초록 ▼

● 신규한 AHL 분해효소 유전자의 발굴 및 신규 AHL 분해효소의 특성화
● 다양한 acyl chain에 대한 기질특이성을 변화시키기 위해 AHL-lactonase의 3차구조 분석을 기반으로 기질 선택성과 관련된 아미노산들에 대한 site-directed mutagenesis 수행 및 변이 효소 구축 및 특성화
● AHL-lactonase 유전자의 knockout 변이주 및 야생주와 phenotype 비교 분석을 통한 세포내 기능 예측
● AHL-lactonase 생산 미생물과 식물의 상호 관련 영향 분석을 통한

● 신규한 AHL 분해효소 유전자의 발굴 및 신규 AHL 분해효소의 특성화
● 다양한 acyl chain에 대한 기질특이성을 변화시키기 위해 AHL-lactonase의 3차구조 분석을 기반으로 기질 선택성과 관련된 아미노산들에 대한 site-directed mutagenesis 수행 및 변이 효소 구축 및 특성화
● AHL-lactonase 유전자의 knockout 변이주 및 야생주와 phenotype 비교 분석을 통한 세포내 기능 예측
● AHL-lactonase 생산 미생물과 식물의 상호 관련 영향 분석을 통한 생태계에서 역할 분석
● 효과적인 식물 세균병 억제 균주 개발을 위해 Bacillus thuringiensis를 이용한 AHL-lactonase의 spore display 균주 개발
● 발굴한 신호물질 분해효소를 이용한 무름병 억제 적용 기술 개발

Abstract ▼

Quorum sensing is a regulatory mechanism employed by bacteria to coordinate the behavior of their community in response to their population density. Bacteria recognize the changes in their population density by sensing the concentration of signal molecules which were accumulated as bacterial cell pr

Quorum sensing is a regulatory mechanism employed by bacteria to coordinate the behavior of their community in response to their population density. Bacteria recognize the changes in their population density by sensing the concentration of signal molecules which were accumulated as bacterial cell proliferates. N-acyl homoserine lactones (AHLs) are communication signals widespread among a number of Gram-negative bacteria. The signal molecules are used for inter-cellular communication, especially for controlling the expression of virulence. AHL-mediated quorum sensing systems playa key role in co-ordinating a wide range of activities implicated in pathogen and host interaction. As AHL-mediated quorum sensing has been shown to be involved in pathogenesis and biofilm formation, the interference of quorum sensing, referred as quorum quenching, have recently received a great deal of attention. Among the quorum quenching strategies, the potent effects of enzyme-based AHL degradation have been identified in a wide diversity of bacteria. AHL-lactonase degrades AHLs by hydrolyzing the lactone ring of AHLs, and produce the corresponding acylhomoserine molecules. A number of organisms also contain genes coding for AHL lactonases or acylase which hydrolyse AHLs into inactive products, thereby blocking the QS systems. Consequently, these enzymes attract intense interest for the development of novel anti-infection therapies.
In this study, the gene for a AHL-acylase from Nocardioides sp. OS4, ahlN, was cloned in E. coli using degenerated PCR and inverse PCR. A comparison of AhlN with known AHL-acylases indicates that the cloned ahlN gene codes the novel AHL-acylase. Also the ahlN gene was expressed heterologously in Bacillus thuringiensis in an active form. The recombinant extracellular AHL-acylase successfully expressed in B. thuringiensis could effectively degrade AHL and effectively reduce the amount of OHHL.
On the basis of molecular structure and catalytic mechanism of AHL lactonase from B. thuringiensis subsp. kurstaki HD263, we mutated the lactonase gene at different positions to study the catalytic activity and substrate specificity of the enzyme. Point mutations were introduced into the AHL-binding hydrophobic channel of the enzyme by site-directed mutagenesis. Compared to wild enzyme, the mutants showed altered activity towards different AHLs. The substrate specificity analysis showed an increase in the activity of mutant enzymes compared to wild, especially in the case of F68L and I73V with respect to the substrates DHL, OHL and OHHL. The structural stability of the enzymes was analyzed using Differential Scannin Calorimetry. Compared to wild enzyme, the thermal stability was seen increased in the case of enzyme I73V. The study mainly focused on unraveling the effect of mutations at the substrate-binding site of AHL lactonase and, thereby understanding the structural features of lactonase which are important for enzyme-substrate interaction.
An AHL-degrading enzyme, AiiA, from B. thuringiensis has been reported to effectively attenuate the virulence of bacteria by quorum quenching. However, very little is known about the role of AiiA in B. thuringiensis itself and in natural ecosystem. Therefore, an aiiA defective mutant was constructed to investigate the role of AiiA. The aiiA mutant showed no detectable AHL degrading activity and was less effective for suppression of soft-rot symptom caused by Erwinia carotovora on the potato slice. The 2D electrophoretic analysis of the aiiA mutant and wild type revealed that 5 spots in the mutant were significantly reduced compared to the wild type. Three spots were identified as camelysin and 2 spots bacillolysin. Camelysin has been reported to affect the activation of insecticidal toxin from B. thuringiensis. These results suggest the close relationship existing between AHL-lactonase and insecticidal toxin. To investigate the role of AiiA in ecosystem, we evaluated population dynamics of the introduced wild type B. thuringiensis and aiiA mutant in the rhizosphere of pepper. On the pepper root, the survival rate of the aiiA mutant significantly decreased over time compared with that of wild type. Interestingly, viable cell count analysis revealed that the bacterial number and composition of E. carotovora were not different between treatments of wild type and the aiiA mutant. These results provide evidence that AiiA can play an important role in rhizosphere competence of B. thuringiensis and bacterial quorum quenching to Gram-negative bacteria without changing bacterial number or composition.
B. thuringiensis, a spore-forming gram-positive bacterium, produces various insecticidal crystal proteins encoded by cry genes, and has been developed as a biological control agent against insect pests in the agriculture. Recently, it has been suggested that the AHL-degrading activity of AiiA might give insecticidal B. thuringiensis the potential to attenuate the virulence of plant pathogenic bacteria. Therefore, we constructed recombinant B. thuringiensis spores displaying AHL-lactonase on the surface of spores. We have successfully displayed the AHL-lactonase on the surface of recombinant B. thuringiensis spores. The displayed AHL-lactonase on the spore is biologically functional. These results indicate that insecticidal B. thuringiensis spores displaying the AHL-lactonase could be used as an efficient biocontrol agent for attenuation of the plant pathogenicity of E. carotovora.

