A novel positive regulatory element for exfoliative toxin A gene expression in Staphylococcus aureus

Author:

Sakurai Susumu1,Suzuki Hitoshi2,Hata Toshiaki3,Yoshizawa Yukio4,Nakayama Ritsuko3,Machida Katsuhiko5,Masuda Shogo6,Tsukiyama Takashi1

Affiliation:

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

Abstract

A 1·4 kb positive regulatory element (ETAexp ) that controls staphylococcal exfoliative toxin A (sETA) transcription was cloned from Staphylococcus aureus. ETAexp 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 ETAexp -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 ETAexp amplified by PCR (1·7 kb eta-ETAexp /pUC9), regardless of the orientation of ETAexp 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-ETAexp /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 ETAexp -deficient eta fragment (pYT3-etaJ3) was 2500–4000 times lower than that of pYT3-etaJ6.

Publisher

Microbiology Society

Subject

Microbiology

Reference32 articles.

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Cited by 2 articles. 订阅此论文施引文献 订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献

1. Lysogenic Conversion in Bacteria of Importance to the Food Industry;Bacteriophages in the Control of Food- and Waterborne Pathogens;2014-04-09

2. Development of a High-Expression System for Staphylococcal Exfoliative Toxin Genes;Journal of Veterinary Medical Science;2011

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