JOURNAL OF BACTERIOLOGY, Mar. 2004, p. 1705–1713 Vol. 186, No. 60021-9193/04/$08.00�0 DOI: 10.1128/JB.186.6.1705–1713.2004Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Role of murE in the Expression of �-Lactam Antibiotic Resistance inStaphylococcus aureus

S. Gardete,1,2 A. M. Ludovice,1 R. G. Sobral,1 S. R. Filipe,2 H. de Lencastre,1,2 and A. Tomasz2*Molecular Genetics Laboratory, Instituto de Tecnologia Química e Biologica da Universidade Nova de Lisboa, 2780 Oeiras,

Portugal,1 and Laboratory of Microbiology, The Rockefeller University, New York, New York 100212

Received 22 October 2003/Accepted 9 December 2003

It was shown earlier that Tn551 inserted into the C-terminal region of murE of parental methicillin-resistantStaphylococcus aureus strain COL causes a drastic reduction in methicillin resistance, accompanied by accu-mulation of UDP-MurNAc dipeptide in the cell wall precursor pool and incorporation of these abnormalmuropeptides into the peptidoglycan of the mutant. Methicillin resistance was recovered in a suppressormutant. The murE gene of the same strain was then put under the control of the isopropyl-�-D-thiogalacto-pyranoside (IPTG)-inducible promoter Pspac. Bacteria grown in the presence of suboptimal concentrations ofIPTG accumulated UDP-MurNAc dipeptide in the cell wall precursor pool. Both growth rates and methicillinresistance levels (but not resistance to other antibiotics) were a function of the IPTG concentration. Northernanalysis showed a gradual increase in the transcription of murE and also in the transcription of pbpB andmecA, parallel with the increasing concentrations of IPTG in the medium. A similar increase in the transcrip-tion of pbpB and mecA, the structural genes of penicillin-binding protein 2 (PBP2) and PBP2A, was alsodetected in the suppressor mutant. The expression of these two proteins, which are known to play critical rolesin the mechanism of staphylococcal methicillin resistance, appears to be—directly or indirectly—under thecontrol of the murE gene. Our data suggest that the drastic reduction of the methicillin MIC seen in the murEmutant may be caused by the insufficient cellular amounts of these two PBPs.

Staphylococcus aureus mutant strain RUSA235 was origi-nally isolated as a member of the large library of Tn551 inser-tional mutants of methicillin-resistant S. aureus (MRSA) strainCOL in which the high and homogeneous level of methicillinresistance was reduced (6). The number of these determinantswith the Tn551 inserts, initially termed fem (factors essentialfor methicillin resistance) or aux (auxiliary) genes, has nowincreased to more than 20 (1, 2, 6, 7). Several of the auxiliarygenes are involved in peptidoglycan biosynthesis or cell wallturnover, some appear to have putative regulatory functions,and others encode proteins with functions as yet unidentified(7). With the possible exception of pbpB, the structural gene ofpenicillin-binding protein 2 (PBP2) (20), it is not clear howthese genes cooperate with the methicillin resistance genemecA (9, 30) in promoting high-level and homogeneous resis-tance to �-lactam antibiotics.

Genetic analysis of mutant RUSA235 identified the target ofTn551 as murE (15), an essential gene of S. aureus (12), en-coding the UDP–N-acetylmuramyl tripeptide synthetase thatcatalyzes the addition of the L-lysine residue to the UDP-linked muramyl dipeptide cell wall precursor. In mutantRUSA235, the insert was 3 bp upstream of the terminationcodon, allowing production of a modified MurE protein withreduced specific activity (15). Biochemical analysis ofRUSA235 demonstrated the accumulation of UDP-MurNAcdipeptide in the cytoplasmic cell wall precursor pool and in-corporation of the dipeptide into the peptidoglycan of themutant (16).

The initial purpose of the studies described in this commu-nication was to examine the possibility that the hypersensitivityof RUSA235 to �-lactam antibiotics is related to some struc-tural or functional defect in the cell wall containing the abnor-mal dipeptide components. Subsequently, the construction of aconditional murE mutant has allowed us to probe in moredetail the role of MurE in cell wall synthesis and drug resis-tance.

