1887

Abstract

To facilitate development of synthetic biology tools for genetic engineering of cyanobacterial strains, we constructed pANS-derived self-replicating shuttle vectors that are based on the minimal replication element of the strain PCC 7942 plasmid pANS. To remove the possibility of homologous recombination events between the shuttle plasmids and the native pANS plasmid, the endogenous pANS was cured through plasmid incompatibility-mediated spontaneous loss. A heterologous toxin–antitoxin cassette was incorporated into the shuttle vectors for stable plasmid maintenance in the absence of antibiotic selection. The pANS-based shuttle vectors were shown to be able to carry a large 20 kb DNA fragment containing a gene cluster for biosynthesis of the omega-3 fatty acid eicosapentaenoic acid. Based on quantitative PCR analysis, there are about 10 copies of pANS and 3 copies of the large native plasmid pANL per chromosome in . Fluorescence levels of GFP reporter genes in a pANS-based vector were about 2.5-fold higher than when in pANL or integrated into the chromosome. In addition to its native host, pANS-based shuttle vectors were also found to replicate stably in the filamentous cyanobacterium sp. strain PCC 7120. There were about 27 copies of a pANS-based shuttle vector, 9 copies of a pDU1-based shuttle vector and 3 copies of an RSF1010-based shuttle vector per genome when these three plasmids co-existed in cells. The endogenous pANS from our laboratory strain was cloned in , re-sequenced and re-annotated to update previously published sequencing data.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.000377
2016-12-21
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/micro/162/12/2029.html?itemId=/content/journal/micro/10.1099/mic.0.000377&mimeType=html&fmt=ahah

