DOI QR코드

DOI QR Code

A Novel Integrative Expression Vector for Sulfolobus Species

  • Choi, Kyoung-Hwa (Department of Microbiology, College of Natural Sciences, Pusan National University) ;
  • Hwang, Sungmin (Department of Microbiology and Cell Science, University of Florida) ;
  • Yoon, Naeun (Department of Microbiology, College of Natural Sciences, Pusan National University) ;
  • Cha, Jaeho (Department of Microbiology, College of Natural Sciences, Pusan National University)
  • 투고 : 2014.05.22
  • 심사 : 2014.07.11
  • 발행 : 2014.11.28

초록

With the purpose of facilitating the process of stable strain generation, a shuttle vector for integration of genes via a double recombination event into two ectopic sites on the Sulfolobus acidocaldarius chromosome was constructed. The novel chromosomal integration and expression vector pINEX contains a pyrE gene from S. solfataricus P2 ($pyrE_{sso}$) as an auxotrophic selection marker, a multiple cloning site with histidine tag, the internal sequences of malE and malG for homologous recombination, and the entire region of pGEM-T vector, except for the multiple cloning region, for propagation in E. coli. For stable expression of the target gene, an ${\alpha}$-glucosidase-producing strain of S. acidocaldarius was generated employing this vector. The malA gene (saci_1160) encoding an ${\alpha}$-glucosidase from S. acidocaldarius fused with the glutamate dehydrogenase ($gdhA_{saci}$) promoter and leader sequence was ligated to pINEX to generate pINEX_malA. Using the "pop-in" and "pop-out" method, the malA gene was inserted into the genome of MR31 and correct insertion was verified by colony PCR and sequencing. This strain was grown in YT medium without uracil and purified by His-tag affinity chromatography. The ${\alpha}$-glucosidase activity was confirmed by the hydrolysis of $pNP{\alpha}G$. The pINEX vector should be applicable in delineating gene functions in this organism.