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions. The bacterial strains andplasmids used in this study are described in Table 1. S. aureus strains were grownin tryptic soy broth (TSB; Difco Laboratories, Detroit, Mich.) with aeration at37°C or on tryptic soy agar (TSA; Difco Laboratories) plates at 37°C. Escherichiacoli strains were grown in Luria-Bertani broth (Difco Laboratories) with aerationat 37°C. Erythromycin (10 �g/ml), chloramphenicol (10 �g/ml), and ampicillin(100 �g/ml) were used as recommended by the manufacturer (Sigma, St. Louis,Mo.) for selection and maintenance of S. aureus and E. coli transformants,respectively.

To measure the growth rate of COLspac::murE, an overnight culture was grownin TSB containing erythromycin (10 �g/ml), chloramphenicol (10 �g/ml), and100 �M isopropyl-�-D-thiogalactopyranoside (IPTG). This starting culture waswashed and resuspended in TSB without IPTG to remove traces of the inducerbefore the culture was diluted to an optical density at 620 nm (OD620) of 0.02 inTSB containing 10 �g of chloramphenicol per ml and supplemented with in-creasing concentrations of IPTG (0, 25, 37.5, 75, and 200 �M). Each culture wasincubated with agitation at 37°C, and the OD620 was monitored.

DNA methods. DNA manipulations were performed by standard methods(24). Restriction enzymes were used as recommended by the manufacturer (NewEngland Biolabs, Beverly, Mass.). Routine PCR amplification was performedwith Tth DNA polymerase (HT Biotechnology, Cambridge, United Kingdom).Wizard Plus Minipreps and Midipreps (Promega, Madison, Wis.) purificationsystems were used for plasmid extraction. PCR and digestion products werepurified with Wizard PCR Preps and Wizard DNA Clean-up systems (Promega).Ligation reactions were performed with T4 ligase (New England Biolabs). DNAsequencing was done at the Rockefeller University Protein/DNA TechnologyCenter by the BigDye terminator cycle sequencing method with either a 3700

* Corresponding author. Mailing address: The Rockefeller Univer-sity, Laboratory of Microbiology, 1230 York Ave., New York, NY10021. Phone: (212) 327-8278. Fax: (212) 327-8688. E-mail: tomasz@mail.rockefeller.edu.

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DNA analyzer for capillary electrophoresis or ABI Prism 377 DNA sequencersfor slab gel electrophoresis.

Determination of antibiotic resistance by population analysis. Overnight cul-tures were plated at various dilutions on TSA plates containing increasing con-centrations of the various antibiotics, and bacterial colonies were counted afterincubation of the plates at 37°C for 48 h as previously described (5). Oxacillin,bacitracin, and cefotaxime were purchased from Sigma. Moenomycin was ob-tained through the courtesy of Aventis Pharma D, DI&A Natural Products(Bridgewater, N.J.).

Analysis of the UDP-linked precursor pool. The UDP-linked cytoplasmicpeptidoglycan precursor pool was extracted by a previously described procedure(16), except that the precursors were separated on a Hypersyl (Runcor Cheshire,United Kingdom) reverse-phase high-performance liquid chromatography(HPLC) octyldecyl silane column (3-�m particle size; 250 by 4.6 mm; 120-A poresize) that was eluted with a linear 5 to 30% methanol gradient in 100 mM sodiumphosphate buffer, pH 2.5, at a flow rate of 0.5 ml/min and assayed for absorbanceat 254 nm.

Cell wall analysis. Cell walls were isolated, the peptidoglycan was purified andhydrolyzed with the M1 muramidase, and the resulting muropeptides were re-duced with borohydride and separated by reverse-phase HPLC as previouslydescribed (3).

Autolytic enzyme extract. Crude autolytic extract was prepared by a methodsimilar to that described previously (29). Strain COL was grown to mid-expo-nential phase in 250 ml of TSB at 37°C with aeration, chilled rapidly, harvestedby centrifugation, washed once in ice-cold 50 mM Tris-HCl (pH 7.5), and ex-tracted with 250 ml of 4% sodium dodecyl sulfate at 4°C for 30 min with stirring.The supernatant was used as the source of autolytic enzymes.

Cell wall hydrolysis in vitro. Purified cell walls were suspended in buffer (50mM Tris-HCl, pH 7.5) to an initial OD620 of 0.5. Lysis was measured as adecrease in OD620 during incubation of wall samples at 37°C with crude lyticenzyme extract (10 mg of protein/ml).

