Genetic and molecular analyses of the gene encoding staphylococcal enterotoxin D

Abstract

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...

The Staphylococcal Enterotoxins

“…The gene for SEA is bacteriophage encoded (Betley and Mekalanos, 1985). In contrast, the gene for SED is encoded by an antibiotic-resistance plasmid (Bayles and Iandolo, 1989). The sequence of the gene that encodes SEJ is on the same plasmid as that for SED (Zhang et al, 1998).…”

Two‐Dimensional Analysis of Exoproteins of Methicillin‐Resistant Staphylococcus aureus (MRSA) for Possible Epidemiological Applications

“…The multiple spots of toxins may be generated in a similar way by the action of own proteases, although we have had no evidence yet. A potent virulence factor, TSST-1, was identified as spot F straight down from the major SEC 3 (45). Collagen adhesin of MF330 was closer to the latter one.…”

“…The first group of exoproteins are alpha-toxin (14), beta-hemolysin (47), gammahemolysin (46), delta-hemolysin (20), and phospholipase C (9). The second group are toxic shock syndrome toxin-1 (TSST-1) (29), enterotoxins (3,4,8,15,22,52), protein A (34), and others (6,31,32,50,53), and the third group contains clumping factor (37) and others (7,12,23,45). More than 30 of these exoproteins that are significantly linked to pathogenesis have been purified to homogeneity, and the genes encoding these exoproteins have been cloned and sequenced (5,48 Abstract: We applied two-dimensional gel electrophoresis (2-DE) to the total exoproteins secreted from pathogenic MRSA strains and identified major protein spots by N-terminal amino acid sequence analysis.…”

Genetic regulation of fatty acid modifying enzyme from Staphylococcus aureus

“…Other investigators have demonstrated the importance of global regulators in controlling the expression of several staphylococcal extracellular proteins [8][9][10][11][12][13][14][15][16][17][18][19][20]. Two of these regulatory elements, the accessory gene regulator (Agr) and the staphylococcal accessory regulator (Sar), control expression of exoproteins by transcriptional control [ 13,141.…”