키워드

참고문헌

  1. Albers SV, Driessen AJM. 2008. Conditions for gene disruption by homologous recombination of exogenous DNA into the Sulfolobus solfataricus genome. Archaea 2: 145-149. https://doi.org/10.1155/2008/948014
  2. Aucelli T, Contursi P, Girfoglio M, Rossi M, Cannio R. 2006. A spreadable, non-integrative and high copy number shuttle vector for Sulfolobus solfataricus based on the genetic element pSSVx from Sulfolobus islandicus. Nucleic Acids Res. 34: e114. https://doi.org/10.1093/nar/gkl615
  3. Berkner S, Grogan D, Albers SV, Lipps G. 2007. Small multicopy, non-integrative shuttle vectors based on the plasmid pRN1 for Sulfolobus acidocaldarius and Sulfolobus solfataricus, model organisms of the (cren-)archaea. Nucleic Acids Res. 35: e88. https://doi.org/10.1093/nar/gkm449
  4. Berkner S, Wlodkowski A, Albers SV, Lipps G. 2010. Inducible and constitutive promoters for genetic systems in Sulfolobus acidocaldarius. Extremophiles 14: 249-259. https://doi.org/10.1007/s00792-010-0304-9
  5. Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254. https://doi.org/10.1016/0003-2697(76)90527-3
  6. Choi KH, Hwang S, Cha J. 2013. Identification and characterization of MalA in the maltose/maltodextrin operon of Sulfolobus acidocaldarius DSM639. J. Bacteriol. 195: 1789-1799. https://doi.org/10.1128/JB.01713-12
  7. Deng L, Zhu H, Chen Z, Liang YX, She Q. 2009. Unmarked gene deletion and host-vector system for the hyperthermophilic crenarchaeon Sulfolobus islandicus. Extremophiles 13: 735-746. https://doi.org/10.1007/s00792-009-0254-2
  8. Farkas J, Chung D, Debarry M, Adams MW, Westpheling J. 2011. Defining components of the chromosomal origin of replication of the hyperthermophilic archaeon Pyrococcus furiosus needed for construction of a stable replicating shuttle vector. Appl. Environ. Microbiol. 77: 6343-6349. https://doi.org/10.1128/AEM.05057-11
  9. Farkas J, Stirrett K, Lipscomb GL, Nixon W, Scott RA, Adams MW, Westpheling J. 2012. Recombinogenic properties of Pyrococcus furiosus strain COM1 enable rapid selection of targeted mutants. Appl. Environ. Microbiol. 78: 4669-4676. https://doi.org/10.1128/AEM.00936-12
  10. Frols S, Ajon M, Wagner M, Teichmann D, Zolghadr B, Folea M, et al. 2008. UV-inducible cellular aggregation of the hyperthermophilic archaeon Sulfolobus solfataricus is mediated by pili formation. Mol. Microbiol. 70: 938-952. https://doi.org/10.1111/j.1365-2958.2008.06459.x
  11. Gudbergsdottir S, Deng L, Chen Z, Jensen JV, Jensen LR, She Q, Garrett RA. 2011. Dynamic properties of the Sulfolobus CRISPR/Cas and CRISPR/Cmr systems when challenged with vector-borne viral and plasmid genes and protospacers. Mol. Microbiol. 79: 35-49. https://doi.org/10.1111/j.1365-2958.2010.07452.x
  12. Hileman TH, Santangelo TJ. 2012. Genetics techniques for Thermococcus kodakarensis. Front. Microbiol. 3: 195.
  13. Jonuscheit M, Martusewitsch E, Stedman KM, Schleper C. 2003. A reporter gene system for the hyperthermophilic archaeon Sulfolobus solfataricus based on a selectable and integrative shuttle vector. Mol. Microbiol. 48: 1241-1252. https://doi.org/10.1046/j.1365-2958.2003.03509.x
  14. Leigh JA, Albers SV, Atomi H, Allers T. 2011. Model organisms for genetics in the domain Archaea: methanogens, halophiles, Thermococcales and Sulfolobales. FEMS Microbiol. Rev. 35: 577-608. https://doi.org/10.1111/j.1574-6976.2011.00265.x
  15. Maaty WS, Wiedenhef t B, Tarlykov P, Schaff N, Heinemann J, Robison-Cox J, et al. 2009. Something old, something new, something borrowed; how the thermoacidophilic archaeon Sulfolobus solfataricus responds to oxidative stress. PLoS One 14: e6964.
  16. Peng N, Xia Q, Chen Z, Liang YX, She Q. 2009. An upstream activation element exerting differential transcriptional activation on an archaeal promoter. Mol. Microbiol. 74: 928-939. https://doi.org/10.1111/j.1365-2958.2009.06908.x
  17. Popov M, Petrov S, Nacheva G, Ivanov I, Reichl U. 2011. Effects of a recombinant gene expression on ColE1-like plasmid segregation in Escherichia coli. BMC Biotechnol. 11: 18. https://doi.org/10.1186/1472-6750-11-18
