1 Department of Molecular Genetics, Kohno Clinical Medicine Research Institute, Shinagawa-ku, Tokyo 140-0001, Japan
2 Division of Bioscience, Graduate School of Environmental Earth Science, Hokkaido University, Kita-ku, Sapporo 060, Japan
3 Department of Molecular Genetics, The Jikei University School of Medicine, Minato-ku, Tokyo 105-8461, Japan
4 Radioisotope Research Center, The Jikei University School of Medicine, Minato-ku, Tokyo 105-8461, Japan
5 Department of Laboratory Medicine, The Jikei University School of Medicine, Minato-ku, Tokyo 105-8461, Japan
6 Department of Microbiology (II), The Jikei University School of Medicine, Minato-ku, Tokyo 105-8461, Japan
Correspondence
Sususmu Sakurai
sakurai-s{at}kcmi.or.jp
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ABSTRACT |
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The GenBank/DDBJ accession number for the sequence reported in this paper is AB070631.
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INTRODUCTION |
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In this paper, we report that the upstream region (ETAexp) of the 5·8 kb eta is required for genetic expression of eta and that the rsETA prepared from an S. aureus transformant into which the recombinant plasmid pYT3-etaJ6 had been introduced was expressed at high levels in the culture supernatant fraction in quantities similar to those produced by E. coli C6008S transformed with petaJ1 as shown by the latex agglutination test. However, the latex agglutination titre of rsETA in the culture supernatant of S. aureus transformed with the recombinant plasmid pYT3-etaJ3 containing the 1·7 kb eta sequence carrying 1·4 kb ETAexp-deficient eta was 25004000 times lower than that of pYT3-etaJ1.
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METHODS |
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To determine exoprotein production S. aureus was cultivated in LB medium (Sigma-Aldrich; Sambrook et al., 1989) at 30 °C for 48 h.
Molecular biology techniques.
Standard techniques were used for DNA isolation, molecular cloning, transformation in E. coli, electrophoresis, and DNA sequencing (Sambrook et al., 1989).
Construction of recombinant plasmids pYT3-etaJ6 and pYT3-etaJ3.
Construction of the 3·4 kb eta-ETAexp/pYT3 (pYT3-etaJ6) and 1·7 kb eta/pYT3 (pYT3-etaJ3) recombinant plasmids was as follows. The 4·2 kb fragment containing the ETAexp gene and the eta gene was obtained by EcoRI digestion of petaJ1. The recombinant plasmid petaJ3 (1·7 kb eta/pUC9) was digested by EcoRI. The 5' protruding ends of the 4·2 kb fragment obtained from petaJ1 digested with EcoRI or the recombinant plasmid petaJ3 digested by EcoRI were removed using the Kilo-Sequence Deletion Kit (Takara Shuzo), and then the 4·2 kb fragment or petaJ3 was digested by HindIII (Fig. 1). The resulting 3·4 kb fragment (3·4 kb eta-ETAexp) or the 1·7 kb eta sequence (etaJ3) carrying 1·4 kb ETAexp-deficient eta was inserted into the shuttle plasmid pYT3 (kindly provided by Professor K. Hiramatsu, Department of Bacteriology, Juntendo University, Japan) cleaved at the SalI site; the 5' protruding ends were converted to blunt ends, and then the fragment was digested with HindIII. Agglutination tests confirmed that E. coli C6008S transformed with pYT3-etaJ6 produced cETA in quantities similar to those produced by E. coli C6008S transformed with petaJ1 (data not shown). However, cETA produced by pYT3-etaJ3 was not detected in the culture supernatant fraction. pYT3-etaJ3 or the pYT3-etaJ6 recombinant plasmid was transformed into S. aureus strain FRI-1169.
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petaJ3 and petaJ1 recombinant plasmids.
In all experiments, we used a recombinant pUC9 plasmid containing a 5·8 kb eta AluI insert (petaJ1) or a 1·7 kb eta AluI insert (petaJ3) prepared according to a previously described procedure (Sakurai et al., 1987; Sakurai et al., 1988
). The petaJ1 recombinant plasmid was constructed by ligating the 5·8 kb eta AluI fragment of the chomosomal DNA from S. aureus ZM, an ETA-producing strain, and a SmaI-cleaved pUC9 with T4 DNA ligase. The petaJ3 recombinant plasmid was constructed by ligating the 1·7 kb fragment carrying the 1·45 kb eta (1·7 kb eta) obtained from the 5·8 kb eta fragment after partial digestion by AluI and SmaI-cleaved pUC9. The production of cETA by ampicillin-resistant E. coli C6008S transformants (Ampr) was assessed with a sandwich ELISA of culture supernatants with rabbit anti-ETA serum, as previously described (Sakurai et al., 1987
, 1998
).
Construction of petaJ2 recombinant plasmid.
