Biofilm 저해제 검출을 위한 biofilm 정량 시스템 및 TraR 단백질의 생산을 위한 유전자 재조합 균주의 개발

유정규 부산대학교 2006 국내석사

Development of biofilm quantification method and production of a quorum sensing activator protein, TraR, in a recombinant E. coli. Junggyu Yu Department of Chemical Engineering, Pusan National University, #30, Changjun-dong, Gumjeoung-ku, Busan 609-735, Korea Abstract Biofilm formation is initiated by cell-to-cell communication mechanism called quorum sensing. Biofilm formation was quantitatively studied using Pseudomonas aeruginosa PAO1(wt) and PAO-JP2(lasI-, rhlI-). It was observed that biofilm was developed better on the surface of polystyrene than that of polypropylene or glass. The amount of surface-attached microorganism could be used to study the effect of a biofilm inhibitor filmbrolide on biofilm formation, but the difference of the amount of microorganisms depending on the presence of fimbrolide was low to be used for the purpose of screening biofilm inhibitors from natural products. TraR is a transcriptional activator in the quorum sensing mechanism of Agrobacterium tumefaciens. In order to produce TraR in large quantity, a recombinant E. coli containing traR under the control of T7 promoter was constructed. The C-terminal of TraR was tagged with His6 residue for Ni2+-affinity purification. The recombinant could produce TraR when incubated with IPTG and TraR could be purified to the electrophoretic pure protein by a single-step affinity chromatograph.

Quorum sensing 저해물질 검색법 개발

김영희 부산대학교 대학원 2002 국내석사

Biofilms are found in many places in nature including the human body and are of great concern because they can result in virulence by the infected microorganisms or cause environmental hazards. They are known to be formed through a unique mechanism called "quorum sensing" which involves membrane-permeant signaling molecules, AHLs (acylated homoserine lactones). The cells in biofilms, are more resistant to antibiotics than their free-living counterpart. de Nys et al. have recently isolated halogenated furanone analogues having antifouling activity from the red algae, Delisea pulchra, that were chemically similar to the AHLs. The isolation of these analogues have prompted research in the discovery of mechanism based quorum sensing inhibitors from natural as well as synthetic products. However, the absence of a sensitive bioassay has hindered the screening of active compounds, since bioactive secondary metabolites are usually present at very low concentrations. The aim of this research was therefore to establish simple, effective and reproducible bioassay systems to detect AHL analogues. A plate bioassay system employing the cross-feeding assay by Fuqua et al using an AHL-responsive A. tumefaciens strain has been developed to identify new AHL-like compounds. By varying the X-gal concentration, incubation time and the sample loading procedure, it was possible to detect low level AHLs up to 10 nM concentrations. The length of the acyl chain of the AHLs was found to affect the sensitivity of this bioassay. The activity was observed to increase when the carbon number in AHLs increased up to 8~12 but decreased with further increase in carbon length. This implies that the binding affinity of AHLs is affected by their lipophilicity A luminometry assay system employing the quorum sensing bacteria V. harveyi was explored for detecting AHL analogues. This system could detect AHLs up to 102nM. The sensitivity of this system seemed to be less than that of the plate bioassay but it was interesting to note that inhibition of luminescence activity was observed by adding AHLs to V. harveyi. This suggests that the AHLs could be quorum sensing inhibitors by competing with the AI-1 in a cell density-dependent regulatory system-1. Moreover, unlike the plate bioassay which could serve as a universal assay system for detection of AHL analogues (whether they are agonists or antagonists), the luminometry assay with V. harveyi seems to be a more specific system. However, similar to the plate bioassay, lipophilicity seemed to affect the binding affinity of AHLs to the receptor. The universal plate bioassay system with A. tumefaciens was also modified into a liquid culture assay system. This system would give more quantitative results by measuring the absorbance intensity caused by the blue color response which is generated from the lacZ gene expression of reporter strain activated by the AHL analogues. AHLs could be detected up to 10-1nM, which makes this the most sensitive system developed so far. Several natural products were fractionated and tested in the above systems. The marine sponge, Petrosia, which has been known to possess antifouling activities did not show activity as a quorum sensing inhibitor which suggests that its activity may have been due to its cytotoxic activity. The significance of this study was in developing sensitive bioassay systems for quorum sensing inhibitors from natural and synthetic products.

Quorum Sensing 저해물질 검색법 개발 및 해양천연물에의 적용

金軟憘 釜山大學校 大學院 2004 국내석사

Quorum sensing is a sophisticated mechanism that allows bacteria to communicate using a signal molecule, most of which are of the N-acyl-homoserine lactone (AHL) structure. The mechanism enables a population of bacteria to regulate gene expression and behavior. Quorum sensing is reported to be involved in bioluminescence, virulence, conjugation, sporulation and biofilm formation. Biofilms has been reported to be involved in harzardous infections in the human body as well as in environmental noxiousness. Recently, halogenated furanones which are chemically similar to AHL isolated from the Australian red algae Delisea pulchra have been reported to have antifouling activity. The isolation of these analogues have prompted research on mechanism-based quorum sensing inhibitors from natural as well as synthetic products. Because the development of detection methods is an important prerequisite for quorum sensing inhibitors, a sensitive liquid culture detection method for AHL and/or its analogues with an AHL-responsive recombinant Agrobacterium tumefaciens strain as a reporter strain has been developed. A blue color response with β-galactosidase activity indicated the presence of AHL or its analogues. This method was more convenient to use and was about 100-fold more sensitive than the conventional agar-plate cross-feeding assay. Moreover, the liquid culture assay system has been optimized by testing various solvents and surfactants. The most appropriate solvent was 1% methanol and surfactant was 0.1% Tween 20 in order to establish a sensitive assay. Fimbrolide, a synthetic bromofuranone analogue showed inhibitory activity in a dose dependent manner in this assay. The feasibility of the liquid culture assay as a detection method for quorum sensing inhibitors from natural products was validated by using an inactive natural products extract spiked with fimbrolide. The agonistic or antagonistic activity of 106 marine products was then examined and revealed 11 samples to be antagonistically active. Further separation to identify the active compound is underway. Moreover, the development of a GFP-based AHL sensor system has beem attempted. The significance of this study is in the optimization of a sensitive liquid culture assay for quorum sensing inhibitors and screening of marine natural products using the assay. Preliminary results have been provided for the development of a new GFP-based AHL sensor systems as well.

