Mutations affecting activity and transport of haemolysin in Escherichia coli (original) (raw)

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 _hly_A 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 _hly_B and _hly_D are provided. Functional HlyC is not required for the transport of the mutant haemolysins. Two site-specific mutations at the N-terminal end of _hly_A (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.

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Authors and Affiliations

1. Institut für Genetik und Mikrobiologie, Universität Würzburg, D-8700, Würzburg, Federal Republic of Germany
   Albrecht Ludwig, Monika Vogel & Werner Goebel

Authors

1. Albrecht Ludwig
2. Monika Vogel
3. Werner Goebel

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Communicated by E. Bautz

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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

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• Received: 29 September 1986
• Issue date: February 1987
• DOI: https://doi.org/10.1007/BF00333579

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