1887

Microbiology Society logo

Volume 156, Issue 11

Revealing the essentiality of multiple archaeal genes using a mutant propagation assay based on an improved knockout method Free

Abstract

Organisms belonging to the Crenarchaeota lineage contain three proliferating cell nuclear antigen (PCNA) subunits, while those in the Euryarchaeota have only one, as for Eukarya. To study the mechanism of archaeal sliding clamps, we sought to generate knockouts for each gene in , a hyperthermophilic crenarchaeon, but failed with two conventional knockout methods. Then, a new knockout scheme, known as marker insertion and target gene deletion (MID), was developed, with which transformants were obtained for each pMID-pcna plasmid. We found that mutant cells persisted in transformant cultures during incubation of pMID-pcna3 and pMID-araS-pcna1 transformants under counter selection. Studying the propagation of mutant cells by semiquantitative PCR analysis of the deleted target gene allele (Δ or Δ) revealed that mutant cells could no longer be propagated, demonstrating that these genes are absolutely required for host cell viability. Because the only prerequisite for this assay is the generation of a MID transformant, this approach can be applied generally to any micro-organisms proficient in homologous recombination.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): 5-FOA, 5′-fluoroorotic acid , AR, allelic replacement , MCI, marker circularization and integration , MID, marker insertion and unmarked target gene deletion , MRL, marker replacement and looping out and PCNA, proliferating cell nuclear antigen
SGM
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.042523-0
2010-11-01
2024-04-26
Loading full text...

Full text loading...

/deliver/fulltext/micro/156/11/3386.html?itemId=/content/journal/micro/10.1099/mic.0.042523-0&mimeType=html&fmt=ahah