The 5·8 kb eta sequence was digested with EcoRI to remove the 3' terminal sequence (Fig. 1). The resulting 4·2 kb segment (etaJ2) was ligated with pUC118 digested with EcoRI, and a recombinant plasmid (petaJ2) was constructed containing the intact eta region, except for the 3' terminal region downstream of the EcoRI site. Agglutination tests confirmed that E. coli C6008S transformed with petaJ2 produced cETA in quantities similar to those produced by E. coli C6008S transformed with petaJ1 (data not shown).
Preparation of deletion mutants.
Deletion mutants for the etaJ2 insertion were made with the Kilo-Sequence Deletion Kit (Takara Shuzo). Five ETAexp deletion mutants were obtained with a sandwich ELISA of culture supernatant with rabbit anti-ETA serum, and their cETA production rates were assessed with the single radial immunodiffusion test or the latex agglutination test. Fig. 2 shows a schematic representation of the ETAJ2 insertion and the deletion mutants (dm-1, -2, -3, -4 and -5).
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Construction of 1·7 kb eta-ETAexp/pUC9 and ETAexp/pUC9 recombinant plasmids.
The inserted sequence on the recombinant pUC9 plasmid containing a 1·7 kb eta at the SmaI insertion site (petaJ3) was digested with HindIII or EcoRI, and a flush end at the HindIII or EcoRI site was created with the Kilo-Sequence Deletion Kit (Takara Shuzo). The primers used to amplify the ETAexp fragment were 5'-CATTTGGATGTCAATGC-3' (primer 1), corresponding to nucleotides 432 to 416 of the ETAexp sequence, and 5'-AACGTTTCTATTTCAAAAC-3' (primer 2), corresponding to nucleotides 946 to 964 of the ETAexp sequence, as counted from the putative initiation codon, residue +1 (ATG). The ETAexp fragment was amplified with 1 µg nucleic acid as the template in a 50 µl reaction mixture of the following composition: 50 mM KCl, 10 mM Tris/HCl (pH 8·3), 1·5 mM MgCl2, 0·01 % (w/v) gelatin, 200 µM of each of the dNTPs, 0·2 µM of each of the ETAexp-specific oligonuleotide primers, and 1·25 units of Taq polymerase (Toyobo). The sample was denatured for 0·5 min at 94 °C, and PCR was performed for 30 cycles at 94 °C for 1·0 min, at 57 °C for 1·0 min and 72 °C for 1·0 min with autoextension in a MiniCycler (MJ Research). A single adenine 3'-end overhang on the 1·4 kb ETAexp sequence amplified with PCR was removed with a Sure Clone Ligation Kit (Amersham Pharmacia Biotech), and the amplified DNA fragments were isolated by 1 % (w/v) agarose gel electrophoresis using DEAE ion-exchange chomatography (DE81, Whatman). The purified PCR products were ligated with recombinant pUC9 plasmid, petaJ3 digested with HindIII or EcoRI, or pUC9 digested with SmaI, and the recombinant plasmids (petaJ4, petaJ5 or ETAexp/pUC9) were constructed. The cETA production rates of petaJ4 and petaJ5 were determined by the single radial immunodiffusion test. ETAexp/pUC9 was used for simultaneous transformation with 1·7 kb eta/pHY300PLK.
Construction of the 1·7 kb eta/pHY300PLK recombinant plasmid.
The insertional sequence of the recombinant pUC9 plasmid containing the 1·7 kb eta sequence at the SmaI site was digested with EcoRI and HindIII, after which the 1·7 kb eta EcoRIHindIII fragment was isolated electrophoretically with Whatman DE81 paper. pHY300PLK was digested with EcoRI and HindIII; the recombinant plasmid (petaJ3-300) was constructed by incubating pHY300PLK with calf intestine alkaline phosphatase followed by ligation with the 1·7 kb eta EcoRIHindIII fragment.
Preparation of crude toxin.
