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Appl Microbiol Biotechnol. 2009 Feb;82(2):303-10. doi: 10.1007/s00253-008-1793-7. Epub 2008 Dec 6.

Analysis of functions in plasmid pHZ1358 influencing its genetic and structural stability in Streptomyces lividans 1326.

Author information

1
Laboratory of Microbial Metabolism and School of Life Science and Biotechnology, Shanghai Jiaotong University, Shanghai 200030, China.

Abstract

The complete DNA sequence of plasmid pHZ1358, a widely used vector for targeted gene disruption and replacement experiments in many Streptomyces hosts, has been determined. This has allowed a detailed analysis of the basis of its structural and segregational instability, compared to the high copy number plasmid pIJ101 of Streptomyces lividans 1326 from which it was derived. A 574-bp DNA region containing sti (strong incompatibility locus) was found to be a determinant for segregational instability in its original S. lividans 1326 host, while the structural instability was found to be related to the facile deletion of the entire Escherichia coli-derived part of pHZ1358, mediated by recombination between 36-bp direct repeats. A point mutation removing the BamHI site inside the rep gene encoding a replication protein (rep*) and/or a spontaneous deletion of the 694-bp region located between rep and sti including the uncharacterized ORF85 (orf85(-)) produced little or no effect on stability. A pHZ1358 derivative (pJTU412, sti(-), rep*, orf85(-)) was then constructed which additionally lacked one of the 36-bp direct repeats. pJTU412 was demonstrated to be structurally stable but segregationally unstable and, in contrast to sti(+) pHZ1358, allowed efficient targeted gene replacement in S. lividans 1326.

PMID:
19066884
DOI:
10.1007/s00253-008-1793-7
[Indexed for MEDLINE]

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