Construction of plasmid pSGII. A DNA fragment containing the ribosome-binding site and the first 311 codons of the murE gene was amplified by PCRwith PfuTurbo DNA polymerase (Stratagene, Heidelberg, Germany) and prim-ers murEspacIA (5�-TAAGATCTACACCGCAATCATTGCCGCC-3�) andmurEspacII (5�-ATTCCCGGGTTGTAGAAAAAGGAGCGGTTCAG-3�).The primers were engineered to carry BglII (murEspacIA) and SmaI(murEspacII) restriction sites (underlined in the primer sequences). Thefollowing PCR conditions were used: 94°C for 4 min; 40 cycles of 94°C for 45s,55°C for 45 s, and 72°C for 1 min; and one final extension step of 72°C for 10min. The purified PCR product was cleaned with a Wizard PCR Preps DNApurification system (Promega), digested with BglII and SmaI, and fused with

the inducible spac promoter present in pMGPI (21), which was also digestedwith BglII and SmaI. The mixture was used to transform E. coli DH5�(Invitrogen, Carlsbad, Calif.) competent cells to obtain plasmid pSGII.

Construction of S. aureus strains with the murE gene under the control of aninducible promoter. Plasmid pSGII was introduced into S. aureus RN4220 elec-trocompetent cells by electroporation with a Gene Pulser apparatus (Bio-Rad,Hercules, Calif.) essentially as previously described (13). The transformationmixture was plated on TSA containing erythromycin (10 �g/ml) and IPTG (300�M). Plasmids pSGII and pMGPII (21) were sequentially transduced to MRSAstrain COL by using phage 80� as previously described (17), except that 100 �MIPTG was added to the media used for preparation of the transducing lysate andfor transduction. Transductants were selected with erythromycin (10 �g/ml) forpSGII and with chloramphenicol (10 �g/ml) for pMGPII. The correct sequenceof pSGII insertion into COL was confirmed.

Experiments in liquid culture were performed by using the following protocol.Cultures were grown overnight at 37°C in TSB supplemented with 100 �M IPTGand the appropriate antibiotic(s). To remove traces of IPTG, bacterial cultureswere centrifuged and the pellet was washed twice with TSB. The cells wereresuspended in the same volume of TSB supplemented with various concentra-tions of IPTG and then used to determine various properties, such as suscepti-bility to antibiotics, growth rates, composition of the UDP-linked precursor pool,and transcription of several genetic determinants.

Determination of susceptibility to different types of antibiotics. Cultures ofCOLspac::murE were spread on the surface of TSA plates supplemented withdifferent concentrations of IPTG (25, 37.5, 75, 150, and 500 �M), and antibioticsusceptibilities were tested with paper disks containing the antibiotics oxacillin (1mg), ciprofloxacin (50 �g), vancomycin (30 �g), D-cycloserine (200 �g), andtetracycline (1 mg). Strain COL was used as a control. The sizes of inhibitionhalos were evaluated after incubation at 37°C for 9.5 h.

Determination of autolysis rates. Triton X-100-stimulated autolysis in glycinebuffer (pH 8.0) was measured as previously described (27). Cells were grownexponentially to an OD620 of about 0.3. The cultures were rapidly chilled on ice,and the cells were washed once with ice-cold distilled water and suspended to anOD620 of 1.0 in 50 mM glycine–0.01% Triton X-100 buffer. Autolysis was mea-sured during incubation at 37°C by monitoring the OD620.

Northern blotting analysis. Cells were grown in TSB or TSB supplementedwith increasing concentrations of IPTG at 37°C to an OD620 of 0.7 to 0.8(log-phase growth), and the RNA was extracted as previously described (28).Next, 7 �g of each RNA sample was analyzed by electrophoresis in a 1.2%agarose gel containing 0.66 M formaldehyde and morpholinepropanesulfonicacid (MOPS; Sigma). The RNA was blotted onto Hybond N� membranes (Am-ersham, Buckinghamshire, United Kingdom) with a turbo blotter alkaline trans-

TABLE 1. Strains and plasmids used in this studya

Strain or plasmid Relevant characteristicsa Source or reference

S. aureus strainsRN4220 Mcs, restriction negative R. NovickCOL Homogeneous Mcr Ems; MIC, 800 �g/ml RUb collectionRUSA235 COL::Tn551-Mcr Emr; MIC, 6.25 �g/ml 6RUSA235-Homo* COL::Tn551-Mcr Emr Homo*; MIC, 400 �g/ml This studyRUSA235 Backcross COL::Tn551-Mcr Emr; MIC, 6.25 �g/ml 16RUSA235-Homo* COL::Tn551-Mcr Emr; MIC, 6.25 �g/ml This studyBackcrossRN4220spac::murE RN4220 with Pspac-murE fusion in the chromosome This studyCOLspac::murE COL with Pspac-murE fusion in the chromosome transformed with pMGPII This studyCOLpMGPII COL transformed with pMGPII 22