Functional analysis of vibrio vulnificus SmcR, a quorum-sensing master regulator, as a novel control target

김승민 서울대학교 대학원 2012 국내박사

Quorum sensing has been implicated as an important signal transduction system regulating the expression of numerous virulence genes in bacterial pathogens. Vibrio vulnificus is a model pathogen for studying many other foodborne pathogens because it causes life-threatening septicemia and gastroenteritis with various potential virulence factors controlled by quorum sensing. Recently, SmcR, a homologue of Vibrio harveyi LuxR, has been identified from V. vulnificus, and proposed as a quorum-sensing master regulator. In the present study, the roles of SmcR during an infectious process were examined in a series of experiments using biofilm cells, and comparing virulence of the smcR mutant with that of the parental wild type. When compared to the smcR mutant, the wild type showed a significant 2-fold increase in biofilm detachment rate in the extended time-course. To determine the genes involved in the biofilm detachment among SmcR-regulated genes, transcriptional profiles of the wild-type and smcR mutant biofilms were analyzed. The differentially expressed genes in the smcR mutant include genes that are known to stimulate biofilm dispersal in other bacteria. Interestingly, the smcR expression was induced upon exposure to intestinal epithelial cells. Thus, the ability of the wild type to detach cells from the biofilms was enhanced as biofilms were exposed to INT-407 human intestinal epithelial cells. On the other hand, the smcR mutant biofilms were not detached even upon exposure to INT-407 cells. In this regard, the defects in biofilm detachment in the smcR mutant resulted in a decrease in both intestinal colonization to new infection sites and histopathological damage in jejunum tissues from the mouse intestine after the intragastric administration of biofilm cells. Furthermore, the LD50 in ICR mice (specific-pathogen free) after intragastric infections of the smcR mutant was approximately 102 times higher than that of the parental wild type, suggesting that the smcR mutant biofilms were impaired in its ability to disperse and colonize new sites in the intestine in vivo. These differences between the wild-type and smcR mutant strain were not observed when planktonic cells were used. Therefore, these results indicate that upon entry into the host intestine, SmcR enables V. vulnificus biofilms to detach to find appropriate sites of infection and initiate a new infection cycle, demonstrating the importance of SmcR in V. vulnificus pathogenesis. After the detachment of cells from the biofilms, flagellum-mediated motility is essential for dispersal of detached cells into the new colonization site. In the present study, the functions of FlhF and regulatory characteristics of the flhF expression of V. vulnificus were investigated. A deletion mutation of FlhF abolished motility, flagella formation, and flagellin synthesis, and introduction of flhF in trans complemented the defects. The flhF mutant revealed decreased expression of the class III and IV flagella genes, indicating that FlhF is a key regulator for the flagella biogenesis of V. vulnificus. The influence of global regulatory proteins on the expression of flhF was examined, and SmcR was found to downregulate the flhF expression at the transcriptional level. SmcR represses the flhF expression only in the stationary phase of growth and exerts its effects by directly binding to the flhF promoter region. Finally, an SmcR binding site, centered at 22.5-bp upstream of the transcription start site, was identified by a DNase I protection assay. The combined results demonstrate that a quorum sensing master regulator SmcR influences the motility and flagella biogenesis of V. vulnificus through modulating the expression of FlhF in a growth phase-dependent manner. During the initial stage of infection immediately after biofilm detachment, smcR expression is repressed because of low cell density, and expression of flhF is allowed, leading to flagella synthesis. The flagella prime V. vulnificus for initial colonization of host intestinal tissue, which is an important step required for the onset of its infectious cycle. In contrast, upon establishing preferred colonization niches with the increase in population density, the necessity of motility is superfluous, even detrimental, for a successful infection of hosts by the bacteria. In fact, flagellins of many enteropathogens have been well characterized as a major inducer as well as a target of host innate immune responses. Therefore, flagellar synthesis needs to be sophisticatedly regulated by quorum sensing regulatory pathways for optimal colonization and disease progression. These previous results led me to confirm that V. vulnificus quorum sensing is essential for the survival and pathogenesis of V. vulnificus. So identification and characterization of small molecules that inhibit quorum sensing are required for delineating novel strategies to control foodborne pathogens with low incidence of bacterial resistance. Therefore, a high throughput screening of small molecule libraries was performed to identify inhibitors of the V. vulnificus quorum sensing. Using a reporter strain PVVMO6_03194::luxAB whose activity entirely depends on SmcR, I identified a small molecule named U-262, 6-(phenylsulfonyl)nicotinonitrile. U-262 suppresses the activities of exoprotease and elastase without inhibiting bacterial growth itself. Attenuated cytotoxic activity, prolonged survival period and alleviated illness in mice were observed after treating V. vulnificus with the chemical. U-262 also decreased the luminescence of V. harveyi and the total protease activities of V. anguillarum which are regulated by quorum sensing, suggesting that it inhibits other quorum sensing of Vibrio spp.. Western blot analysis demonstrated that the chemical decreases the cellular level of SmcR in a dose dependent manner, indicating that the upper quorum-sensing signaling cascade is inhibited by the chemical. Meanwhile, E. coli dual plasmid system and EMSA showed that specific interactions between U-262 and SmcR resulted in the reduced DNA binding activity of SmcR, implying that U-262 affects both the cellular amount and the activity of SmcR. Taken together, these results suggested that U-262 is a novel anti-microbial agent inhibiting the quorum sensing of Vibrio spp. without the antibiotic resistance. It will be useful to protect food from the Vibrio spp. and help to enhance a public health.

