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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Altschul SF, Madden TL, Schaffer AA, Zhang JH, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402
Altschul SF, Koonin EV (1998) Iterated profile searches with PSI-BLAST—a tool for discovery in protein databases. Trends Biochem Sci 23:444–447
Bai LQ, Li L, Xu H, Minagawa K, Yu Y, Zhang Y, Zhou X, Floss HG, Mahmud T, Deng Z (2006) Functional analysis of the validamycin biosynthetic gene cluster and engineered production of validoxylamine A. Chem Biol 13:387–397
Bierman M, Logan R, Obrien K, Seno ET, Rao RN, Schoner BE (1992) Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene 116:43–49
Chen S, Huang X, Zhou XF, Bai LQ, He J, Jeong KJ, Lee SY, Deng ZX (2003) Organizational and mutational analysis of a complete FR-008/candicidin gene cluster encoding a structurally related polyene complex. Chem Biol 10:1065–1076
Deng ZX, Kieser T, Hopwood DA (1988) “Strong incompatibility” between derivatives of the Streptomyces multi-copy plasmid pIJ101. Mol Gen Genet 214:286–294
Ewing B, Hillier L, Wendl MC, Green P (1998) Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res 8:175–185
Gordon D, Abajian C, Green P (1998) Consed: a graphical tool for sequence finishing. Genome Res 8:195–202
Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580
Harvey BM, Mironenko T, Sun YH, Hong H, Deng ZX, Leadlay PF, Weissman KJ, Haydock SF (2007) Insights into polyether biosynthesis from analysis of the nigericin biosynthetic gene cluster in Streptomyces sp. DSM4137. Chem Biol 14:703–714
He XY, Ou HY, Yu Q, Zhou XF, Wu J, Liang JD, Zhang W, Rajakumar K, Deng ZX (2007) Analysis of a genomic island housing genes for DNA S-modification system in Streptomyces lividans 66 and its counterparts in other distantly related bacteria. Mol Microbiol 65:1034–1048
Hu ZH, Bao K, Zhou XF, Zhou Q, Hopwood DA, Kieser T, Deng ZX (1994) Repeated polyketide synthase modules involved in the biosynthesis of a heptaene macrolide by Streptomyces sp. FR-008. Mol Microbiol 14:163–172
Ishikawa J, Hotta K (1999) FramePlot: a new implementation of the frame analysis for predicting protein-coding regions in bacterial DNA with a high G plus C content. FEMS Microbiol Lett 174:251–253
Katz E, Thompson CJ, Hopwood DA (1983) Cloning and expression of the tyrosinase gene from Streptomyces antibioticus in Streptomyces lividans. J Gen Microbiol 129:2703–2714
Kendall KJ, Cohen SN (1988) Complete nucleotide-sequence of the Streptomyces lividans plasmid pIJ101 and correlation of the sequence with genetic properties. J Bacteriol 170:4634–4651
Kieser T, Hopwood DA, Wright HM, Thompson CJ (1982) pIJ101, a multi-copy broad host-range Streptomyces plasmid: functional analysis and development of DNA cloning vectors. Mol Gen Genet 185:223–238
Kieser T, Bibb MJ, Chater KF, Butter MJ, Hopwood DA (2000) Practical Streptomyces genetics: a laboratory manual. John Innes Foundation, Norwich, UK
Liang J, Wang Z, He X, Li J, Zhou X, Deng Z (2007) DNA modification by sulfur: analysis of the sequence recognition specificity surrounding the modification sites. Nucleic Acids Res 35:2944–2954
MacNeil DJ, Gewain KM, Ruby CL, Dezeny G, Gibbons PH, MacNeil T (1992) Analysis of Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel integration vector. Gene 111:61–68
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Schaffer AA, Aravind L, Madden TL, Shavirin S, Spouge JL, Wolf YI, Koonin EV, Altschul SF (2001) Improving the accuracy of PSI-BLAST protein database searches with composition-based statistics and other refinements. Nucleic Acids Res 29:2994–3005
Sun YH, Zhou XF, Liu J, Bao K, Zhang GM, Tu GQ, Kieser T, Deng ZX (2002) ‘Streptomyces nanchangensis’, a producer of the insecticidal polyether antibiotic nanchangmycin and the antiparasitic macrolide meilingmycin, contains multiple polyketide gene clusters. Microbiology 148:361–371
Sun YH, Hong H, Samborskyy M, Mironenko T, Leadlay PF, Haydock SF (2006) Organization of the biosynthetic gene cluster in Streptomyces sp. DSM 4137 for the novel neuroprotectant polyketide meridamycin. Microbiology 152:3507–3515
Wang LR, Chen S, Xu TG, Taghizadeh K, Wishnok JS, Zhou XF, You DL, Deng ZX, Dedon PC (2007) Phosphorothioation of DNA in bacteria by dnd genes. Nat Chem Biol 3:709–710
Weber JM, Leung JO, Maine GT, Potenz RHB, Paulus TJ, Dewitt JP (1990) Organization of a cluster of erythromycin genes in Saccharopolyspora erythraea. J Bacteriol 172:2372–2383
Zhou XF, He XY, Li AY, Lei F, Kieser T, Deng ZX (2004) Streptomyces coelicolor A3(2) lacks a genomic island present in the chromosome of Streptomyces lividans 66. Appl Environ Microbiol 70:7110–7118
Zhou XF, He XY, Liang JD, Li AY, Xu TG, Kieser T, Helmann JD, Deng ZX (2005) A novel DNA modification by sulphur. Mol Microbiol 57:1428–1438
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
- Streptomyces lividans
- Gene replacements