Staphylococcal enterotoxins cause the intoxication staphylococcal food poisoning syndrome (3). The enterotoxins are classified by serological criteria into five major groups (A to E, referred to as SEA to SEE, respectively); SEC can be further subdivided into SEC1, SEC2, and SEC3 (3,29). Cross-reactivity has been observed between SEA and SEE and between SEB and the SECs (9,18,19,40).The protein sequences of SEA, SEB, and SEC1 and nucleotide sequences of their genes (entA, entB, and entC1, respectively) have been determined (6,7,11,12,16,33). Comparison of the amino acid sequences of SEA, SEB, and SEC1 has demonstrated that these proteins are not only related to each other but also to type A streptococcal pyrogenic exotoxin (SPE A) (6,7,14,16,44). SPE A, like the enterotoxins, is mitogenic and pyrogenic and enhances endotoxic shock (1, 3).The genes for entA and SPE A (speA) are encoded by lysogenic phages in Staphylococcus aureus and Streptococcus pyogenes, respectively (5,15,43). The relationship between these phages is not known. The entB and entC, genes are linked to the same plasmid in at least one Staphylococcus aureus strain (2). Another S. aureus strain has an entD-encoding plasmid (K. W. Bayles, and J. J. Iandolo, Abstr. Annu. Meet. Am. Soc. Microbiol., 1987, B187, p. 56).Previously, we have shown (6) that DNA isolated from SEE-producing (EntE+) S. aureus strains has homology with the entA gene. In this report, cloning and nucleotide sequence determinations of the homologous DNA demonstrated that it is the entE gene. The entE gene has 84% nucleotide sequence homology with the entA gene. Comparison of amino acid sequence data obtained from nucleotide and peptide analyses suggest that SEE is synthesized as a precursor of 257 amino acid residues (molecular weight; 29,358) and is processed to yield a mature form of 230 amino acid residues (molecular weight, 26,425). Amino acid sequence comparisons also agreed with the relationships suggested by serological data (9,18,19,40). SEE is more closely related to SEA than it is to SEB, SEC1, or SPE A. * Corresponding author. MATERIALS AND METHODSBacterial strains, plasmids, phages, and culture conditions. S. aureus MJB265 was strain RN450 that was lysogenized with the entA-converting phage FRI337-1 (5 ) contained pBR322, pMJB9, and pMJB38, respectively. pMJB9 is a pBR322 derivative that contains a 2.5-kilobase-pair (kb) HindIII fragment insert encoding a functional entA gene. pMJB38 is a pBR322 derivative that contains a 624-base-pair fragment (designated A-624) that contains only entA structural gene sequences. E. coli strains were routinely grown in LB (21) containing 100 p,g of ampicillin per ml.The propagation of E. coli phage M13 derivatives (24, 27, 42) and E. coli JF626, which was used for M13 phage propagation (obtained from Jeff Felton), has been described previously (35).DNA isolation procedures. Genomic DNA was isolated from S. aureus protoplasts (37). Staphylococcal DNA probes for hybridization reactions were obtained as follows. Phage FRI337-1 was prop...

A variant enterotoxin gene, referred to as sezA+, has been identified. Staphylococcus aureus FRI1106, a staphylococcal enterotoxin type D producer (Sed+), contained Hindlll fragments of 3.8 and 9.4 kilobase pairs (kbp) that hybridized in Southern blot analysis to a probe containing only staphylococcal enterotoxin type A structural gene sequences. Presumably, probe A-624 hybridized to the 9.4-kbp HindIll fragment because of the sequence homology between sea+ and sed+. This 9.4-kbp HindIII fragment, which was part of a staphylococcal plasmid, was isolated and ligated into an Escherichia coli plasmid vector; Sed+ E. coli recombinant clones were isolated. The 3.8-kbp HindUl fragment was shown to be part of a viable lysogenic bacteriophage, and it contained sezA+. This sezA4-containing fragment was cloned into E. coli, and its DNA sequence was determined. Examination of the nucleotide sequence revealed a 771-bp region that contained an open reading frame with 85 and 77% nucleotide and derived amino acid sequence identities with sea' and staphylococcal enterotoxin type A, respectively. This open reading frame has 83 to 50% nucleotide sequence identities with the other types of staphylococcal enterotoxin genes. sezA+ was shown to be transcribed into stable mRNA.However, the sezA' mRNA was not translated into an enterotoxinlike protein because it lacks an appropriate translation initiation codon. 1614 on August 3, 2020 by guest http://iai.asm.org/ Downloaded from