RESUMO
We have recently reported [Kafala, B., Sasarman, A., 1994. Can. J. Microbiol. 40, 651 657] the cloning and sequencing of the Staphylococcus aureus hemB gene. This gene purportedly encodes the delta-aminolevulinic acid dehydratase of the heme pathway. In this present communication, we report the sequences and identities of three putative hem genes. Two of these genes are located immediately upstream from hemB. Complementation analysis of Escherichia coli and Salmonella typhimurium hemC and hemD mutants and the comparison of the Sa nucleotide sequences with those of Bacillus subtilis and Ec showed that these two open reading frames, ORF1 and ORF2, are likely to be the hemC gene coding for porphobilinogen deaminase and the hemD gene coding for uroporphyrinogen III synthase, respectively. The third hem gene, hemL, is located immediately downstream of hemB, and encodes glutamate 1-semialdehyde 2,1-aminotransferase. Sequencing of the region which extends past hemL indicates that no further hem genes are located downstream of hemL. In Sa, hemC, hemD, hemB and hemL are proposed to constitute a hem cluster encoding enzymes required for the synthesis of uroporphyrinogen III from glutamate 1-semialdehyde (GSA).
Assuntos
Proteínas de Bactérias/genética , Proteínas de Escherichia coli , Genes Bacterianos , Heme/biossíntese , Hidroximetilbilano Sintase , Staphylococcus aureus/genética , Uroporfirinogênio III Sintetase/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Teste de Complementação Genética , Transferases Intramoleculares/genética , Dados de Sequência Molecular , Peso Molecular , Família Multigênica , Fases de Leitura Aberta/genética , Sintase do Porfobilinogênio/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Staphylococcus aureus/enzimologia , Uroporfirinogênio III Sintetase/química , Uroporfirinogênios/biossínteseRESUMO
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.
