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Analysis and toxic overexpression in Escherichia coli of a staphylococcal gene encoding isoleucyl-tRNA synthetase
A F Chalker1, J M Ward, A P Fosberry
1Department of Biotechnology, SmithKline Beecham Pharmaceuticals, Brockham Park, Surrey, UK.
Abstract:
We have cloned and sequenced the Staphylococcus aureus Oxford ileS gene which encodes isoleucyl-tRNA synthetase (Ile-RS), the target for the antibiotic mupirocin. The gene was identified by hybridisation to oligodeoxyribonucleotide probes derived from internal Ile-RS amino acid (aa) sequences. The 2754-bp open reading frame encodes a 918-aa protein of 105 kDa which is homologous to other known Ile-RS from Gram- bacteria, archaebacteria, yeast and protozoa. Motifs which have been implicated in the functioning of the active site are strongly conserved. The gene was engineered for high-level expression in Escherichia coli. Ile-RS overproduction was toxic to the E. coli host, the magnitude of its observed effects being strain-dependent.