We have cloned the Staphylococcus aureus entB gene in Escherichia coli, using pBR322 as the vector plasmid; however, no detectable staphylococcal enterotoxin B (SEB) was produced by the E. coli clones. When the entB gene was placed downstream from the strong X phage promoter, PR9SEB was synthesized at readily detectable levels in E. coli. Interestingly, mature SEB was almost exclusively present in the cytoplasmic fraction. The SEB precursor was found associated with the cell membrane. The entB gene was introduced back into S. aureus, and the clones were shown to produce SEB. The entB gene has been located to a 2.1-kilobase-pair region. Maxam-Gilbert sequencing of a part of the entB gene yielded a DNA sequence that corresponds to the known amino acid sequence of SEB. Southern hybridization experiments showed that the entB gene was present on identical restriction fragments in the chromosomes of SEB-producer strains. The entB gene is absent from SEB-nonproducer strains.Staphylococcal enterotoxins are exoproteins produced by certain strains in culture media and in foods (1). These toxins are the causative agents of staphylococcal food poisoning. Staphylococcus aureus enterotoxins have been classified into five serological groups, A, B, C, D, and E (1, 2). Staphylococcal enterotoxin B (SEB) has been purified and studied in detail by several groups (3-7). SEB consists of a single polypeptide chain and has a molecular weight of 28,500. The complete amino acid sequence of SEB has been reported by Bergdoll's group (4). SEB is synthesized as a precursor, processed, and transported across the membrane to give the mature extracellular toxin (8).A number of recent studies have attempted to identify the SEB gene (entB) (9-14). Several SEB-producing (SEB+) strains, such as DU4916, 592, and COL, carry a 26-kilobasepair (kb) penicillin resistance plasmid (pSN3) and a 4.4-kb tetracycline resistance plasmid (pSN1). These plasmids are not involved in SEB production (11). Strains DU4916 and 592 carry an additional 1.3-kb plasmid, pSN2, whereas strains COL and S6 do not carry this plasmid (10)(11)(12)15). Studies with pSN2-negative, SEBW strains have clearly demonstrated that the entB gene is chromosomal (11,12). In pSN2-positive, SEB+ strains, there is contradictory evidence as to the role of this plasmid in SEB production. Previous studies in our laboratory showed that the pSN2 plasmid does not carry the entB gene and is not involved in SEB production (14). However, experiments involving transformation and protoplast fusion techniques performed by other investigators have suggested that pSN2 provides regulatory functions essential for SEB synthesis (12,13,15). Transformation, transduction, and mutation analyses have suggested that the entB gene is structurally unstable and possibly a mobile genetic element (9-13).In this communication we describe the cloning and expression of the entB gene in Escherichia coli and S. aureus. MATERIALS AND METHODSBacterial Strains. The bacterial strains used in this study are described in Ta...