcTTTATTATGATATTcATcTcTTTTTAATGGAAATAAAAGTGcGATTAATGTGATAATACAG

. RBS +1 ORF145 . . .TTACGTTAATTAAAAAAATAAAATGCAAGGAGAGGTAATATGCTAACTGTATATGGACATAGAGGATTACCTAGTAAAGCTCCGGAAAATACAATTGCAT

M L T V Y G H R G L P S K A P E N T I A S

110F1CATAAAGCTGCTTCAGAAGTAGAAGGTATAAACTGGTTGGAGTTAGATGTTGCAATTACAAAAGATGAACAACTGATTATCATTCATGATGATTATTTF K A A S E V E G I N W L E L D V A I T K D E Q L I I I H D D Y L

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E R T T N M S G E I T E L N Y D E I K D A S A G S W F G E K F K D

. . ...... -.GAA,CATTGCCAACTTTCGATGATGTAGTAAAAATAGCAAATGAATATAATATGAATTAAATGTAGAATTAAAAGGTATTACTGGACCGAATGGACTAGE H L P T F D D V V K I A N E Y N M N L N V E L K G I T G P N G L A

CACTTTCTAAAAGTATGGTTAAGCAAGTGGAAGAACAATTAACAAACTTAAATCAGAATCAAGAAGAGCTCATTTAAGCTTTAATGTTGTGCTTGTTAAAL S K S M V K Q V E E Q L T N L N Q N Q E E L I

CTTGCAGAAGAAATCATGCCATAATATAACAGAGCAGTTATATTCCATACAACTTCGTTTCGTGAAGACTGGAGAACACTTTAGATTACTGTAATGCTA

AATAGTAAACACTGAAGATGCCAAACTTACTAAAGCAAAAGTAAAAATGGTAAAAGAAGCGGGTTATGAATTGAACGTATGGACTGTAAACAAACCAGC

ACGTGCAAACCAACTTGCTAATTGGGGAGTTGATGGTATCTTTACAGACAATGcAGATAAAATGGTGcATTTGTCTCAATAGAAAGTTAGAGGTGAGTCT

TACGTTTCAGTGACGGTAGACTTACCTTTAACATGTTACATACTAAAAAATTAATTTGAATAAGAAAGAGAGACATATATGAAATACGATGATTTTATAG

TAGGAGAAACATTCAAACAAAAaGCTTCATATTACAGAAGAAGAAATTATCCAATTTGCAACAACTTTTGATCCTCAATATATGCATATAGATAAAGA

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2001 AAAAGCAGAACAAAGTAGATTTAAAGGTATCATTGcATCT 2040

Borfl4S 1 .... LTVYGHRGLPSKAPENTIASFKAASEVEGINWLELDVAITKDEQLIIIHDDYLERTTNMSGEITELNYDEIKDASAGSWFGEKFKDEHLPTFDD 95

UgpQ 1 MSN9PYPRIVA9HRGGGKLAPENTLASIDVGAKY.GHKMIEFDAKLSKDGEIFLLHDDNLERTSNGWGVAGELNW9DLLRVDAGSWYSKMFKGEPLPLLSQ 99

orfl45 96 VVKIANEYNINLNVELKGITGPNGL ....... ....... ALSKSMVKQVE.EQLTNLNQNQEEL ........ ............................ 144

UgpQ 100 VAERCREHGMANIEIKPTTGTGPLTG!0VALAAELWAGSTPPLLSSFEIDALEAAQQAAPELPRGLLLDEWRDDWRELTARLGCVSIHLNHKLLNKAR 199

FIG. 2. (A) Nucleotide sequence of the 2,040-bp hypervariable region of BB270. The two 16-nt perfect inverted repeats (IR) and the 10dru's analyzed in this report are indicated by arrows and named on the basis of their start points. The G+C-rich region is boxed. The potentialribosomal binding site (RBS) and putative -10 region are indicated. The initiation codon of ORF145 is marked as + 1. The translated productis given below the sequence. (B) Amino acid sequence alignment of ORF145 with ugpQ using the gap program of the University of WisconsinGenetics Computer Group. Gap weight, 3,000; length weight, 0.100. Amino acid identity, 34.965%; amino acid similarity, 55.944%. Aminoacid identities are shown by lines, and conservative amino acid substitutions are shown by dots.

