Characterization and Cloning of the Gene Encoding Autoregulator Receptor Protein from Streptomyces longwoodensis

Streptomyces longwoodensis로부터 Autoregulator Receptor Protein 유전자의 클로닝 및 특성

  • Yeo Soo-Hwan (The Center for Traditional Microorganism Resources, Keimyung University) ;
  • Lee Sung-Bong (Department of Microbiology, College of Natural Science, Keimyung University) ;
  • Kim Hyun-Soo (Department of Microbiology, College of Natural Science, Keimyung University)
  • 여수환 (전통미생물자원개발 및 산업화연구센터) ;
  • 이성봉 (계명대학교 자연과학대학 미생물학과) ;
  • 김현수 (계명대학교 자연과학대학 미생물학과)
  • Published : 2005.06.01

Abstract

For screening of autoregulator receptor gene from Streptomyces longwoodensis, PCR was performed with primers of receptor gene designed on the basis of amino acid sequences of autoregulator receptor proteins with known function. PCR products were subcloned into the BamHIsite of pUC19 and transformed into the E. coli $DH5{\alpha}$. The isolated plasmid from transformant contained the fragment of 100 bp, which was detected on $2\%$ gel after BamHI treatment. The insert, 100 bp PCR product, was confirmed as the expected internal segment of gene encoding autoregulator receptor protein by sequencing. Southern and colony hybridizations with the 100 bp fragment as a probe allowed to select a genomic clone of S. longwoodensis, pSLT harboring a 4.4 kb SphI fragment. Nucleotide sequencing analyses revealed a 651 bp open reading frame(ORF) were isolated protein showing moderate homology ($35{\sim}46\%$) with the ${\Gamma}$-butyrolactone autoregulator receptors from Streptomyces sp., and this ORF was named sltR The sltR/pET-17b plasmid was constructed to overexpress the recombinant SltR protein (rSltR) in E. coli BL21 (DE3)/pLysS, and the rSltR protein was purified to homogeneity by DEAE-Sephacel column chromatography, and DEAE-5PW chromatography (HPLC). The molecular mass of the purified rSltR protein was 55 kDa by HPLC gel-filtration chromatography and 28 kDa by SDS-PAGE, indicating that the rSltR protein is present as a dimer. A binding assay with tritium-labeled autoregulators revealed that the rSltR has clear binding activity with a A-factor type autoregulator as the most effective ligand.

공시균인 S. longwoodensis IFO 14251의 autoregulators 및 receptor gene 탐색의 일환으로 기존의 Streptomyces속 receptor gene의 공통배열을 primer로 이용하여 PCR을 수행하였다. 예상되는 100 bp 크기의 단편을 pUC19 vector에 ligation하여 E. coli $DH5{\alpha}$에 transformation한 후, plasmid를 분리하여 BamHI을 처리하여 $2\%$ agarose gel에 전기영동한 결과, pUC19 외에 receptor gene PCR product가 100 bp 위치에 존재하는 것을 확인하였다. 형질전환된 plasmid로 PCR을 수행한 후, 염기배열을 결정하여 분석한 결과, Streptomyces sp. 유래의 receptor gene의 일부분임이 확인되었다. 따라서 S. longwoodensis IFO 14251에는 lysocellin 생산에 관여한다고 추정되는 autoregulator receptor protein을 코드하는 유전자가 존재할 것으로 예상되어 100 bp의 PCR product를 probe로 이용하여 Southern 및 colony hybridization을 통하여 4.4 kb의 SphI 단편을 가지는 plasmid(pSLT)를 제작하였고 이를 sequencing한 결과, Streptomyces 속 유래의 autoregulator receptor 단백질을 코드하는 유전자와 3개의 open reading frame(651 bp)을 확인하여 sltR이라 명명하였다. 유전자 해석 결과, 기존의 autoregulator receptor proteins과 비교시 $35{\sim}46\%$의 homology를 나타내었다. 재조합 단백질의 발현을 위해, sltR/pET-l7b plasmid를 제작하고 E. coli BL21(DE3)/pLysS을 host cell로 이용하여 재조합 단백질을 발현시켰다. SltR 재조합 단백질의 정제는 DEAE-Sephacel column chromatography와 DEAE-5PW chromatography(HPLC)를 통해 수행하였다. Gel filtration chromatography(HPLC, 55 kDa)와 SDS-PAGE(28 kDa)를 통해 분자량을 확인한 결과, 재조합 단백질은 dimer형태로 존재하는 것으로 확인되었다. 또한, 결합활성에 있어 A-factor type의 autoregulator에 대해 가장 강한 결합활성을 나타내었다.

