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.
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Acknowledgements
The authors wish to thank the National Science Foundation of China, the Ministry of Science and Technology (973 and 863 programs), the Ministry of Education of China, and the Shanghai Municipal Council of Science and Technology for the research support. We are very grateful to Professor Peter F. Leadlay, FRS for his critical reading of the manuscript.
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Sun, Y., He, X., Liang, J. et al. Analysis of functions in plasmid pHZ1358 influencing its genetic and structural stability in Streptomyces lividans 1326. Appl Microbiol Biotechnol 82, 303–310 (2009). https://doi.org/10.1007/s00253-008-1793-7
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DOI: https://doi.org/10.1007/s00253-008-1793-7