that the DNA length polymorphism of the staphylococcalmec determinant is due to the presence of a very unusualcluster of 10 long direct repeats with a best minimal repeatedsequence of40 nt. We termed this novel structure on the mecdeterminant of S. aureus dru and the DNA sequence inwhich the dru's are located the dru element. We calculated aconsensus sequence on the basis of the best probability thata certain nucleotide would occur at the same position withinthe different dru's. We did not determine the 5' and 3' endsof dru, and it is to be handled as a circular sequence. Thedru's are contiguous, with exception of a 1-nt shift betweendru481 to dru522 and dru722 to dru763. dru441, dru481, anddru562 are identical and, like dru682, have 95% homology tothe calculated consensus sequence. As for other repeatedsequences in prokaryotes (19), the function and perpetuationof these elements remain to be established. An additionalintriguing observation of the hypervariable region was madeby analyzing its G+C content. As shown in Fig. 3A, wefound a DNA fragment with an unusual base composition 5'of the dru element (from nt 280 to 380), namely, a high G+Ccontent of 47%. This value is considerably higher than the

mean for a fragment of staphylococcal DNA of the samelength (approximately 31%). This G+C-rich fragment waspreceded by a long perfect inverted repeat of 16 nt.Whether the dru element is functionally connected to

IS431mec or is a residual of multiple imprecise insertion andexcision events of other mobile DNA elements is not known.The search for similarities in the GenBank-EMBL DNASequence Data Library revealed that the dru's and the druelement are not related to any known sequences. The druelement is an intriguing feature of mec DNA, and its biolog-ical function, its impact on Mcr regulation or virulence, andits role in the evolution of multiple drug resistance in Mcrstaphylococci remain to be investigated. The hypervariableregion seemed to have no observable function or to affectdirectly the expression of Mcr, since the cloned 4-kb HindIIIfragment, isolated from two different sources, which carriedonly mecA but not ORF145 or dru, was able to confer Mcr tosusceptible S. aureus strains (32, 45). Interestingly, a regionof length polymorphism associated with a cluster of directrepeats and an insertion sequence has been recently de-scribed for Mycobacterium tuberculosis complex strains

AGAACGGACTACAAATATGTCCGGGGAAATAACTGAATTGAATTATGATGAAATTAAAGATGCTTCTGCAGGATCTTGGTTTGGTGAAAAATTCAAAGAT

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dru441 CTAAGTAA&ATTGCAGLTA&QAGGTTTGTTAAA&GCAGTTdru481 CTAAGTAAAATTGCAG&TAAGAGGTTTGTTAAAAGCAGTTdru522 TCAGTAAAATTACAGGA?A&GAGGTAGCTTAAAAGCAGTTdru562 CTAAGTAAAATTGCAGATAAGAGT TGTTAAA&GCAGTTdru602 CTCAGTAA&ATTGCAG&TA&GAGGTACGTTAAAAGCAATTdrl642 CCATGCAAAATTGCTGQTA&GQGGTAAGTTAAA&GCAGTTdrLu82 CTCAGTAAMATTGCAG TAAGAGGTACGTTAAAAGCAGTTdru722 CTAGGCAALATTGCAGLYA&QAGG GATCGTTA&AAGCAGdru763 TCTCAGTA&AATTCTGATA¢GGGGTAAGTTAAAAGCAATCdru803 CTAAGTAAAATTGCAGATAAGGGGTACAGAAAAACTAGAC

dru-consensus: CTAAGTAAAATTGCAGATAAGAGGTAAGTTAAAAGCAGTTFIG. 3. Alignment of 10 dru sequences. The sequences of 10

