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Vibrio fluvialis oligopeptide permease (oppA) 유전자 deletion에 의한 생리적 특성

Characterization of Physiological Properties in Vibrio fluvialis by the Deletion of Oligopeptide Permease (oppA) Gene

  • Ahn Sun Hee (Department of Biotechnology and Bioengineering, Pukyong National University) ;
  • Lee Eun Mi (Department of Biotechnology and Bioengineering, Pukyong National University) ;
  • Kim Dong Gyun (Department of Biotechnology and Bioengineering, Pukyong National University) ;
  • Hong Gyoung Eun (Department of Biotechnology and Bioengineering, Pukyong National University) ;
  • Park Eun Mi (Department of Biotechnology and Bioengineering, Pukyong National University) ;
  • Kong In Soo (Department of Biotechnology and Bioengineering, Pukyong National University)
  • 발행 : 2006.02.01

초록

미생물이 이용할 수 있는 nitrogen source는 di-, tri-, oli- go-peptide 또는 amino acid의 형태로 세포내로 uptake되어 대사과정에 사용되고 있다. 이와같은 peptide는 특이한 transport system에 의해서 이동되고 있는데 oligo peptide(Opp) transport system에는 binding protein, permease protein, energy 생성을 위한 ATP 분해에 관여하는 protein 이 관여하고 있으며 염색체 상에서 이들 단백질들은 operon 형태의 유전자로부터 발현되고 있다. 본 연구는 gram 음성 세균이며 수해양 서식 세균인 V, fluvialis로부터 얻어진 Opp operon 유전자 가운데 oligopeptide binding protein을 coding하고 있는 oppA 유전자가 deletion된 mutant를 사용하여 여러 환경변화에 따른 생육을 wild type과 비교한 연구 결과 이다. 생육을 위한 완전배지인 brain heart infusion (BHI) 배지와 최소배지인 M9 minimal 배지를 사용한 결과 OppA protein의 생성 결핍에 따라 초기 및 대수증식기 과정 중에는 mutant의 생육이 늦어지고 있으나 Opp system이 아닌 다른 peptide전달 경로로 추정되는 system을 이용하여 대수 증식기 후반에서는 wild type과 거의 같은 생육 형태를 보여 주고 있었다. pH의 변화에 따른 생육은 pH 7에서는 생육정도가 비슷하였으나 약알칼리 부근에서는 oppA mutant의 생육이 wild type에 비하여 낮아지고 있었다. 또한 5 mM $H_2O_2$를 사용하여 $OD_{600}=1.2$농도의 세포들에 대한 영향을 검토한 결과 두 균 모두 높은 생존율을 보여 주었으며 이는 대수증식기 세포들을 사용한 결과와는 매우 다른 형태를 보여 주고 있었다. 항생제 내성에 대한 연구에서는 mutant가 streptomycin과 tetracycline 에 대해서는 wild type과는 다르게 매우 낮은 농도에서도 생육이 되고 있지 않으나 polymyxin B에 대해서는 wild type과 같이 $10{\mu}g/ml$의 농도에서도 잘 자라고 있었다.

Oligopeptide is known to be an essential nitrogen nutrient for bacterial growth. Oligopeptide can be transported into cytoplasm by a specific transport system, Opp system. Opp system is composed of five proteins, which are transcribed by an operon. These are responsible for oligopeptide binding protein (OppA), permease (OppB and OppC) and energy generation system (OppD and OppF), respectively. Previously, we isolated the opp operon from Vibrio fluvialis and constructed the oppA mutant by allelic exchange method. In this study, we investigated the growth pattern and biofilm production under the different growth condition. When the cells were cultivated using brain heart infusion(BHI) medium, the wild type was faster than the mutant in growth during the exponential phase. However, it showed that the growth pattern of two strains in M9 medium is very similar. The growth of wild type showed better than that of the mutant grown at pH 8. At pH 7, there was no an obvious difference in growth. After 5 mM $H_2O_2$ was treated to the cells $(OD_{600}=1.2)$, the cell survival was examined. The oppA mutation did not affect in survivability. In the presence of $10{\mu}g/ml$ polymyxin B, the biofilm production of the oppA mutant was higher than that of the wild type.