Inhibition of Acyl-homoserine Lactone-mediated Quorum Sensing by Novel Phenolic Compounds of Nymphaea tetragona (Water Lily) : Nymphaea tetragona의 페놀 화합물에 의한 아실호모세린락톤-중계 쿼럼 센싱의 억제

Hossain, Akil Md 경북대학교 대학원 2016 국내박사

쿼럼 센싱(QS)이란 생존을 위해 세균이 정보를 공유하거나 유전자 발현 조절 등을 통해 세균의 정족수 밀도와 관련된 신호전달 체계이다. 이러한 신호전달 체계를 이용하여 세균은 정보를 공유하고 유전자 발현을 조절한다. 세균은 QS를 이용하여 바이오필름의 형성, 운동성, 병원성 인자 활성 같은 다양한 표현형 조절을 한다. 여러가지 병원성 인자의 활성을 통하여 숙주세포와 세균의 상호관계를 설정한다. 식물은 매우 다양하면서 구체적인 생물학적 활동들로 구성되어 있기 때문에 수천년 동안 의약소재로 사용되고 있다. 따라서 본 연구의 목적은 QS억제제를 탐색하고 개발하여 세균이 항생제 저항성을 갖는 것을 막고, 궁극적으로는 이러한 소재를 이용하여 병원성 세균 감염증를 제어하는 것이다. Nymphaea tetragona의 50% 메탄올 추출물 (NTME)에 대하여 QS 억제효과가 있는지 그리고 세균의 병원성을 효울적으로 제어할 수 있는지 조사하였다. 먼저 NTME 단독 용매 분획물질과 기존의 항생제와의 항균 효과를 본 연구에서 조사하였다. 한편, QS 억제에 가장 강력한 추출 분획 물질을 확인하였으며 활성화합물도 분석 및 확인하였다. 최종적으로 NTME에 다량 포함되어 있는 메틸 갈레이트와 피로갈롤을 포함한 5가지 페놀 화합물의 효과를 확인하였다.수련추출물은 농도에 비례하여 항균활성을 보였고 NTME는 강력한 QS 및 QS 관련 세균의 병원성 발현을 억제하였다. 그리고 본 연구에서 NTME는 in vitro나 in vivo에서 독성이 확인 되지 않았다. NTME의 아세테이트 분획물(EFNTE)은 좋은 항균 활성을 보여 주었다. S. typhimurium에 대한 EFNTE-Tylosin과 EFNTE-streptomycin의 Fractional Inhibitory Concentration Index는 각각 상승 및 상가효과를 보였다. 분획물질 중 EFNTE와 부탄올 분획물에서 확실한 QS 억제 효과를 보였다. 페놀 함유 항산화물질은 EFNTE와 부탄올 분획물의 주요 페놀화합물로 5가지 성분중에서 메틸갈레이트만이 정량적 분석에서 QS의 억제 효과를 보였다. 이 화합물은 아실호모세린락톤(AHL) 합성 및 활성이 모두 억제를 하였다. 또한 P. aeruginosa의 바이오필름 형성 및 QS 관련 병원성 발현도 억제하였다. 메틸갈레이트는 P. aeruginosa의 유전자 lasI/R, rhlI/R 및 pqsA의 발현도 억제를 하였다. 이 연구의 결과, 메틸갈레이트는 QS 조절을 통한 치료제로써 가능성을 보였다. 이러한 결과는 최초의 보고로 메틸칼레이트가 병원성 세균의 QS-제어 표현형을 억제하는 QS 조절 물질로의 AHL 합성과 AHL 활성작용을 동시에 억제한다는 기전을 구명하였다. 따라서 본 논문에서는 항생제 내성 및 병원 세균의 병원성 감소시키기 위하여 항쿼럼센싱 물질을 수련추출물에서 탐색하여 주요 성분인 메틸갈레이트를 확인하였고 이 성분에 대한 항퉈럼센싱의 기전을 분자생물학적인 방법으로 밝혀 최초의 활성기전을 구명하였다. 이 결과는 새로운 항생제 내성 획득을 억제하고 병원성 발현을 조절하는 소재로 활용이 가능할 것으로 기대된다.

Role of N-Acyl Homoserine Lactone based Quorum-Sensing in Serratia glossinae strain GS2 and Its Influence the Reaction of Plants to Rhizobacteria : N-Acyl Homoserine Lactone에 기초한 쿼럼센싱이 Serratia glossinae GS2 균주 및 식물과 근권세균에 미치는 영향

