Cloning and sequence analysis of the hemB gene of Staphylococcus aureus.

Abstract

The hemB gene is a member of the family of genes encoding enzymes of the porphyrin biosynthetic pathway and codes for the enzyme porphobilinogen synthase, which is responsible for the conversion of delta-aminolevulinic acid to porphobilinogen. To clone the hemB gene of Staphylococcus aureus we used Tn917-mediated transposon mutagenesis. Tn917 confers resistance to erythromycin and is carried by plasmid pTV1ts, which has thermosensitive replication. Hem mutants were selected by growth in the presence of kanamycin and erythromycin at 43 degrees C. Preliminary identification of the hem mutants was based on their dwarf colony growth, which could be restored to normal by hemin. DNA extracted from one of the hem mutants was digested with several restriction endonucleases and hybridized to a probe representing the XbaI-AvaI end of Tn917. A BglII-EcoRI fragment of 4.5 kb gave a positive signal and was cloned into pUC18. Transformants were identified by colony hybridization with the Tn917 probe. The positive clones were sequenced, starting from the transposon end. The results allowed us to identify an open reading frame whose nucleotide sequence presented a homology of 63% to the sequence of the hemB gene of Bacillus subtilis and of 55% to the sequence of the hemB gene of Escherichia coli K12. No other nucleotide sequences, except those belonging to known hemB genes, presented significant homologies to our sequence. The cloning of the hemB gene of S. aureus was confirmed by the ability of the gene to complement a hemB mutant of E. coli K12. To our knowledge, this is the first report of the cloning of a hem gene in S. aureus.