E. coli DH5� recA endA1 gyrA96 thi-1 hsdR17 supE44 relA1 �80 �lacZ�M15 BethesdaResearchLaboratories

PlasmidspMGPI S. aureus integrative vector with IPTG-inducible Pspac promoter and lacI

gene; Apr Emr21

pMGPII S. aureus replicative plasmid containing lacI gene; Apr Cmr 21pSGII pMGPI with murE ribosome binding site and first 311 codons fused to

Pspac promoter, Apr EmrThis study

a Oxacillin MICs are given. Apr, ampicillin resistant; Emr, erythromycin resistant; Cmr, chloramphenicol resistant.b RU, Rockefeller University.

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fer system (Schleicher & Schuell, Inc., Keene, N.H.) with SSC20X. The PCR-amplified DNA probes were labeled with [�-32P]dCTP (Amersham LifeSciences, Piscataway, N.J.) with a Ready to Go labeling kit (Amersham) andhybridized under high-stringency conditions. The blots were subsequentlywashed and autoradiographed.

Membrane purification. Membrane proteins were prepared from bacterialcultures as previously described (25). Proteins were quantified with a DcProteinassay kit (Bio-Rad Laboratories).

Western blotting analysis. For detection of PBP2A in the membrane proteinfraction, 10 and 30 �g of each membrane protein preparation was resolved on8% acrylamide gels and transferred to nitrocellulose membranes by Westernblotting as previously described (31). Incubation with a monoclonal antibodyagainst PBP2A of an MRSA strain (Eli Lilly & Co., Indianapolis, Ind.) wascarried out with the ECL Western blot analysis system (Amersham) (31).

RESULTS

Selective suppression of �-lactam antibiotic resistance inTn551 murE mutant RUSA235. The susceptibilities of parentalstrain COL and mutant RUSA235 to bacitracin (MIC, 50 �g/ml), moenomycin (MIC, 0.25 �g/ml), and vancomycin (MIC,1.5 �g/ml) were the same, while resistance to �-lactam antibi-otics was drastically reduced. The oxacillin MIC went from 800to 6 �g/ml, and that of cefotaxime went from 400 to 25 �g/ml.These findings indicate that the inhibitory effect of the murEmutation was selective to �-lactam antibiotics and did notaffect inhibitors of earlier steps in cell wall biosynthesis.

Recovery of high-level �-lactam resistance in Homo* deriv-atives of mutant RUSA235. The population analysis profile ofmethicillin resistance showed a heterogeneous phenotype. Themethicillin MIC was reduced in 99.9% of the cells of strainRUSA235 cultures—from a MIC of 800 �g/ml in the parentalstrain to a MIC of 6 �g/ml in the mutant. Nevertheless, suchcultures also contained subpopulations of bacteria that re-tained the parental level of oxacillin resistance. Such so-calledHomo* subpopulations (8) were present with a frequency of10�5 to 10�7 in mutant cultures. Homo* colonies picked fromthe agar plate gave rise to virtually homogeneous cultures withhigh-level methicillin resistance. When Homo* cultures werebackcrossed into parental strain COL (by selection for theTn551 marker, i.e., erythromycin resistance), the transductantsshowed the phenotype of the original RUSA235 mutant, indi-cating that the recovery of high-level antibiotic resistance inthe Homo* cells was due to a compensatory (suppressor) mu-tation(s) distant from the Tn551 insertion (Fig. 1).

Accumulation of dipeptide cell wall precursors in Homo*cultures. The relative amounts of UDP–N-acetylmuramyldipeptides and UDP–N-acetylmuramyl pentapeptide werecompared in the cell wall precursor pools of parental strainCOL, mutant 235, and Homo* cultures (Fig. 2). The relativeamount (expressed as a percentage of all UDP-linked cell wallprecursors) of muramyl dipeptides decreased from 18.5% inRUSA235 to about 8% in the Homo* extract, while muramylpentapeptides increased from 55% in the mutant to about 67%in the Homo* extract. The corresponding values in parentalstrain COL were 5% dipeptides and 71% pentapeptide.

