Identification of genetic determinants for the hemolytic activity of Streptococcus agalactiae by ISS1 transposition.

Streptococcus agalactiae is a poorly transformable bacterium and studies of molecular mechanisms are difficult due to the limitations of genetic tools. Employing the novel pGh9:ISS1 transposition vector we generated plasmid-based mutant libraries of S. agalactiae strains O90R and AC475 by random chromosomal integration. A screen for mutants with a nonhemolytic phenotype on sheep blood agar led to the identification of a genetic locus harboring several genes that are essential for the hemolytic function and pigment production of S. agalactiae. Nucleotide sequence analysis of nonhemolytic mutants revealed that four mutants had distinct insertion sites in a single genetic locus of 7 kb that was subsequently designated cyl. Eight different open reading frames were identified: cylX, cylD, cylG, acpC, cylZ, cylA, cylB, and cylE, coding for predicted proteins with molecular masses of 11, 33, 26, 11, 15, 35, 32, and 78 kDa, respectively. The deduced amino acid sequence of the protein encoded by cylA harbors a conserved ATP-binding cassette (ABC) motif, and the predicted proteins encoded by cylA and cylB have significant similarities to the nucleotide binding and transmembrane proteins of typical ABC transporter systems. Transcription analysis by reverse transcription-PCR suggests that cylX to cylE are part of an operon. The requirement of acpC and cylZABE for hemolysin production of S. agalactiae was confirmed either by targeted mutagenesis with the vector pGh5, complementation studies with pAT28, or analysis of insertion elements in naturally occurring nonhemolytic mutants.