  18. Reilly MS, Grogan DW. 2001. Characterization of intragenic recombination in a hyperthermophilic archaeon via conjugational DNA exchange. J. Bacteriol. 183: 2943-2946. https://doi.org/10.1128/JB.183.9.2943-2946.2001
  19. Santangelo TJ, Cubonová L, Reeve JN. 2008. Shuttle vector expression in Thermococcus kodakaraensis: contributions of cis elements to protein synthesis in a hyperthermophilic archaeon. Appl. Environ. Microbiol. 74: 3099-3104. https://doi.org/10.1128/AEM.00305-08
  20. Sato T, Fukui T, Atomi H, Imanaka T. 2003. Targeted gene disruption by homologous recombination in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. J. Bacteriol. 185: 210-220. https://doi.org/10.1128/JB.185.1.210-220.2003
  21. Sato T, Fukui T, Atomi H, Imanaka T. 2005. Improved and versatile transformation system allowing multiple genetic manipulations of the hyperthermophilic archaeon Thermococcus kodakaraensis. Appl. Environ. Microbiol. 71: 3889-3899. https://doi.org/10.1128/AEM.71.7.3889-3899.2005
  22. Schelert J, Dixit V, Hoang V, Simbahan J, Drozda M, Blum P. 2004. Occurrence and characterization of mercury resistance in the hyperthermophlic archaeon Sulfolobus solfataricus by use of gene disruption. J. Bacteriol. 186: 427-437. https://doi.org/10.1128/JB.186.2.427-437.2004
  23. Schelert J, Drozda M, Dixit V, Dillman A, Blum P. 2006. Regulation of mercury resistance in the crenarchaeote Sulfolobus solfataricus. J. Bacteriol. 188: 7141-7150. https://doi.org/10.1128/JB.00558-06
  24. She Q, Singh RK, Confalonieri F, Zivanovic Y, Allard G, Awayez MJ, et al. 2009. The complete genome of the crenarchaeon Sulfolobus solfataricus P2. Proc. Natl. Acad. Sci. USA 98: 7835-7840.
  25. Szabo Z, Sani M, Groeneveld M, Zolghadr B, Schelert J, Albers SV, et al. 2007. Flagellar motility and structure in the hyperthermoacidophilic archaeon Sulfolobus solfataricus. J. Bacteriol. 189: 4305-4309. https://doi.org/10.1128/JB.00042-07
  26. Waege I, Schmid G, Thumann S, Thomm M, Hausner W. 2010. Shuttle vector-based transformation system for Pyrococcus furiosus. Appl. Environ. Microbiol. 76: 3308-3313. https://doi.org/10.1128/AEM.01951-09
  27. Wagner M, Berkner S, Ajon M, Driessen AJ, Lipps G, Albers SV. 2009. Expanding and understanding the genetic toolbox of the hyperthermophilic genus Sulfolobus. Biochem. Soc. Trans. 37: 97-101. https://doi.org/10.1042/BST0370097
  28. Wagner M, van Wolferen M, Wagner A, Lassak K, Meyer BH, Reimann J, Albers SV. 2012. Versatile genetic tool box for the crenarchaeote Sulfolobus acidocaldarius. Front. Microbiol. 3: 214.
  29. Worthington P, Hoang V, Perez-Pomares F, Blum P. 2003. Targeted disruption of the $\alpha$-amylase gene in the hyperthermophilic archaeon Sulfolobus solfataricus. J. Bacteriol. 185: 482-488. https://doi.org/10.1128/JB.185.2.482-488.2003
  30. Zhang C, Guo L, Deng L, Wu Y, Liang Y, Huang L, She Q. 2010. Revealing the essentiality of multiple archaeal pcna genes using a mutant propagation assay based on an improved knockout method. Microbiology 156: 3386-3397. https://doi.org/10.1099/mic.0.042523-0
  31. Zhang C, Whitaker RJ. 2012. A broadly applicable gene knockout system for the thermoacidophilic archaeon Sulfolobus islandicus based on simvastatin selection. Microbiology 158: 1513-1522. https://doi.org/10.1099/mic.0.058289-0
  32. Zheng T, Huang Q, Zhang C, Ni J, She Q, Shen Y. 2012. Development of a simvastatin selection marker for a hyperthermophilic acidophile, Sulfolobus islandicus. Appl. Environ. Microbiol. 78: 568-574. https://doi.org/10.1128/AEM.06095-11
  33. Zolghadr B, Weber S, Szabo Z, Driessen AJ, Albers SV. 2007. Identification of a system required for the functional surface localization of sugar binding proteins with class III signal peptides in Sulfolobus solfataricus. Mol. Microbiol. 64: 795-806. https://doi.org/10.1111/j.1365-2958.2007.05697.x

피인용 문헌

  1. Isolation and Molecular Identification of Auxotrophic Mutants to Develop a Genetic Manipulation System for the Haloarchaeon Natrinema sp. J7-2 vol.2015, pp.None, 2015, https://doi.org/10.1155/2015/483194