Functional analysis of the escherichia coli haemolysin and its export mechanism.

Haemolytic activity produced by E.coli strains carrying the recombinant plasmid pLG570 was investigated. This plasmid contains a haemolytic determinant subcloned from the wild-type E.coli LE2001, isolated from a human urinary tract infection. Quantitative assays for haemolytic activity and for the separation of soluble subcellular compartments were developed. These revealed the absence of any detectable periplasmic pool of haemolytic activity in strains expressing the intact haemolytic determinant. Restriction mapping the haemolytic determinant enabled the identification, subcloning and independent expression from separate plasmids of the hly genes. The production of secreted haemolytic activity was shown to require the expression of four contiguous genes, hlyC, hlyA (the haemolysin polypeptide), hlyB and hlyD. Quantitative assays of haemolytic activity were undertaken on subcellular fractions of strains expressing subsets of hly genes. This enabled the following observations. HlyC is required for activation, but not for secretion, of HlyA. Both HlyB and HlyD are required for secretion of HlyA. Absence of any combination of HlyC, HlyB and/or HlyD did not result in periplasmic accumulation of HlyA. Thus it was concluded that the secretion of haemolysin involves tightly coupled translocation through both bacterial membranes without release into the periplasm. Identification of a "nucleotide binding site" in HlyB implied that at least part of this protein was exposed at the cytoplasmic face of the bacterial inner membrane. A topogenic domain at the C-terminal of HlyA was identified. Truncation of hlyA, resulting in the deletion of 27 amino acids from the C-terminal of HlyA, was shown to produce a haemolytic polypeptide which was not secreted. In addition, HlyA derived polypeptides, expressed from subcloned 3' portions of the hlyA gene, were shown to be secreted. Thus it was concluded that the C-terminal 113 amino acids of HlyA apparently include all the information required for interaction with the hly secretion machinery.