The complete nucleotide sequence of the enterotoxin B gene from Staphylococcus aureus, as well as the 5' and 3' flanking regions, was determined. Starting from an ATG initiator codon, an open reading frame encoded the enterotoxin B precursor that consisted of 266 amino acids (Mr,31,400). The 5' terminal portion of the gene encodes a signal peptide 27 amino acids long. The deduced amino acid sequence matched, with a few exceptions, the published amino acid sequence of enterotoxin B. The structural gene was flanked on the 5' side by a promoter-like sequence and on the 3' side by a palindromic structure followed by a thymine-rich region that resembled a transcription termination signal. Downstream from the entB gene were two overlapping open reading frames corresponding to 134 and 185 amino acids in the opposite orientation. The signal sequence of the enterotoxin B precursor resembled that of other secreted proteins found in other bacteria.Staphylococcus aureus produces a number of extracellular proteins that are associated with its pathogenicity. One such group of proteins is composed of the enterotoxins that are involved in staphylococcal food poisoning (1). Staphylococcal enterotoxins are classified into six serological groups: A, B, C1, C2, D, and E. These enterotoxins have similar structural and biological properties (1).Staphylococcal enterotoxin B (SEB) has been studied in detail. SEB has been purified to homogeneity and consists of 239 amino acids in a single polypeptide chain with a molecular weight of 28,336 (5, 11). The complete amino acid sequence of SEB was determined by amino acid analysis (11). There is extensive homology between the amino acid sequence of SEB and that of enterotoxin C1, which also consists of 239 amino acids (26). A precursor to SEB having an approximate molecular weight of 32,000 (34) was identified as a membrane component of SEB-producing S. aureus strains. A SEB precursor was also identified in the membranes of Escherichia coli strains carrying the cloned enterotoxin B gene (entB) (22). The mechanism of action of SEB is not fully understood, but it probably causes vomiting and diarrhea by its emetic action on the abdominal viscera (33).Considerable interest has been directed toward an understanding of the mechanism of SEB production in S. aureus. Several studies have suggested the involvement of a small plasmid, pSN2, in SEB synthesis (6, 28-30). However, the determination of the complete nucleotide sequence of pSN2 and cloning of the entB gene have demonstrated that this plasmid is not involved in SEB production (12,22).In a previous report we have described the cloning and expression of the entB gene in S. aureus and E. coli (22). Studies on the expression of the entB gene in E. coli have shown that the gene is expressed only when transcribed from a strong E. coli promoter (22). Here we report the complete nucleotide sequence of the entB gene from S. aureus S6, including the 5' and 3' flanking regions. The structural gene consists of 798 nucleotides that encode the SEB precurso...

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...

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Dihydroxyacetone-phosphate:acyl coenzyme A acyltransferase (EC 2.3.1.42) was solubilized and partially purified from guinea pig liver crude peroxisomal fraction. The peroxisomal membrane was isolated after osmotic shock treatment and the bound dihydroxyacetone-phosphate acyltransferase was solubilized by treatment with a mixture of KCl-sodium cholate. The solubilized enzyme was partially purified by ammonium sulfate fractionation followed by Sepharose 6B gel filtration. The enzyme was purified 1200-fold relative to the guinea pig liver homogenate and 80- to 100-fold from the crude peroxisomal fraction, with an overall yield of 25-30% from peroxisomes. The partially purified enzyme was stimulated two- to fourfold by Asolectin (a soybean phospholipid preparation), and also by individual classes of phospholipid such as phosphatidylcholine and phosphatidylglycerol. The kinetic properties of the enzyme showed that in the absence of Asolectin there was a discontinuity in the reciprocal plot indicating two different apparent Km values (0.1 and 0.5 mM) for dihydroxyacetone phosphate. The Vmax was 333 nmol/min/mg protein. In the presence of Asolectin the reciprocal plot was linear, with a Km = 0.1 mM and no change in Vmax. The enzyme catalyzed both an exchange of acyl groups between dihydroxyacetone phosphate and palmitoyl dihydroxyacetone phosphate in the presence of CoA and the formation of palmitoyl [3H]coenzyme A from palmitoyl dihydroxyacetone phosphate and [3H]coenzyme A, indicating that the reaction is reversible. The partially purified enzyme preparation had negligible glycerol-3-phosphate acyltransferase (EC 2.3.1.15) activity.