dru's were aligned and named on the basis of their start points(compare Fig. 2A). These dru sequences were identified by search-ing the 2,040-bp sequence with the calculated dru consensus and byusing a stringency of 60%. The possible dru consensus sequence wasdetermined by calculating the probability that a nucleotide would beat the same position within the 40-bp dru sequences. The calculatedprobabilities were C, 87%; T, 82%; A, 56%; A, 70%; G, 87%; T,64%; A, 88%; A, 95%; A, 96%; A, 93%; T, 90%; T, 93%;G, 71%; C,85%; A, 77%; G, 87%; A, 87%; T, 88%; A, 88%; A, 100%; G, 89%;A,72%;G,84%;G,94%;T,82%;A,48%;T,40%;G,81%;T,83%;T, 97%; A, 92%; A, 98%; A, 95%; A, 100%; G, 93%; C, 88%; A,93%; G, 92%; T, 94%; and T, 94%. We did not define the precise 5'and 3' ends of the dru consensus sequence, and it should thereforebe considered as a circular string of probable positions for the givennucleotides.

(19). Hermans et al. have shown further that this region is ahot spot for integration of insertion elements (IS987) in thechromosome (19). These results corroborate the possiblerole of the dru's as a hot spot for attracting IS elements andresistance determinants in staphylococci.

Additionally, we identified ORF145, which codes for aprotein with an amino acid sequence comparable to that ofthe glycerophosphoryl diester phosphodiesterase (UgpQ) ofE. coli (9, 24, 43). We found that transcription of ORF145in coagulase-negative and -positive Mcr staphylococci wasconstitutive. Furthermore, by Northern blot analysis usingthe ORF145-specific probe, we detected two differentmRNAs. One, 600 nt long, had approximately the sizepredicted for ORF145. The longer mRNA was not alwaysdetectable, and the hybridization signal was variable amongthe various RNA preparations and strains. The role and

FIG. 4. Northern blot analysis of total RNA of Mcr S. aureusBB270 and BB565 to detect transcripts encoded by ORF145. TotalRNA was prepared from culture samples taken at different timesafter induction with 0.5 ,ug of methicillin per ml. After blotting, theRNA was probed with the insert of recombinant plasmid pBBB83,which carries ORF145. Mcr S. aureus BB270 was in lanes a to c, andMcr S. aureus BB565 was in lanes d to f. Lanes: a, 35 minuninduced; b, 35 min induced; c, 70 min induced; d, 35 minuninduced; e, 35 min induced; f, 70 min induced.

FIG. 5. Detection of the ORF145-encoded product. Proteinsencoded by pBBB30, the plasmid carrying the hypervariable regionand its flanking sequences, were expressed in E. coli CSR603.Lanes: a, CSR603 transformed with pBBB30; b, as a control, strainCSR603 transformed with cloning vector pTZ18R. The only pre-dominant band detectable was a 16.5-kDa protein.

nature of this 1-kb transcript are currently being investi-gated. The contribution of ORF145 to Mcr expression instaphylococci remains to be elucidated. It would be interest-ing to know whether ORF145 has a biologically activefunction in Mcr staphylococci and whether it can interactwith native staphylococcal phosphate metabolism.The nature of the staphylococcal mec determinant has yet

to be determined. It may be that the mec determinantcontains genes or gene fragments of chromosomal originfrom other species. Furthermore, the overall organization ofgene clusters of different species seems to be conservedwithin smaller or larger sections of the chromosome. In E.coli, the ugp operon (min 76) maps close to genes for cellwall-metabolizing enzymes, a striking similarity to the orga-nization of mec, where PBP2' is located next to ORF145, augpQ-related enzyme (2, 9). One could then speculate that asection(s) of the mec determinant might be of chromosomalorigin and has been truncated and mobilized, presumably bythe dru element or IS431.

ACKNOWLEDGMENTSWe thank W. Boos for sending us strain Brz495, J. Tommassen

for discussion, and K. Dyke for the very helpful critique. We alsothank G. Martinetti for critical reading of the manuscript and AnniStrassle for technical assistance.

This study was supported by Swiss National Science Foundationgrant 32-26278.89.