References

  1. Allers T., Mevarech M. 2005; Archaeal genetics – the third way. Nat Rev Genet 6:58–73
     [Google Scholar]
  2. Auernik K. S., Maezato Y., Blum P. H., Kelly R. M. 2008; The genome sequence of the metal-mobilizing, extremely thermoacidophilic archaeon Metallosphaera sedula provides insights into bioleaching-associated metabolism. Appl Environ Microbiol 74:682–692
     [Google Scholar]
  3. Barry E. R., Bell S. D. 2006; DNA replication in the archaea. Microbiol Mol Biol Rev 70:876–887
     [Google Scholar]
  4. Berquist B. R., DasSarma P., DasSarma S. 2007; Essential and non-essential DNA replication genes in the model halophilic archaeon, Halobacterium sp. NRC-1. BMC Genet 8:31
     [Google Scholar]
  5. Cann I. K., Ishino S., Hayashi I., Komori K., Toh H., Morikawa K., Ishino Y. 1999; Functional interactions of a homolog of proliferating cell nuclear antigen with DNA polymerases in Archaea. J Bacteriol 181:6591–6599
     [Google Scholar]
  6. Chen L., Brugger K., Skovgaard M., Redder P., She Q., Torarinsson E., Greve B., Awayez M., Zibat A. other authors 2005; The genome of Sulfolobus acidocaldarius, a model organism of the Crenarchaeota. J Bacteriol 187:4992–4999
     [Google Scholar]
  7. Contursi P., Jensen S., Aucelli T., Rossi M., Bartolucci S., She Q. 2006; Characterization of the Sulfolobus host–SSV2 virus interaction. Extremophiles 10:615–627
     [Google Scholar]
  8. Daimon K., Kawarabayasi Y., Kikuchi H., Sako Y., Ishino Y. 2002; Three proliferating cell nuclear antigen-like proteins found in the hyperthermophilic archaeon Aeropyrum pernix: interactions with the two DNA polymerases. J Bacteriol 184:687–694
     [Google Scholar]
  9. Deng L., Zhu H., Chen Z., Liang Y. X., She Q. 2009; Unmarked gene deletion and host-vector system for the hyperthermophilic crenarchaeon Sulfolobus islandicus. Extremophiles 13:735–746
     [Google Scholar]
  10. Dionne I., Bell S. D. 2005; Characterization of an archaeal family 4 uracil DNA glycosylase and its interaction with PCNA and chromatin proteins. Biochem J 387:859–863
     [Google Scholar]
  11. Dionne I., Nookala R. K., Jackson S. P., Doherty A. J., Bell S. D. 2003; A heterotrimeric PCNA in the hyperthermophilic archaeon Sulfolobus solfataricus. Mol Cell 11:275–282
     [Google Scholar]
  12. Doré A. S., Kilkenny M. L., Jones S. A., Oliver A. W., Roe S. M., Bell S. D., Pearl L. H. 2006; Structure of an archaeal PCNA1–PCNA2–FEN1 complex: elucidating PCNA subunit and client enzyme specificity. Nucleic Acids Res 34:4515–4526
     [Google Scholar]
  13. Grabowski B., Kelman Z. 2003; Archeal DNA replication: eukaryal proteins in a bacterial context. Annu Rev Microbiol 57:487–516
     [Google Scholar]
  14. Grúz Pisani F. M., Shimizu M., Yamada M., Hayashi I., Morikawa K., Nohmi T. 2001; Synthetic activity of Sso DNA polymerase Y1, an archaeal DinB-like DNA polymerase, is stimulated by processivity factors proliferating cell nuclear antigen and replication factor C. J Biol Chem 276:47394–47401
     [Google Scholar]
  15. Hutton R. D., Roberts J. A., Penedo J. C., White M. F. 2008; PCNA stimulates catalysis by structure-specific nucleases using two distinct mechanisms: substrate targeting and catalytic step. Nucleic Acids Res 36:6720–6727
     [Google Scholar]
  16. Imamura K., Fukunaga K., Kawarabayasi Y., Ishino Y. 2007; Specific interactions of three proliferating cell nuclear antigens with replication-related proteins in Aeropyrum pernix. Mol Microbiol 64:308–318
     [Google Scholar]
  17. Indiani C., O'Donnell M. 2006; The replication clamp-loading machine at work in the three domains of life. Nat Rev Mol Cell Biol 7:751–761
     [Google Scholar]
  18. Jonuscheit M., Martusewitsch E., Stedman K. M., 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
     [Google Scholar]
  19. Kawarabayasi Y., Hino Y., Horikawa H., Jin-no K., Takahashi M., Sekine M., Baba S., Ankai A., Kosugi H. other authors 2001; Complete genome sequence of an aerobic thermoacidophilic crenarchaeon, Sulfolobus tokodaii strain7. DNA Res 8:123–140
     [Google Scholar]
  20. Kiyonari S., Takayama K., Nishida H., Ishino Y. 2006; Identification of a novel binding motif in Pyrococcus furiosus DNA ligase for the functional interaction with proliferating cell nuclear antigen. J Biol Chem 281:28023–28032
     [Google Scholar]
  21. Kiyonari S., Uchimura M., Shirai T., Ishino Y. 2008; Physical and functional interactions between uracil-DNA glycosylase and proliferating cell nuclear antigen from the euryarchaeon Pyrococcus furiosus. J Biol Chem 283:24185–24193
     [Google Scholar]
  22. Lu S., Li Z., Wang Z., Ma X., Sheng D., Ni J., Shen Y. 2008; Spatial subunit distribution and in vitro functions of the novel trimeric PCNA complex from Sulfolobus tokodaii. Biochem Biophys Res Commun 376:369–374
     [Google Scholar]
  23. Matsumiya S., Ishino Y., Morikawa K. 2001; Crystal structure of an archaeal DNA sliding clamp: proliferating cell nuclear antigen from Pyrococcus furiosus. Protein Sci 10:17–23
     [Google Scholar]
  24. Mayanagi K., Kiyonari S., Saito M., Shirai T., Ishino Y., Morikawa K. 2009; Mechanism of replication machinery assembly as revealed by the DNA ligase–PCNA–DNA complex architecture. Proc Natl Acad Sci U S A 106:4647–4652
     [Google Scholar]
  25. Miyata T., Suzuki H., Oyama T., Mayanagi K., Ishino Y., Morikawa K. 2005; Open clamp structure in the clamp-loading complex visualized by electron microscopic image analysis. Proc Natl Acad Sci U S A 102:13795–13800
     [Google Scholar]
  26. Moldovan G. L., Pfander B., Jentsch S. 2007; PCNA, the maestro of the replication fork. Cell 129:665–679
     [Google Scholar]
  27. Naryzhny S. N. 2008; Proliferating cell nuclear antigen: a proteomics view. Cell Mol Life Sci 65:3789–3808
     [Google Scholar]
  28. Peng N., Xia Q., Chen Z., Liang Y. X., She Q. 2009; An upstream activation element exerting differential transcriptional activation on an archaeal promoter. Mol Microbiol 74:928–939
     [Google Scholar]
  29. Reno M. L., Held N. L., Fields C. J., Burke P. V., Whitaker R. J. 2009; Biogeography of the Sulfolobus islandicus pan-genome. Proc Natl Acad Sci U S A 106:8605–8610
     [Google Scholar]
  30. Roberts J. A., Bell S. D., White M. F. 2003; An archaeal XPF repair endonuclease dependent on a heterotrimeric PCNA. Mol Microbiol 48:361–371
     [Google Scholar]
  31. Sambrook J., Russell D. W. 2001 Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  32. 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
     [Google Scholar]
  33. She Q., Singh R. K., Confalonieri F., Zivanovic Y., Allard G., Awayez M. J., Chan-Weiher C. C., Clausen I. G., Curtis B. A. other authors 2001; The complete genome of the crenarchaeon Sulfolobus solfataricus P2. Proc Natl Acad Sci U S A 98:7835–7840
     [Google Scholar]
  34. She Q., Zhang C., Deng L., Peng N., Chen Z., Liang Y. X. 2009; Genetic analyses in the hyperthermophilic archaeon Sulfolobus islandicus. Biochem Soc Trans 37:92–96
     [Google Scholar]
  35. Tsurimoto T. 1999; PCNA binding proteins. Front Biosci 4:D849–D858
     [Google Scholar]
  36. Worthington P., Hoang V., Perez-Pomares F., Blum P. 2003; Targeted disruption of the α-amylase gene in the hyperthermophilic archaeon Sulfolobus solfataricus. J Bacteriol 185:482–488
     [Google Scholar]
  37. Xing G., Kirouac K., Shin Y. J., Bell S. D., Ling H. 2009; Structural insight into recruitment of translesion DNA polymerase Dpo4 to sliding clamp PCNA. Mol Microbiol 71:678–691
     [Google Scholar]
  38. Yang H., Chiang J. H., Fitz-Gibbon S., Lebel M., Sartori A. A., Jiricny J., Slupska M. M., Miller J. H. 2002; Direct interaction between uracil-DNA glycosylase and a proliferating cell nuclear antigen homolog in the crenarchaeon Pyrobaculum aerophilum. J Biol Chem 277:22271–22278
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.042523-0
Loading
Revealing the essentiality of multiple archaeal pcna genes using a mutant propagation assay based on an improved knockout method
Microbiology 156, 3386 (2010); https://doi.org/10.1099/mic.0.042523-0
/content/journal/micro/10.1099/mic.0.042523-0
/content/journal/micro/10.1099/mic.0.042523-0
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Most cited Most Cited RSS feed

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