Summary auto-generated
This study describes the cloning, sequencing, and genetic mapping of the aroA gene from Staphylococcus aureus strain 8325-4. The aroA gene, which encodes 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, was isolated on a 3 kb HindIII fragment and later re-cloned on a 6.2 kb EcoRI fragment. The gene was shown to be part of an operon with aroB and aroC, establishing the gene order as aroC-aroB-aroA. Sequence analysis revealed the aroA gene encodes a 404-amino acid protein with significant homology to EPSP synthases from E. coli and B. subtilis. An aroA::Tc' insertion mutant was constructed via allelic replacement, and genetic mapping using this mutant confirmed the chromosomal location within SmaI fragment A, with gene order thy-aroA-tyrB. Although the insertion mutant required aromatic amino acids for growth, it remained independent of p-aminobenzoic acid (PAB), likely due to expression of a truncated protein from the 5' insertion. Notably, the aroA::Tc' mutant was not attenuated in mouse infection models, possibly explained by retained PAB prototrophy. Attempts to create a complete null mutation in aroA were unsuccessful.
Key findings
- The S. aureus aroA gene is organized in an operon with aroB and aroC genes in the order aroC-aroB-aroA, distinct from the unlinked arrangement found in enteric bacteria.
- The aroA::Tc' insertion mutant requires aromatic amino acids but remains independent of p-aminobenzoic acid (PAB) due to a 5' insertion allowing truncated protein expression.
- EPSP synthase from S. aureus shares 43% identity with B. subtilis aroE and 29% identity with E. coli aroA gene products.
- The aroA mutant was not attenuated in mouse infection models, contrasting with other bacterial aroA mutants that show reduced virulence.
- A complete null aroA mutant could not be generated, suggesting the gene may be essential for S. aureus growth.
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Abstract
Summary: The aroA gene of Staphylococcus aureus 8325-4 was cloned. Sequence analysis and the phenotype of directed plasmid insertions 5" to aroA suggest that aroA is located in an operon and that it maps 3" to the aroC and aroB genes. A revised consensus sequence for the aroA gene product EPSP synthase binding site for its substrate (phosphoenolpyruvate) and an inhibitor (glyphosate) is proposed. An aroA insertion mutant isolated by allelic replacement was employed in genetic mapping experiments which demonstrated the gene order thy aroA tyrB in SmaI fragment A of the S. aureus 8325-4 chromosome. The aroA::Tcr mutant required aromatic amino acids but remained independent of p-aminobenzoic acid (PAB). This could be due to the insertion being located close to the 5" end of the gene, allowing expression of a truncated protein. The PAB independence may explain the finding that the mutant was not attenuated in mouse infection experiments. It was not possible to isolate a null mutant in aroA.