Applied Microbiology and Biotechnology

, Volume 82, Issue 2, pp 303–310

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

Authors

  • Yuhui Sun
    • Laboratory of Microbial Metabolism and School of Life Science and BiotechnologyShanghai Jiaotong University
    • Department of BiochemistryUniversity of Cambridge
  • Xinyi He
    • Laboratory of Microbial Metabolism and School of Life Science and BiotechnologyShanghai Jiaotong University
  • Jingdan Liang
    • Laboratory of Microbial Metabolism and School of Life Science and BiotechnologyShanghai Jiaotong University
  • Xiufen Zhou
    • Laboratory of Microbial Metabolism and School of Life Science and BiotechnologyShanghai Jiaotong University
    • Laboratory of Microbial Metabolism and School of Life Science and BiotechnologyShanghai Jiaotong University
Applied Genetics and Molecular Biotechnology

DOI: 10.1007/s00253-008-1793-7

Cite this article as:
Sun, Y., He, X., Liang, J. et al. Appl Microbiol Biotechnol (2009) 82: 303. doi:10.1007/s00253-008-1793-7

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.

Keywords

pIJ101pHZ1358Streptomyces lividansGene replacements

Copyright information

© Springer-Verlag 2008