정병권 경북대학교 대학원 2017 국내박사

세균의 밀도 증가가 일정한 공간 내에서 한계에 이를 때 작은 크기의 신호전달물질이 분비되기 시작한다. 이러한 현상을 quorum-sensing (QS) 이라 부르는데, 많은 그람 음성세균에서는 N-acyl homoserine lactone (AHL)을 신호전달 물질로써 사용하고 있다. 신호전달물질은 세포의 밀도와 분포도에 따라서 조절되는데, 이는 다양한 유전자의 발현을 조절하는 역할을 하기도 한다. 이러한 QS 시스템은 각각의 세균 단일 종 내에서 조절되기도 하지만, 최근에는 식물과 같은 고등생물과의 상호작용에 의해서 그 활성이 조절된다는 것이 보고되고 있다. 그리하여 본 연구의 목적은 먼저 참깨 근권토양으로부터 분리되었고, QS 신호전달물질과 식물생장 촉진활성을 가지는 식물 생장 촉진 근권세균(PGPR)인 Serratia glossinae GS2 균주의 유전자 조작을 통해 QS이 이 세균의 능력에 미치는 영향을 분석하는 것이며, 또한 QS 신호 전달물질을 식물 근권에 처리하여, 식물의 생장 및 근권생태계에 미치는 영향을 분석하는 것이다. 먼저 참깨 근권토양으로부터 S. glossinae GS2 균주를 분리할 수 있었는데, 이 균주는 식물생장촉진 호르몬인 indole-3-acetic acid (IAA), 신호전달물질인 AHLs, 식물스트레스 완화물질인 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, 그리고 뿌리에 흡착하는데 필수적인 요소인 biofilm을 생산하는 것으로 확인되었다. 그리하여 GS2 균주의 다양한 능력을 검증하고 유전적 특성을 조사하기 위해 유전자 수준에서 분석한 결과, 하나의 염색체 DNA와 2개의 plasmid를 보유하고 있는 것으로 확인되었으며, 그 크기는 각각 6,100,511 bp, 131,551 bp, 93,737 bp로 확인되었다. 또한 유전자 annotation을 통해 AHL synthase, 신호전달물질 receptor protein, IAA, ACC deaminase, motility, biofilm 생산에 관여하는 다양한 유전자가 존재하는 것으로 확인되었다. 분석된 유전정보를 바탕으로 하여, AHL synthase, receptor protein 유전자를 유전체에서 deletion시킨 후 transcriptome, proteome, phenome 분석을 통해 QS system이 GS2 균주 내에서 어떠한 활성을 조절하는지에 대해 조사하였다. 먼저 phenotype microarray를 통해 에너지원 이용능을 분석한 결과, 주로 탄소원 이용능이 돌연변이 균주에서 비교적 wild-type 균주에 비해 감소하는 것으로 나타났으며, transcriptome과 proteome 분석을 통해 식물생장촉진에 관여하는 IAA, ACC deaminase, acetoin, biofilm 생산능 또한 그 활성이 감소하는 것으로 확인되었다. 뿐만 아니라 근권으로 이동하는데 필수요소인 이동성에 관여하는 다양한 편모합성 유전자들도 QS에 의해 조절 되는 것으로 확인됨에 따라, GS2 균주의 QS system은 광범위하게 유전자를 조절하는 것으로 확인되었다. 그리고, QS 신호전달 물질인 AHL이 식물과 근권세균에 미치는 영향을 분석하기 위해 총 10종의 AHL을 식물근권에 처리하여 식물생장측정 및 군집분석, meta-transcriptome을 분석하였다. 그 결과 신호전달물질 중 OHL과 DHL이 식물의 줄기, 뿌리 신장 및 엽록소 함량과 생채중량을 비교적 증가시키는 것으로 확인되었다. 또한 AHL이 근권세균의 군집구성에 미치는 영향을 분석한 결과, 종의 풍부성과 다양성을 증가시키지는 않았지만, Pseudomonas, Rhizobium, Bradyrhizobium, Sinorhizobium 속과 같은 PGPR 세균들의 밀도가 증가하는 것으로 나타났다. 뿐만 아니라 식물생장촉진 호르몬, 항생물질 생산 등 식물의 생장에 영향을 미치는 다양한 물질들이 AHL 처리에 의해서 발현이 유도되는 것으로 나타났는데, 이러한 현상은 AHL이 근권에 처리됨으로써 이에 반응하는 세균의 밀도가 특이적으로 증가할 뿐만 아니라 유전자 발현을 촉진시키는 것으로 추측된다. 따라서 세균의 신호전달물질인 AHL은 단일 종이 아닌 다수의 세균 종에 영향을 미칠 뿐만 아니라 유전자 발현 조절을 통해 식물의 생장을 촉진시킬 수 있었으며, 이러한 연구 결과를 통해 세균의 QS system이 식물-미생물 간의 상호작용에 중요한 역할을 할 수 있다는 것이 증명되었다. When the bacterial population reaches a threshold, the microbial cells begin to release small signaling molecule mediated sensing response pathways. This effect has been defined as quorum-sensing (QS). In many Gram-negative bacteria the synthesis of autoinducers of the N-acyl homoserine lactone (AHL) type is tightly regulated in response to cell density. These metabolites are released into the cellular environment to sense the quality of the ecological niche in terms of diffusion space and the density and distribution of their own population. This environmental sensing mechanism helps to adapt the regulation of their gene expression to the given conditions in their habitat and thus optimizes the fitness of the population. Although QS has primarily been studied in the context of single species, the expression of QS systems may be manipulated by the activities of other bacteria within complex microbial consortia which employ different QS signals and by higher organisms. Hence, the objectives of this study were to investigate the quorum sensing and PGPR activities of Serratia glossinae strain GS2 isolated from the sesame rhizosphere and to evaluate its ability to promote rice growth. Additionally, we present new evidence that AHL signal molecules influence the rhizobacterial community and gene regulation in the rice rhizosphere. For this study, Serratia glossinae strain GS2 was isolated from sesame rhizospheric soil. This bacterium showed indole-3-acetic acid (IAA) and AHLs production, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity, and biofilm formation. To know about the genomics level of strain GS2, we sequenced the complete genome of this strain using PacBio RSII platform. The complete genome features of S. glossinae strain GS2 were consist of a single circular chromosome with two circular plasmids. The size of the chromosome, plasmid 1, and plasmid 2 were 6,100,511 bp, 131,551 bp, and 93,737 bp, respectively. Analysis of genome of strain GS2 revealed the presence of AHL synthase (gloI) and its receptor protein, indole-3-acetic acid synthesis, ACC deaminase, motility, and biofilm formation-related genes. Therefore, to investigate the effect of QS on metabolic regulation, we were performed the transcriptome, proteome, and phenome in S. glossinae GS2 wild-type, ΔgloI mutant, and ΔgloR mutant strains. In order to acquire a global understanding of the metabolic capabilities of S. glossinae strain GS2 wild-type and mutants cellular processes, we were analyzed Phenotype microarray analysis. Using PM plates 1 to 952 different compounds were tested, including 190 carbon sources, 380 nitrogen sources, 65 phosphorus sources, 30 sulfur sources, 95 nutrient supplements, 96 different osmolytes, and 96 different pH conditions. In this work, we shed light on the metabolic features between wild-type and mutant strains. Besides, all values of PGP trait-related features, including IAA, ACC deaminase, acetoin, and biofilm formation, was also decreased at the transcription and translation level. Several genes involved in expression and assembly of flagella, as well as motility and chemotaxis, were up-regulated by quorum sensing. We were able to confirm the flagellar class I, II, and III genes using transcriptome, microscopy with staining, and swarming motility test. The ΔgloI and ΔgloR mutant produces fewer flagella than does the wild-type strain, which is consistent with the down-regulation of flhD, fliA, and fliC in this strain. There is also decreased transcription of motA and motB in the ΔgloI and ΔgloR mutant strain, which is restored by complementing with the cloned gloI and gloR gene. This study expands the knowledge on QS regulation in S. glossinae, showing that QS is a global regulatory system that controls not only genes involved in pathogenesis but also genes involved in bacterial metabolism, protein biosynthesis, and cell growth, and PGP ability, among other functions. Additionally, Studies of transkingdom signaling between plants and bacteria based on N-acyl homoserine lactone (AHL) are just beginning to reveal its diverse role in healthy plants and the field of plant-microbe interactions will undoubtedly provide good examples of the molecular mechanisms involved in the interaction. Here, we could observe a certain tendency of growth promotion after AHL in waito-c rice plant. Similarly the shoot length and chlorophyll content was significantly increased after DHL application. Besides OHL application, root length, chlorophyll content, and plant weight was more increased than other AHL application. Throughout our analyses, we found differences in the bacterial community structures of the samples collected from the ten AHLs applied rhizospheric soil. The results showed that the OTU richness and Shannon indices of the bacterial communities in rhizospheric soil treated with AHLs were not different from water only treatment. Nonetheless, the density of beneficial bacteria such as Pseudomonas, Rhizobium, Bradyrhizobium, Sinorhizobium genera was significantly increased by AHLs application. In this regard, we were also confirmed that transcription of various genes, relating plant hormones, antibiotics, and bacterial quorum-sensing (QS), was reliably detected by AHL application. In accordance with these findings of apparent influences of AHLs and possibly also their effect on rhizobacterial community and gene transcription, we were able to confirm bacteria and metabolite composition change by AHLs application. Our results suggest that QS is very important regulatory mechanism for plant-microbe interaction.

