No 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...

posure of Staphylococcus aureus MF 31 to sublethal temperatures produced a temporary change in the salt tolerance and growth of the organism. After sublethal heat treatment at 55 C for 15 min, more than 99% of the viable population was unable to reproduce on media containing 7.5% NaCl. The data presented demonstrate that thermal injury, in part, occurred owing to changes in the cell membrane, which allowed soluble cellular components to leak into the heating menstruum. When the cells were placed in a limiting medium, complete recovery did not occur, regardless of the incubation time. The temperature and the pH which produced the optimal rate of recovery were similar to those described previously for the multiplication of uninjured cells. However, the rate of recovery as well as the unchanging total count during recovery indicated that cell multiplication was not a factor during the recovery process. The nutrient requirements for the complete recovery of injured cells consisted of a solution containing an energy source, such as glucose, a mixture of amino acids, and phosphate. The use of the metabolic inhibitors, penicillin, cycloserine, 2,4-dinitrophenol, and chloramphenicol, did not inhibit recovery. Actinomycin D, however, completely suppressed recovery. This result implied that ribonucleic acid synthesis was particularly involved; this inference was substantiated by radio tracer experiments. The rate at which label was incorporated in the nucleic acid fraction paralleled that of recovery and the return of salt tolerance. Although an exaggerated lag phase is a cultural response to thermal injury (21, 22), it can more accurately be described as a recovery period. As defined by Harris (15), "it is the period of 'getting back' organisms from their (sublethal) environment." This is usually accomplished by incubating the treated organisms in a nutrient environment. The use of metabolites or some type of nutrient medium for post-treatment incubation of damaged cells was first reported by Heinmetz, Taylor, and Lehman (17). They demonstrated that heattreated organisms produced higher counts on minimal media if they were previously incubated in 0.2% solutions of various metabolites. Garvie (13), on the other hand, refuted these data by demonstrating that it was "virtually impossible" to purify media with respect to a nitrogen source. ' This report is from a dissertation submitted by the senior author in partial fulfillment of the requirements for the Ph.D. degree in Food Science.

No abstract

No abstract

Staphylococcus aureus PS54 manifests no lipase (geh) activity. This is due to the insertion of bacteriophage L54a DNA into the geh structural gene. The nucleotide sequence of this 2,968-base-pair DNA fragment was determined. Lipase deduced from the nucleotide sequence is a polypeptide of 690 amino acids which extends from nucleotide 706 to 2776.Many strains of staphylococci produce a true lipase or glycerol ester hydrolase (EC 3.1.1.3). The activity of the staphylococcal lipase gene is negatively regulated by bacteriophage lysogenization, also known as lysogenic conversion (3, 21 385(1 x SSC is 0.15 M NaCI and 15 mM sodium citrate [pH 7.0]) containing 0.1% sodium dodecyl sulfate. The membrane was dried at 80°C for 10 min and then autoradiographed as we described previously (4).DNA sequence analysis. Various restriction endonuclease fragments of the geh element were cloned into the M13 bacteriophage derivatives mpl8 or mpl9 (33) and propagated in Escherichia coli JM103. DNA sequencing was carried out by the dideoxy chain termination method of Sanger et al. (22). A computer-assisted sequence analysis was carried out with Seqaid (19), a software package kindly provided by Donald J. Roufa, Kansas State University.Deletion mutagenesis. Plasmids pLI210 and pLI211 containing the 2.9-kilobase (kb) insert with the lipase gene from S. aureus PS54C (11) were linearized by endonuclease digestion at the unique BamHI site and further digested with BAL 31 exonuclease to obtain various-length deletions from either end of the 2.9-kb geh insert. The digests were then phenol extracted, ethanol precipitated, ligated with T4 DNA ligase, and transformed into competent E. coli LE392 (11). Transformants were selected on L-broth plates containing 10 ,ug of chloramphenicol per ml. A panel of plasmids with various size deletions in the geh fragment was obtained from the transformants. Size estimates were made by agarose gel electrophoresis of minilysates of clones after linearization of the plasmids by restriction enzyme digestion. Alternatively, plasmids pLI210 or pLI211 were digested with restriction enzymes to delete specific sections of DNA and then religated. RESULTSDeletion mutagenesis. We reported earlier (11) that plasmids pLI210 and pLI211 carry a 2,968-base-pair (bp) DNA fragment containing the lipase gene (geh) of S. aureus PS54C which expressed lipase activity both in E. coli and S. aureus. To further localize the geh gene, various plasmids containing deletions at either end of the 2.9-kb fragment were generated. These deletions are schematically shown in Fig. 1 along with an indication of the effect of the deletion of lipase activity. Up to 500 bp could be removed from the left end of the fragment and up to 80 bp could be removed from the right end without influencing activity. Larger deletions at either end of the insert resulted in loss of enzymatic activity.ONA sequence of the geh gene. The strategy used for nucleotide sequencing is shown in Fig. 2. Each restriction fragment was subcloned into bacteriophages M13 mpl8 or ...

The DNA encoding the exfoliative toxin A gene (eta) of Staphylococcus aureus was cloned into bacteriophage Xgtll and subsequently into plasmid pLI50 on a 1,391-base-pair DNA fragment of the chromosome. Exfoliative toxin A is expressed in the Escherichia coli genetic background, is similar in length to the toxin purified from culture medium, and is biologically active in an animal assay. The nucleotide sequence of the DNA fragment containing the gene was determined. The protein deduced from the nucleotide sequence is a polypeptide of 280 amino acids. The mature protein is 242 amino acids. The DNA sequence of the exfoliative toxin B gene was also determined. Corrections indicate that the amino acid sequence of exfoliative toxin B is in accord with chemical sequence data.The exfoliative toxins A and B (ETA and ETB) of Staphylococcus aureus are the causative agents of staphylococcal scalded-skin syndrome (11). They possess the same biological activity (12), but they are immunologically distinct proteins (11, 12). The two forms also differ in amino acid composition, amino acid sequence, and heat resistance (13). In addition, eta is expressed from the chromosome, whereas etb is of plasmid origin (14,21,29).To study the mode of action and the molecular properties of these two proteins, we set out to clone and sequence both genes. Recently, we reported the cloning and sequencing of the etb gene (9, 10). In this communication, we report the cloning and DNA sequence of eta. O'Toole and Foster (17) also reported the cloning of eta and in the accompanying paper (18) report their sequence of the gene. Our data and their report contain identical DNA sequence determinations as well as general conclusions regarding the expression of the eta gene. eta is expressed in the Escherichia coli background and is biologically active in the neonatal mouse assay. Furthermore, the sequence of the protein deduced from the DNA sequence is identical to the published sequence of the N-terminal region of the ETA molecule (12). MATERIALS AND METHODSBacterial strains and plasmids. S. aureus UT0002, a phage group II staphylococcal scalded-skin syndrome clinical isolate (20), was used as the source of library DNA. E. coli Y1090 was used in the Protoclone system (Promega Biotec Co., Madison, Wis.) as a recipient for bacteriophage Xgtll packaging carried out according to the manufacturer's instructions. E. coli LE392 was used for transformation and propagation of plasmids and cloned DNA (16).* Corresponding author.

No abstract