목차 Contents

• 표지...1
• 제출문...2
• 보고서 요약서...3
• 요약문...4
• SUMMARY...7
• 목차...10
• 제 1 장 연구개발과제의 개요...12
• 제 1 절 연구개발의 목적 및 필요성...12
• 제 2 절 연구개발의 범위...12
• 제 2 장 국내외 기술개발 현황...13
• 제 1 절 세균의 정족수 인식 (Quorum sensing) 시스템 연구...13
• 1. 신호물질 (Autoinducer) 연구...13
• 2. Quorum sensing과 세균병...15
• 3. Quorum sensing에 의한 Biofilm 형성 및 항생제 내성...15
• 제 2 절 새로운 항감염제 개발을 위한 Anti-Quorum sensing 전략...15
• 1. QS 신호전달 교란을 위한 AHL 유사체...17
• 제 3 절 AHL 신호물질의 효소적 분해를 통한 Quorum quenching 및 이를 이용한 무름병제어...18
• 1. 효소적 분해를 통한 신호물질의 불활성화...18
• 2. Quorum quenching 효소를 이용한 식물 세균병 억제...18
• 3. AHL-lactonase 의 세포내 및 생태계에서의 기능연구...19
• 4. 국내 연구 현황...21
• 제 3 장 연구개발수행 내용 및 결과...22
• 제 1 절 신규 AHL분해효소 및 유전자 발굴...22
• 1. Nocardioides sp. OS4의 AHL 분해능 조사...22
• 2. Nocardioides sp. OS4 유래 AHL 분해효소의 특성화...22
• 3. AHL 분해효소 유전자의 cloning 및 ahlN 유전자 분석...24
• 4. Bacillus thuringiensis에서 ahlN의 heterologous expression...28
• 5. 재조합 AhlN의 활성과 기질 특이성...29
• 6. AhlN 발현에 의한 E. carotovora의 병발 억제 효과...30
• 제 2 절 AHL-lactonase의 기질 선택성과 관련된 다양한 변이효소의 구축 및 특성화...31
• 1. Site-directed mutagenesis를 통한 B. thuringiensis subsp. kurstaki AHL-lactonase 변이 효소 구축 및 정제...31
• 2. AHL-lactonase 변이단백질의 고발현 및 분리정제...33
• 3. 다양한 길이의 acyl side chain을 가진 AHLs에 대한 기질특이성 변화...35
• 4. I73V 변이단백질의 Differential Scanning Calorimetry 분석...38
• 제 3 절 AHL-lactonase 의 세포내 및 생태계에서의 기능 규명...39
• 1. B. thuringiensis subsp. israelensis aiiA 유전자 knockout 변이주 제조...39
• 2. B. thuringiensis 야생주와 aiiA 변이주의 phenotype 비교 분석...40
• 3. B. thuringiensis 야생주와 aiiA knockout 변이주의 proteome 분석...41
• 4. 근권 생태계에서의 population dynamics분석을 통한 B. thuringiensis 유래 AHL-lactonase의 기능연구...42
• 5. AHL-lactonase의 B. thuringiensis spore 표면 발현을 이용한 병발 억제 연구...44
• 가. B. thuringiensis spore에 AHL-lactonase의 발현 확인...44
• 나. AiiA 단백질의 spore 표면발현 및 active form 확인...44
• 다. Spore의 보관기간, 온도별 활성변화 조사...45
• 라. AHL-lactonase를 표면발현 시킨 B. thuringiensis spore를 이용한 병발 억제 test...46
• 제 4 장 목표달성도 및 관련분야에의 기여도...47
• 1. 연구개발의 최종목표...47
• 2. 연차별 연구개발 목표 및 내용...47
• 3. 계획대비 달성도...48
• 4. 위 연구목표(총연구기간)에서 중요도 순으로 4-5개 목표 추출 및 가중치 부여...49
• 제 5 장 연구개발결과의 활용계획...50
• 제 6 장 연구개발과정에서 수집한 해외과학기술정보...51
• 제 7 장 참고문헌...52