천연물에서 Quorum Sensing 저해제 탐색

김태우 부산대학교 2007 국내석사

Cell-to-cell communication by means of small signal molecules not only is of vital importance to multicelled organisms functional coordination among family members of single-celled organisms such as bacteria. Cell density-dependent signal transduction, quorum sensing (QS), involves in the synthesis and detection of low molecular-weight molecules known as autoinducers. Inhibitors of bacterial quorum sensing systems offer potential treatment of infections with highly virulent or multidrug-resistant agents. N-acylhomoserine lactones, known as autoinducers, are widely conserved signal molecules present in quorum-sensing systems of many Gram-negative bacteria. We studied the inhibition effector on autoinducers which are induced by Pseudomonas syringae pv. tabaci (ATCC 11528). P. syringae pv. tabaci produced N-(3-oxohexanoyl)-L-homoserine lactone and N-(3-oxooctanoyl)-L- homoserine lactone which were detected their ability by using an Agrobacterium tumefaciens NT1 biosensor strain containing a traI::lacZ fusion. Using thin layer chromatography (TLC) which detects the homoserine lactone types and amounts, our study explains the novel materials inhibited the N-acyl homoserine lactone. The novel materials which quenched quorum sensing system were purified the fraction of 83 min peak from cabbage, leek and onion in recycling preparative HPLC. The common fraction quenched the quorum sensing of A. tumefaciens NT1 biosensor strain in ABMM containing X-gal. The common fraction did not inhibit the growth of A. tumefaciens NT1. So the novel materials in natural products are estimated a kind of antagonists. We will study on the structure of common fraction which is purified from natural products by GC, LC-MS and NMR.

생물막 억제를 위한 Quorum Quenching 균주의 탐색 및 Pseudomonas sp. 1A1 유래 AHL-Acylase 유전자들의 특성규명

이치호 배재대학교 대학원 2013 국내석사

Quorum sensing (QS) is cell-to-cell communication system, which is used by many bacteria to regulate diverse gene expression in response to changes in population density. Bacteria recognize the changes in population density by sensing the concentration of signal molecules, N-acyl homoserine lactones (AHLs). As AHL-mediated quorum sensing plays a key role in biofilm formation, the interference of quorum sensing, referred to as quourm quenching has received a great deal of attention. In addition, the effective reduction of biofouling by quorum quenching enzymes that inactivate the AHLs has been reported. In this study, we isolated diverse AHL degrading bacteria from Sihwa-lake and Tancheon in order to develop a new strategy for preventing biofilm formation on membrane surface. An enrichment culture containing AHL as the sole carbon source was used for the screening of quorum quenching bacteria. Diverse strains including Pseudomonas sp., Burkholderia sp., Bacillus sp., Rhodococcus sp., Stenotrophomonas sp., Micrococcus sp., etc. were identified by 16S rDNA sequence analysis. AHL-degrading activity of a novel strain named Pseudomonas sp. 1A1, and its characteristics were investigated. The species showed extracellular quorum quenching activity, hence it was speculated that the organism may be carrying AHL-degrading genes. The presence of three homologous AHL-acylase genes were confrimed by degenerate PCR and in silico analysis. The three AHL acylase genes of Pseudomonas sp. 1A1 were cloned and expressed in E. coli and Bacillus thuringiensis. They also showed a high similarity with a known AHL-acylase genes from Pseudomonas aeruginosa PAO1. The amino acid sequence of these three genes showed 52%, 63% and 69% identities with that of pvdQ, quiP and hacB, respectively, from P. aeruginosa PAO1. Finally, confirmation of inhibition of biofilm formation through co-culture method were done. Quorum sensing은 세포밀도에 의한 다양한 유전자의 발현 조절과 관련된 세포 간 커뮤니케이션 (cell-to-cell communication) 기작이다. 이를 위해 박테리아는 신호분자인 N-acylhomoserine lactones (AHLs)의 농도를 감지하여 세포밀도의 변화를 인식한다. AHL을 매개로한 quorum sensing은 박테리아의 생물막 (biofilm) 형성에 매우 중요한 역할을 하며, 따라서 quorum sensing 메커니즘을 억제하는 quorum quenching은 많은 관심을 받고 있다. Quorum quenching 효소에 의해 AHL을 불화성화 시켜 생물오염을 (biofouling) 효과적으로 감소시키는 연구가 보고되어있다. 본 연구에서는 하‧폐수 처리를 위한 생물막반응기 (membrane bioreactor, MBR) 표면에 생물막 형성을 방지하기 위한 새로운 전략을 개발하기 위해 시화호와 탄천의 sample에서 AHL을 분해하는 다양한 박테리아를 분리하였다. 이러한 AHL을 분해하는 미생물의 16S rDNA 염기 서열 분석한 결과 4균주의 Pseudomonas sp.와 Burkholderia sp., Bacillus sp., Rhodococcus sp., Stenotrophomonas sp., Micrococcus sp.에 속하는 각 1균주씩을 확보하였다. 그 중 AHL 분해 활성이 가장 우수한 균주를 Pseudomonas sp. 1A1으로 명명하여, quorum quenching과 관련된 특성을 조사하였다. Pseudomonas sp. 1A1은 세포외 배양액에서 quorum quenching 활성을 보였으며, degenerate PCR 및 in silico 분석을 통하여 Pseudomonas aeruginosa PAO1의 3종류의 AHL-acylase인 pvdQ, quiP, hacB와 상동성을 보이는 유전자의 존재를 확인하였다. Pseudomonas sp. 1A1로부터 3종류의 AHL-acylase로 추정되는 유전자를 cloning하여 E. coli와 Bacillus thuringiensis에서 발현됨을 확인하였다. Cloning한 유전자의 분석결과 각각의 AHL-acylase 유전자는 이미 보고된 P. aeruginosa PAO1유래의 PvdQ, QuiP와 HacB의 추정 아미노산 서열과 각각 52%, 63%와 68%가 일치하였다. 궁극적으로, 생물막을 형성하는 Pseudomonas sp. 균주와 재조합 균주와의 co-culture 실험을 통하여 구축된 재조합 AHL-acylase를 발현시킨 대장균에 의해 생물막 형성이 억제되었다.

