Tn551-mediated insertional inactivation of the fmtB gene encoding a cell wall-associated protein abolishes methicillin resistance in Staphylococcus aureus
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(2) H. Komatsuzawa et al. genetic background. Although most factors affecting methicillin resistance were identified by Tn551-mediated insertional inactivation followed by screening for susceptibility to methicillin, the presence of Triton X-100 in the screening procedure yielded a different class or genes as exemplified by the lipophilic fmt gene.12 The same screening procedure has now allowed us to identify a novel cell wall-associated factor which affects both methicillin resistance and Triton X-100 susceptibility.. Materials and methods Bacterial strains and growth conditions The bacterial strains and plasmids used in this study are listed in Table I. S. aureus and Escherichia coli were grown. in trypticase soy broth (TSB, Becton-Dickinson Microbiology Systems, Cockeysville, MD, USA) and Luria Bertani broth, respectively. Erythromycin (EM; 30 mg/L), chloramphenicol (CP; 10 mg/L), or ampicillin (100 mg/L) were added when necessary to maintain the transposon in the chromosomal DNA or plasmids in the cell.. Transposon mutagenesis Different dilutions of a 30°C overnight culture of S. aureus COL harbouring the temperature-sensitive plasmid pRN3208 that carries transposon Tn551 (ermB) were plated on erythromycin-containing plates and incubated for 48 h at 42°C. Colonies growing on these plates were screened for integration of Tn551 into the chromosome. Table I. Bacterial strains and plasmids used in this study Strain or plasmid. Relevant characteristics. Origin or reference. Strains S. aureus KSA8 COL COL (pRN3208) NCTC10443 RN450 KSA8 TS111 COL TS111 COL TS111-1 NCTC10443 TS111 RN450 TS111 BB591 HK9696 E. coli XL-1 Blue. homogeneous Mcr, mec homogeneous Mcr, mec COL with pRN3208[Rep(Ts)] homogeneous Mcr, mec laboratory strain, Mcs fmtB::Tn551 (transduction from COL-TS111), Mcs, EMr, mec mutagenized fmtB::TN551, Mcs, EMr, mec fmtB::Tn551 (backcross from COL-TS111), Mcs, EMr, mec fmtB::Tn551 (transduction from COL-TS111), Mcs, EMr, mec fmtB::Tn551 (transduction from COL-TS111) glmM::Tn551 of NCTC8325 glmM::Tn551 of COL (transduction from BB591), mec. clinical isolate ref. 10 ref. 10 clinical isolate. rec1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1ac [F proAB lacIqZM15 Tn10 (Tetr)] hsdS gal (lambdacIts857 ind1 Sam7 nin5 lacUV5-T7 gene 1). ref. 44. BL21 Plasmids pRN3208 pUC19 pGC2 pLI50 pFLAG MAC pHK4011 pHK4014 pHK4069 pHK 4357 pHK4176 pHK4251 pHK4148 pHK4203. Tn551(ermB), Cdr, rep-ts cloning vector, Ampr shuttle vector, Ampr in E. coli, Cmr in S. aureus shuttle vector, Ampr in E. coli, Cmr in S. aureus expression vector (Eastman Kodak Company) pUC19/4 kb HindIII-PstI fragment containing Tn551 left junction and its flanking region of KSA8-TS111 pUC19/8.9 kb XbaI fragment (fmtB) of KSA8 pGC2/8.9 kb XbaI fragment (fmtB) of KSA8 pGC2/7.8 kb PCR fragment (fmtB) of COL pLI50/1.8 kb PCR fragment (glmM) of COL pLI50/1.8 kb glmM fragment 7.8 kb fragment (fmtB) of COL pFLAG MAC/partial fmtB fragment (repeated sequence) pFLAG MAC/glmM fragment. 422. this study this study this study this study this study ref. 7 this study. ref. 27. ref. 10 ref. 45 ref. 46 ref. 47 this study this study this study this study this study this study this study this study this study.