REFERENCES1. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Mooreet, J. G.

Seidman, J. A. Smith, and J. G. Struhl. 1987. Current protocolsin molecular biology. Greene Publishing Associates and Wiley-Interscience Press, New York.

2. Bachmann, B. J. 1990. Linkage map of Escherichia coli K-12,edition 8. Microbiol. Rev. 54:130-197.

3. Barberis-Maino, L., B. Berger-Bachi, H. Weber, W. D. Beck,

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and F. H. Kayser. 1987. IS431, a staphylococcal insertionsequence-like element related to IS26 from Proteus vulgaris.Gene 59:107-113.

4. Barberis-Maino, L., C. Ryffel, F. H. Kayser, and B. Berger-Bachi. 1990. Complete nucleotide sequence of IS431mec inStaphylococcus aureus. Nucleic Acids Res. 18:5548.

5. Beck, W. D., B. Berger-Bachi, and F. H. Kayser. 1986. Addi-tional DNA in methicillin-resistant Staphylococcus aureus andmolecular cloning of mec-specific DNA. J. Bacteriol. 165:373-378.

6. Berger-Bachi, B., L. Barberis-Maino, A. Strassle, and F. H.Kayser. 1989. FemA, a host mediated factor essential formethicillin resistance in Staphylococcus aureus: molecularcloning and characterization. Mol. Gen. Genet. 219:263-269.

7. Berger-Bachi, B., and M. L. Kohler. 1983. A novel site on thechromosome of Staphylococcus aureus influencing the level ofmethicillin resistance: genetic mapping. FEMS Microbiol. Lett.20:305-309.

8. Berger-Bachi, B., A. Striassle, and F. H. Kayser. 1989. Naturalmethicillin resistance in comparison with that selected by in-vitro drug exposure in Staphylococcus aureus. J. Antimicrob.Chemother. 23:179-188.

9. Brzoska, P., and W. Boos. 1988. Characteristics of a ugp-encoded and phoB-dependent glycerophosphoryl diester phos-phodiesterase which is physically dependent on the Ugp trans-port system of Escherichia coli. J. Bacteriol. 170:4125-4135.

10. Byrne, M. E., T. G. Littlejohn, and R. A. Skurray. 1990.Transposons and insertion sequences in the evolution of multi-resistant Staphylococcus aureus, p. 165-174. In R. P. Novick(ed.), Molecular biology of the staphylococci. VCH PublishersInc., New York.

11. Chambers, H. F., G. Archer, and M. Matsuhashi. 1989. Low-level methicillin resistance in strains of Staphylococcus aureus.Antimicrob. Agents Chemother. 33:424-428.

12. Church, G. M., and W. Gilbert. 1984. Genomic sequencing.Proc. Natl. Acad. Sci. USA 81:1991-1995.

13. ElSolh, N., J. Alliquet, V. G. Loucle, and P. Mazodier. 1990.Nucleotide sequence of a staphylococcal plasmid gene, vgb,encoding a hydrolase that inactivates the B components ofvirginamycin-like antibiotics, p. 617-622. In R. P. Novick (ed.),Molecular biology of the staphylococci. VCH Publishers Inc.,New York.

14. Galetto, D., J. Frogatt, J. Kornblum, and G. Archer. 1988.Analysis of methicillin resistance genes in staphylococci usingDNA probes, abstr. A-59, p. 10. Abstr. 88th Annu. Meet. Am.Soc. Microbiol. 1988. American Society for Microbiology,Washington, D.C.

15. Gillespie, M. T., B. R. Lyon, L. S. L. Loo, P. R. Matthews, P. R.Stewart, and R. A. Skurray. 1987. Homologous direct repeatsequences associated with mercury, methicillin, tetracyclineand trimethoprim resistance determinants in Staphylococcusaureus. FEMS Microbiol. Lett. 43:165-171.

16. Gilmann, M. Z., and M. J. Chamberlin. 1983. Developmentaland genetic regulation of Bacillus subtilis genes transcribed bysigma-28-RNA polymerase. Cell 35:285-293.

