Summary
By introduction of site-specific deletions, three regions in HlyA were identified, which appear to be involved in pore formation by Escherichia coli haemolysin. Deletion of amino acids 9–37 at the N-terminus led to a haemolysin which had an almost threefold higher specific activity than wild-type and formed pores in an artificial asolectin lipid bilayer with a much longer lifetime than those produced by wild-type haemolysin. The three hydrophobic regions (DI–DIII) located between amino acids 238–410 contributed to pore formation to different extents. Deletion of DI led to a mutant haemolysin which was only slightly active on erythrocyte membranes and increased conductivity of asolectin bilayers without forming defined pores. Deletions in the two other hydrophobic regions (DII and DIII) completely abolished the pore-forming activity of the mutant haemolysin. The only polar amino acid in DI, Asp, was shown to be essential for pore formation. Removal of this residue led to a haemolysin with a considerably reduced capacity to form pores, while replacement of Asp by Glu or Asn had little effect on pore formation. A deletion mutant which retained all three hydrophobic domains but had lost amino acids 498–830 was entirely inactive in pore formation, whereas a shorter deletion from amino acids 670–830 led to a mutant haemolysin which formed abnormal minipores. The conductivity of these pores was drastically reduced compared to pores introduced into an asolectin bilayer by wild-type haemolysin. Based on these data and structural predictions, a model for the pore-forming structure of E. coli haemolysin is proposed.
Similar content being viewed by others
References
Benz R, Janko K, Boos W, Läuger P (1978) Formation of large, ion-permeable membrane channels by the matrix protein (porin) of Escherichia coli. Biochim Biophys Acta 511:305–319
Benz R, Schmid A, Wagner W, Goebel W (1989) Pore formation by the Escherichia coli hemolysin: evidence for an associationdissociation equilibrium of the pore-forming aggregates. Infect Immun 57:887–895
Bhakdi S, Mackman N, Nicaud J-M, Holland IB (1986) Escherichia coli hemolysin may damage target cell membranes by generating transmembrane pores. Infect Immun 52:63–69
Boehm DF, Welch RA, Snyder IS (1990) Domains of Escherichia coli hemolysin (HlyA) involved in binding of calcium and erythrocyte membranes. Infect Immun 58:1959–1964
Chakraborty T, Schmid A, Notermans S, Benz R (1990) Aerolysin of Aeromonas sobria: evidence for formation of ion-permeable channels and comparison with alpha-toxin of Staphylococcus aureus. Infect Immun 58:2127–2132
Eberspächer B, Hugo F, Bhakdi S (1989) Quantitative study of the binding and hemolytic efficiency of Escherichia coli hemolysin. Infect Immun 57:983–988
Eisenberg D, Schwarz E, Komaromy M, Wall R (1984) Analysis of membrane and surface protein sequences with the hydrophobic moment plot. J Mol Biol 179:125–142
Erb K, Vogel M, Wagner W, Goebel W (1987) Alkaline phosphatase which lacks its own signal sequence becomes enzymatically active when fused to N-terminal sequences of Escherichia coli haemolysin (HlyA). Mol Gen Genet 208:88–93
Felmlee T, Pellett S, Welch RA (1985) Nucleotide sequence of an Escherichia coli chromosomal hemolysin. J Bacteriol 163:94–105
Glaser P, Sakamoto H, Bellalou J, Ullmann A, Danchin A (1988) Secretion of cyclolysin, the calmodulin-sensitive adenylate eyclase-haemolysin bifunctional protein of Bordetella pertussis. EMBO J 7:3997–4004
Goebel W, Hedgpeth J (1982) Cloning and functional characterization of the plasmid-encoded hemolysin determinant of Escherichia coli. J Bacteriol 151:1290–1298
Hess J, Wels W, Vogel M, Goebel W (1986) Nucleotide sequence of a plasmid-encoded hemolysin determinant and its comparison with a corresponding chromosomal hemolysin sequence. FEMS Microbiol Lett 34:1–11
Koronakis V, Cross M, Senior B, Koronakis E, Hughes C (1987) The secreted hemolysins of Proteus mirabilis, Proteus vulgaris, and Morganella morganii are genetically related to each other and to the alpha-hemolysin of Escherichia coli. J Bacteriol 169:1509–1515
Kraig E, Dailey T, Kolodrubetz D (1990) Nucleotide sequence of the leukotoxin gene from Actinobacillus actinomycetemcomitans: homology to the alpha-hemolysin/leukotoxin gene family. Infect Immun 58:920–929
Kramer W, Drutsa V, Jansen H-W, Kramer B, Pflugfelder M, Fritz H-J (1984) The gapped duplex DNA approach to oligonucleotide-directed mutation construction. Nucleic Acids Res 12:9441–9456
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Lo RYC, Strathdee CA, Shewen PE (1987) Nucleotide sequence of the leukotoxin genes of Pasteurella haemolytica A1. Infect Immun 55:1987–1996
Ludwig A, Vogel M, Goebel W (1987) Mutations affecting activity and transport of haemolysin in Escherichia coli. Mol Gen Genet 206:238–245
Ludwig A, Jarchau T, Benz R, Goebel W (1988) The repeat domain of Escherichia coli haemolysin (HlyA) is responsible for its Ca2+-dependent binding to erythrocytes. Mol Gen Genet 214:553–561
Menestrina G (1988) Escherichia coli hemolysin permeabilizes small unilamellar vesicles loaded with calcium by a single-hit mechanism. FEBS Lett 232:217–220
Menestrina G, Mackman N, Holland IB, Bhakdi S (1987) Escherichia coli haemolysin forms voltage-dependent ion channels in lipid membranes. Biochim Biophys Acta 905:109–117
Nicaud J-M, Mackman N, Gray L, Holland IB (1985) Characterisation of H1yC and mechanism of activation and secretion of haemolysin from E. coli 2001. FEBS Lett 187:339–344
Oropeza-Wekerle RL, Müller E, Kern P, Meyermann R, Goebel W (1989) Synthesis, inactivation, and localization of extracellular and intracellular Escherichia coli hemolysins. J Bacteriol 171:2783–2788
Pellett S, Boehm DF, Snyder IS, Rowe G, Welch RA (1990) Characterization of monoclonal antibodies against the Escherichia coli hemolysin. Infect Immun 58:822–827
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Strathdee CA, Lo RYC (1987) Extensive homology between the leukotoxin of Pasteurella haemolytica A1 and the alpha-hemolysin of Escherichia coli. Infect Immun 55:3233–3236
Strathdee CA, Lo RYC (1989) Cloning, nucleotide sequence, and characterization of genes encoding the secretion function of the Pasteurella haemolytica leukotoxin determinant. J Bacteriol 171:916–928
Vogel M, Hess J, Then I, Juarez A, Goebel W (1988) Characterization of a sequence (hlyR) which enhances synthesis and secretion of hemolysin in Escherichia coli. Mol Gen Genet 212:76–84
Welch RA (1987) Identification of two different hemolysin determinants in uropathogenic Proteus isolates. Infect Immun 55:2183–2190
Author information
Authors and Affiliations
Additional information
Communicated by E.K.P. Butz
Rights and permissions
About this article
Cite this article
Ludwig, A., Schmid, A., Benz, R. et al. Mutations affecting pore formation by haemolysin from Escherichia coli . Mol Gen Genet 226, 198–208 (1991). https://doi.org/10.1007/BF00273604
Issue Date:
DOI: https://doi.org/10.1007/BF00273604