Genetic Analysis of Different Resistance Mechanisms Against the Herbicidal Antibiotic Phosphinothricyl-Alanyl-Alanine
Streptomyces viridochromogenes Tü494, the producer of phosphinothricyl-alanyl-alanine (Ptt), is sensitive to its own antibiotic. Two phenotypically discernible Ptt-resistant S. viridochromogenes mutants, ES1 and ES2, were isolated and employed to clone resistance genes. Thus, two different DNA fragments both conferring Ptt resistance could be detected. The DNA regions including the resistance genes were sequenced and the gene products were investigated. The first gene (pat) encodes a phosphinothricin N-acetyltransferase which inactivates the antibiotically effective component phosphinothricin. Following modification of the 5’ region, the pat gene was transferred into plants and phosphinothricin-resistant transgenic plants were obtained. The second gene, a glutamine synthetase (GS) gene mediated Ptt resistance in multi-copy state only. The gene product is heat-labile, and the deduced amino acid sequence was shown to be highly homologous to eucaryotic and to Rhizobiaceae GSII-type enzymes. Therefore the gene was named glnII. Southern hybridizations with different Streptomyces strains suggest that they all carry two types of GS genes, g1nA and g1nII.
KeywordsGlutamine Synthetase Glutamine Synthetase Gene NcoI Fragment Clone Resistance Gene Promoter Probe Plasmid
Unable to display preview. Download preview PDF.
- Alijah, R., Dorendorf, J., Müller, S., Pühler, A., and Wohlleben, W., 1990, Analysis of phosphinothricyl-alanyl-alanine biosynthesis in Streptomyces viridochromogenes Tü494: Characterization of non-producing mutants and isolation of a biosynthetic gene, manuscript in preparation.Google Scholar
- Behrmann, I., Hillemann, D., Pühler, A., Strauch, E., and Wohlleben, W., 1990, Overexpression of a Streptomyces viridochromogenes gene (g1nII) encoding an eucaryotic-like glutamine synthetase confers resistance against the antibiotic phosphinothricyl-alanyl-alanine, J. Bacteriol., manuscript submittedGoogle Scholar
- Broer, I., Arnold, W., Wohlleben, W., and Pühler, A., 1989, The Phosphinothricin N-Acetyltransferase Gene as a Selection Marker for Plant Genetic Engineering, in: “Proceedings Appl. Plant Mol. Biol.,” G. Galling, ed., pp. 240–246, Zentralstelle far Weiterbildung, Braunschweig.Google Scholar
- Kondo, Y., Shomura, T., Ogawa, Y., Tsuruoka, T., Watanabe, H., Totukawa, K., Suzuki, T., Moriyama, C., Yoshida, J., Inouye, S., and Niída, T., 1973, Studies on a new antibiotic SF-1293, I. Isolation and physicochemical and biological characterization of SF-1293 substances, Sci. Rep. Meiji Seika, 13: 34–41.Google Scholar
- Labes, G., Simon, R., and Wohlleben, W., 1990, A rapid method for the analysis of plasmid content and copy number in various Streptomyces grown on agar plates, Nucleic Acids Res., 18(8), in press.Google Scholar
- Thompson, C. J., Movva, N. R., Tizard, R., Crameri, R., Davies, J., Lauwereys, M., and Botterman, J., 1987, Characterization of the herbicide-resistance gene bar from Streptomyces hygroscopicus, EMBO 6: 2519–2523.Google Scholar
- Wohlleben, W., Muth, G., Birr, E., and Pühler, A., 1986, A vector system for cloning in Streptomyces and E. coli, in: “Sixth Int. Sym. on Actinomycetes Biology,”G. Szabó, S. Biró, and M. Goodfellow, eds., pp. 99–101, Akadiémiai Kiadó, Budapest.Google Scholar
- Wohlleben, W., Arnold, W., Bissonnette, L., Pelletier, A., Tanguay, A., Roy, P. H., Gamboa, G. C., Barry, G. F., Aubert, E., Davies, J., and Kagan, S. A., 1989, On the evolution of Tn21-like multiresistance transposons: Sequence analysis of the gene (aacCl) for gentamicin acetyltransferase-3-I (AAC(3)-I), another member of the Tn21-based expression cassette, Mol. Gen. Genet., 217: 202–208.PubMedCrossRefGoogle Scholar
- Ward, J. M., Janssen, G. R., Kieser, T., Bibb, M. J., Buttner, M. J., and Bibb, M. J., 1986, Construction and characterization of a series of multi-copy promoter probe plasmid vectors for Streptomyces using the aminoglycoside phosphotransferase gene from Tn5 as indicators, Mol. Gen. Genet., 203: 468–478.PubMedCrossRefGoogle Scholar