References

  1. Berla B. M., Pakrasi H. B. 2012; Upregulation of plasmid genes during stationary phase in Synechocystis sp. strain PCC 6803, a cyanobacterium. Appl Environ Microbiol 78:5448–5451 [View Article][PubMed]
    [Google Scholar]
  2. Brahamsha B. 1996; A genetic manipulation system for oceanic cyanobacteria of the genus Synechococcus . Appl Environ Microbiol 62:1747–1751[PubMed]
    [Google Scholar]
  3. Castets A.-M., Houmard J., Tandeau de Marsac N. 1986; Is cell motility a plasmid-encoded function in the cyanobacterium Synechocystis 6803?. FEMS Microbiol Lett 37:277–281 [View Article]
    [Google Scholar]
  4. Chen A. H., Afonso B., Silver P. A., Savage D. F. 2012; Spatial and temporal organization of chromosome duplication and segregation in the cyanobacterium Synechococcus elongatus PCC 7942. PLoS One 7:e47837 [View Article][PubMed]
    [Google Scholar]
  5. Chen Y., Holtman C. K., Magnuson R. D., Youderian P. A., Golden S. S. 2008; The complete sequence and functional analysis of pANL, the large plasmid of the unicellular freshwater cyanobacterium Synechococcus elongatus PCC 7942. Plasmid 59:176–192 [View Article][PubMed]
    [Google Scholar]
  6. Clerico E. M., Ditty J. L., Golden S. S. 2007; Specialized techniques for site-directed mutagenesis in cyanobacteria. Methods Mol Biol 362:155–171 [View Article][PubMed]
    [Google Scholar]
  7. Elhai J., Wolk C. P. 1988; Conjugal transfer of DNA to cyanobacteria. Methods Enzymol 167:747–754[PubMed] [CrossRef]
    [Google Scholar]
  8. Elhai J., Vepritskiy A., Muro-Pastor A. M., Flores E., Wolk C. P. 1997; Reduction of conjugal transfer efficiency by three restriction activities of Anabaena sp. strain PCC 7120. J Bacteriol 179:1998–2005[PubMed] [CrossRef]
    [Google Scholar]
  9. Encinas D., Garcillán-Barcia M. P., Santos-Merino M., Delaye L., Moya A., de la Cruz F. 2014; Plasmid conjugation from proteobacteria as evidence for the origin of xenologous genes in cyanobacteria. J Bacteriol 196:1551–1559 [View Article][PubMed]
    [Google Scholar]
  10. Gendel S. M. 1987; Instability of Tn5 inserts in cyanobacterial cloning vectors. J Bacteriol 169:4426–4430[PubMed] [CrossRef]
    [Google Scholar]
  11. Golden S. S., Sherman L. A. 1983; A hybrid plasmid is a stable cloning vector for the cyanobacterium Anacystis nidulans R2. J Bacteriol 155:966–972[PubMed]
    [Google Scholar]
  12. Golden S. S., Sherman L. A. 1984; Optimal conditions for genetic transformation of the cyanobacterium Anacystis nidulans R2. J Bacteriol 158:36–42[PubMed]
    [Google Scholar]
  13. Golden S. S., Brusslan J., Haselkorn R. 1987; Genetic engineering of the cyanobacterial chromosome. Methods Enzymol 153:215–231[PubMed] [CrossRef]
    [Google Scholar]
  14. Griese M., Lange C., Soppa J. 2011; Ploidy in cyanobacteria. FEMS Microbiol Lett 323:124–131 [View Article][PubMed]
    [Google Scholar]
  15. Hays S. G., Ducat D. C. 2015; Engineering cyanobacteria as photosynthetic feedstock factories. Photosynth Res 123:285–295 [View Article][PubMed]
    [Google Scholar]
  16. Hu B., Yang G., Zhao W., Zhang Y., Zhao J. 2007; MreB is important for cell shape but not for chromosome segregation of the filamentous cyanobacterium Anabaena sp. PCC 7120. Mol Microbiol 63:1640–1652 [View Article][PubMed]
    [Google Scholar]
  17. Koksharova O. A., Wolk C. P. 2002; Genetic tools for cyanobacteria. Appl Microbiol Biotechnol 58:123–137[PubMed] [CrossRef]
    [Google Scholar]
  18. Kopfmann S., Hess W. R. 2013; Toxin–antitoxin systems on the large defense plasmid pSYSA of Synechocystis sp. PCC 6803. J Biol Chem 288:7399–7409 [View Article][PubMed]
    [Google Scholar]
  19. Kuhlemeier C. J., van Arkel G. A. 1987; Host–vector systems for gene cloning in cyanobacteria. Methods Enzymol 153:199–215[PubMed] [CrossRef]
    [Google Scholar]
  20. Kuhlemeier C. J., Borrias W. E., van den Hondel C. A., van Arkel G. A. 1981; Vectors for cloning in cyanobacteria: construction and characterization of two recombinant plasmids capable of transformation of Escherichia coli K12 and Anacystis nidulans R2. Mol Gen Genet 184:249–254 [View Article][PubMed]
