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Bordetella bronchiseptica의 alcaligin siderophore 생합성 유전자인 alcA에 관한 연구

Studies of an alcA Gene Involved in Alcaligin Siderophore Biosynthesis in Bordetella bronchiseptica

  • 황호순 (부산대학교 자연과학대학 생명과학부) ;
  • 김영희 (부산대학교 자연과학대학 생명과학부) ;
  • 김삼웅 (부산대학교 자연과학대학 생명과학부) ;
  • 유종언 (부산대학교 자연과학대학 생명과학부) ;
  • 유아영 (부산대학교 자연과학대학 생명과학부) ;
  • 강호영 (부산대학교 자연과학대학 생명과학부) ;
  • 이태호 (부산대학교 자연과학대학 생명과학부)
  • Hwang, Ho-Soon (Division of Biological Sciences, College of Natural Sciences, Pusan National University) ;
  • Kim, Young-Hee (Division of Biological Sciences, College of Natural Sciences, Pusan National University) ;
  • Kim, Sam-Woong (Division of Biological Sciences, College of Natural Sciences, Pusan National University) ;
  • Yu, Jong-Earn (Division of Biological Sciences, College of Natural Sciences, Pusan National University) ;
  • Yoo, Ah-Young (Division of Biological Sciences, College of Natural Sciences, Pusan National University) ;
  • Kang, Ho-Young (Division of Biological Sciences, College of Natural Sciences, Pusan National University) ;
  • Lee, Tae-Ho (Division of Biological Sciences, College of Natural Sciences, Pusan National University)
  • 발행 : 2006.12.01

초록

돼지 위축성 비염과 개의 kennel cough의 원인균인 B. bronchiseptica는 각 숙주의 상부 호흡기관의 점막에 집락을 형성하는 병원균으로서 철이 부족한 환경에서 hydroxamate type의 alcaligin이 라는 siderophore를 생산한다. Alcaligin의 생합성에 관련하는 구조유전자 중 alcA 유전자의 기능을 밝히고자 alcA 결손돌연변이주 구축을 통하여 확인하였다. alcA 유전자 결손 돌연변이를 위해 0.6 kb alcA 5' flanking DNA와 0.7 kb alcA 3' flanking DNA fragment들을 pCP1.11을 주형으로 하여 PCR법으로 증폭한 후, 5' flanking과 3' flanking DNA가 연결된 재조합 suicide vector pDMl을 구축하여 세포 접합을 통해 B. bronchiseptica로 도입시켰다. 도입된 pDM1으로부터 allelic exchange법에 의해 alcA 유전자가 결손된 돌연변이주 B. bronchiseptica H1을 얻을 수 있었다. B. bronchiseptica H1은 야생형인 B. bronchiseptica에 비하여 alcaligin siderophore를 거의 생성하지 못하였다. alc 오페론 중 promoter와 alcA 유전자만을 가지는 재조합 플라스미드를 B. bronchiseptica Hl에 도입하였을 때 alcaligin siderophore의 생산이 회복됨을 확인할 수 있었다. 이상의 결과로부터 alcA 유전자가 alcaligin 생합성에서 매우 중요한 역할을 수행하는 것을 알 수 있었다.

Bordetella bronchiseptica, the agent of swine atrophic rhinitis and kennel cough in dogs, is a mucosal pathogen and produces the hydroxamate type alcaligin siderophore under iron-limited conditions. Genes involved in alcaligin siderophore biosynthesis are contained in an alcABCDE operon. In order to provide direct evidence for the role of AlcA in alcaligin biosynthesis, we needed a B. bronchiseptica mutant carrying alcA gene deletion. A 0.6 kb alcA 5'-flanking and 0.7kb 3'-flanking DNA fragments were PCR amplified with the use of pCP1.11 as a template DNA. The 5'-and 3'-flanking DNA fragments were joined in a suicide plasmid, resulting in a recombinant suicide plasmid pDM1. After introduction of pDM1 into B. bronchiseptica by conjugation, the allelic exchange technique was performed and a B. bronchiseptica alcA deletion mutant, named B. bronchiseptica H1, was obtained. The mutant strain produced reduced amount of siderophore as expected. When a plasmid containing complete alcA gene was transformed back into the mutant, the complemented mutant recovered ability of siderophore production. These results indicated that AlcA is one of essential components for the alcaligin siderophore biosynthesis. The mutant strains obtained in this study will be used in the further studies for the biochemical function of AlcA.

키워드

참고문헌

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