Characterization of QuoruQuenching N-Acyl-Homoserine Lactonases from Bacillus thuringiensis and Arthrobacter sp.

박순양 연세대학교 2003 국내박사

Quorum-sensing is a signaling mechanism that controls diverse biological functions, including virulence via N-acyl-homoserine lactone (AHL) signal molecules in gram-negative bacteria. AHL-mediated signaling mechanisms are widespread and highly conserved in many pathogenic bacteria. Therefore, it has been postulated that the biological inactivation of AHL could be a target for control of pathogen. Recently, the aiiA gene encoding an AHL lactonase has been cloned from Bacillus sp. 240B1. During investigations in the course of the ongoing B. thuringiensis subsp. morrisoni genome project, an aiiA homologue was found. Furthermore, aiiA homologue genes were widespread in 16 subspecies of B. thuringiensis, and exhibited high homology of 90-96% compared with the Bacillus sp. 240B1 aiiA gene in deduced amino acid sequence. Among the subspecies of B. thuringiensis having an aiiA gene, aizawai, galleriae, kurstaki, kyushuensis, ostriniae, and subtoxicus were shown to degrade AHL. It was observed that recombinant Escherichia coli producing AiiA proteins also had AHL-degrading activity, and could also attenuate the plant-pathogenicity of Erwinia carotovora. These results indicate that insecticidal B. thuringiensis strains might have potential to compete with gram-negative bacteria in natural ecosystems by AHL-degrading activity. Also, as a source of AHL-degrading enzymes, novel bacteria were screened by their ability to utilize OHHL as the sole carbon source. Among four isolates, strain IBN110, identified as Arthrobacter sp., was found to grow rapidly on OHHL, and exhibit degrading activity towards various AHLs with different lengths and acyl side chain substitutions. Coculture with the Arthrobacter sp. IBN110 and E. carotovora significantly reduced both the AHL amount and pectate lyase activity in coculture medium, suggesting the possibility of applying Arthrobacter sp. IBN110 to the control of AHL-producing pathogenic bacteria. The ahlD gene from Arthrobacter sp. IBN110 encoding the enzyme catalyzing AHL degradation was cloned, and found to encode a protein of 273 amino acids showing AHL-lactonase activity. Although a comparison of AhlD with other known AHL-lactonases revealed relatively low identities (21-26%) in the deduced amino acid sequences, the HXDH"H"D motif was conserved in all the AHL-lactonases, suggesting that this motif is essential for AHL-lactonase activity. From a genome database search based on the conserved motif, putative AhlD-like lactonase genes were found in several other bacteria, and AHL-degrading activities observed in Klebsiella pneumoniae and Bacillus stearothermophilus. Furthermore, it was verified that ahlK, an ahlD homologue gene, encodes AHL-degrading enzyme in K. pneumoniae. Accordingly, the results suggest the possibility that AhlD-like AHL-lactonases could exist in many other microorganisms. Quorum sensing이란 세균들이 자신이 합성하는 저분자 신호물질의 축척을 통해 개체군의 밀도를 인식하여 특정 유전자의 발현을 조절하는 일련의 현상을 지칭한다. 그람음성세균의 대표적인 신호물질은 N-acyl-homoserine lactones (AHLs)이며, 이들은 공생, 항생제 생산, 생물막형성 등과 같은 세균들의 다양한 생리 현상들의 조절에 관여한다. 특히 동식물 병원성 세균들의 병발 인자들이 AHL에 의해 조절되기 때문에, AHL의 생물학적 분해는 항균 치료제 개발을 위한 대상이 되어왔다. 본 연구에서는 quorum sensing 차단을 통한 병발 억제를 위하여 다양한 AHL 분해 유전자 및 효소를 분리하여 특성을 조사하였다. 최근에, AHL 분해 효소를 코딩하는 aiiA 유전자가 최초로 Bacillus sp. 240B1 균주로부터 cloning되었다. aiiA homologue가 Bacillus thuringiensis subsp. morrisoni 의 게놈 프로젝트를 수행하는 중 게놈 염기서열에서 발견되었다. AiiA homologue는 B. thuringiensis의 16 아종들에도 확인 할 수 있었고, 이들과 AiiA와 아미노산 서열에서의 상동성은 90-96%였다. aiiA homologue를 가지고 있는 B. thuringiensis 아종들 중, aizawai, galleriae, kurstaki, kyushuensis, ostriniae와 subtoxicus 균주로부터 AHL 분해능을 확인하였다. 또한 AiiA를 발현하는 재조합 대장균으로부터 AHL 분해능을 관찰하였고, 이 재조합 대장균은 식물병원성 균주인 Erwinia carotovora N98의 병발을 억제하였다. 이 결과는 살충제의 기능을 가지고 있는 B. thuringiensis가 신호물질 분해능에 의하여 그람음성 세균과도 경쟁할 수 있음을 보여준다. 신규한 AHL 분해 유전자를 찾기 위하여 OHHL을 유일 탄소원으로 이용하는 균주들을 증균배양으로 분리하였다. Arthrobacter sp.로 동정된 4개 분리균 중 IBN110 strain이 OHHL을 포함하는 최소배지에서 가장 빠른 성장을 보였고, 다양한 종류의 AHL들에 대한 분해능을 보였다. Arthrobacter sp. IBN110과 E. carotovora N98의 coculture를 통해 배지중의 OHHL 양과 병원성 인자인 pectate lyase 활성이 크게 감소됨을 확인하였다. 이 결과부터 Arthrobacter sp. IBN110가 AHL을 생산하는 병원성 균주들을 제어 할 수 있을 것으로 기대되었다. AHL 분해 단백질 유전자인 ahlD를 Arthrobacter sp. IBN110로부터 cloning하였고, 273의 아미노산으로 구성되어 있는 이 단백질은 AHL lactonase 활성을 보였다. AhlD는 기존에 알려진 AHL lactonases인 AiiA, AiiA homologue, AttM 과 아미노산 서열에서 26% 이하의 낮은 상동성을 보였다. 그러나 HXDH-H-D 모티프는 모든 lactonases에 잘 보존이 되어있어 이 모티프가 lactonase 활성에 필수적임을 알 수 있었다. HXDH-H-D 모티프를 이용한 genome database 분석 결과 몇몇 다른 세균들에서 AhlD-like lactonase 유전자를 발견하였고, Klebsiella pneumonia와 Bacillus stearothermophilus로부터 AHL 분해능을 확인하였다. 더구나 K. pneumonia로부터 ahlD 유사 유전자인 ahlK가 AHL 분해 효소를 코딩하고 있음을 확인하였다. 따라서 이들 결과들로부터 AhlD-like lactonase가 다양한 세균들에 분포 되어 있을 것으로 기대된다.