Keywords

References

  1. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254 https://doi.org/10.1016/0003-2697(76)90527-3
  2. Chater, K. F. 1993. Genetics of differentiation in Streptomyces. Annu. Rev. Microbiol. 47: 685-713 https://doi.org/10.1146/annurev.mi.47.100193.003345
  3. Champness, W. C. and K. F. Chater. 1994. Regulation and integration of antibiotic production and morphological differentiation in Streptomyces sp. In Regulation of Bacterial Differentiation. P. J. Piggot, C. P. Moran, and P. Youngman. (eds.), Washington, D. C; American Society for Microbiology. USA
  4. Grafe, U., W. Shade, I. Eritt, W. F. Fleck, and L. Radics. 1982. A new inducer of anthracycline biosynthesis from Streptomyces viridochromogenes. J. Antibiot. 35: 1722-1723 https://doi.org/10.7164/antibiotics.35.1722
  5. Horinouchi, S., O. Hara, and T. Beppu. 1983. Cloning of a pleiotropic gene that positively controls biosynthesis of Afactor, actinorhodin, and prodigiosin in Streptomyces coelicolor A3(2) and Streptomyces lividans. J. Bacteriol. 155: 1238-1248
  6. Horinouchi, S., Y. Kumada, and T. Beppu. 1984. Unstable genetic determinant of A-factor biosynthesis in streptomycinproducing organisms: cloning and characterization. J. Bacteriol. 158: 481-487
  7. Khokhov, A. S., I. I. Tavalova, L. N. Borisova, S. A. Pliner, L. N. Shevchenko. Kolnitskaya, N. S. Ivkina, and I. A. Rapoport. 1967. A-factor responsible for the biosynthesis of streptomycin by a mutant strain of Actinomycetes streptomycini. Dokalady Akademii Nauk SSSR 177: 232
  8. Kim, H. S., H. Tada, T. Nihira, and Y. Yamada. 1990. Purification and characterization of virginiae butanolide Cbinding protein, a possible pleiotropic signal-transducer in Streptomyces virginiae. J. Antibiot. 43: 692-706 https://doi.org/10.7164/antibiotics.43.692
  9. Kim, H. S. and S. Y. Kang. 1994. General microbiology, physiology and metabolism; Induction of secondary metabolites by virginiamycin inducing factor, virginiae butanolide C. Kor. J. Appl. Microbiol. Biotechnol 22: 459-466
  10. Kim, H. S., T. Nihira, H. Tada, M. Yanagimoto, and Y. Yamada. 1989. Identification of binding protein of virginiae butanolide C, an autoregulator in virginiamycin production from Streptomyces virginiae. J. Antibiot. 42: 769-778 https://doi.org/10.7164/antibiotics.42.769
  11. Kinoshita, H., H. Ipposhi, S. Okamoto, H. Nakano, T. Nihira, and Y. Yamada. 1997. Butyrolactone autoregulator receptor protein (BarA) as a transcriptional regulator in Streptomyces virginiae. J. Bacteriol. 179: 6986-6993
  12. Kondo, K., Y. Higuchi, S. Sakuda, T. Nihira, and Y. Yamada. 1989. New virginiae butanolides from Streptomyces virginiae. J. Antibiot. 42: 1873-1876 https://doi.org/10.7164/antibiotics.42.1873
  13. Laemmli, L. K. 1970. Cleavage structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685 https://doi.org/10.1038/227680a0
  14. Lee, C. K., Y. Kamitani, T. Nihira, and Y. Yamada. 1996. Identification and in vivo funtional analysis Streptomyces virginiae. J. Bacteriol 181: 3293-3297
  15. Miyake, K., T. Kuzuyma, S. Horinouchi, and T. Beppu. 1990. The A-factor binding protein of Streptomyces griseus negatively controls streptomycin and sporulation. J. Bacteriol. 172: 3003-3008
  16. Nakano, H., E. Takehara, T. Nihira, and Y. Yamada. 1998. Gene replacement analysis of the Streptomyces virginiae barA gene encoding the butyrolactone autoregulator receptor reveals that BarA acts as a Repressor in virginiamycin biosynthesis J. Bacteriol. 180: 3317-3322
  17. Nihira, T., Y. Shimizu, H. S. Kim, and Y. Yamada. 1988. Structure-activity relationships of virginiae butanolide C, an inducer of virginiamycin production in Streptomyces virginiae. J. Antibiot. 41: 1828-1837 https://doi.org/10.7164/antibiotics.41.1828
  18. Okamoto, S., K. Nakamura, T. Nihira, and Y. Yamada. 1995. Virginiae butanolide binding protein from Streptomyces virginiae. J. Biol. Chem. 270: 12319-12326 https://doi.org/10.1074/jbc.270.20.12319
  19. Ruengjitchatchawalya, M., T. Nihira, and Y Yamada. 1995. Purification and characterization of the IM-2-binding protein from Streptomyces sp. strain FRI-5. J. Bacteriol. 177: 551-557
  20. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A laboratory manual (ed.) 2, Cold spring harbor laboratory, Cold spring harbor. New York. USA
  21. Waki, M., T. Nihira, and Y. Yamada. 1997. Cloning and characterization of the gene (far A) encoding the receptor for an extracellular regulatory factor (IM-2) from Streptomyces sp. strain FRI-5. J. Bacteriol.179: 5131-5137
  22. Yamada, Y, T. Nihira, and S. Sakuda. 1997. Butyrolactone autoregulators inducers of virginiamycin in Streptomyces virginiae: their structures, biosynthesis, receptor proteins, and induction of virginiamycin biosynthesis. Biotechnology of antibiotics, Marcel Dekker, Inc., New York, pp. 63-79
  23. Yamada, Y., K. Sugimura, K. Kondo, M. Yanagimoto, and H. Okada. 1987. The structure of inducing factors for virginiamycin in Streptomyces virginiae. J. Antibiot. 40: 496 https://doi.org/10.7164/antibiotics.40.496