17. Grubb, W. B., and D. I. Annear. 1972. Spontaneous loss ofmethicillin resistance in Staphylococcus aureus at room-tem-perature. Lancet ii:1257.

18. Hartman, B. J., and A. Tomasz. 1984. Low-affinity penicillin-binding protein associated with ,-lactam resistance in Staphy-lococcus aureus. J. Bacteriol. 158:513-516.

19. Hermans, P. W. M., D. van Soolingen, E. M. Bik, P. E. W. deHaas, J. W. Dale, and J. D. A. van Embden. 1991. Insertionelement IS987 from Mycobacterium bovis BCG is located in ahot-spot integration region for insertion elements in Mycobac-terium tubercolosis complex strains. Infect. Immun. 59:2695-2705.

20. Hulton, C. S. J., C. F. Higgins, and P. M. Sharp. 1991. ERICsequences: a novel family of repetitive elements in the genomesof Escherichia coli, Salmonella typhimurium and other entero-bacteria. Mol. Microbiol. 5:825-834.

21. Inglis, B., P. R. Matthews, and P. R. Stewart. 1988. Theexpression in Staphylococcus aureus of cloned DNA encoding

methicillin resistance. J. Gen. Microbiol. 134:1465-1469.22. Inglis, B., P. R. Matthews, and P. R. Stewart. 1990. Induced

deletions within a cluster of resistance genes in the mec regionof the chromosome of Staphylococcus aureus. J. Gen. Micro-biol. 136:2231-2239.

23. Kanda, K., E. Suzuki, K. Hiramatsu, T. Oguri, H. Miura, T.Ezaki, and T. Yokota. 1991. Identification of a methicillin-resistant strain of Staphylococcus caprae from a human clinicalspecimen. Antimicrob. Agents Chemother. 35:174-176.

24. Kasahara, M., K. Makino, M. Amemura, and A. Nakata. 1989.Nucleotide sequence of the ugpQ gene encoding glycerophos-phoryl diester phosphodiesterase of Escherichia coli K-12.Nucleic Acids Res. 17:2854.

25. Lyon, B. R., and R. A. Skurray. 1987. Antimicrobial resistanceof Staphylococcus aureus: genetic basis. Microbiol. Rev. 51:88-134.

26. Maniatis, T., E. F. Fritsch, and J. E. Sambrook. 1982. Molecularcloning: a laboratory manual. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.

27. Matthews, P. R., K. Reed, and P. R. Stewart. 1987. The cloningof chromosomal DNA associated with methicillin and otherresistances in Staphylococcus aureus. J. Gen. Microbiol. 133:1919-1929.

28. Matthews, P. R., and P. R. Stewart. 1988. Amplification of asection of chromosomal DNA in methicillin-resistant Staphylo-coccus aureus following growth in high concentrations of me-thicillin. J. Gen. Microbiol. 134:1455-1464.

29. Miller, J. H. 1972. Experiments in molecular genetics. ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.

30. Patel, A. H., T. J. Foster, and P. A. Pattee. 1989. Physical andgenetic mapping of the protein A gene in the chromosome ofStaphylococcus aureus 8325-4. J. Gen. Microbiol. 135:1799-1807.

31. Poston, S. M., and F.-L. LiSawHee. 1991. Genetic characteriza-tion of resistance to metal ions in methicillin-resistant Staphy-lococcus aureus: elimination of resistance to cadmium, mercuryand tetracycline with loss of methicillin resistance. J. Med.Microbiol. 34:193-201.

32. Ryffel, C. Unpublished data.33. Ryffel, C., F. H. Kayser, and B. Berger-Bachi. Submitted for

publication.34. Ryffel, C., W. Tesch, I. Birch-Machin, P. E. Reynolds, L.

Barberis-Maino, F. H. Kayser, and B. Berger-Bachi. 1990.Sequence comparison of mecA genes isolated from methicillin-resistant Staphylococcus aureus and Staphylococcus epidermi-dis. Gene 94:137-138.