The recombinant plasmid was transformed into E. coli C6008S; the transformant was grown for 18 h at 37 °C in 500 ml 2xTY broth containing 50 µg ampicillin ml1, then centrifuged at 8000 g for 20 min at 4 °C. The culture supernatant and the precipitated cell fractions were used to prepare toxin solution, as reported previously (Sakurai et al., 1998). Five hundred millilitres of culture supernatant was saturated by adding 342 g ammonium sulfate with stirring on ice, followed by centrifugation at 8000 g for 20 min at 4 °C (ammonium-sulfate-precipitated culture supernatant fraction). The precipitated cell fractions were suspended in 10 ml distilled water, sonicated with an ultrasonic disruptor (UD 201, Tomy Seiko) on ice, and then centrifuged at 14 000 g for 20 min at 4 °C. The resulting supernatant was saturated by the addition of 8·5 g ammonium sulfate with stirring on ice followed by centrifugation at 14 000 g for 20 min at 4 °C (ammonium-sulfate-precipitated cell extract fraction). The ammonium-sulfate-precipitated fractions were dissolved in 20 ml distilled water and dialysed against 2 l distilled water at 4 °C for 16 h with a VT 801 dialysis tube (molecular sieving of 8000) while being stirred with a magnetic stirrer at 4 °C overnight. The dialysate was lyophilized, and the dried material was dissolved in 3 ml distilled water and subjected to the single radial immunodiffusion test and immunoblot analysis. The ETA-producing strain S. aureus ZM was grown at 37 °C for 48 h in 500 ml TY medium, as reported previously (Kondo et al., 1973
), and then centrifuged at 8000 g for 20 min at 4 °C. Five hundred millilitres of the culture supernatant was saturated by the addition of 342 g ammonium sulfate with stirring on ice, followed by centrifugation at 8000 g for 20 min at 4 °C. The ammonium-sulfate-precipitated culture supernatant fraction from S. aureus ZM (sETA) was dissolved in 20 ml distilled water and dialysed against 2 l distilled water, after which the dialysate was lyophilized as described above. The dried material was dissolved in 3 ml distilled water and then used for immunoblot analysis. The methods of isolation and purification of sETA and the immunization procedure were as described previously (Kondo et al., 1973
).
Single radial immunodiffusion test of cETA.
A single radial immunodiffusion test was performed on a glass slide with 3 ml 1 % (w/v) agarose gel in 10 mM Tris/HCl0·5 M NaCl, pH 7·5, containing rabbit anti-sETA serum. The concentration of antiserum in the agarose gel was adjusted to 1 : 75, and the plate was examined for the formation of turbid haloes around the wells with the ammonium-sulfate-precipitated cell extract fraction in a moisture chamber at room temperature.
Immunoblot analysis of cETA.
A 10 µl sample of the culture supernatant, the ammonium-sulfate-precipitated culture supernatant fraction, and the ammonium-sulfate-precipitated cell extract fraction of petaJ1 and petaJ3 were used to assess production of cETA. The ammonium-sulfate-precipitated culture supernatant fraction from S. aureus ZM (sETA) was used as a control. Samples were separated with SDS-PAGE according to the method of Laemmli (1970), electrophoretically transferred to PVDF membranes using the Semi-Dry Blot Apparatus (ATTO) according to the manufacturer's instructions, and then incubated with rabbit anti-sETA IgG followed by a goat anti-rabbit IgGhorseradish peroxidase conjugate. sETA was purified with a procedure previously reported (Kondo et al., 1973
) and used as the molecular mass standard.
Northern blot analysis of RNA from petaJ1 and petaJ3.
RNA from petaJ3 or petaJ1 transformants that had grown to the early stationary phase in 2xTY medium at 37 °C was extracted with the RNeasy Mini Kit (QIAGEN) according to the manufacturer's instructions. A 30 µg sample of RNA was electrophoresed on a gel containing 1·0 % (w/v) agarose, 20 mM MOPS, pH 7·0, and 2·2 M formaldehyde (Sambrook et al., 1989). The RNA was transferred to a nylon membrane (Amersham Pharmacia Biotech) with a model 785 vacuum blotter (Bio-Rad). The filters were prehybridized and then hybridized with digoxigenin (DIG; Roche Diagnostics)-labelled 1·7 kb eta prepared according to the manufacturer's instructions. Signals were visualized with a DIG Luminescent Detection Kit (Roche Diagnostics) according to the manufacturer's instructions.
Assay of rsETA by latex agglutination test.
S. aureus was cultured in LB medium at 30 °C for 48 h, and the culture supernatant was used for the latex agglutination test, performed with a latex agglutination kit (Denka-Seiken) according to the manufacturer's instructions.
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RESULTS |
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Immunoblot analysis of cETA
On immunoblotting, cETA produced by E. coli C6008S transformed with petaJ1 was identical in size to the purified sETA from S. aureus ZM (Fig. 4, lane 1). cETA was expressed at high levels in the culture supernatant (Fig. 4
, lane 4), the ammonium-sulfate-precipitated culture supernatant fraction (Fig. 4
, lane 2), and the ammonium-sulfate-precipitated cell extract fraction from E. coli C6008S transformed with petaJ1 (Fig. 4
, lane 6). The amount of cETA produced by petaJ1 (Fig. 4
, lane 2) was almost the same as that produced by the control S. aureus ZM strain (data not shown). However, the cETA in E. coli C6008S transformed with petaJ3 could not be detected in the culture supernatant (Fig. 4
, lane 5), the ammonium-sulfate-precipitated culture supernatant fraction (Fig. 4
, lane 3), or the ammonium-sulfate-precipitated cell extract fraction (Fig. 4
, lane 7).