(3) Characterization of S. aureus fmtB gene and loss of the plasmid moiety by replica plating on plates containing either erythromycin or 50 mg/L of cadmium. The curing efficiency of the plasmid was approximately 1 105. The mutants thus obtained were screened for decreased oxacillin resistance in the presence of 0.02% Triton X-100 as described earlier.12 One mutant unable to grow on 16 mg/L of oxacillin in the presence of Triton X-100, strain COL-TS111, was kept for further analysis.. pUC19. Finally, a corresponding 8.9 kb XbaI fragment covering the Tn551 insertional region was isolated from wild-type KSA8. DNA sequencing of the fragment was performed using synthesized primers. Since we originally isolated the mutant from S. aureus COL, we determined the DNA sequence of the corresponding regions of S. aureus COL and COL-TS111 using PCR fragments as template DNA with the same primers as used for S. aureus KSA8 DNA sequencing.. Transduction Transductions were performed with phage 80 alpha26 using COL-TS111 as donor and S. aureus COL, KSA8, NCTC10443 or RN450 as recipients. Transductants were selected for growth on 30 mg/L of erythromycin.. MICs Minimum inhibitory concentrations and population analysis of various antibiotics were determined by a microdilution method as described before.25 Population analysis profiles were determined by plating aliquots of an overnight culture on plates containing various concentrations of oxacillin or Triton X-100. Colonies were counted after 48 h incubation at 37°C.24. DNA manipulations Routine DNA manipulations, Southern blots and hybridization, and DNA sequencing were performed essentially as described elsewhere.27 Restriction enzymes and shrimp alkaline phosphatase were purchased from Boehringer– Mannheim Biochemica (Tokyo, Japan), and T4 DNA ligase was from New England BioLabs (Beverly, MA, USA). Pulsed-field gel electrophoresis (PFGE) was carried out as described.28 Hybridization was performed by means of a chemiluminescent procedure (ECL direct labelling kit or 3-oligolabelling kit; Amersham Life Science, Buckinghamshire, UK). DNA sequences of both strands were determined by the dideoxy chain termination method with an Autoread sequencing kit (Pharmacia Biotech., Tokyo, Japan). PCR reagents were from Boehringer–Mannheim, and PCR was performed with the GeneAmp PCR System 2400 (Perkin Elmer, Norwalk, USA).. Cloning of Tn551 insertional region The 4 kb PstI–HindIII fragment containing the left portion of Tn551 and its flanking region from S. aureus (KSA8TS111 chromosomal DNA was identified using the oligonucleotide probe, 5-AGC GCC TAC GGG GAA TTT GT-3 derived from the known Tn551 sequence.29 PstI– HindIII digests of KSA8-TS111 chromosomal DNA were ligated into pUC19 and transformed into E. coli XL-1 Blue. The desired clone was identified from this library by colony hybridization. The cloned fragment was in turn used to probe an XbaI library of S. aureus KSA8 strain cloned into. Primer extension Total cellular RNAs were extracted from S. aureus COL by the procedure of Cheung et al.30 using a FastRNA kit (BIO 101, Vista, CA, USA). Primer extension was carried out with the AMV Reverse Transcriptase Primer Extension System (Promega, Madison, WI, USA). The synthetic oligonucleotide primer (5-TTTTCTGATACTAAATTTTTGTTGTCT-3) was end labelled with [32P]-ATP. The labelled primer was then mixed with 100 g or 50 g of RNA. Annealing was at 58°C for 20 min, then at room temperature for 10 min, and extension was at 42°C for 30 min. The reaction mixture was loaded on to a 6% polyacrylamide gel, together with sequencing reaction samples using the same primer. After electrophoresis, the gel was dried and exposed to a Fuji imaging plate (Fuji Photo Film Co., Tokyo, Japan), then detected with a BAS2000 scanner.. Complementation experiments For complementation experiments we cloned the 8.9 kb XbaI fragment of S. aureus KSA8 that contained the corresponding fmtB gene into the shuttle plasmid pGC2, resulting in plasmid pHK4069. The fmtB gene from S. aureus COL was amplified by PCR with the primers 5-CTGATGAAGATGCTGAAAGA-3 and 5-GTTATGTATGAAAGGAGTA-3, generating a 7.8 kb fragment which was cloned into plasmid pGC2, resulting in plasmid pHK4357. The glmM gene was amplified from S. aureus COL with the primers 5-GTTAAACAGAAAGGTAGT-39 and 5-CGTTAAAAACACAAAGCA-3 and cloned into pLI50, to produce plasmid pHK4176. Finally the insert of pHK4357 was inserted downstream of glmM into the pHK4176 plasmid, resulting in plasmid pHK4251, which thus contained both the glmM and fmtB genes.. Antiserum To analyse the protein expression of FmtB and GlmM, we generated antiserum against FmtB and GlmM. First, we prepared the recombinant proteins of FmtB and GlmM by using the E. coli FLAG Expression System (Eastman Kodak Company, New Haven, CT, USA). The repeated sequence region of the fmtB gene was amplified by PCR. 423.
