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RISS 인기검색어
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톱다리개미허리노린재 (Riptortus pedestris)공생균인 Burkholderia의 purine 생합성 유전자가 공생에 미치는 역할 규명
Riptortus pedestris-Burkholderia의 공생 시스템은 공생박테리아인 Bur-kholderia의 유전자 조작이 가능하므로써, 공생의 분자 메커니즘을 연구할 수 있는 유망한 실험적인 모델이다. Transposon 돌연변이를 이용하여, 본 연구자는 숙주와의 공생은 유지하나, 공생기관을 덜 발달하게 하는 돌연변이 균주를 발견하였다. 돌연변이 된 유전자를 확인한 결과, 박테리아의 purine 생합성에 관련된 purL 유전자임을 밝혔다. purL 돌연변이 균주의 in vitro 성장 장애와 adenine과 adenosine에 의존하는 성장은 purine 생합성에 기능이 중단된 사실을 알 수 있었다. 또한 purL 돌연변이 균주는 biofilm 형성을 형성하지 않았으며 purine 유도체를 보충해주었음에도 불구하고 biofilm 형성의 결함을 회복하지 않았다. purL 돌연변이 균주가 숙주인 곤충에 접종 되었을 때, purL 돌연변이 균주는 처음에 곤충의 장에 접종은 되었으나, 정상 접종수를 유지하지 못하였다. purL 돌연변이 균주의 낮은 접종수는 초기 약충에서 장의 crypt 발달에 영향을 미치고, 중, 후기 약충에서는 숙주인 곤충의 몸무게와 몸길이를 감소시켰다. 그리고 purine 생합성 경로에서 purL 다음 효소로 작용하는 purM의 돌연변이 균주도 purL과 같은 표현형을 in vivo와 in vitro에서 나타나는 사실을 확인하였고, 이로부터 purL은 확실히 purine 생합성에 관련된 유전자임을 확인할 수 있었다. 그 결과 Burkholderia의 purine 생합성관련 유전자는 숙주인 곤충의 장에서 공생균의 증식능력, 초기 장의 crypt 발달 및 곤충의 몸무게와 몸길이 성장에 중요하다는 것을 증명하였다. Riptortus-Burkholderia symbiosis system is a promising experimental model to study molecular mechanisms involved in insect-bacterium symbiotic association due to the availability of genetically manipulated Burkholderia symbiont. By utilizing transposon mutagenesis, we found a symbiosis-deficient mutant that is able to colonize but induce less developed host symbiotic organ. The disrupted gene was identified as purL involved in bacterial purine biosynthesis. In vitro growth impairment of the purL mutant and its growth dependency to adenine and adenosine suggested the functional disruption of purine synthesis gene. The purL mutant also showed defect in biofilm formation, which was not rescued by the purine derivatives. When the purL mutants were infected to host insect, they colonized the symbiotic organ initially, but failed to keep a normal infection density. The low level of infection density of the purL mutant attenuated gut crypt development in early instar and decreased fitness values throughout nymphal stages. Another purine biosynthesis mutant, ΔpurM, also revealed the similar phenotypes like the purL mutant in vitro and in vivo, confirming the purL phenotypes are attributed to the disrupted purine biosynthesis. These results demonstrate that Burkholderia purine biosynthesis genes are critical for the accommodation of symbiotic bacteria in the symbiotic organ and hence for the host crypt development and fitness.
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In order to understand the molecular interactions between hosts and symbiotic bacteria, we need to address how symbiotic bacteria can colonize to hosts’ niches. Also, it should be addressed how symbiotic bacteria overcome the host defense system to colonize in their niches. In my laboratory, my colleagues have intensively studied about the symbiotic molecular interactions between Burkholderia symbiotic bacterium and its host insect, Riptortus pedestris. Since this insect is known to acquire symbiotic bacteria from environments every generation, it will be a good model to study of how Burkholderia bacteria can colonize in the host niche and of how this bacterium overcomes host defense system. To achieve colonization in host insects, symbiotic bacteria firstly should pass through host saliva, in which several antimicrobial substances that are known to be released to defend their bodies from invading pathogenic microbes including symbiotic bacteria. Until this time, there is no report about how symbiotic bacteria can survive in the antimicrobial environmental conditions of host insects and what kinds of molecules of symbiotic bacteria is involved in making resistance against host antimicrobial substance of salivary glands. To screen this molecule from Burkholderia symbiotic bacteria, transposon mutagenesis method was employed. Among 1440 transposon-inserted bacteria, I found that one transposon-inserted mutant bacterium was susceptible to the lysate of Riptortus salivary gland and purified antibacterial protein (named rip-trialysin) from salivary gland. When transposon insertion site was determined, bamC, a gene of Burkholderia lipoproteins, was disrupted by transposon insertion, resulting in to be susceptible to the lysate of host salivary gland. I further confirmed this susceptibility by constructing bamC deletion mutant by homologous recombination method. Also, the bamC deletion mutant and transposon-inserted mutant showed increased outer membrane permeability, suggesting that bamC gene was deeply involved in maintaining outer membrane stability of Burkholderia symbiotic bacteria. These results demonstrate that bamC of Burkholderia gut symbiont is an important gene for maintaining a symbiotic relationship between R. pedestris and Burkholderia.
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