Analysis and toxic overexpression in Escherichia coli of a staphylococcal gene encoding isoleucyl-tRNA synthetase.
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.
Chalker
A F
AF
Department of Biotechnology, SmithKline Beecham Pharmaceuticals, Brockham Park, Surrey, UK.
Ward
J M
JM
Fosberry
A P
AP
Hodgson
J E
JE
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D015342:DNA Probes / Q000138:chemical synthesis; D004269:DNA, Bacterial / Q000302:isolation & purification; D004926:Escherichia coli / Q000254:growth & development; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics*; D005816:Genetic Complementation Test; D015698:Genomic Library; D007533:Isoleucine-tRNA Ligase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009876:Operon / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics
D015342:DNA Probes; D004269:DNA, Bacterial; D007533:Isoleucine-tRNA Ligase