Naslov
DNA rearrangements in the terminal regions of the linear chromosome of Streptomyces rimosus

Autori
Andrea Paravić

Vrsta, podvrsta i kategorija rada
Ocjenski radovi, doktorska disertacija

Fakultet
Dem Fachbereich Biologie

Mjesto
Kaiserslautern, Germany

Datum
15.11

Godina
2001

Stranica
123

Mentor
Cullum, John i Hranueli, Daslav

Ključne riječi
Streptomyces rimosus; linear chromosome; terminal regions; DNA rearrangements

Sažetak
Streptomyces rimosus is used commercially for production of the antibiotic oxytetracycline (OTC). The strain is very instable and gives rise to numerous morphological variants, which were classified according to OTC resistance in three classes (Gravius et al., 1993). Recently physical map of the chromosome S. rimosus R6-501 was constructed (Pandza et al., 1997) showing that the 8 Mb chromosome is linear and similar in size and organization to that of other Streptomycetes. In S. rimosus there is also a linear plasmid pPZG101 of 387 kb in length with long terminal inverted repeats. Previous work has shown that in the S. rimosus strain MV25 the replacement of one chromosome end with one end of the linear plasmid pPZG101 has occurred (Gravius et al., 1994). Although the site of integration was precisely localised (Pandza, 1996) the large scale structure of integration was not clear. This work has shown that MV25 consists of a mixed population. A component of the population that has lost the otc cluster and posses a single 480 kb AseI junction fragment (MV25W derivatives) was isolated. The presence of more then one AseI junction fragment and only one BfrI and XbaI junction fragment in the MV25 population was explained. Strains 612, 613 and 615 (OTC blocked mutants derived from S. rimosus R6-65) have integrated parts of linear plasmid pPZG102 (Gravius et al., 1994). Hybridisation experiments showed that the plasmid and chromosomal integration sites in 612, 613 and 615 are different from those in MV25. The possible structure of integration events was presented as a model. Transformants that arose from experiments to disrupt the otcD1-ORF1 gene were analysed. Pulsed field gel electrophoresis (PFGE) analysis and hybridisation experiments have shown that they carry a variety of DNA deletions and amplifications. Spontaneous revertants that produce OTC and have a dark brown appearance were isolated from Class II mutants. The Class II mutants of S. rimosus have deleted about 700 kb of the chromosome including one end and the otc gene cluster. The deletion of the OTC cluster results in the loss of production and sensitivity to OTC because the resistance genes are in the cluster. The analysis of the revertants has shown that they fell into two classes; one that is indistinguishable morphologically from the parent strain and shows the same XbaI and AseI restriction pattern and the second one which shows better sporulation and different XbaI and AseI restriction patterns. A model to explain reversion of deletion mutants in S. rimosus was proposed. To facilitate analysis of DNA rearrangements in the terminal regions a rpslL cassette on a BamHI fragment was constructed.

Izvorni jezik
Engleski

Znanstvena područja
Prehrambena tehnologija

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