    [Google Scholar]
  21. Kuhlemeier C. J., Thomas A. A., van der Ende A., van Leen R. W., Borrias W. E., van den Hondel C. A., van Arkel G. A. 1983; A host–vector system for gene cloning in the cyanobacterium Anacystis nidulans R2. Plasmid 10:156–163 [View Article][PubMed]
    [Google Scholar]
  22. Lau R. H., Doolittle W. F. 1979; Covalently closed circular DNAs in closely related unicellular cyanobacteria. J Bacteriol 137:648–652[PubMed]
    [Google Scholar]
  23. Lau R. H., Sapienza C., Doolittle W. F. 1980; Cyanobacterial plasmids: their widespread occurrence, and the existence of regions of homology between plasmids in the same and different species. Mol Gen Genet 178:203–211 [View Article][PubMed]
    [Google Scholar]
  24. Lee M. H., Scherer M., Rigali S., Golden J. W. 2003; PlmA, a new member of the GntR family, has plasmid maintenance functions in Anabaena sp. strain PCC 7120. J Bacteriol 185:4315–4325 [View Article][PubMed]
    [Google Scholar]
  25. Liu X., Wang D., Wang H., Feng E., Zhu L., Wang H. 2012; Curing of plasmid pXO1 from Bacillus anthracis using plasmid incompatibility. PLoS One 7:e29875 [View Article]
    [Google Scholar]
  26. Marraccini P., Bulteau S., Cassier-Chauvat C., Mermet-Bouvier P., Chauvat F. 1993; A conjugative plasmid vector for promoter analysis in several cyanobacteria of the genera Synechococcus and Synechocystis . Plant Mol Biol 23:905–909 [View Article][PubMed]
    [Google Scholar]
  27. Matsunaga T., Takeyama H., Nakamura N. 1990; Characterization of cryptic plasmids from marine cyanobacteria and construction of a hybrid plasmid potentially capable of transformation of marine cyanobacterium, Synechococcus sp., and its transformation. Appl Biochem Biotechnol 24–25:151–160 [View Article][PubMed]
    [Google Scholar]
  28. Miyake M., Nagai H., Shirai M., Kurane R., Asada Y. 1999; A high-copy-number plasmid capable of replication in thermophilic cyanobacteria. Appl Biochem Biotechnol 77–79:267–275[PubMed] [CrossRef]
    [Google Scholar]
  29. Niederholtmeyer H., Wolfstädter B. T., Savage D. F., Silver P. A., Way J. C. 2010; Engineering cyanobacteria to synthesize and export hydrophilic products. Appl Environ Microbiol 76:3462–3466 [View Article][PubMed]
    [Google Scholar]
  30. Pecota D. C., Kim C. S., Wu K., Gerdes K., Wood T. K. 1997; Combining the hok/sok, parDE, and pnd postsegregational killer loci to enhance plasmid stability. Appl Environ Microbiol 63:1917–1924[PubMed]
    [Google Scholar]
  31. Rubin B. E., Wetmore K. M., Price M. N., Diamond S., Shultzaberger R. K., Lowe L. C., Curtin G., Arkin A. P., Deutschbauer A. et al. 2015; The essential gene set of a photosynthetic organism. Proc Natl Acad Sci U S A 112:E6634E6643 [View Article][PubMed]
    [Google Scholar]
  32. Ruffing A. M. 2011; Engineered cyanobacteria: teaching an old bug new tricks. Bioeng Bugs 2:136–149 [View Article][PubMed]
    [Google Scholar]
  33. Sarsekeyeva F., Zayadan B. K., Usserbaeva A., Bedbenov V. S., Sinetova M. A., Los D. A. 2015; Cyanofuels: biofuels from cyanobacteria. Reality and perspectives. Photosynth Res 125:329–340 [View Article][PubMed]
    [Google Scholar]
  34. Schmetterer G., Wolk C. P. 1988; Identification of the region of cyanobacterial plasmid pDU1 necessary for replication in Anabaena sp. strain M-131. Gene 62:101–109 [View Article][PubMed]
    [Google Scholar]
  35. Scholz P., Haring V., Wittmann-Liebold B., Ashman K., Bagdasarian M., Scherzinger E. 1989; Complete nucleotide sequence and gene organization of the broad-host-range plasmid RSF1010. Gene 75:271–288 [View Article][PubMed]
    [Google Scholar]
  36. Shulse C. N., Allen E. E. 2011; Widespread occurrence of secondary lipid biosynthesis potential in microbial lineages. PLoS One 6:e20146 [View Article][PubMed]
    [Google Scholar]
  37. Sode K., Tatara M., Takeyama H., Burgess J. G., Matsunaga T. 1992; Conjugative gene transfer in marine cyanobacteria: Synechococcus sp., Synechocystis sp. and Pseudanabaena sp. Appl Microbiol Biotechnol 37:369–373 [View Article][PubMed]
