Naslov
Transposon mutagenesis in Streptomyces rimosus

Autori
Horvat, Lada Ivana ; Paravić, Andrea ; Pandža, Suada ; Hranueli, Daslav ; Cullum, John

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Zbornik sažetaka priopćenja, Šestog kongresa biologa Hrvatske / Đ. Huber - Zagreb : Hrvatsko biološko društvo, 1997, 79-80

Skup
Šesti kongres biologa Hrvatske

Mjesto i datum
Opatija, Hrvatska, 22.09.1997. - 26.09.1997

Vrsta sudjelovanja
Poster

Vrsta recenzije
Domaća recenzija

Ključne riječi
Transposon; mutagenesis; Streptomyces rimosus

Sažetak
Transposons are versatile tools for the genetic manipulation of bacteria. One of their uses is the provision of portable restriction sites for physical mapping. We recently developed a physical map of the 8 Mb linear chromosome of S. rimosus, the producer of a clinically-important antibiotic oxytetracycline and wanted to use transposons for further studies. The temperature-sensitive replication plasmid pUKG403 which carries Tn5424 (Irnich & Cullum, Biotechnol. Lett., 16, 437-442, 1994) was introduced into S. rimosus MV1 by electroporation. pUKG403 was stably maintained in S. rimosus and showed loss of the transposon thiostrepton resistance marker on growth at 37 oC as previously seen in S. lividans. Transposition events could be isolated by selecting thiostrepton resistance after growth at 37 oC and included a number of auxotrophs. However, loss of plasmid vector sequences at 37 oC could not be directly monitored using the pUKG403 neomycin resistance gene (aphII) because S. rimosus is naturally resistant to neomycin. We therefore constructed the plasmid pPZG200, by introducing an erythromycin resistance (mls) gene into the unique BglII site of pUKG403. Using pPZG200 the elimination of vector sequences from S. rimosus cells could be monitored by testing thiostrepton resistant (Tn5424 marker) colonies for loss of erythromycin resistance (vector marker). Pulse-field gel electrophoresis analysis of the genomic DNA of auxotrophic transposants confirmed the positions of transpositions.

Izvorni jezik
Engleski

Znanstvena područja
Prehrambena tehnologija

POVEZANOST RADA