engleski

Construction of New pZG-Bifunctional Plasmids and their Structural Stability in Streptomyces

New Streptomyces-Escherichia coli bifunctional vectors were constructed with the aim to study their maintenance in Streptomyces. The plasmids were based on pIJ101 derivatives - pIJ350 and pIJ303 - ligated to E.coli plasmids pBR322, pBR328, Bluescribe M13- and pUC18 at the PstI site. In our previous report on that subject the structural instability of a bifunctional plasmid pZG1, consisting of pIJ350 and pBR322 replicons were described. In an attempt construct bifunctional vectors which would be staly maintained in Streptomyces, changes in Streptomyces and E.coli parts of pZG1 were introduced. The pBR322 part of pZG1 was replaced by pBR328, what resulted in the formation of pZG3.1 and pZG3.2 (pBR328 in two orientations in respect to pIJ350). Another change was introduced by ligating pIJ350 to Bluescribe M13 and pUC18 at the Pst1 site, to form shuttle vectors pZG5 and pZG6, respectively, which were rather stably maintained in Streptomyces. Since pIj350 is derivative of pIJ101 lacking minus origine of replication (M-O) or initiation site (ssi) for the second strand synthesis, it belongs to the group of plasmids described in gram-positive bacteria which replicate via ssDNA intermediate, probably by rolling-circle replication. In these plasmids both homologous and illegitimate recombinations are greatly stimulated, which may lead to deletions. We have presented experimental evidence that pZG1 accumulated ssDNA in Streptomyces. To gain additional evidence that the replication of pZG1 in Streptomyces via sand is at least one of the causes of its high structural instability, pIJ303 having intact M-O (ssi) was used in construction of pZG4.1 and pZG4.2. Plasmid pBR322 was inserted into the PSTI at the beginning of the "spread" region (pZG4.2) of pIJ303. These plasmids were indeed more stable in Streptomyces than pZG1. The structural stability of new pZG-plasmids will be discussed in respect to the origin of plasmid DNA used for Streptomyces transformation ; to the mode of plasmid replication in Streptomyces, and to the host strain and transforming colony age.

Characterization of the ribosomal RNA operon rrnF from Streptomyces rimosus

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