FIG. 1. Heterogeneous expression of oxacillin resistance in S. au-reus mutant RUSA235 carrying a Tn551 insert in murE. Expression ofoxacillin resistance was determined by population analysis as describedin Materials and Methods. Overnight cultures were plated on TSA oron TSA containing increasing concentrations of oxacillin. Plates wereincubated for 48 h at 37°C, and the CFU were counted. Symbols:parental strain COL, Œ; mutant RUSA235, E; RUSA235 backcross, �;RUSA235-Homo*, ■; RUSA235-Homo* backcross, F.

FIG. 2. Alterations in the composition of the cytoplasmic cell wallprecursor pool in murE mutant RUSA235. Cytoplasmic peptidoglycanprecursors isolated from parental strain COL, mutant RUSA235, andderivative RUSA235-Homo* were prepared and analyzed by HPLC asdescribed in Materials and Methods. The elution profiles of extractsfrom the parental strain (top), mutant RUSA235 (middle), and theHomo* derivative of the mutant (bottom) are shown. Cell wall pre-cursors were identified by mass spectrometry. 1, UDP-MurNAc; 2,UDP–MurNAc–L-Ala; 3, UDP–MurNAc–L-Ala–D-Glu; 4, UDP–Mur-NAc–L-Ala–D-Glu–D-Ala–D-Ala pentapeptides. The component witha retention time of 80 min is vancomycin (26), which was used toinduce accumulation of wall precursors. Abs, absorbance.

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Cell wall peptidoglycan composition of cultures grown in thepresence of sub-MICs of oxacillin. Previous work has alreadydemonstrated the basic abnormality of the peptidoglycan ofRUSA235, namely, the appearance of two abnormal disaccha-ride dipeptide derivatives that differed from one another in thepresence of isoglutamine or free glutamic acid residues (16).Figure 3 compares the muropeptide compositions of the pep-tidoglycans prepared from parental strain COL, mutant strainRUSA235, and its Homo* derivative, each grown either inTSB or in TSB supplemented with oxacillin at 5 �g/ml. Thisantibiotic concentration was previously shown to inhibit all ofthe native PBPs of S. aureus, leaving the mecA product PBP2Aas the only transpeptidase to catalyze the cross-linking of mu-ropeptides (4). The HPLC profiles of the three peptidoglycanswere very similar; there was no evidence of the relative enrich-ment of the peptidoglycan in the abnormal dipeptide compo-nents when bacteria were grown with oxacillin in the medium.

Purified cell walls prepared from strain COL, mutantRUSA235, and its Homo* derivative grown in the presence orabsence of oxacillin showed comparable rates of cell wall deg-radation when treated with crude autolysin extract. The samestrains also showed similar relative rates of autolysis when

suspensions of the bacteria were exposed to the detergentTriton X-100 (Fig. 4).

Placing murE under the control of the inducible Pspac pro-moter. Since murE is an essential gene, the impact of reducedlevels of MurE on �-lactam resistance was tested by putting thetranscription of the murE gene under the control of a promoterthat can be induced with IPTG. The method of constructionand the structure of the Pspac-controlled murE gene are shownin Fig. 5. For tighter repression of the Pspac promoter, multiple-copy plasmid pMGPII carrying the lacI repressor gene was alsointroduced into strain COL.

Cultures of strain COL in which the expression of murE wasinducible with IPTG were grown at different concentrations ofthe inducer. Overnight cultures of the bacteria grown in thepresence of optimal concentrations of IPTG were centrifuged,washed twice with TSB, and resuspended in IPTG-free me-dium before being diluted to an OD620 of 0.02 in TSB con-taining 10 �g of chloramphenicol per ml (in order to ensurethe presence of plasmid pMGPII) and supplemented with in-creasing concentrations of IPTG. The growth rate of the cul-tures was followed by monitoring the rate of increase in OD.

FIG. 3. Changes in the muropeptide composition of the peptidoglycan of murE mutant RUSA235. Muropeptide composition was determinedin peptidoglycans isolated from parental strain COL, mutant RUSA235, and derivative RUSA235-Homo* grown in either TSB (A) or TSBcontaining oxacillin at 5 �g/ml (B). Peptidoglycan was isolated and hydrolyzed with muramidase, and the resulting muropeptides were separatedby HPLC as described in Materials and Methods. Top, muropeptide pattern of parental strain COL; middle, muropeptide profile of mutantRUSA235; bottom, muropeptide profile of derivative RUSA235-Homo*. Abs, absorbance.

