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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.

Three broad-host-range plasmids (pRK290, pSa4 and pKT230) and one native Pasteurella haemolytica plasmid (pPH33) were used in transformation experiments with P. haemolytica strains T179 (serotype A1), Y216 (serotype A2) and its capsular-deficient variant Y216/NS1. No transformants were detected with either heat-shock or freeze-thaw techniques. However, by electroporation, all P. haemolytica strains were transformed by pPH33 but not by pRK290 or pSa4. The highest frequency obtained was 91 x 10(4) transformants per microgram of pPH33 DNA with P. haemolytica strain Y216/NS1. Although pPH33 itself was non-transmissible by conjugation, it could be mobilized from Escherichia coli, using the transfer function of the IncP plasmid pRK2013, into P. haemolytica at a frequency of 0.3-2.2 x 10(-3) per recipient cell.

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At least three different forms of staphylococcal a-toxin have been shown to exist: soluble active a-toxin (a 3S), soluble inactive a-toxin (al2s), and insoluble inactive aggregate. Aggregation to the insoluble, biologically inactive form could be induced by brief heating to 60 C. The aggregate was dissociated by treatment with 8 M urea with reappearance of biological activity. Subsequent removal of urea by dialysis resulted in some spontaneous reaggregation to the insoluble state. The supernatant

1. A method is described for the isolation of pure mesosomal membrane fractions from Micrococcus lysodeikticus. 2. Plasmolysis of cells, before wall digestion, was necessary for effective mesosome release. 3. The effect of mild shearing forces, temperature and time upon the release of mesosomal membrane from protoplasts was investigated. 4. The optimum yield of mesosomal membranes from stable protoplasts was achieved at 10mm-Mg(2+). 5. Mesosomal membrane vesicle fractions prepared at differing Mg(2+) concentrations above 10mm were similar in chemical composition. 6. Comparison of the properties of peripheral and mesosomal membrane fractions revealed major differences in the distribution of protein components, membrane phosphorus, mannose and dehydrogenase activities between the two fractions. 7. Only cytochrome b(556) was detected in mesosomal membranes, whereas peripheral membranes contained a full complement of cytochromes. 8. Preliminary investigations suggested the localization of an autolytic enzyme(s) in the mesosomal vesicles. 9. The anatomy of mesosomal and peripheral membrane have been compared by the negative-staining and freeze-fracture technique. 10. The results are discussed in relation to a plausible role for the mesosome.

PLATE VITECHNIQUES for studying the mode of action of bacterial toxins at the molecular level are fast becoming valuable and necessary tools for the determination of the role of these agents as virulence factors in diseases of man and animals. The mechanism of action of Escherichia coli haemolysin is poorly understood. Although enough evidence now exists to conclude that calcium ions are required for haemolytic activity, only two groups of investigators have attempted to study the kinetics of erythrocyte lysis by E. coli a-haemolysin.The findings of Zwadyk and Snyder (1971) and Short and Kurtz (1971) indicated that lysis of sheep erythrocytes was dependent on haemolysin concentration, pH and temperature. Less haemolysis was observed as the concentration of red cells (RBC) was increased. It has been suggested that a complex consisting of haemolysin, calcium ions and RBC must be maintained until the lytic event (Short and Kurtz, 1971). These authors found that the addition of ethylenediaminetetraacetic acid (EDTA) at any time during the haemolytic reaction prevented subsequent haemolysis. They also noted that neither reducing agents nor lecithin or cholesterol affected a-haemolysin.Both groups of workers used methods that required centrifugation of reaction mixtures at different times, followed by spectrophotometric estimation of haemoglobin. In these processes there are unavoidable delays during which haemolysin-affected cells may be lysed.In this study, the turbidity of reaction mixtures was monitored continuously at 650 nm in a controlled-temperature spectrophotometer. By this technique it was possible to make rate measurements on haemolysis curves. MATERIALS AND METHODSHaemolysin production and purification. The preparations of E. coli u-haemolysin used in this study were produced in glucose-nutrient-broth medium and were purified by the three-stage method of Rennie and Arbuthnott (1974). Because it was more readily available, stage-I1 u-haemolysin was used for most assays. However, in each experiment, purified stage-III a-haemolysin was tested to confirm the results obtained with less pure preparations. (Rennie and Arbuthnott, 1974). Kinetic studies were carried out with silica cells of 1-cm light path in a controlled-temperature Unicam SP800 spectrophotometer, to which was attached a Unicam SP22 chart recorder (Pye-Unicam, Cambridge, England) set at a five-times multiplication factor. The spectrophotometer was adjusted so that extinction (E) was monitored at a constant wavelength of 650 nm.A 0.7% suspension of sheep erythrocytes (SRBC) was made in Veronal buffer, pH 7.3 (Cruickshank, 1969), containing lOmM CaCl2 (VC buffer). The spectrophotometer and chart recorder were then calibrated on a linear scale with the SRBC suspension. The contents of a 1-cm cell, containing 1-4 ml of VC buffer and 0.1 ml of 0.7% SRBC, were mixed by inversion and read at E 6 5 0 . The zero controls of the spectrophotometer and chart recorder were adjusted if necessary such that the E650 was 0.40. This represented 100% erythrocyte ...