Overexpression of a Streptomyces viridochromogenes gene (glnII) encoding a glutamine synthetase similar to those of eucaryotes confers resistance against the antibiotic phosphinothricyl-alanyl-alanine.
Phosphinothricyl-alanyl-alanine (PTT), also known as bialaphos, contains phosphinothricin, a potent inhibitor of glutamine synthetase (GS). A 2.75-kilobase NcoI fragment of the Streptomyces viridochromogenes PTT-resistant mutant ES2 cloned on a multicopy vector mediated PTT resistance to S. lividans and to S. viridochromogenes. Nucleotide sequence analysis of the 2.75-kb NcoI fragment revealed the presence of three open reading frames. Open reading frame 3 was termed glnII since significant similarity was found between its deduced amino acid sequence and those from GS of eucaryotes and GSII of members of the family Rhizobiaceae. Subcloning experiments showed that PTT resistance is mediated by overexpression of glnII encoding a 37.3-kilodalton protein of 343 amino acids. A three- to fourfold increase in gamma-glutamyltransferase activity could be observed in S. lividans transformants carrying the glnII gene on a multicopy plasmid. For S. viridochromogenes it was shown that PTT resistance conferred by the 2.75-kb NcoI fragment was dependent on its multicopy state. GS activity encoded by glnII was found to be heat labile. Southern hybridization with seven different Streptomyces strains suggested that they all carry two types of GS genes, glnA and glnII.
Behrmann
I
I
Lehrstuhl für Genetik, Fakultät für Biologie, Universität Bielefeld, Federal Republic of Germany.
Hillemann
D
D
Pühler
A
A
Strauch
E
E
Wohlleben
W
W
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001483:Base Sequence; D015139:Blotting, Southern; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004352:Drug Resistance, Microbial / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D005974:Glutamate-Ammonia Ligase / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D009943:Organophosphorus Compounds / Q000494:pharmacology*; D010957:Plasmids; D012093:Replicon; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013302:Streptomyces / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*
D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D009943:Organophosphorus Compounds; C003122:bialaphos; D005974:Glutamate-Ammonia Ligase