키워드

참고문헌

  1. Borezee, E., E. Pellegrini and P. Berche. 2000. OppA of Listeria monocytogenes, an oligopeptide-binding protein required for bacterial growth at low temperature and involved in intracellular survival. Infect. Immun. 68, 7069-7077 https://doi.org/10.1128/IAI.68.12.7069-7077.2000
  2. Cundell, D. R., B. J. Pearce, J. Samderls, A. M. Naughton and H. R. Masure. 1995. Peptide permease from Streptococcus pneumoniae affect adherence to eukaryotic cells. Infect. Immun. 63, 2493-2498
  3. Darmstadt, G. L., L. Mentele, A. Podbielski and C. E. Rubens. 2000. Role of group A Streptococcal virulence factors in adherence to keratinocytes. Infect. Immun. 68, 1215-1221 https://doi.org/10.1128/IAI.68.3.1215-1221.2000
  4. Detmers, F. J. M., F. C. Lanfermeijer and B. Poolman, 2001. Peptides and ATP binding cassette peptide transporters. Res. Microbiol. 152, 245-258 https://doi.org/10.1016/S0923-2508(01)01196-2
  5. Goodell, E. W. and C. F. Higgins. 1987. Uptake of cell wall peptides by Salmonella typhimurium and Escherichia coli. J. Bacteriol. 169, 3861-3865 https://doi.org/10.1128/jb.169.8.3861-3865.1987
  6. Higgins, C. F. 1992. ABC transporters : from microorganisms to man. Ann. Rev. Cell Biol. 8, 67-113 https://doi.org/10.1146/annurev.cb.08.110192.000435
  7. Lazazzera, B. 2001. The intracellular function of extracellular signaling peptides. Peptides 22, 1519-1527 https://doi.org/10.1016/S0196-9781(01)00488-0
  8. Lee. E. M., S. H. Ahn, J. H. Park, J. H. Lee, S. C. Ahn and I. S. Kong. 2004. Identification of oligopeptide permease(opp) gene cluster in Vibrio fluvialis and characterization of biofilm production by oppA knockout mutation. FEMS Microbiol. Lett. 240 21-30 https://doi.org/10.1016/j.femsle.2004.09.007
  9. Leonard, B. A., A. Podbielski, P. J. Hedberg and G. M. Dunny. 1996. Enterococcus faecalis pheromone binding protein, PrgZ, recruits a chromosomal oligopeptide permease system to import sex pheromone cCF10 for induction of conjugation. Proc. Natl. Acad. Sci. 93, 260-264
  10. Lin, B., S. A. Short, M. Eskildsen, M. S. Klempner and L. T. Hu. 2001. Functional testing of putative oligopeptide permease (Opp) proteins of Borrelia burgdorferi : a complementation model in opp- Escherichia coli. Biochim. Biophys. Acta 1499, 222-231 https://doi.org/10.1016/S0167-4889(00)00121-X
  11. Payne, J. W. and M. W. Smith. 1994. Peptide transport by microorganisms. Adv. Microb. Physiol. 36, 1-80 https://doi.org/10.1016/S0065-2911(08)60176-9
  12. Perego, M, C. F. Higgins, S. R. Pearce, M. P. Gallagher and J. A. Hoch. 1991. The oligopeptide transport system of Bacillus subtilis plays a role in the initiation of sporulation. Mol. Microbiol. 5, 173-185 https://doi.org/10.1111/j.1365-2958.1991.tb01838.x
  13. Podbielski, A., B. Pohl, M. Woischnik, C. Korner, K. H. Schmidt, E. Rozdinski and B. A. Leonard. 1996. Molecular characterization of group A streptococcal (GAS) oligopeptide permease (Opp) and its effect on cystein protease production. Mol. Microbiol. 21, 1087-1099 https://doi.org/10.1046/j.1365-2958.1996.661421.x
  14. Renee, M. G., A. Seth and N. D. Connel. 2000. A peptide permease mutant of Mycobacterium bovisBCG resistant to the toxic peptides glutathione and s-nitrosoglutathione. Infect. Immun. 68, 429-436 https://doi.org/10.1128/IAI.68.2.429-436.2000
  15. Rudner, D. Z., J. R. LeDeaux, K. Ireton and A. D. Grossman. 1991. The Spo0K locus of Bacillus subtilis is homologous to oligopeptide permease locus and is required for sporulation and competence. J. Bacteriol. 173, 1388-1398 https://doi.org/10.1128/jb.173.4.1388-1398.1991
  16. Solomon, J. M., R. Magnuson, A. Srivastava and A. D. Grossman. 1995. Convergent sensing pathways mediate response to two extracellular competence factors in Bacillus subtilis. Genes Dev. 9, 547-558 https://doi.org/10.1101/gad.9.5.547
  17. Tam, R. and M. T. Saier. 1993. Structural, functional, and evolutionary relationships among extracellular solute-binding receptors of bacteria. Microbiol. Rev. 57, 320-346