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The gene (entD) encoding staphylococcal enterotoxin D (SED) has been located on a 27.6-kilobase penicillinase plasmid designated pIB485. This plasmid was present in all SED-producing strains tested. The entD gene was cloned on a 2.0-kilobase DNA fragment and was expressed in Escherichia coli. Sequence analysis of this fragment revealed an open reading frame that encoded a 258-amino-acid protein that possessed a 30-amino-acid signal peptide. The 228-amino-acid mature polypeptide had a molecular weight of 26,360 and contained a high degree of sequence similarity to the other staphylococcal enterotoxins. S1 nuclease mapping showed that transcription of entD was initiated 266 nucleotides upstream from the translation start codon. The entD gene was also shown to be activated by the staphylococcal regulatory element known as agr. (22), is thought to be a defective bacteriophage or an integrated plasmid (22). A large penicillinase plasmid containing entB and the gene (entC) encoding staphylococcal enterotoxin C1 (SEC1) was also reported (1), but this appears to be a unique isolate. Finally, the gene (entE) encoding staphylococcal enterotoxin E (SEE) has recently been analyzed and proposed to be associated with a defective bacteriophage (13).To study staphylococcal enterotoxin D (SED), we cloned and sequenced its structural gene (entD) and the flanking DNA. entD was shown to reside on a large penicillinase plasmid designated pIB485. The deduced amino acid sequence of SED shows that it is highly related to the other enterotoxins. In addition, an analysis of the 5'-flanking sequence by S1 nuclease mapping revealed that transcription of the entD gene in Staphylococcus aureus is initiated 266 bases upstream from the translation start codon. MATERIALS AND METHODSBacterial strains and growth conditions. All bacterial strains are listed in Table 1 Ethidium bromide curing. Plasmids were eliminated by growth in ethidium bromide as described previously (15). Cells were inoculated into 2.5 ml of brain-heart infusion (BHI) broth containing increasing concentrations of ethidium bromide ranging from 3.2 x 10-6 to 2.5 x 10-5 M. The cultures were agitated for 18 to 24 h in the dark at 37°C. The highest concentration of ethidium bromide in which bacteria grew was used to plate cells onto nonselective BHI agar. Ensuing colonies were replica plated to BHI agar and BHI agar containing penicillin (5 p.g/ml); clones were scored for the loss of penicillin resistance.Hybridization analysis. Plasmid DNA (1.0 p.g) prepared from 12 different SED-producing strains was digested with 1.0 U of the restriction endonuclease EcoRI at 37°C for 1 h. Electrophoresis of the digested DNA (0.2 p.g) was carried out in a 0.6% agarose-TBE (0.089 M Tris, 0.089 M boric acid, 2.5 mM EDTA) gel at 6 V/cm. The DNA fragments were electrophoretically transferred to GeneScreen Plus at 6 V in blotting buffer (10 mM Tris [pH 7.8], 5 mM sodium acetate, 0.5 mM EDTA). The membrane was then incubated in hybridization buffer (1% sodium dodecyl sulfate [SDS], 1 M NaCl, 10% dext...

posure of Staphylococcus aureus MF 31 to sublethal temperatures produced a temporary change in the salt tolerance and growth of the organism. After sublethal heat treatment at 55 C for 15 min, more than 99% of the viable population was unable to reproduce on media containing 7.5% NaCl. The data presented demonstrate that thermal injury, in part, occurred owing to changes in the cell membrane, which allowed soluble cellular components to leak into the heating menstruum. When the cells were placed in a limiting medium, complete recovery did not occur, regardless of the incubation time. The temperature and the pH which produced the optimal rate of recovery were similar to those described previously for the multiplication of uninjured cells. However, the rate of recovery as well as the unchanging total count during recovery indicated that cell multiplication was not a factor during the recovery process. The nutrient requirements for the complete recovery of injured cells consisted of a solution containing an energy source, such as glucose, a mixture of amino acids, and phosphate. The use of the metabolic inhibitors, penicillin, cycloserine, 2,4-dinitrophenol, and chloramphenicol, did not inhibit recovery. Actinomycin D, however, completely suppressed recovery. This result implied that ribonucleic acid synthesis was particularly involved; this inference was substantiated by radio tracer experiments. The rate at which label was incorporated in the nucleic acid fraction paralleled that of recovery and the return of salt tolerance. Although an exaggerated lag phase is a cultural response to thermal injury (21, 22), it can more accurately be described as a recovery period. As defined by Harris (15), "it is the period of 'getting back' organisms from their (sublethal) environment." This is usually accomplished by incubating the treated organisms in a nutrient environment. The use of metabolites or some type of nutrient medium for post-treatment incubation of damaged cells was first reported by Heinmetz, Taylor, and Lehman (17). They demonstrated that heattreated organisms produced higher counts on minimal media if they were previously incubated in 0.2% solutions of various metabolites. Garvie (13), on the other hand, refuted these data by demonstrating that it was "virtually impossible" to purify media with respect to a nitrogen source. ' This report is from a dissertation submitted by the senior author in partial fulfillment of the requirements for the Ph.D. degree in Food Science.

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