(4) H. Komatsuzawa et al. with two primers (5-CAAGCGAAACAAGATATTATCCAA-3 and 5-GCTTGGTTCGCTTTAGGTTTA-3), and the full glmM gene was amplified with primers (5-ATGGGAAAATATTTTGGT-3 and 5-GCTTGGTTCGCTTTAGGTTTA-3) from KSA8 chromosomal DNA, then cloned into the pFLAG MAC vector (Eastman Kodak Company) to generate pHK4148 and pHK4203, respectively. These plasmids were electroporated into E. coli BL21, and used to produce the recombinant proteins. The recombinant proteins were purified from E. coli lysates with the anti-FLAG affinity gel according to the manufacturer’s manual. The purified proteins were used to immunize rabbits. The antiserum was diluted 1000-fold for immunoblotting.. Extraction of total proteins from S. aureus cells and fractionation of cells S. aureus cells growing to the early stationary phase were collected by centrifuguation at 10 000g. After washing with phosphate-buffered saline (PBS), cells were suspended in PBS containing 1 mM phenylmethylsulphonylfluoride (PMSF) and digested with lysostaphin (final concentration 100 mg/L) for 30 min at 37°C. After centrifugation at 10 000g, the supernatant was used as crude fraction. Various fractions of S. aureus cells were prepared as follows: (i) culture supernatant was obtained by centrifugation at 10 000g for 30 min and concentrated 60 times by 80% saturated ammonium sulphate precipitation; (ii) cell wall extracts and (iii) cytoplasmic fractions were obtained as follows: cells were suspended in digestion buffer (30% raffinose in 0.05 M Tris (pH 7.5) with 0.145 M NaCl) containing 1 mg of lysostaphin (Sigma Chemical Co., St Louis, MO, USA), 100 g of DNase (Sigma) and PMSF (1 mM). The cell mixture was incubated for 1 h at 37°C. Protoplasts were removed by centrifugation at 8000g for 10 min, and the supernatant was used as cell wall extract. The protoplasts were lysed in PBS, and used as cytoplasmic fraction.. Results Isolation of a Tn551 insertion mutant with reduced oxacillin resistance Within 2000 Tn551 insertionally inactivated S. aureus COL mutants which had lost the plasmid moiety, one clone, designated COL-TS111, showed reduced resistance to oxacillin in the presence as well as the absence of Triton X100. Backcrosses of the Tn551 insertion from COL-TS111 into COL resulted in transductants of identical phenotype as COL-TS111, proving the linkage of the Tn551 insertion with reduced oxacillin and Triton X-100 resistance. One representative transductant, strain COL-TS111-1, was kept for further analysis. Transductions of the Tn551 insert in two further, genetically distinct MRSA strains, KSA8 and NCTC10443, also yielded oxacillin-susceptible transductants, represented here by strains KSA8-TS111 and NCTC104433-TS111, respectively. Transductions of the Tn551 insert in the susceptible strain S. aureus RN450, resulting in strain RN450-TS111, had no apparent effects on its oxacillin susceptibility (data not shown). Attempts to transduce Tn551 in other different susceptible S. aureus isolates failed. The Tn551 insertion was mapped to the SmaI-I fragment of S. aureus COL was determined by probing a Southern blot of an SmaI digest of S. aureus COL separated by PFGE with a Tn551 probe (data not shown).. Antibiotic susceptibility of the Tn551 mutants The Tn551 insertion drastically reduced the methicillin resistance of strains KSA8-TS111, COL-TS111, and NCTC10443-TS111 to various β-lactam antibiotics such as methicillin, cefoxitin, and imipenem, whereas