    [Google Scholar]
  38. Takeyama H., Grant Burgess J., Sudo H., Sode K., Matsunaga T. 1991; Salinity-dependent copy number increase of a marine cyanobacterial endogenous plasmid. FEMS Microbiol Lett 90:95–98 [View Article]
    [Google Scholar]
  39. Taton A., Lis E., Adin D. M., Dong G., Cookson S., Kay S. A., Golden S. S., Golden J. W. 2012; Gene transfer in Leptolyngbya sp. strain BL0902, a cyanobacterium suitable for production of biomass and bioproducts. PLoS One 7:e30901 [View Article][PubMed]
    [Google Scholar]
  40. Taton A., Unglaub F., Wright N. E., Zeng W. Y., Paz-Yepes J., Brahamsha B., Palenik B., Peterson T. C., Haerizadeh F. et al. 2014; Broad-host-range vector system for synthetic biology and biotechnology in cyanobacteria. Nucleic Acids Res 42:e136 [View Article][PubMed]
    [Google Scholar]
  41. Thiel T. 1994; Genetic analysis of cyanobacteria. In The Molecular Biology of Cyanobacteria , pp. 581–611 Edited by Bryant D. A. Dordrecht, Netherlands: Springer; [CrossRef]
    [Google Scholar]
  42. Trevors J. T. 1986; Plasmid curing in bacteria. FEMS Microbiol Lett 32:149–157 [View Article]
    [Google Scholar]
  43. Unterholzner S. J., Poppenberger B., Rozhon W. 2013; Toxin–antitoxin systems: Biology, identification, and application. Mob Genet Elements 3:e26219 [View Article][PubMed]
    [Google Scholar]
  44. van den Hondel C. A., Keegstra W., Borrias W. E., van Arkel G. A. 1979; Homology of plasmids in strains of unicellular cyanobacteria. Plasmid 2:323–333 [View Article][PubMed]
    [Google Scholar]
  45. van den Hondel C. A., Verbeek S., van der Ende A., Weisbeek P. J., Borrias W. E., van Arkel G. A. 1980; Introduction of transposon Tn901 into a plasmid of Anacystis nidulans: preparation for cloning in cyanobacteria. Proc Natl Acad Sci U S A 77:1570–1574 [View Article][PubMed]
    [Google Scholar]
  46. Van der Plas J., Oosterhoff-Teertstra R., Borrias M., Weisbeek P. 1992; Identification of replication and stability functions in the complete nucleotide sequence of plasmid pUH24 from the cyanobacterium Synechococcus sp. PCC 7942. Mol Microbiol 6:653–664 [View Article][PubMed]
    [Google Scholar]
  47. Watanabe S., Ohbayashi R., Kanesaki Y., Saito N., Chibazakura T., Soga T., Yoshikawa H. 2015; Intensive DNA replication and metabolism during the lag phase in cyanobacteria. PLoS One 10:e0136800 [View Article]
    [Google Scholar]
  48. Wolk C. P., Fan Q., Zhou R., Huang G., Lechno-Yossef S., Kuritz T., Wojciuch E. 2007; Paired cloning vectors for complementation of mutations in the cyanobacterium Anabaena sp. strain PCC 7120. Arch Microbiol 188:551–563 [View Article][PubMed]
    [Google Scholar]
  49. Xu Y., Alvey R. M., Byrne P. O., Graham J. E., Shen G., Bryant D. A. 2011; Expression of genes in cyanobacteria: adaptation of endogenous plasmids as platforms for high-level gene expression in Synechococcus sp. PCC 7002. Methods Mol Biol 684:273–293 [View Article][PubMed]
    [Google Scholar]
  50. Yang Y., Huang X. Z., Wang L., Risoul V., Zhang C. C., Chen W. L. 2013; Phenotypic variation caused by variation in the relative copy number of pDU1-based plasmids expressing the GAF domain of Pkn41 or Pkn42 in Anabaena sp. PCC 7120. Res Microbiol 164:127–135 [View Article][PubMed]
    [Google Scholar]
  51. Yano S., Kawata Y., Kojima H., Yano S. 1995; Salinity-dependent copy number change of endogenous plasmids in sp. strain PCC 7002. Curr Microbiol 31:357–360 [CrossRef]
    [Google Scholar]
  52. Yu J., Liberton M., Cliften P. F., Head R. D., Jacobs J. M., Smith R. D., Koppenaal D. W., Brand J. J., Pakrasi H. B. 2015; Synechococcus elongatus UTEX 2973, a fast growing cyanobacterial chassis for biosynthesis using light and CO(2). Sci Rep 5:8132
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.000377
Loading
/content/journal/micro/10.1099/mic.0.000377
Loading

Data & Media loading...

Supplements

Supplementary File 1

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error