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The DNA encoding the exfoliative toxin A gene (eta) of Staphylococcus aureus was cloned into bacteriophage Xgtll and subsequently into plasmid pLI50 on a 1,391-base-pair DNA fragment of the chromosome. Exfoliative toxin A is expressed in the Escherichia coli genetic background, is similar in length to the toxin purified from culture medium, and is biologically active in an animal assay. The nucleotide sequence of the DNA fragment containing the gene was determined. The protein deduced from the nucleotide sequence is a polypeptide of 280 amino acids. The mature protein is 242 amino acids. The DNA sequence of the exfoliative toxin B gene was also determined. Corrections indicate that the amino acid sequence of exfoliative toxin B is in accord with chemical sequence data.The exfoliative toxins A and B (ETA and ETB) of Staphylococcus aureus are the causative agents of staphylococcal scalded-skin syndrome (11). They possess the same biological activity (12), but they are immunologically distinct proteins (11, 12). The two forms also differ in amino acid composition, amino acid sequence, and heat resistance (13). In addition, eta is expressed from the chromosome, whereas etb is of plasmid origin (14,21,29).To study the mode of action and the molecular properties of these two proteins, we set out to clone and sequence both genes. Recently, we reported the cloning and sequencing of the etb gene (9, 10). In this communication, we report the cloning and DNA sequence of eta. O'Toole and Foster (17) also reported the cloning of eta and in the accompanying paper (18) report their sequence of the gene. Our data and their report contain identical DNA sequence determinations as well as general conclusions regarding the expression of the eta gene. eta is expressed in the Escherichia coli background and is biologically active in the neonatal mouse assay. Furthermore, the sequence of the protein deduced from the DNA sequence is identical to the published sequence of the N-terminal region of the ETA molecule (12). MATERIALS AND METHODSBacterial strains and plasmids. S. aureus UT0002, a phage group II staphylococcal scalded-skin syndrome clinical isolate (20), was used as the source of library DNA. E. coli Y1090 was used in the Protoclone system (Promega Biotec Co., Madison, Wis.) as a recipient for bacteriophage Xgtll packaging carried out according to the manufacturer's instructions. E. coli LE392 was used for transformation and propagation of plasmids and cloned DNA (16).* Corresponding author.

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A complete amino acid analysis is presented for staphylococcal enterotoxin B. The protein is composed solely of amino acids and is particularly rich in aspartic acid and lysine. It has no free sulfhydryl groups and only one disulfide bridge. On the basis of the amino acid composition the molecular weight is 35,380, the isoionic point is 8.70, and the partial specific volume is Q WJtaphylococcal enterotoxin B is the emetic material elaborated by the S-6 strain of Staphylococcus aureus. A purification procedure permitting the isolation of large amounts of an immunologically and physically homogeneous material is described in an accompanying paper (Schantz, et al., 1965). The toxin is devoid of any lipid, carbohydrate, or nucleic acid. This report presents a complete amino acid analysis of the protein, using the automatic amino acid analyzer, and a comparison with an earlier, largely microbiological analysis (Hibnick and Bergdoll, 1959). It also describes the identification of the amino-(N-) and carboxyl-(C-) terminal amino acids of the toxin, and quantitation of these residues. The implications these analyses have on the structure and conformation of the protein are discussed. Materials and MethodsMaterials. The enterotoxin was prepared according to the method of Schantz et al. (1965). It was stored as the lyophilized powder in a deep freeze. All the solvents used in chromatography were reagent grade. Anhydrous hydrazine was prepared by distillation under reduced pressure in a nitrogen atmosphere from sodium hydroxide pellets and was stored in a desiccator in foilcovered tubes. Benzaldehyde was freshly distilled before use. Dioxane was distilled over sodium, frozen, and stored in a deep freeze. Phenyl isothiocyanate was purified by distillation in vacuo. Amino Acid AnalysisPreparation of Samples. Samples containing approximately 2 mg/ml protein were pipetted into 40-ml Pyrex freeze-drying ampoules. An equal volume of concen-* From the

Methods for the production and isolation of exfoliative toxin are described. Fermentation conditions were established under which large quantities of the crude material can be produced. Column chromatography methods, including carboxymethyl cellulose and hydroxyapatite, were utilized to purify the toxic protein. The pure toxin had a molecular weight of 26,000 as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The pure toxin is a simple protein composed of 17 amino acids. Tests for carbohydrate and for alpha- and beta-hemolysin were negative. The mean effective dose of the purified toxin was 0.5 mug per newborn mouse.