susceptibilities of non-β-lactam inhibitors like fosfomycin, bacitracin, and vancomycin and to protein synthesis inhibitors like chloramphenicol and tetracycline were not affected by the insertion (data not shown).. Chemicals and reagents Triton X-100 was purchased from Nacalai tesque (Kyoto, Japan). Oxacillin, methicillin, bacitracin, vancomycin, N-acetylglucosamine and glucosamine were from Sigma. Cefoxitin, imipenem, chloramphenicol, fosfomycin and tetracycline were from Daiichi Seiyaku (Tokyo, Japan), Banyu Seiyaku (Tokyo, Japan), Sankyo (Tokyo, Japan), Wako Chemicals (Osaka, Japan) and Lederle Japan (Tokyo, Japan), respectively.. Nucleotide sequence accession number The nucleotide sequence discussed in this work will appear in DDBJ, EMBL and GenBank under accession numbers ABO15223 for S. aureus KSA8 fmtB and ABO25716 for S. aureus COL.. Population analysis The three parent MRSA strains, KSA8, COL and NCTC10443, showed high and almost homogeneous resistance to oxacillin, whereas the Tn551 insertion abolished their oxacillin resistance level to values 0.06 mg/L of oxacillin. Only a very small subpopulation, 1 in 104 cells in S. aureus KSA8 and COL, and 1 in 106 cells in NCTC10443, displayed higher, heterogeneous resistance, which, however, did not reach the values of the highest resistance seen in the corresponding clones of the parent subpopulation (Figure 1). Interestingly, resistance to Triton X-100 was also substantially reduced below 0.2% in the mutants. In addition, the mutants produced a small subpopulation resistant to high concentrations of Triton X-100, comprising 1 in 103 cells in S. aureus KSA8 and. 424.
(5) Characterization of S. aureus fmtB gene c. 1 in 105 cells in S. aureus COL and NCTC10443. Common to all mutants was the complete abolition of basal oxacillin resistance levels, whereas the size and profile of the subpopulation able to grow on oxacillin and Triton X-100 were reproducible, apparently strain-dependent characteristics. However, in the susceptible S. aureus RN450 both oxacillin resistance and Triton X-100 resistance remained unaltered by the Tn551 insertion (data not shown).. Cloning and sequencing of the Tn551 insertion region Starting from the sequences obtained from the 8.9 kb XbaI fragment of S. aureus KSA8 covering the Tn551 insertion. site, a primer pair was designed to amplify a corresponding 7.8 kb region in S. aureus COL which was subsequently sequenced. The first 5 223 bp of the sequence matched perfectly with the downstream sequence following the phosphoglucosamine mutase gene glmM (GenBank accession no. Y15477) (Figure 2). It was followed by an open reading frame (orf1) coding for a putative 2478 amino acid protein of a deduced mass of 263 kD with a calculated isoelectric point of 4.29. A putative Shine Dalgarno sequence GGAGGA was identified 7 bp upstream of the ATG start codon. Primer extension analysis revealed that the orf1 transcript initiates at an adenine residue located 14 bp 5 to the orf1 start codon (Figure 3). The Tn551 had inserted at. Figure 1. Population analysis of the parent strains () and their Tn551-inserted mutants (). Overnight cultured S. aureus cells were plated on TSA containing serial dilutions of oxacillin or Triton X-100 and were incubated at 37°C for 48 h.. Figure 2. Restriction map of the fmtB region of S. aureus COL. The boxed arrows represent the ORFs and the orientation of transcription. The arrow of Tn551 represents its orientation.. 425.
