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  • 1
    Electronic Resource
    Electronic Resource
    Oligomerization of Escherichia coli haemolysin (HlyA) is involved in pore formation (1993)
    Springer
    Molecular genetics and genomics 241 (1993), S. 89-96 
    Details
    ISSN: 1617-4623
    Keywords: Haemolysin ; Escherichia coli ; Oligomerization of HlyA ; Pore formation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Coexpression of pairs of nonhaemolytic H1yA mutants in the recombination-deficient (recA) strain Escherichia coli HB101 resulted in a partial reconstitution of haemolytic activity, indicating that the mutation in one H1yA molecule can be complemented by the corresponding wild-type sequence in the other mutant HlyA molecule and vice versa. This suggests that two or more HlyA molecules aggregate prior to pore formation. Partial reconstitution of the haemolytic activity was obtained by the combined expression of a nonhaemolytic HlyA derivative containing a deletion of five repeat units in the repeat domain and several nonhaemolytic HlyA mutants affected in the pore-forming hydrophobic region. The simultaneous expression of two inactive mutant HlyA proteins affected in the region at which HlyA is covalently modified by HlyC and the repeat domain, respectively, resulted in a haemolytic phenotype on blood agar plates comparable to that of wild-type haemolysin. However, complementation was not possible between pairs of HlyA molecules containing site-directed mutations in the hydrophobic region and the modification region, respectively. In addition, no complementation was observed between HlyA mutants with specific mutations at different sites of the same functional domain, i.e. within the hydrophobic region, the modification region or the repeat domain. The aggregation of the HlyA molecules appears to take place after secretion, since no extracellular haemolytic activity was detected when a truncated but active HlyA lacking the C-terminal secretion sequence was expressed together with a non-haemolytic but transport-competent HlyA mutant containing a deletion in the repeat domain.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00280205
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  • 2
    Electronic Resource
    Electronic Resource
    Mutations affecting activity and transport of haemolysin in Escherichia coli (1987)
    Springer
    Molecular genetics and genomics 206 (1987), S. 238-245 
    Details
    ISSN: 1617-4623
    Keywords: E. coli ; Haemolysin ; Mutants ; Functional domains of HlyA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: 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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00333579
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  • 3
    Electronic Resource
    Electronic Resource
    Mutations affecting pore formation by haemolysin from Escherichia coli (1991)
    Springer
    Molecular genetics and genomics 226 (1991), S. 198-208 
    Details
    ISSN: 1617-4623
    Keywords: Haemolysin ; Escherichia coli ; Pore formation ; Site-specific mutation ; Lipid bilayer
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: 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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00273604
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