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Figure 6 shows that the rate of bacterial growth was propor-tional to the concentration of IPTG in the medium.

The same overnight cultures were also used to determine theeffects of different IPTG concentrations on the expression ofoxacillin resistance. The cultures were spread on agar supple-mented with different concentrations of IPTG. A paper diskcontaining 1 mg of oxacillin was placed in the middle of theagar plates, and the diameter of the inhibition zone was deter-mined after incubation of the plates at 37°C. Figure 7 showsthat the diameter of the inhibition zone decreased in parallelwith the increasing IPTG concentrations in the agar medium.The effect of IPTG was specific for �-lactam resistance; nochanges in the diameters of zones of inhibition due to tetracy-cline, ciprofloxacin, vancomycin, or D-cycloserine were de-tected as a function of the concentration of IPTG in the agarmedium (data not shown).

Accumulation of UDP–N-acetylmuramyl dipeptides in cul-tures grown in suboptimal concentrations of IPTG. Bacterialcultures were grown in TSB supplemented with different con-centrations of IPTG. When the OD of the cultures reached 0.5,bacterial cultures were centrifuged and the composition of thecell wall precursor pool was determined as described in Mate-

rials and Methods. Cultures growing in the presence of increas-ing concentrations of IPTG showed a gradual increase in theamount of UDP-muramyl pentapeptide and a gradual de-crease in the amounts of UDP–N-acetylmuramyl dipeptides inthe cell wall precursor pool. For instance, the relative amountof the dipeptides decreased from 42 to 8% as the concentra-tion of IPTG increased from 37.5 to 200 �M while the relativeamount of muramyl pentapeptides increased from 27 to about66% under the same growth conditions (Fig. 8).

Transcription of murE and methicillin resistance genesmecA and pbpB in cultures growing with various concentra-tions of IPTG. Figure 9A through C show the Northern anal-ysis of the transcription of murE and two genetic determinantsknown to be associated with �-lactam antibiotic resistance in S.aureus: mecA and pbpB, the structural determinants of PBP2Aand PBP2, respectively. Cultures of COLspac::murE were grownat four different concentrations of IPTG, and transcriptionlevels were determined. In each one of the cases, the levels oftranscription appeared to be a function of the IPTG concen-tration in the medium.

Led by these observations, we also determined the transcrip-tion of mecA and pbpB in strain COL, mutant RUSA235, and

FIG. 4. Susceptibility of murE mutant cells and cell walls to autolytic degradation in vitro and in vivo. Cell walls prepared from strains COL(open columns), RUSA235 (dark gray columns), and RUSA235-Homo* (light gray columns) grown in the absence (A) or presence (B) of oxacillinat 5 �g/ml were tested for susceptibility to in vitro degradation by autolysins extracted from strain COL (see Materials and Methods). (C) Culturesof strains COL (open columns), RUSA235 (dark gray columns), and RUSA235-Homo* (light gray columns) grown in TSB were suspended in aTriton X-100 lysis buffer to an initial OD of 1.0, and the rates of autolysis were monitored as described in Materials and Methods. Data representthe means of two or three independent experiments.

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its Homo* derivative. Reduced transcription in the mutant anda return to a higher degree of expression in the Homo* deriv-ative were noted (Fig. 9). The transcription levels of murF andpta (phosphate acetyltransferase)—two genes selected as con-trols—did not change in these three strains (Fig. 9).

DISCUSSION

Earlier data on the phenotype of murE insertional mutantRUSA235 pointed to abnormalities of cell wall peptidoglycanstructure as the possible cause of the drastic reduction in thelevel of oxacillin resistance. In the cytoplasmic cell wall pre-cursor pool of the murE mutant, there was an accumulation ofUDP–N-acetylmuramyl dipeptides that then found their wayinto the polymerized cell wall of the mutant (16). The disac-charide dipeptides must be incorporated into the peptidogly-can through transglycosylation and will remain unavailable forcross-linking since they are missing both the critical diaminoacid component and the C-terminal D-Ala–D-Ala residue.

