Inactivation of the alpha-haemolysin gene of Staphylococcus aureus 8325-4 by site-directed mutagenesis and studies on the expression of its haemolysins

Microb Pathog. 1986 Apr;1(2):125-38. doi: 10.1016/0882-4010(86)90015-x.

Abstract

S. aureus strain 8325-4 was shown to produce alpha-, beta-, delta- and gamma-haemolysins by haemolytic assays and immunoblotting. Hybridization experiments indicated that a single copy of the alpha-haemolysin gene (hla) resides in the chromosome. Site-directed mutagenesis was used to inactivate the hla gene. This gene, which had previously been cloned in E. coli, was inactivated in vitro by inserting a fragment carrying an erythromycin resistance marker. Shuttle plasmids were constructed and transformed into 8325-4 and non-haemolytic recombinants enriched by a plasmid incompatibility technique. A previously isolated Tn551 insertion defective in alpha-haemolysin was not located in hla. It had pleiotropic defects in expression of alpha-, beta- and delta-haemolysins. Expression of alpha-haemolysin from a plasmid-located hla gene was very low. In contrast, hla-erm mutants were deficient only in alpha-haemolysin and allowed high level expression of the plasmid-borne hla gene. The Tn551 insertion is probably located in a gene encoding a positive regulatory element required for expression of several exoproteins. An hla-erm mutant was less virulent than the otherwise isogenic 8325-4 hla+ strain in a mouse peritonitis model, confirming that alpha-haemolysin is an important virulence factor.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Toxins / genetics*
  • DNA, Bacterial / genetics
  • Gene Expression Regulation
  • Genes, Bacterial
  • Hemolysin Proteins / genetics*
  • Mutation
  • Sequence Homology, Nucleic Acid
  • Staphylococcus aureus / genetics*
  • Staphylococcus aureus / pathogenicity
  • Virulence

Substances

  • Bacterial Toxins
  • DNA, Bacterial
  • Hemolysin Proteins
  • staphylococcal alpha-toxin