Evaluation of cis-2-Decenoic acid and vanillin as quorum sensing compounds for biofouling reduction in membrane bioreactor process

송원중 건국대학교 대학원 2021 국내박사

Water pollution is increasing due to industrialization and urbanization, and the water demand is increasing rapidly with industrial development and increase of population. In addition, the application of the membrane process is increasing due to the social demand for high quality water. Membrane bioreactor (MBR) for wastewater treatment has been widely applied due to the high quality of treated water and several advantages of the process compared to traditional biological treatment processes. However, membrane contamination must be considered in order to apply the membrane process. Among them, biofouling occupies the highest proportion of membrane contamination occur in the membrane process and is difficult to control. Many methods of controlling the biofilm on the membrane surface have been studied and applied. Since the biofilm control methods focus on controlling particulate matter and organic matter, pretreatment process enhancement, backwashing, and washing enhancement have been applied. To reduction of biofouling, disinfection and oxidation processes using chlorine have been applied. Biofilm can be suppressed through this control method, but microorganisms essential in the MBR process are also affected. Therefore, it is necessary to research on a capable technology of effectively controlling biofilm without affecting microorganisms essential to the process. Studies on the factors that determine the properties of extracellular polymeric substances (EPS) generated during biofilm formation have been considerably conducted. The existence of a quorum sensing system was revealed as a communication system that promotes the generation of in vitro metabolites. N-acyl homoserine lactone (AHL) is known as the language used in the QS system. Recently, research on the diffusible signal factor (DSF) system as a higher level system of the AHL system has been actively conducted. In this study, a control method for biofouling reduction in membrane process was developed by using cis-2-Decenoic acid (CDA), which acts on the DSF system and vanillin, which acts on the AHL system to inhibit biofilm formation. CDA and vanillin were applied to the lab-scale MBR system, and changes in biofilm formation and MBR operation characteristics were evaluated. In order to evaluate the applicability of CDA to MBR, this study was conducted on the strains. The effect of CDA-induced biofilm dispersion of single strain and multi species was confirmed through CLSM analysis. Biofilm dispersion increased as the applied CDA concentration increased. EPS reduction by CDA was also reduced by 70% in single strain condition and 46% in multi strain condition by 300nM CDA. In conditions with MBR sludge, the biofilm on the membrane surface also decreased as the concentration of CDA increased. As a result of quantitative CLSM image analysis, it was confirmed that all indicators decreased. It was determined that biofilm control in MBR could be possible through the results of biofilm dispersion of CDA for the strains. For MBR studies using QSC, CDA and vanillin were applied. The MBR operation was extended by the QSC in each condition. The EPS results on the membrane surface also confirmed the dispersion and reduction of biofilm by CDA. The total EPS decreased by 11.4% by the CDA application, and the EPS reduction by 15% was possible through the additional application of vanillin. Through the results of the MBR operation, the possibility of CDA application for CDA biofouling control was confirmed. In addition, synergy with vanillin showed improved biofouling reduction effect compared to CDA application. However, However, EPS characteristics under CDA with vanillin conditions were different from CDA application, and it seems necessary to study the interaction of each QS mechanism for understanding. When vanillin is applied, the decrease in the T-N and T-P removal of MBR is due to the antibacterial properties of vanillin. Because of these properties, it is considered that performance degradation should be considered for simultaneous application of CDA and vanillin. To confirm the effect of QSC in an environment where biofilm formation is promoted by