We hypothesized that the drastic and selective reduction ofresistance to �-lactam antibiotics may be related to the incor-poration of abnormal muropeptides causing some sort of struc-tural defect in the mutant cell wall. One may envision severalspecific scenarios. For instance, growth in the presence of�-lactam antibiotics may further increase the relative propor-tion of the abnormal dipeptide component to such a degreethat it begins to jeopardize the structural stability of the cellwall. Mutant cell walls in which cross-linking is further inhib-ited by oxacillin may be hypersensitive to autolytic degrada-tion. Increased sensitivity of poorly cross-linked staphylococcalcell walls to enzymatic degradation in vitro has been described(22). The abnormal dipeptide components may be recognizedin �-lactam-treated bacteria as signals for a suicidal activationof enzymes that catalyze cell wall turnover.

Experimental tests of these hypotheses (described in thispaper) gave negative results. Growth of the mutant bacteria inthe presence of sub-MICs of oxacillin did not increase theincorporation of dipeptides into the peptidoglycan. Mutantand parental cell walls prepared from normal or oxacillin-treated bacteria were shown to have virtually identical sensi-

FIG. 5. Construction of strain COLspac::murE with the murE gene under the control of an inducible promoter. Only the uninterrupted copy ofmurE under the control of the Pspac promoter produces a functional protein.

FIG. 6. Control of the growth rate of S. aureus strain COL by therate of transcription of murE. A culture of strain COLspac::murE grownovernight at 37°C in TSB supplemented with 100 �M IPTG and theappropriate antibiotics was centrifuged, washed twice with TSB, resus-pended in the same volume of TSB, and distributed into test tubescontaining TSB supplemented with various concentrations of IPTGand chloramphenicol (10 �g/ml). Growth of the cultures was moni-tored as described in Materials and Methods.

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tivities to degradation by autolytic extracts in vitro. Actually,the rate of autolysis induced by the detergent Triton X-100 wasslightly slower in the mutant compared to that in the parentalcells. Furthermore, there was no difference in cell wall lysissensitivity between mutant RUSA235 and its Homo* deriva-tive, in which high-level methicillin resistance was restored.

These data do not support our initial hypothesis that the

mechanism of reduced �-lactam resistance was related to thestructurally defective cell wall produced in the murE mutant.

In mutant RUSA235, the transposon is inserted 3 bp fromthe C terminus of the gene (15), resulting in the production ofan abnormal protein that has reduced specific catalytic activitycompared to the enzyme from parental cells (data not shown).In order to test the impact of inhibition of MurE on �-lactam

FIG. 7. Effect of murE transcription on oxacillin resistance. Strain COLspac::murE was plated on TSA supplemented with increasing concentra-tions of IPTG (25 [A], 37.5 [B], 75 [C], 150 [D], and 500 [E] �M). The sizes of inhibition halos around paper disks containing 1 mg of oxacillinwere measured after incubation at 37°C for 9.5 h. The values under the panels are the diameters of inhibition zones.

FIG. 8. Effect of murE transcription on the composition of the cytoplasmic peptidoglycan precursor pool. Cytoplasmic cell wall precursors wereextracted from strain COLspac::murE grown with different concentrations of IPTG and were analyzed as described in Materials and Methods.Numbers 1 through 4 identify the cell wall precursors listed in the legend to Fig. 2. Abs, absorbance.

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resistance, we put the transcription of the gene under thecontrol of an inducible promoter.

The introduction of this new experimental system provideda striking confirmation of the importance of MurE for theexpression of oxacillin resistance. Not only was the growth rateof the cultures with the controllable murE gene a function ofthe IPTG concentration in the medium, but the level of ox-acillin resistance also showed clear dependence on the tran-scription levels of murE and cultures grown at suboptimalconcentrations of IPTG showed accumulation of UDP–N-acetylmuramyl dipeptides in the cell wall precursor pool. Thus,the basic microbiological and biochemical observations ob-tained in the Tn551 mutant RUSA235 were reproduced inparental strain COL by experimental modulation of the tran-scription of murE.

Unexpectedly, and most interestingly, the controlled rate oftranscription of murE also brought along parallel changes inthe transcription levels of mecA and pbpB, two genes that playa central role in methicillin resistance.