(6) H. Komatsuzawa et al. nt 976, interrupting the N-terminal part of orf1 (Figure 2). We gave orf1 the designation fmtB (factor that affects the methicillin resistance levels in the presence and absence of Triton X-100); consequently we renamed the first fmt gene identified earlier by the same screening method12 as fmtA. Since Tn551 insertions sometimes result in the deletion or mutation of DNA close to the insertion site, we checked both Tn551 flanking sequences in COL-TS111, but no rearrangements of any sort had occurred.. fmtB encodes a cell surface protein with a multiple repeat domain. Figure 3. Primer extension analysis of fmtB transcription of S. aureus COL. One hundred micrograms (lane 1) and 50 g (lane 2) of total RNA were used. ACGT shows the sequence of the corresponding region of the fmtB. Transcription start is indicated with an asterisk.. Figure 2 shows a schematic representation of the fmtB region in S. aureus COL. The central part of FmtB, starting with the amino acid at position 687 and ending at amino acid 1993, contains 17 tandemly repeated sequences of 75 amino acids. From the first to the twelfth repeat, four amino acids are perfectly conserved (Figure 4), whereas the last five repeats show less similarity. A cell wall-sorting motif, LPXTG, was identified at the C terminus of FmtB in S. aureus COL. Comparison of the FmtB aa sequence with. Figure 4. Comparison of the amino acid sequences among the repeated units in the FmtB protein in S. aureus COL. Amino acid identities are indicated by box shadows and similarities by shadows. Numbers of R1–R12 at the left represent the repeat units.. 426.
(7) Characterization of S. aureus fmtB gene Table II. MICs of oxacillin for S. aureus COL and its derivatives Strain COL COL-TS111 COL-TS111 pHK4069 (fmtB of KSA8) COL-TS111 pHK4357 (fmtB of COL) COL-TS111 pHK4176 (glmM) COL-TS111 pHK4151 (glmM fmtB). plasmid pHK4176, and almost complete restoration of the oxacillin resistance was obtained with the plasmid pHK4151 covering both glmM and fmtB (Table II).. MIC of oxacillin (mg/L). Identification and localization of FmtB by Western blots. 512 0.13 0.13 0.13 128 256. known protein sequences in DDJB data banks revealed similarity to the EF protein in Streptococcus suis31 and Lmp1 in Mycoplasma hominis.32. Complementation experiments The fmtB mutation in COL-TS111 could be complemented in trans neither with the wild-type fmtB allele of S. aureus KSA8 by introduction of plasmid pHK4069 nor with that of S. aureus COL in plasmid pHK4357. The oxacillin susceptibility remained as low in both complemented strains as in COL-TS111 (Table II). Unexpectedly, though, a 1000fold increase in the MIC of oxacillin was obtained by complementation of COL-TS111 with the glmM gene in. Figure 5. Western blot of fractions from S. aureus strain COL. The fractionation method is described in Materials and methods. Samples were prepared from S. aureus COL (lanes 1–4), COLTS111 (lane 5), COL-TS111 having pHK4357 (fmtB) (lane 6), and resolved by SDS–PAGE in 5% polyacrylamide gel, then subjected to Western blotting. Immunodetection was performed using anti-FmtB serum as primary serum. Lanes: 1, culture supernatant; 2, cytoplasmic fraction; 3, cell wall extract; 4–6, whole cell lysate.. Antibodies raised against the repeat domain of FmtB revealed in Western blots a protein band migrating with an apparent size of 310 kDa located mainly in the cell wall fraction of S. aureus COL and in small amounts in the supernatant, but not in the membrane or cytoplasmic fraction. It disappeared in the fmtB mutant COL-TS111 (Figure 5). This suggested that the immunoreactive high molecular mass protein was FmtB, although its molecular weight estimated from SDS–PAGE did not fully correspond with that calculated from the nucleotide sequence, as often seen with membrane or cell wall proteins. When COL-TS111 was complemented with the plasmid pHK4357 (fmtB), the FmtB band appeared again as a strong band (Figure 5), although oxacillin resistance was not restored. The FmtB was overproduced in that strain and appeared mainly in the cell wall fraction, but some was also found in the supernatant and in the cytoplasm (data not shown).. GlmM expression in the fmtB mutant Since not the fmtB but the glmM gene restored the wildtype phenotype in the fmtB mutant, Western blots were performed with antiserum raised against GlmM to see if fmtB inactivation affects GlmM production. However, S. aureus COL and COL-TS111 produced similar amounts of a 50 kDa protein corresponding in its size to the GlmM protein (49.2 kDa)33 (Figure 6), suggesting that fmtB. Figure 6. Western blot of whole cell lysates of S. aureus COL and its derivative strains. Samples were resolved by SDS–PAGE in 10% polyacrylamide gel, then subjected to Western blotting. Immunodetection used anti-GlmM serum as primary serum. Lanes: 1, recombinant GlmM; 2, COL; 3, COL-TS111; 4, COL (glmM::Tn551) having pHK4176; 5, COL (glmM::Tn551).. 427.