stress, CDA was applied to MBR for treatment of saline wastewater, and the synergistic effect of biofilm formation inhibition with vanillin was also evaluated. In the saline environment, the change in EPS due to the applied QSC showed a difference. Unlike the result of CDA application, the condition including vanillin had a relatively higher bound EPS. This is believed to be due to the increase in EPS by microorganisms as the antibacterial properties of vanillin acted as stress along with the stress caused by salt. The application of QSC reduced total EPS. When applied to CDA, EPS decreased by about 9%, and biofilm inhibition due to additional application of vanillin reduced total EPS by 18%. In addition, the HMW-EPS fraction in EPS decreased due to the saline environment, which was attributed to incomplete decomposition of organic substances and microbial polymers and accumulation of intermediate products. As a result of this study, it was confirmed that the change in the composition of the biofilm on the membrane surface of the MBR process and the extension of the operation were possible by QSC. It was proved that biofouling can be reduced by QSC application. This result suggests that the MBR process could be another biological contamination control method. As a technology for advanced biofouling control by QS compound, follow-up studies such as application method and continuous evaluation through long-term operation are required to be applied to actual MBR process. 산업화와 도시화로 물 오염이 가중되고 물 수요량은 급격히 증가하여 사용 가능한 물이 부족해지고 있다. 또한 최근 높은 수준의 수질에 대한 사회적 요구와 맞물려 멤브레인 공정의 적용이 증가되고 있는 추세이다 폐수의 처리를 위한 멤브레인 생물 반응기 (MBR)는 기존 생물학적 처리공정 대비 높은 처리 수질과 더불어 공정이 갖는 여러 장점 인해 널리 적용되고 있다. 그러나 멤브레인 공정을 적용하기 위해서는 막오염을 고려해야 한다. 그 중 biofouling은 멤브레인 공정에서 일어나는 막 오염 중에 가장 높은 비중을 차지하며 제어하기 어렵다. 막 표면의 biofilm을 제어하는 많은 방법이 연구되고 적용되었다. Biofilm 제어기술은 입자상물질 및 유기물 제어에 초점이 맞춰져 있어 전처리 공정강화, 역세척 및 세정강화 등이 적용되어 왔다. biofouling의 경우는 위의 기술에 덧붙여 염소 등을 이용한 소독 및 산화공정 적용 등이 적용되어 왔으며, 이러한 제어 방법을 통해 biofilm의 억제가 가능하지만, MBR 공정에서 필수적인 미생물 역시 이에 영향을 받게 된다. 때문에 공정에 필수적인 미생물에 영향을 미치지 않고 효과적으로 biofilm의 제어가 가능한 기술의 연구가 필요하다. 생물막 (biofilm) 형성 시 발생되는 체외대사물질, 즉 extracellular polymeric substance (EPS) 특성을 결정하는 인자들에 대한 연구가 상당히 발전되었다. 체외대사물질 발생을 촉진하는 의사소통체계로 정족수감지 (quorum sensing)시스템의 존재가 밝혀졌으며. QS시스템에 사용되는 언어로 N-acyl homoserine lactone (AHL)이 알려졌다. 최근에는 AHL시스템의 상위체계로 diffusible signal factor (DSF)시스템 연구가 활발한 상황이다. 본 연구는 AHL시스템에 작용하여 biofilm 형성을 저해하는 vanillin과 DSF시스템에 작용하는 cis-2-Decenoic acid (CDA)를 사용하여 정족수감지 시스템 발현을 방해함으로써 막여과에서의 biofouling 저감기술을 개발하고, 수처리 분야에 확대할 수 있는 EPS성분 변화기작을 연구하고자 한다. CDA와 vanillin을 lab-scale MBR system에 적용하였으며, biofilm formation의 변화와 MBR 운전 특성 변화에 대해 평가하였다. CDA의 MBR 적용 가능성 평가를 위해 균주를 대상으로 한 연구를 진행하였다. CDA에 의한 단일 균주 및 복합 균주의 biofilm 분산 효과는 적용 농도가 증가함에 따라 biofilm 분산 정도가 증가하는 것을 CLSM 분석을 통해 확인하였다. CDA에 의한 EPS 감소는 300nM의 CDA에 의해 단일 균주 조건에서 70 %, 복합 균주 조건에서 46 % 감소하였다. MBR sludge 적용 조건 역시 CDA의 농도가 증가함에 따라 멤브레인 표면의 biofilm이 감소했으며, CLSM image 정량 분석 결과, total biomass를 포함한 모든 지표에서 감소를 확인했다. QSC를 이용한 MBR 연구는 CDA와 vanillin을 적용하였다. 각 실험 조건에서 주입된 QS compound에 의해 MBR 가동 기간은 연장되었다. 막 표면의 EPS 분석결과에서도 CDA에 의한 biofilm의 분산 및 저감을 확인하였다. CDA의 적용에 의해 total EPS는 11.4% 감소하였으며, vanillin의 추가 적용을 통해 15%의 EPS 저감이 가능했다. MBR 연구결과를 통해 CDA의 biofouling control 적용 가능성을 확인했다. 또한 vanillin과의 시너지는 단독적용 대비 향상된 biofouling 저감 효과를 나타냈다. 다만 CDA와 vanillin의 동시 적용에 의한 EPS 특성은 CDA 단독 적용과 차이가 있었으며, 이해를 위해서는 각 QS mechanism의 상호작용에 대한 연구가 필요해 보인다. Vanillin 적용시 MBR의 T-N, T-P 제거율 저하는 vanillin이 가지고 있는 항균 특성에 의한 것으로, CDA와 vanillin의 동시적용은 이러한 성능 저하를 고려하여야 될 것으로 판단된다. 스트레스로 인한 biofilm 형성이 촉진되는 환경에서의 CDA 효과를 확인하기 위해 염폐수를 처리를 위한 MBR에 CDA를 적용하였으며, vanillin과의 biofilm 형성 억제 상승효과 역시 평가하였다. Salt stress가 존재하는 환경에서, 적용된 QSC에 의한 EPS 변화는 차이를 나타냈다. QSC의 적용으로 total EPS는 감소했다. CDA 적용 조건의 total EPS는 약 9 % 감소했으며, vanillin 추가 적용으로 인한 biofilm 억제는 total EPS를 18 % 감소시켰다. CDA 적용 결과와 달리 vanillin 추가 조건은 상대적으로 높은 bound EPS를 가졌다. 이는 salt에 의한 stress와 함께 vanillin의 항균성이 stress로 작용하여 미생물이 발생시키는 EPS가 증가했기 때문으로 판단된다. 또한 saline 환경으로 인해 EPS 내 HMW-EPS 분획이 감소하였으며, 이는 유기 물질과 미생물 생성 중합체의 불완전 분해와 중간 생성물의 축적이 원인으로 판단된다. 본 연구결과, QSC 적용에 의해 MBR process의 막표면에 형성되는 biofilm의 구성변화 및 운전 기간의 연장이 가능함을 확인하였으며, biofouling 저감이 가능함을 입증하였다. 이 결과는 MBR 공정의 또 다른 생물학적 오염 제어 방법이 될 수 있음을 시사한다. QS compound에 의한 biofouling control method가 향상된 기술로서 실제 MBR 공정에 적용되기 위해서는 적용 방식, 장기간 운전을 통한 지속적인 평가 등의 후속 연구가 필요할 것으로 판단된다.