A new group of serotype F bacteriophages of Staphylococcus aureus has been found which mediates the simultaneous triple-lysogenic conversion of enterotoxin A, staphylokinase and beta-lysin. The phages were recovered fro methicillin-resistant strains of S. aureus isolated in Irish hospitals between 1971 and 1988 and from strain PS42-D, which has been used as the propagating strain for the S. aureus typing phage 42D since before 1965. The molecular mechanism of triple conversion mediated by three of these phages was determined by molecular cloning, restriction endonuclease site mapping and hybridization analysis, and compared with the mechanism of beta-lysin and staphylokinase conversion mediated by the serotype F, double-converting phase phi 13. THe genetic determinants mediating expression of enterotoxin A (entA) and staphylokinase (sak) were cloned from the DNA of the triple-converting phage and expression of the cloned determinants detected in Escherichia coli and S. aureus. The entA and sak determinants were closely linked in the phage DNA adjacent to the phage attachment site (attP) in each case and furthermore, the sak determinant of phage phi 13 was also located near its attP. The restriction maps of the entA-, sak- and attP-containing DNA regions of the three triple-converting phages were very similar to each other and to the corresponding sak- and attP- containing DNA region of phage phi 13. Hybridization analysis using a cloned beta-lysin determinant (hlb) and cloned attP-containing DNA fragments as probes demonstrated that beta-lysin conversion mediated by the triple-converting phages and phage phi 13 was caused by insertional inactivation of the chromosomally encoded hlb determinant by orientation-specific integration of phage DNA following lysogenization.

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Comparison of hemolytic activity and chromate-releasing activity of partially purified preparations of staphylococcal a-toxin indicated the presence of a lytic factor other than a-toxin. This lytic release factor (RF) was isolated from the preparations and was shown to be active against both lipid spherules and erythrocytes. Heat-purified a-toxin (HP a-toxin) disrupted spherules, with the formation of fragments which always showed the presence of ring structures similar in dimensions (ca. 90 A) to pure a 12S-toxin. The interaction of HP a-toxin with spherules was accompanied by loss of hemolytic activity and adsorption of toxic protein. The a 12S-toxin, although only weakly hemolytic, was shown to be lytic for spherules. An a 12S-free toxin rapidly disrupted spherules, with formation of fragments with attached rings similar in dimensions to the a 12S molecule. Lipid monolayer experiments showed that HP a-toxin could penetrate lipid monolayers by virtue of a hydrophobic interaction. Effects of HP a-toxin on rabbit and human erythrocyte ghosts were similar to its effects on spherules, in that rings appeared on membrane fragments. Toxin-lysed rabbit erythrocytes showed similar rings on the resulting membrane fragments. However, rings were not seen on toxin-treated rabbit erythrocytes in the prelytic lag phase; this result and the fact that human erythrocytes are largely insensitive to a-toxin were interpreted as evidence against a lytic mechanism involving ring formation as the primary event. Rings were interpreted as toxin polymers similar to a 12S molecules, formed from specifically orientated active toxin molecules at the surface of lipid structures. Possible mechanisms for toxin lysis of spherules and erythrocytes are discussed.

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PLATE VTHE haemolytic activity of Escherichia coli has been investigated by only a few authors. Although there are many reports in the literature about haemolytic E. coli, most authors have merely noted that haemolytic strains were isolated. Lovell and Rees (1960) and Smith (1963) first demonstrated the production of an extracellular haemolysin (designated a-haemolysin by Smith) in an alkaline meat-extract broth. Smith (1963) showed also that a cell-associated haemolysin (designated ,6-haemolysin) was produced by some strains. Other investigators (Snyder and Koch, 1966; Snyder and Zwadyk, 1969; Short and Kurtz, 1971) observed extracellular haemolytic activity when a strain of serogroup 0 6 was grown in chemically defined medium.There is little information about the purification of E. coli a-haemolysin. Zwadyk and Snyder (1971), like Lovell and Rees (1960), were unable to recover haemolytic activity after dialysis of ammonium-sulphate precipitates. Short and Kurtz (1971) found that two peaks of haemolytic activity appeared after chromatography of culture filtrates on Sephadex G-200 and Sepharose 6B. However, they gave no indication of the extent of purification and the activity of their starting material was low.Only one author, Smith (1963), has investigated the biological properties of a-haemolysin. He observed that the toxin was active against a range of erythrocyte species, that large amounts of haemolysin were required to kill mice and rabbits after intravenous injection, and that haemolytic culture filtrates did not cause dermonecrosis in the skin of rabbits or guinea-pigs. The haemolysin was unreactive after intragastric administration to rabbits.In the present study we have attempted to clarify some of the deficiencies of previous work. Our main objectives were as follows: (a) to produce large amounts of high-titre a-haemolysin, (b) to purify and characterise this agent, and (c) to investigate the biological properties of a-haemolysin in preparations with high specific activities. In an accompanying paper, the kinetics of erythrocyte lysis by a-haemolysin are reported (Rennie, Freer and Arbuthnott, 1974).