The synthesis and secretion of the toxic exoprotein alpha-haemolysin of E. coli PM152 is coded by the transmissible plasmid pHly152 (41 x 10(6) dalton) as shown by the transformation of the plasmid DNA and the isolation of mutants that are specifically altered in the synthesis and transport of haemolysin. These mutants were obtained by chemical mutagenesis and insertion of the ampicillin transposon (Tn3) into pHly152. Tn3 transposition was also used for the identification and the location of the cistrons on pHly152 essential for haemolysis. The EcoRI and HindIII fragments of the haemolytic plasmid pHly152 were cloned and used for the complementation of the haemolysis negative Tn3 insertion mutants. A DNA segment of 3.2 x 10(6) dalton could be thus identified which consists of at least three clustered cistrons necessary for haemolysis. Two of these cistrons are required for the formation of active haemolysin. At least one other cistron seems to be involved in the secretion of active haemolysin through the outer membrane of E. coli. The gene products determined by these cistrons were identified in minicells of E. coli. Their molecular properties were determined and their possible function in the formation and secretion of haemolysin will be discussed.

Hemolytic Escherichia coli cells were found to synthesize and secrete significant amounts of hemolysin into a mineral salt-glucose medium containing hemoglobin. The release of de novo-synthesized hemolysin was stopped in the presence of energy metabolism inhibitors such as 2,4-dinitrophenol, sodium azide, or potassium cyanide, resulting in an accumulation of intracellular hemolysin. A similar effect was observed in the presence of procaine, a neuroactive drug which inhibits the processing of exoproteins. Small amounts of hemolysin were secreted into the medium within approximately 10 min of inhibition of protein synthesis by chloramphenicol. This represented the final release of preformed periplasmic hemolysin en route to secretion through the outer membrane and was not caused by adsorption of extemal hemolysin to the cell surface. This secretion was not energy dependent but was inhibited above pH 8 and at low temperatures (10 to 200C). We concluded that two transport processes are involved in hemolysin secretion. De novo-synthesized hemolysin is extruded by an energy-dependent process through the cytoplasmic membrane and probably requires processing. In the periplasmic space a small internal pool ofpreforned hemolysin is accumulated temporarily before being transported through the outer membrane. Release of hemolysin through the outer membrane does not require energy or de novo protein synthesis.Bacteria are able to produce a large variety of extracellular toxic proteins (1, 3). Among these, the cytolytic toxins or cytolysins (1) have attracted much attention as pathogenic factors, particularly in gram-positive bacteria (1). These proteins cause damage to membranes by mechanisms which are only partially understood (1). A related exotoxin, hemolysin, is also produced by some Escherichia coli strains (21,22). Recent studies in our laboratory have shown that its production by E. coli strain PM152 (8) is controlled by a cluster of three plasmid-borne cistrons determining not only the synthesis of biologically active hemolysin but also a function(s) required for its secretion through the outer membrane (15). There is now evidence that this gene cluster is common to many if not all he-

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Haploid plants were cultured from ‘Kentucky 31’ tall fescue (Festuca arundinacea Schreb.). Field‐grown plants were cut at the soil surface when the panicles showed about 3 cm above the flag leaf. The plants were kept in a flask of tap water and preconditioned in darkness at 5 C before cultures were made. Florets attached to about 2.5 cm of panicle tissue were cultured on modified Murashige and Skoog medium containing 2 mg of 2,4‐dichlorophenoxyacetic acid (2,4‐D)/liter. The panicle segments served as nurse tissue. Loose proliferations of cells emerged from several anthers after about 5 weeks. Some plantlets became apparent at about 7 weeks. More than 30 green plantlets formed. There were no albino plantlets. Examination of metaphase cells in root and shoot tips showed that 22 of the 23 plants examined had the haploid chromosome number, n = 21. The plants grew vigorously after transfer to pots in a plant growth chamber.