It has been well established that in MRSA cultures even lowconcentrations of �-lactam antibiotics can fully acylate andinactivate the normal complement of PBPs. In strain COL,with an oxacillin MIC of more than 400 �g/ml, as little as 2 to

5 �g of oxacillin per ml was shown to block the formation ofmost oligomeric muropeptides, resulting in the production of apeptidoglycan that was primarily composed of muropeptidemonomers and dimers plus a small amount of trimeric mu-ropeptides (4). According to our current model, in staphylo-cocci growing in the presence of oxacillin, cell wall biosynthesisis mainly catalyzed by two proteins, methicillin resistance pro-tein PBP2A, providing transpeptidase activity, and PBP2, pro-viding the essential transglycosylase activity (20). The observa-tions described in this communication indicate that thetranscription rates of the genetic determinants of these twocritical resistance proteins—mecA and pbpB—depend on therate of transcription of murE, the structural gene of an enzymethat is involved with a step in the synthesis of the UDP-linkedcell wall precursors. Apparently, the reduced rate of murEtranscription in the bacteria brought along a parallel decline inthe transcription of the two PBP-encoding genes and loss ofoxacillin resistance. Analysis by Western blotting demon-strated that suppressor mutants of RUSA235 in which normallevels of oxacillin resistance were recovered contained largeramounts of PBP2A than did the mutant bacteria (Fig. 9).

Together, these data suggest that the mechanism by which�-lactam resistance is suppressed in murE mutants is related to

FIG. 9. Transcription analysis of selected S. aureus genes in cultures of promoter-controlled murE and Tn551 murE mutants. Northern analysiswas used to determine the levels of transcription of mecA (A), pbpB (B), and murE (C) in cultures of COL and COLspac::murE grown in TSBsupplemented with different concentrations of IPTG. Lanes: 1, COL; 2 through 5, COLspac::murE grown in the presence of 25 (lane 2), 37.5 (lane3), 75 (lane 4), and 200 (lane 5) �M IPTG. (D to G) Northern blotting analysis of the mecA (D), pbpB (E), pta (F), and murF (G) genes fromparental strain COL (lane 1), the RUSA235 murE mutant (lane 2), and the RUSA235-Homo* derivative (lane 3). (H) Western blotting analysisof the amounts of PBP2A in parental strain COL (lane 1), the RUSA235 murE mutant (lane 2), and the RUSA235-Homo* derivative (lane 3).The analysis was done for 10 and 30 �g of total protein. The reasons for the smaller molecular size of the reactive band in panel C, line 1, are notclear. A similar change in molecular size has been observed before in spac control promoters (21). The change may involve some rearrangementaround the initiation site of the promoter.

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a deficit in the cellular amounts of PBP2A and PBP2—twoproteins that form the key components of the �-lactam resis-tance mechanism in S. aureus. Dependence of the methicillinMIC on the cellular amounts of PBP2A is not without prece-dent. In clinical isolates of MRSA that carry the specific chro-mosomal or plasmid-borne regulatory genes, the oxacillin MICdepends on the rate of transcription of mecA and/or the cel-lular amounts of PBP2A (23).

The apparently coordinate regulation of the transcription oftwo PBPs, PBP2A and PBP2, by the rate of transcription of agene (murE) involved with the biosynthesis of cell wall precur-sors has interesting implications beyond the context of �-lac-tam resistance. Our data imply that PBP2 and PBP2A may beunstable—either in the functional or in the topographic sense.The selective localization of PBP2 at the sites of staphylococcalcell division has recently been demonstrated (19). It is conceiv-able that deposition of this essential protein at the zone of anew cell division requires the production of new molecules ofthis protein, in coordination with the rate of cell wall biosyn-thesis, a control site of which may be at the transcription ofmurE. Whether the transcriptional control is directly exertedthrough the MurE protein or through the change in the con-centration of cell wall precursors, e.g., through the change inthe concentration of the UDP-MurNAc pentapeptide cell wallprecursor, remains to be determined. The participation of cellwall precursors in the control of expression of penicillinase-based �-lactam resistance and also in the control of the processof recycling of cell wall components has already been demon-strated in E. coli (10, 11, 14, 18).

ACKNOWLEDGMENTS

Partial support for this work was provided by a grant from the U.S.Public Health Service (RO1 AI37275) to Alexander Tomasz and byFCT Project 34872/99 from the Fundacao para a Ciencia e Tecnologia,Portugal, awarded to Herminia de Lencastre. Susana Gardete and RitaSobral were supported by grants SFRH/BD/3137 and -3138 2000, re-spectively, from the Fundacao para a Ciencia e Tecnologia.

We gratefully acknowledge the help of Shang Wei Wu (The Rock-efeller University) with Northern and Western blotting analysis andKeiko Tabei (Wyeth Research) for assistance with mass spectrometryanalysis of cell wall precursor pools. Moenomycin was obtained by thecourtesy of Aventis Pharma D, DI&A Natural Products.

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