Summary
Temperature-sensitive mutants that exhibit an altered haemolytic phenotype were isolated from Escherichia coli harbouring the plasmid pHly152. Complementation with recombinant plasmids carrying one of the four hly genes (C, A, B or D) allowed localization of the hly ts mutations. A ts mutation in hlyC leads to a pro→leu exchange in amino acid position 53 of HlyC. Two ts mutations in HlyA were found in positions 312 (ser→pro) and 315 (thr→ile). Both amino acid exchanges are located in the same hydrophobic domain of HlyA which extends from amino acids 299 to 327. Two different mutations were introduced by site-specific mutagenesis in this hlyA domain: one by an exchange of ala, val to asp, glu (positions 313, 314) altering the hydrophobicity of this region and another which removes most of this hydrophobic portion. Both mutants have entirely lost the haemolytic activity but the mutant haemolysins are still efficiently transported across both membranes when hlyB and hlyD are provided. Functional HlyC is not required for the transport of the mutant haemolysins. Two site-specific mutations at the N-terminal end of hlyA (one at amino acid position 2 leading to a thr→pro exchange and another deleting ile and thr at positions 4 and 5) also do not affect the transport of the altered haemolysins. The thr→pro exchange enhances the haemolytic activity of the corresponding mutant, whereas the ile, thr deletion exhibits little or no effect on the haemolytic activity. Removal of the last 37 amino acids from the C-terminal end of HlyA leads to a truncated haemolysin which retains its haemolytic activity but is not secreted by the HlyB and HlyD transport system.
Similar content being viewed by others
References
Berger H, Hacker J, Juarez A, Hughes C, Goebel W (1982) Cloning of the chromosomal determinants encoding hemolysin production and mannose resistant hemagglutination in Escherichia coli. J Bacteriol 152:1241–1247
Felmlee T, Pellett S, Lee EY, Welch RA (1985a) Escherichia coli hemolysin is released extracellularly without cleavage of a signal peptide. J Bacteriol 163:88–93
Felmlee T, Pellett S, Welch RA (1985b) Nucleotide sequence of an Escherichia coli chromosomal hemolysin. J Bacteriol 163:94–105
Goebel W, Hedgpeth J (1982) Cloning and functional characterization of the plasmid-encoded hemolysin determinant of Escherichia coli. J Bacteriol 151:1290–1298
Goebel W, Royer-Pokora B, Lindenmaier W, Bujard H (1974) Plasmids controlling synthesis of hemolysin in Escherichia coli: molecular properties. J Bacteriol 118:964–973
Gonzalez-Carrero MI, Zabala JC, de la Cruz F, Ortiz JM (1985) Purification of α-hemolysin from an overproducing E. coli strain. Mol Gen Genet 199:106–110
Hacker J, Hughes C, Hof H, Goebel W (1983) Cloned hemolysin genes from Escherichia coli that cause urinary tract infection determine different levels of toxicity in mice. Infect Immun 42:57–63
Härtlein M, Schießl S, Wagner W, Rdest U, Kreft J, Goebel W (1983) Transport of hemolysin by Escherichia coli. J Cell Biochem 22:87–97
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
Kramer W, Drutsa V, Jansen HW, Kramer B, Pflugfelder M, Fritz HJ (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
Mackman N, Holland IB (1984) Functional characterization of a cloned haemolysin determinant from E. coli of human origin, encoding information for the secretion of a 107 K polypeptide. Mol Gen Genet 196:129–134
Mackman N, Nicaud JM, Gray L, Holland IB (1985a) Genetical and functional organisation of the Escherichia coli haemolysin determinant 2001. Mol Gen Genet 201:282–288
Mackman N, Nicaud JM, Gray L, Holland IB (1985b) Identification of polypeptides required for the export of haemolysin 2001 from E. coli. Mol Gen Genet 201:529–536
Minshew BH, Jorgensen J, Counts GW, Falkow S (1978) Association of hemolysin production, haemagglutination of human erythrocytes and virulence for chicken embryos of extraintestinal Escherichia coli isolates. Infect Immun 20:50–54
Nicaud JM, Mackman N, Gray L, Holland IB (1985) Characterisation of HlyC and mechanism of activation and secretion of haemolysin from E. coli 2001. FEBS Lett 187:339–344
Noegel A, Rdest U, Goebel W (1981) Determination of the functions of hemolytic plasmid pHly152 of Escherichia coli. J Bacteriol 145:233–247
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Wagner W, Vogel M, Goebel W (1983) Transport of hemolysin across the outer membrane of Escherichia coli requires two functions. J Bacteriol 154:200–210
Welch RA, Hull R, Falkow S (1983) Molecular cloning and physical characterization of a chromosomal hemolysin from Escherichia coli. Infect Immun 42:178–186
Zoller MJ, Smith M (1982) Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA. Nucleic Acids Res 10:6487–6500
Author information
Authors and Affiliations
Additional information
Communicated by E. Bautz
Rights and permissions
About this article
Cite this article
Ludwig, A., Vogel, M. & Goebel, W. Mutations affecting activity and transport of haemolysin in Escherichia coli . Mol Gen Genet 206, 238–245 (1987). https://doi.org/10.1007/BF00333579
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00333579