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...
Anthrax is a fatal infection of humans and livestock that is caused by the gram-positive bacterium Bacillus anthracis. The virulent strains of B. anthracis are encapsulated and toxigenic. In this paper we describe the development of a PCR technique for identifying spores of B. anthracis. Two 20-mer oligonucleotide primers specific for the capB region of 60-MDa plasmid pXO2 were used for amplification. The amplification products were detected by using biotin-and fluorescein-labeled probes in a novel dual-probe hybridization format. Using the combination of PCR amplification and dual-probe hybridization, we detected two copies of the bacterial genome. Because the PCR assay could detect a minimum of 100 unprocessed spores per PCR mixture, we attempted to facilitate the release of DNA by comparing the effect of limited spore germination with the effect of mechanical spore disruption prior to PCR amplification. The two methods were equally effective and allowed us to identify single spores of B. anthracis in PCR mixtures.
Polymerase chain reactions (PCRs) for the capsule and oedema factor genes of Bacillus anthracis were used to assess methods for detecting B. anthracis spores. Untreated spore preparations were found to contain significant amounts of extracellular template DNA which probably accounted for observed amplification from these preparations without spore lysis. Germination of spores with suitable media allowed the detection of less than 10 spores in a PCR test. Mechanical disruption of spores with glass or zirconia beads yielded similar results to germination but in a much shorter time. The techniques described should improve the detection by PCR of B. anthracis and other sporulating bacteria.
The chromosomal location of the enterotoxin B gene in Staphylococcus aureus is unknown. Southern hybridization analysis of the chromosomal DNA from several enterotoxin B (SEB)-producing strains has shown that at least 26.8 kilobases (kb) of DNA is associated with the enterotoxin B gene (entB). We have found that one end of the entB element is located approximately 1.5 kb downstream of the entB gene. The chromosomal region adjacent to this end of the entB element was found to be homologous in several SEB-producing (SEB+) and SEB-nonproducing (SEB-) S. aureus strains. The chromosomes of all the SEB+ strains studied were homologous for at least 24 kb upstream of the entB gene. Some naturally occurring SEB-strains lacked the entire entB element, while others showed variable homology to the region upstream of the entB gene. These data suggest that the entB gene is part of a discrete genetic element that is at least 26.8 kb in size.Staphylococcal enterotoxins are a group of extracellular proteins that are responsible for staphylococcal food poisoning. These enterotoxins are similar in their structure and mechanism of action and are classified into six serological groups, designated A, B, C1, C2, D, and E (2). The exact mechanism of action of these toxins is not fully understood, but they probably cause vomiting and diarrhea by an emetic action on the abdominal viscera and by inhibition of water absorption in the intestine (2, 4, 31).Staphylococcal enterotoxin B (SEB) consists of a single polypeptide chain and has a molecular weight of 28,336 (14, 17). A precursor to SEB having a molecular weight of 32,000 has been identified (17,25,32). The mature toxin consists of 239 amino acid residues and shares nucleotide and amino acid sequence homology with staphylococcal enterotoxin C1 and streptococcal pyrogenic exotoxin A (15, 34). The enterotoxin B gene (entB) has been cloned from Staphylococcus aureus S6, and its nucleotide sequence was reported (17,25).Early studies on the genetics of SEB production demonstrated that the entB gene cotransduced with the markers for methicillin resistance and tetracycline resistance at a high frequency (9). In addition, cotransduction of the entB gene with a small plasmid, pSN2, was also observed (27-29). However, the entB gene is not linked to any of these resistance markers (19,30) and has been shown to be a chromosomally linked trait (25,27,28). In addition, transformation and hybridization analyses have demonstrated that pSN2 does not play any role in SEB production (18). Earlier experiments involving transformation, transduction, and mutation analysis suggest that the entB gene is structurally unstable and possibly part of a mobile genetic element such as a phage or a transposon (4,9,18,25,28,29). In this paper we present DNA hybridization data suggesting that the entB gene is part of a discrete genetic element. MATERIALS AND METHODSBacterial strains and plasmids. The bacterial strains and plasmids used in this study are listed in Table 1.Preparation of chromosomal and plasmid DNA. Ch...
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