Molecular cloning and nucleotide sequence of the group B streptococcal hemolysin

Genomic DNA from a Streptococcus agalactiae strain and pUC8 were used to clone the genetic determinant of the group B streptococcal hemolysin. The smallest recombinant plasmid, which enables its E. coli hosts to express beta-hemolysis on sheep blood agar or in blood broth, was found to contain a 3.9 kb insert of streptococcal DNA. A restriction endonuclease map was established for the enzymes Cla I, EcoRI, Hind II, Hind III and Sau I. Transposon mutagenesis experiments with Tn5 extinguished hemolytic activity when Tn5 was inserted at 1.7 kb or 1.9 kb downstream the 5' end of the streptococcal fragment between the Hind II and EcoRI sites. The complete 3.9 kb fragment was sequenced on both strands by the dideoxy method of Sanger. It was shown to contain 4 open reading frames; one of 690 bp, including the area of transposon insertion mentioned above, has a potential promoter region upstream. The deduced amino acid sequence of 230 residues shows a hydrophobic signal sequence of 19 amino acids at its N-terminus.