Isolation and Characterization of Pyrimidine Auxotrophs from the Hyperthermophilic Archaeon Sulfolobus acidocaldarius DSM 639

국문 초록 (Abstract)

고세균 Sulfolobus acidocaldarius의 기능유전체학 연구를 위하여 피리미딘 생합성 유전자군의 pyrEF 유전자에 근거한 피리미딘 영양요구주를 구축하였다. 원균주는 정상적인 pyrEF 존재하에서 5-fluoroorotic acid를 첨가하면 성장이 불가능하나 피리미딘 영양요구주는 성장이 가능한 원리를 활용하였다. 자외선을 이용하여 얻어진 5-FOA 첨가에 저항성을 갖는 돌연변이주를 얻었으며, 두 돌연변이주 KH1U와 KH2U는 각각 pyrE 유전자 부분의 점돌연변이와 삽입돌연변이를 갖는 돌연변이주임을 알 수 있었다. 이 두 돌연변이 균주는 5-FOA의 첨가에 의하여 이 세포를 사멸시킬 수 있는 능력이 사라짐을 확인하였다. 정상적인 pyrEF 유전자를 갖는 Sulfolobus-E. coli 플라스미드를 이용하여 보완실험을 수행한 결과 KH2U 돌연변이주는 다시 5-FOA에 대한 저항성을 잃어버렸으며, 배지 내에 피리미딘의 첨가가 없어도 생존할 수 있는 능력을 보여주는 원균주와 같은 표현형으로 회귀함을 확인하였다. 이 연구는 차후 고세균 Sulfolobus acidocaldarius의 유전자 불활성화를 통한 유전학연구에 효율적인 도구로 사용되기에 유용한 연구로 생각된다.

다국어 초록 (Multilingual Abstract)

To study the functional genomic analysis of a crenachaeon Sulfolobus acidocaldarius, we have constructed an auxotrophic mutant based on pyrEF, which encodes the pyrimidine biosynthetic enzymes orotate phosphoribosyltransferase and orotidine-5’-monophosphate decarboxylase. S. acidocaldarius was shown to be sensitive to 5-fluoroorotic acid (5-FOA), which can be selected for mutations in pyrEF genes within a pyrimidine biosynthesis cluster. Spontaneous 5-FOA-resistant mutants by ultraviolet, KH1U and KH2U, were found to contain two point mutations and a frame shift mutation in pyrE, respectively. Mutations at these sites from KH1U and KH2U decreased the activity of orotate phosphoribosyl-transferase encoded by the pyrE gene and blocked the degradation of 5-FOA into toxic 5-FOMP and 5-FUMP that kill the cells. Therefore, KH1U and KH2U were uracil auxotrophs. Transformation of Sulfolobus-Escherichia coli shuttle vector pC bearing pyrEF genes from S. solfataricus P2 into S. acid-ocaldarius mutant KH2U restored 5-FOA sensitivity and overcame the uracil auxotrophy. This study establishes an efficient genetic strategy towards the systematic knockout of genes in S. acidocaldarius.

목차 (Table of Contents)

• Introduction
• Materials and Methods
• Results and Discussion
• Acknowledgements
• References
• 초록

참고문헌 (Reference)

1 Deng, L., "Unmarked gene deletion and host–vector system for the hyperthermophilic crenarchaeon Sulfolobus islandicus" 13 : 735-746, 2009

2 Marintchev, A, "Translation initiation: structures, mechanisms and evolution" 37 : 197-284, 2004

3 Blaby, I. K., "Towards a systems approach in the genetic analysis of archaea: Accelerating mutant construction and phenotypic analysis in Haloferax volcanii" 2010 : 426239-, 2010

4 Worthington, P, "Targeted disruption of the α-amylase gene in the hyperthermophilic archaeon Sulfolobus solfataricus" 185 : 482-488, 2003

5 Yurist-Doutsch, S., "Sweet to the extreme: protein glycosylation in archaea" 68 : 1079-1084, 2008

6 Brock, T. D., "Sulfolobus: A new genus of sulfur-oxidizing bacteria living at low pH and high temperature" 84 : 54-68, 1972

7 Berkner, S., "Small multicopy, non-integrative shuttle vectors based on the plasmid pRN1 for Sulfolobus acidocaldarius and Sulfolobus solfataricus, model organisms of the (cren-)archaea" 35 : e88-, 2007

8 Aravalli, R. N, "Shuttle vectors for hyperthermophilic archaea" 1 : 183-192, 1997

9 Eichler, J, "Posttranslational protein modification in archaea" 69 : 393-425, 2005

10 Berkner, S, "Mutation and reversion frequencies of different Sulfolobus species and strains" 12 : 263-270, 2008

11 Sambrook, J., "Molecular cloning: a laboratory manual, 3rd ed" Cold Spring Harbor Laboratory Press 2001

12 Geiduschek, E. P, "MicroReview: Archaeal transcription and ts regulators" 56 : 1397-1407, 2005

13 Sato, T., "Improved and versatile transformation system allowing multiple genetic manipulations of the hyperthermophilic archaeon Thermococcus kodakaraensis" 71 : 3889-3899, 2005

14 Kurosawa, N, "Homologous recombination of exogenous DNA with the Sulfolobus acidocaldarius genome: Properties and uses" 253 : 141-149, 2005

15 Peck, R. F., "Homologous gene knockout in the archaeon Halobacterium salinarum with ura3 as a counterselectable marker" 35 : 667-676, 2000

16 Lindstrom, E. B, "High efficiency of plating of the thermophilic sulfur-dependent archaebacterium Sulfolobus acidocaldarius" 55 : 3020-3021, 1989

17 Berkner, S, "Genetic tools for Sulfolobus spp.: vectors and first applications" 190 : 217-230, 2008

18 Facciotti, M. T., "General transcription factor specified global gene regulation in archaea" 4630-4635, 2007

19 Pritchett, M. A., "Development of a markerless genetic exchange method for Methanosarcina acetivorans C2A and its use in construction of new genetic tools for methanogenic archaea" 70 : 1425-1433, 2004

20 Barry, E. R, "DNA replication in the archaea" 70 : 876-887, 2006

21 Ellen, A., "Comparative study of the extracellular proteome of Sulfolobus species reveals limited secretion" 14 : 87-98, 2010

22 Allers, T, "Archaeal genetics: the third way" 6 : 58-73, 2005

23 Jun, S. H., "Archaeal RNA polymerase and transcription regulation" 46 : 27-40, 2011

24 Cannio, R., "An autonomously replicating transforming vector for Sulfolobus solfataricus" 180 : 3237-3240, 1998