Some Factors Affecting Production and Assay of Escherichia coli Haemolysins

Abstract

SUMMARYThe amount of haemolysin produced by Escherichia coli grown under various gas phases was determined by the amount of growth obtained under these conditions. Calcium or strontium was required for activation of haemolysin. The haemolytic reaction was stopped by sodium citrate. The loss of haemolytic activity after incubation with trypsin and chymotrypsin indicated that at least the active group of the haemolytic molecule is protein or a peptide. I N T R O D U C T I O NSeveral studies have shown discordant results about the production and assay of Escherichia coli haemolysins. This paper attempts to clarify the effects of various gas phases on the production of haemolysin and to demonstrate a cation requirement for activation of the haemolysin. METHODS Organism.A haemolysin-producing strain of Escherichia coli type 06 (Iowa Stock Culture, ISC, no. 447), originally obtained from a patient with pyelonephritis, was used. A standard inoculum was obtained as previously described (Snyder & Koch, I 966).Measurement of haemolytic activity. Total haemolysin content of the cultures was obtained by two-fold dilution of whole cultures. Filterable haemolysin was determined in the same manner with culture filtrates passed through a Millipore filter (0.45 mp porosity). The amount of haemolysin/ml. culture or filtrate was determined as previously described (Snyder & Koch, 1966).Measurement of growth andpH value. Turbidometric measurements of growth were obtained with a Spectronic 20 spectrophotometer at a wavelength of 625 mp. The pH value of the culture was determined with a Beckman Zeromatic II pH meter. Preparation of media.Beef heart for infusion medium (BHI) was prepared from Difco Beef Heart for infusion as described by the manufacturer. When glucose was added to the media, I ml. of a Millipore-filtered solution 20 % (w/v) glucose was added to IOO ml. autoclaved media. In media buffered with phosphate, 0.23 g. KBHPOl and 0.078 g. KHBPOI were added to each IOO ml. of medium before sterilization.Alkaline meat-extract broth was prepared as described by Smith (1963).Incubation of cultures. Anaerobic growth was obtained by using Brewer jars. The jars were flushed with nitrogen three times and filled with hydrogen, a palladium catalyst being used for removal of residual oxygen. Incubation in a COB atmosphere

E. coli <FONT FACE=Symbol>a</FONT>-hemolysin: a membrane-active protein toxin

“…Other divalent cations (e.g., Mg 2+ , Mn 2+ , Zn 2+ , Cd 2+ ) do not support HlyA activity. The data are in accordance with early observations of Ca 2+ requirements (57)(58)(59). However, other reports appeared to suggest that Ca 2+ was not required for the hemolytic action (60)(61)(62).…”

Production and properties of haemolysins from clinical isolates of the Proteeae

“…The haemolytic activity of E. coli strains is generally calcium dependent (Bamforth and Dudgeon, 1952;Snyder and Zwadyk, 1969;Short and Kurtz, 1971 ; and heat labile (Love11 and Rees, 1960;Smith, 1963;Snyder and Koch, 1966;Jorgensen et al, 1976). Since, however, the heat lability of the haemolysin is influenced by calcium-being more stable in its absence (Snyder and Koch, 1966)-it is easy to understand why the calciumindependent haemolysin of Proteus spp.…”

The hemolysin of escherichia coli

“…These studies collectively indicated that the hemolytic reaction involves an initial lag phase followed by a phase of hemolysis. A requirement for calcium ions for the hemolytic process was reported (55,59,61). Based on a study of hemolysis kinetics, it was concluded that lysis followed one-hit characteristics (38).…”