35. Sancar, A., A. M. Hack, and W. D. Rupp. 1979. Simple methodfor the identification of plasmid-coded proteins. J. Bacteriol.137:692-693.

36. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequenc-ing with chain-terminating inhibitors. Proc. Nati. Acad. Sci.USA 74:5463-5467.

37. Skinner, S., B. Inglis, P. R. Matthews, and P. R. Stewart. 1988.Mercury and tetracycline resistance genes and flanking repeatsassociated with methicillin resistance on the chromosome ofStaphylococcus aureus. Mol. Microbiol. 2:289-297.

38. Song, M. D., M. Wachi, M. Doi, F. Ishino, and M. Matsuhashi.1987. Evolution of an inducible penicillin-target protein inmethicillin-resistant Staphylococcus aureus by gene fusion.FEBS Lett. 221:167-171.

39. Staeheli, P., P. Danielson, 0. Haller, and J. G. Sutcliffe. 1986.Transcriptional activation of mouse Mx gene by type I inter-feron. Mol. Cell. Biol. 6:4770-4774.

40. Tesch, W., C. Ryffel, A. Strassle, F. H. Kayser, and B. Berger-Bachi. 1990. Evidence of a novel staphylococcal mec-encodedelement (mecR) controlling expression of penicillin-binding pro-tein PBP2'. Antimicrob. Agents. Chemother. 34:1703-1706.

41. Tesch, W., A. Strassle, B. Berger-Bachi, D. O'Hara, P. Rey-nolds, and F. H. Kayser. 1988. Cloning and expression ofmethicillin resistance from Staphylococcus epidermidis inStaphylococcus carnosus. Antimicrob. Agents Chemother. 32:1494-1499.

on May 18, 2016 by guest

http://jb.asm.org/

Dow

nloaded from

7422 RYFFEL ET AL. J. BACTERIOL.

42. Tommassen, J. K. (State University of Utrecht). Personal com-mhinication.

43. Tommassen, J., K. Eiglmeier, S. T. Cole, P. Overduin, T. J.Larson, and W. Boos. 1991. Characterization of two genes, glpQand ugpQ, encoding glycerophosphoryl diester phosphodi-esterases of Escherichia coli. Mol. Gen. Genet. 226:321-327.

44. Trees, D. L., and J. J. landolo. 1988. Identification of aStaphylococcus aureus transposon (Tn4291) that carries themethicillin resistance gene(s). J. Bacteriol. 170:149-154.

45. Ulbukata, K., R. Nonoguchi, M. Matsuhashi, and M. Konno.1989. Expression and inducibility in Staphylococcus aureus ofthe mecA gene, which encodes a methicillin-resistant S. aureus-specific penicillin-binding protein. J. Bacteriol. 171:2882-2885.

46. Ubukata, K., R. Nonoguchi, M. Matsuhashi, M. D. Song, and M.Konno. 1989. Restriction maps of the regions coding for methi-cillin and tobramycin resistances on chromosomal DNA inmethicillin-resistant Staphylococcus aureus. J. Antimicrob.

Agents Chemother. 33:1624-1626.47. Ubukata, K., N. Yamashita, and M. Konno. 1985. Occurrence of

a ,-lactam-inducible penicillin-binding protein in methicillin-resistant staphylococci. Antimicrob. Agents Chemother. 28:397-403.

48. Utsui, Y., and T. Yokota. 1985. Role of an altered penicillin-binding protein in methicillin- and cephem-resistant Staphylo-coccus aureus. Antimicrob. Agents Chemother. 28:397-403.

49. Wada, A., Y. Katayama, K. Hiramatsu, and T. Yokota. 1991.Southern hybridization analysis of the mecA deletion frommethicillin-resistant Staphylococcus aureus. Biochem. Bio-phys. Res. Commun. 176:1319-1325.

50. Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. ImprovedM13 phage cloning vector and host strains: nucleotide se-quences of the M13mpl8 and M13mpl9 vectors. Gene 33:103-119.

on May 18, 2016 by guest

http://jb.asm.org/

Dow

nloaded from