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DISCUSSION |
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To test the hypothesis that the ETAexp gene product binds to the eta promoter region and activates transcription, we developed a model of the secondary and tertiary structure of the ETAexp gene product predicted by computer analysis of the ETAexp nucleotide sequence. The most striking feature of the ETAexp protein is a helixturnhelix motif that is present in several other previously characterized DNA-binding proteins (Pabo & Sauer, 1984). Many DNA-binding proteins recognize dyad symmetrical sequences, and an inverted sequence is present between the 10 region corresponding to the consensus sequence of the E. coli promoter and the SD sequence of etaJ3 (Sakurai et al., 1987
). These findings suggest that the ETAexp protein binding to the promoter region is a necessary step in the transcriptional activation of the eta promoter.
When petaJ4 or petaJ5 was cloned into E. coli, the transformant produced a large amount of cETA on the single radial immunodiffusion test regardless of the orientation of the ETAexp insert (Fig. 6). These findings suggest that ETAexp activates the promoter of etaJ3 as a transcriptional activator. However, when ETAexp/pUC9 and the petaJ3-300 recombinant plasmid were simultaneously cloned into E. coli, the latex agglutination titre of the ammonium-sulfate-precipitated cell extract fraction from the transformant was 5000 to 10 000 times lower than that of petaJ1 (data not shown), suggesting that ETAexp must be located nearby upstream or downstream of the eta gene.
In the above latex agglutination experiment, the rsETA prepared from an S. aureus transformant into which the recombinant plasmid pYT3-etaJ6 had been introduced was expressed at high levels in the culture supernatant fraction in quantities similar to those produced by E. coli C6008S transformed with petaJ1. However, the latex agglutination titre of rsETA in the culture supernatant of S. aureus FRI-1169 transformed with pYT3-etaJ3 (containing the 1·7 kb eta with the 1·4 kb ETAexp-deficient eta) was 25004000 times lower than that of pYT3-etaJ6. pYT3-etaJ6 introduced into S. aureus clinical isolates that produce -toxin, TSST-1, staphylococcal enterotoxin B (SEB) and SAK had no effect on the rate of production of
-toxin, SEB, TSST-1, SAK, protein A or coagulase (data not shown). These results indicate that a fuctional ETAexp element is required for expression of the eta gene, although we did not examine the production of other exoproteins, such as serine protease, nuclease and
-haemolysin. An accessory gene regulator (agr), a chomosomal locus of S. aureus, is required for the high-level post-exponential-phase expression of several exoproteins, such as
-haemolysin, serine protease, TSST-1,
-haemolysin, nuclease,
-haemolysin and enterotoxin B (Recsei et al., 1986
; Morfeldt et al., 1988
). The agr gene has also been cloned in E. coli, and its nucleotide sequence has been determined (Peng et al., 1988
; Janzon et al., 1989
). Our analysis has shown that the nucleotide sequence of ETAexp is distinct from the nucleotide sequence of three global regulatory loci of S. aureus (agr, sar and Mgr; data not shown). In this experiment, the ETA produced by five deletion mutants of ETAexp in the 5·8 kb eta sequence and the 1·7 kb eta sequence was detected only with ELISA; however, the cETA produced by the deletion mutants and the 1·7 kb eta did not form precipitation haloes on the single radial immunodiffusion test (Fig. 6
, well 2), and their exfoliative activity could not be detected with an in vivo assay (data not shown) performed with a method reported previously (Kondo et al., 1973
). These results indicate that the integrity of ETAexp in the upstream region of eta is required for high expression of the eta gene. Our Southern blot analysis using eta and ETAexp probes showed that eta and ETAexp are located only on the 4 kb HindIII fragment of chomosomal DNA from ETA-producing strains of S. aureus (data not shown). The SAK gene is located on a staphylococcal serotype B bacteriophage (Kondo & Fujise, 1977
), and the nucleotide sequences of the lytA gene in staphylococcal serotype B phage 80, the eta gene and the SAK gene have homologous downstream regions (Bon et al., 1997
). Yoshizawa et al. (2000)
have reported that when the restriction-minus, non-ET-producing S. aureus 1039 strain was lysogenized with the temperate phage
-ZM-1 from an ETA-producing ZM strain, clones of 6 of 10 lysogens produced ETA and the eta gene fragment could be amplified with PCR. Why the temperate phage
-ZM-1 carries both the eta gene and ETAexp is unknown. ETAexp may act as an activator of some other gene or gene(s) in the phage genome. In a recent preliminary experiment, we found that the ammonium-sulfate-precipitated cell extract fraction from the E. coli transformed with the petaJ1 recombinant plasmid binds to the 451 bp promoter fragment obtained from the 1·7 kb eta after digestion with RsaI. We are now isolating and characterizing the ETAexp protein.
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Received 2 December 2002;
revised 9 December 2003;
accepted 16 December 2003.
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