(8) H. Komatsuzawa et al. inactivation does not reduce GlmM production. In the control strain BB591, a glmM::Tn551 mutant, this band was absent as expected (Figure 6).. Phenotypic complementation of fmtB mutants with N-acetylglucosamine and glucosamine Although GlmM production was not affected by the fmtB inactivation, oxacillin resistance in COL-TS111 could apparently be restored by a plasmid carrying the glmM gene, resulting presumably in GlmM overproduction, due to the copy number effect of the plasmid. GlmM is a phosphoglucosamine mutase catalysing the formation of glucosamine-1-phosphate, a precursor of the cytoplasmic steps of peptidoglycan biosynthesis.34 Inactivation of glmM leads, similarly to fmtB inactivation, to reduced oxacillin resistance.33,34 Interestingly, fmtB inactivation had a stronger effect on oxacillin resistance than did glmM inactivation. The MIC of oxacillin against COL-TS111 was 0.25 mg/L compared with 4 mg/L in strain HK9696, which was constructed here by transduction of glmM::Tn551 into S. aureus COL (Table III). We analysed the effect of adding the potential substrates of the glucosamine mutase GlmM to the growth medium. Indeed, oxacillin resistance was strongly increased 30 to 400-fold in both mutants by external addition of either N-acetylglucosamine or glucosamine to the growth medium (Table III). A very slight increase in the oxacillin MIC was also seen in the parent with those substrates. No effects on MIC values were generated by addition of similar concentrations of glucose, fructose or mannosamine, nor of glutamine to the medium in the same strains (data not shown).. Discussion Insertion of transposon Tn551 into fmtB, a gene that codes for a cell wall-associated protein of as yet unknown function, abolishes oxacillin resistance in a dramatic way, making MRSA strains hypersusceptible to all β-lactams, though it still allowed the formation of highly resistant subclones. Except for S. aureus RN450, transductions of fmtB::Tn551. in other susceptible S. aureus strains were unsuccessful for as yet unknown reasons. This was despite Southern blots with a selection of different S. aureus strains revealing fmtB-specific sequences in all susceptible and methicillinresistant S. aureus strains tested (data not shown). Viability of fmtB mutants in a susceptible background may be diminished, or the few transductants appearing may carry secondary mutations counteracting fmtB inactivation. The FmtB protein is probably not directly involved in methicillin resistance, since resistance was not restored in fmtB mutants by complementation with plasmids carrying either S. aureus COL or KSA8 fmtB allele, although the complementing COL fmtB allele produced large amounts of FmtB that were found in the cell wall fraction. Curiously the fmtB sequence in S. aureus KSA8 strain coded for a truncated FmtB protein without a cell wall-spanning domain and cell wall anchor, and, accordingly, S. aureus KSA8 strain had a smaller FmtB protein which appeared in the cytoplasm and supernatant only (data not shown). Nevertheless, the finding that fmtB inactivation in KSA8TS111 resulted in the same reduction of oxacillin and Triton X-100 resistance as in COL-TS111 provided a further indication that the intact FmtB protein itself plays no direct role in oxacillin resistance. The reduction in oxacillin resistance is therefore presumably due to a polar effect of the Tn551 insert on distant genes. Immediately upstream of fmtB and separated by less than 400 bases is the end of the glmM gene followed by a rho-independent transcription terminator. The fmtB inactivation had no effects on glmM transcription (data not shown) nor on production of GlmM. All transcripts covering glmM and the glmM operon were apparently too short to cover also the fmtB gene.33 However, since chromosomal fmtB transcripts could repeatedly and by different means not be made visible in Northern blots owing to degradation (data not shown), we cannot rule out that a few long transcripts may cover glmM and fmtB. The phosphoglucosamine mutase, GlmM, catalyses the formation of glucosamine-1phosphate, a precursor of N-acetylglucosamine (GlcNAc) which is a constituent of the disaccharide backbone of the peptidoglycan. Interestingly, both the overproduction of. Table III. Effect of N-acetylglucosamine and glucosamine on the MIC of oxacillin for S. aureus COL and its derivatives MIC of oxacillin (mg/L) Substrate. COL. COL fmtB::Tn551. None N-acetylglucosamine (1 mM) (10 mM) (100 mM) Glucosamine (1 mM) (10 mM). 512 512 1024 1024 512 1024. 0.25 64 64 128 16 32. 428. COL glmM::Tn551 4 64 64 128 64 64.
