A novel positive regulatory element for exfoliative toxin A gene expression in Staphylococcus aureus.
A 1.4 kb positive regulatory element (ETA(exp)) that controls staphylococcal exfoliative toxin A (sETA) transcription was cloned from Staphylococcus aureus. ETA(exp) is located upstream of the cloned 5.8 kb eta gene (etaJ1) obtained from the chomosomal DNA of S. aureus ZM, the standard ETA-producing strain. The cETA prepared from an Escherichia coli transformant into which the recombinant plasmid petaJ1 (5.8 kb eta/pUC9) had been introduced was expressed at high levels in the culture supernatant and the ammonium-sulfate-precipitated culture supernatant fraction as shown by immunoblotting and the single radial immunodiffusion test. However, cETA produced by the recombinant plasmid petaJ3 containing the 1.7 kb eta sequence (etaJ3) with a 1.45 kb ETA(exp)-deficient eta fragment (1.7 kb eta/pUC9) obtained from the 5.8 kb eta sequence by subcloning was not detected in either the culture supernatant or the ammonium-sulfate-precipitated culture supernatant fraction (167-fold concentrate of the culture supernatant) by immunoblotting or the single radial immunodiffusion test. A large amount of cETA was produced by the 1.7 kb eta sequence when it was linked to ETA(exp) amplified by PCR (1.7 kb eta-ETA(exp)/pUC9), regardless of the orientation of ETA(exp) insertion. Northern blot hybridization showed lower levels of the transcripts of the 1.7 kb eta sequence than of the 5.8 kb eta sequence. The rsETA prepared from an S. aureus transformant into which the recombinant plasmid 3.4 kb eta-ETA(exp)/pYT3 (pYT3-etaJ6) had been introduced was expressed at high levels in the culture supernatant fraction as shown by the latex agglutination test. However, the agglutination titre in the culture supernatant fraction of rsETA produced by the recombinant plasmid (1.7 kb eta/pYT3) containing the 1.7 kb eta sequence carrying the 1.4 kb ETA(exp)-deficient eta fragment (pYT3-etaJ3) was 2500-4000 times lower than that of pYT3-etaJ6.
Sakurai
Susumu
S
Department of Molecular Genetics, Kohno Clinical Medicine Research Institute, Shinagawa-ku, Tokyo 140-0001, Japan.
Suzuki
Hitoshi
H
Division of Bioscience, Graduate School of Environmental Earth Science, Hokkaido University, Kita-ku, Sapporo 060, Japan.
Hata
Toshiaki
T
Department of Molecular Genetics, The Jikei University School of Medicine, Minato-ku, Tokyo 105-8461, Japan.
Yoshizawa
Yukio
Y
Radioisotope Research Center, The Jikei University School of Medicine, Minato-ku, Tokyo 105-8461, Japan.
Nakayama
Ritsuko
R
Department of Molecular Genetics, The Jikei University School of Medicine, Minato-ku, Tokyo 105-8461, Japan.
Machida
Katsuhiko
K
Department of Laboratory Medicine, The Jikei University School of Medicine, Minato-ku, Tokyo 105-8461, Japan.
Masuda
Shogo
S
Department of Microbiology (II), The Jikei University School of Medicine, Minato-ku, Tokyo 105-8461, Japan.
Tsukiyama
Takashi
T
Department of Molecular Genetics, Kohno Clinical Medicine Research Institute, Shinagawa-ku, Tokyo 140-0001, Japan.
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D005083:Exfoliatins / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D015151:Immunoblotting; D005779:Immunodiffusion; D007841:Latex Fixation Tests; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA; D013211:Staphylococcus aureus / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D014158:Transcription, Genetic; D014169:Transformation, Bacterial
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D005083:Exfoliatins