Inhibition of isoleucyl-transfer ribonucleic acid synthetase in Echerichia coli by pseudomonic acid

Abstract

The mode of action of the antibiotic pseudomonic acid has been studied in Escherichia coli. Pseudomonic acid strongly inhibits protein and RNA synthesis in vivo. The antibiotic had no effect on highly purified DNA-dependent RNA polymerase and showed only a weak inhibitory effect on a poly(U)-directed polyphenylalanine-forming ribosomal preparation. Chloramphenicol reversed inhibition of RNA synthesis in vivo. Pseudomonic acid had little effect on RNA synthesis in a regulatory mutant, E. coli B AS19 RC(rel), whereas protein synthesis was strongly inhibited. In pseudomonic acid-treated cells, increased concentrations of ppGpp, pppGpp and ATP were observed, but the GTP pool size decreased, suggesting that inhibition of RNA synthesis is a consequence of the stringent control mechanism imposed by pseudomonic acid-induced deprivation of an amino acid. Of the 20 common amino acids, only isoleucine reversed the inhibitory effect in vivo. The antibiotic was found to be a powerful inhibitor of isoleucyl-tRNA synthetase both in vivo and in vitro. Of seven other tRNA synthetases assayed, only a weak inhibitory effect on phenylalanyl-tRNA synthetase was observed; this presumably accounted for the weak effect on polyphenylalanine formation in a ribosomal preparation. Pseudomonic acid also significantly de-repressed threonine deaminase and transaminase B activity, but not dihydroxyacid dehydratase (isoleucine-biosynthetic enzymes) by decreasing the supply of aminoacylated tRNA(Ile). Pseudomonic acid is the second naturally occurring inhibitor of bacterial isoleucyl-tRNA synthetase to be discovered, furanomycin being the first.

Characterization of the stringent and relaxed responses of Streptococcus equisimilis

“…For amino acid starvation experiments, the isoleucine content of the lowphosphate medium was reduced to 1/10 the normal concentration (i.e., 10 g/ ml), and pseudomonic acid (Beecham Pharmaceuticals), an inhibitor of isoleucyl-tRNA synthetase (21), was added at 200 to 300 ng/ml when the cultures had reached an OD 600 of 0.25. To achieve energy source exhaustion, the glucose concentration of the low-phosphate medium was reduced to 0.07%, causing the cessation of exponential growth at OD 600 values ranging from 0.25 to 0.32.…”

Studies of the GTPase domain of archaebacterial ribosomes

“…To this end, the two methanogens were grown in low-phosphate medium (see Methods) with [32P]phosphate (100 pCi ml-', final) and, after one mass doubling, pseudomonic acid was added (50 pg m1-I). This antibiotic interferes with aminoacylation of tRNA"' competitively with respect to isoleucine [29] and is a potent inhibitor of the growth of methanogens [9]. When extracts were prepared from such cells using 1 M formic acid, no (p)ppGpp was detected although, under similar conditions, E. coli B (relA ') accumulated massive amounts of these materials (data not given).…”

Identification of a 60 kb region of the chromosome of Pseudomonas fluorescens NCIB 10586 required for the biosynthesis of pseudomonic acid (mupirocin)

“…It inhibits isoleucyl-tRNA synthetase, by blocking the formation of the enzyme. Ile-AMP complex, thus preventing the incorporation of isoleucine into growing peptide chains (Hughes & Mellows, 1978. Its primary use has been in the treatment of skin infections by members of the bacterial genus Stapblococczrs (e.g.…”