(9) Characterization of S. aureus fmtB gene GlmM and the addition of either glucosamine or GlcNAc to the growth medium restored oxacillin resistance almost completely in fmtB mutants. This suggests that fmtB inactivation results in a shortage of either glucosamine-1phosphate or N-acetylglucosamine-1-phosphate, which are precursors of UDP-GlcNAc.35 A parallel pathway besides GlmM has been postulated for the synthesis of glucosamine-1-phosphate.33 Since GlmM production is apparently not affected by fmtB inactivation the parallel pathway may be the target of fmtB inactivation. Either by supplying fmtB mutants with the corresponding substrates or by increasing the GlmM concentration, the desired UDPGlcNAc precursors are made available at sufficient concentrations again by a bypass reaction. The mechanism triggered by fmtB inactivation is not clear yet. On the one hand fmtB inactivation seems to act in cis, since trans-complementation is inefficient; on the other hand there are apparently no effects on the transcription and translation of the proximal glmM operon. Instead there may be effects on the downstream region. Further experiments are being performed to elucidate this interesting problem. Downstream of fmtB no transcriptional terminator could be identified and the next orf separated by 1 kb is transcribed divergently. The FmtB protein itself may have interesting properties. The region of FmtB which showed a similarity to EF protein in S. suis31 and Lmp1 in M. hominis32 is within repeated sequences of 75 amino acids (Figure 4). These proteins are thought to be associated with pathogenesis.31,32 Like EF protein, the molecular mass of FmtB calculated from sequence analysis was different from that estimated from SDS–PAGE (Figure 5). It has been observed that highly charged proteins often migrate anomalously in SDS gels.31,36 Production of proteins with multiple repeats has been reported in some bacteria.37–41 Multiple repeats are thought to be involved in binding of the protein to specific ligands.37–41 In some cases the amino acid sequences in repeats are highly conserved, whereas other cases show limited homology with variations.41–43 The repeat variation has commonly been observed in the M protein of Streptococcus pyogenes,42,43 the glucosyltransferase of Streptococcus downei and S. mutans,41 and the toxin A of Clostridium difficile41 The 17 amino acid repeated region of FmtB also shows weak similarity among the repeated sequences, but the role of the repeated region remains unknown. Further biochemical research into FmtB will elucidate the association with methicillin resistance.. Acknowledgements This work was supported by a grant-in-aid for Encouragement of Young Scientists (grant no. 10770119) from the Ministry of Education, Science, Sports and Culture of Japan, and Health Sciences Research Grants for Research on Emerging and Re-emerging Infectious Diseases from the. Ministry of Health and Welfare of Japan. P. Glanzmann and B. Berger-Bächi were supported by Swiss National Science Foundation Grant 31-52239.97. References 1. Fontana, R. (1985). Penicillin-binding proteins and the intrinsic resistance to beta-lactams in Gram-positive cocci. Journal of Antimicrobial Chemotherapy 16, 412–16. 2. Reynolds, P. E. & Fuller, C. (1986). Methicillin resistant strains of Staphylococcus aureus; presence of an identical additional penicillin-binding protein in all strains examined. FEMS Microbiology Letters 33, 251–4. 3. Ubukata, K., Nogorochi, R., Matsuhashi, M. & Knno, M. (1989). 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