Insertion of the erythromycin-resistance transposon Tn551 into the Staphylococcus aureus chromosome at a site which maps between the purB and ilv loci has a pleiotrophic effect on the production of a number of extracellular proteins. Production of alpha, beta and delta hemolysin, toxic shock syndrome toxin (TSST-1) and staphylokinase was depressed about fifty-fold while protein A production was elevated twenty-fold. Hybridization analysis showed that the defect in expression of TSST-1 and alpha hemolysin was at the transcriptional level. Inability of the mutant strain to express either a cloned TSST-1 gene or the chromosomal gene indicates that the transposon has inactivated a trans-active positive control element. This element has been designated agr for accessory gene regulator.

A 1.5-kilobase-pair fragment of DNA that contains the lysostaphin gene from Staphylococcus simulans and its flanking sequences has been cloned and completely sequenced. The gene encodes a preproenzyme of Mr 42,000.The NH2-terminal sequence of the preproenzyme is composed of a signal peptide followed by seven tandem repeats of a 13-amino acid sequence. Conversion of prolysostaphin to the mature enzyme occurs extracellularly in cultures of S. simulans and involves removal of the NH2-terminal portion of the proenzyme that contains the tandem repeats. The high degree of homology of the repeats suggests that they have arisen by duplication of a 39-base-pair sequence of DNA. In S. simulans, the lysostaphin gene is present on a large ,B-lactamase plasmid.Lysostaphin is a cell wall-degrading enzyme secreted by a single known strain of Staphylococcus simulans (NRRL B-2628) isolated by Schindler and Schuhardt (1,2). The enzyme lyses practically all known staphylococcal species but it is inactive against bacteria of all other genera (1, 3, 4). Although its catalytic properties are not well-characterized, lysostaphin apparently hydrolyzes polyglycine cross-links present in the peptidoglycan of the staphylococcal cell wall (5). The enzyme is a monomer of 000 and is reported to contain zinc (6). Lysostaphin production occurs in stationary-phase cultures grown under certain conditions and appears to be coordinated with production of other extracellular enzymes, including a protease and a hexosaminidase (7). Producing cultures are resistant to the enzyme, while cultures grown under at least some nonproducing conditions are sensitive (8). It is not clear whether resistance to lysostaphin in S. simulans results from the action of an immunity product(s) or whether it occurs naturally under certain conditions. Alterations in sensitivity to the enzyme may be due to changes in the amino acid composition of the peptidoglycan (8, 9).In this paper, we show that the lysostaphin gene is present on a large penicillinase plasmid and encodes a preproenzyme of Mr ==42,000. Conversion of prolysostaphin to the mature enzyme occurs extracellularly in cultures of S. simulans and involves removal of the NH2-terminal portion of the proenzyme, which contains seven tandem repeats of a 13-amino acid sequence. MATERIALS AND METHODSMaterials. Restriction enzymes, T4 DNA ligase, Escherichia coli DNA polymerase, and ribonuclease were from Boehringer Mannheim; M13 pentadecamer primer was from New England Biolabs; goat antibodies to rabbit IgG were from Miles-Yeda (Rehovot, Israel); calf intestine alkaline phosphatase and 5-bromo-4-chloroindolyl phosphate were from Sigma; and lysostaphin was from Mead Johnson.Preparation of DNA. S. simulans grown to midlogarithmic phase on 0.5 liter of CAA medium (8) was harvested by centrifugation, washed with 50 mM Tris.HCl/5O mM EDTA, pH 7.8, and suspended in 100 ml of this buffer containing lysostaphin (50 gg/ml) and lysozyme (0.5 mg/ml). After 2 hr at 37TC, Pronase (1 mg/ml) and NaDodSO4 (0.6%) were added and ...

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