• 미생물학회지
• /
• 제50권2호
• /
• pp.128-136
• /
• 2014

Acyl homoserine lactone (AHL) 분해효소인 lactonase는 높은 기질 특이성을 지니기 때문에 경제적이고 효율적인 쿼럼 저해 기술로 이용될 가능성을 지니고 있다. 본 연구에서는 Chromobacterium violaceum CV026과 Agrobacterium tumefaciens NTL4를 바이오센서로 이용하여 biofouling이 일어난 역삼투막 시료로부터 쿼럼 센싱과 관련된 생물막 형성을 억제하는 6종의 균주를 분리 연구하였다. 분리된 균주는 모두 Bacillus 속으로 동정되었으며, AHL 분자의 acyl 사슬 길이나 치환 종류에 상관 없이 쿼럼 저해활성을 보여주었다. 균주들은 Pseudomonas aeruginosa PAO1에 의한 생물막 형성을 46.7-58.3% 정도 감소시켰으며 이 때 저해물질은 열처리에 민감한 특성을 보여주었다. 분리 균주 중 RO1S-5를 이용하여 N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12 AHL)과 반응시킨 결과, 상응하는 acyl homoserine (3-oxo-C12-HS)이 생성되는 것을 LC-MS로 확인하여 쿼럼 저해가 lactonase 활성에 의한 것임을 규명하였다. AHL 물질에 대한 높은 특이성 등을 감안할 때 분리 균주 RO1S-5는 생물막 형성과 관련된 질병이나 산업공정 중 발생하는 biofouling을 해결하는데 유용하게 쓰일 수 있을 것으로 기대된다.

• Journal of Microbiology and Biotechnology
• /
• 제30권6호
• /
• pp.937-945
• /
• 2020

N-acyl-homoserine lactone (AHL)-mediated quorum sensing (QS) plays a major role in development of biofilms, which contribute to rise in infections and biofouling in water-related industries. Interference in QS, called quorum quenching (QQ), has recieved a lot of attention in recent years. Rhodococcus spp. are known to have prominent quorum quenching activity and in previous reports it was suggested that this genus possesses multiple QQ enzymes, but only one gene, qsdA, which encodes an AHL-lactonase belonging to phosphotriesterase family, has been identified. Therefore, we conducted a whole genome sequencing and analysis of Rhodococcus sp. BH4 isolated from a wastewater treatment plant. The sequencing revealed another gene encoding a QQ enzyme (named jydB) that exhibited a high AHL degrading activity. This QQ enzyme had a 46% amino acid sequence similarity with the AHL-lactonase (AidH) of Ochrobactrum sp. T63. HPLC analysis and AHL restoration experiments by acidification revealed that the jydB gene encodes an AHL-lactonase which shares the known characteristics of the α/β hydrolase family. Purified recombinant JydB demonstrated a high hydrolytic activity against various AHLs. Kinetic analysis of JydB revealed a high catalytic efficiency (k_cat/K_M) against C4-HSL and 3-oxo-C6 HSL, ranging from 1.88 x 10⁶ to 1.45 x 10⁶ M^-1 s^-1, with distinctly low K_M values (0.16-0.24 mM). This study affirms that the AHL degrading activity and biofilm inhibition ability of Rhodococcus sp. BH4 may be due to the presence of multiple quorum quenching enzymes, including two types of AHL-lactonases, in addition to AHL-acylase and oxidoreductase, for which the genes have yet to be described.

• Journal of Applied Biological Chemistry
• /
• 제48권4호
• /
• pp.183-186
• /
• 2005

Arabidopsis AHL gene encodes a 3'(2')-phosphoadenosine 5'-phosphate (PAP)-specific phosphatase that plays a role in the sulfate activation pathway. We complemented E. coli cysQ mutant defective in cysteine biosynthesis with the AHL gene. AHL cDNA was cloned into the prokaryotic expression vector pKK388-1 and transformed into the bacterial mutant. Since cysQ mutant is a leaky cysteine auxotroph only under aerobic conditions, the bacteria were grown in liquid media with vigorous shaking to provide more aeration. In cysteine-free medium, cysQ mutant and the mutant harboring empty vector did not grow well, whereas cells harboring AHL cDNA exhibited significantly improved growth with doubling time of approximately 3 h. cysQ is known to encode a 3'(2'),5'-diphosphonucleoside 3'(2')-phosphohydrolase (DPNPase). However, our data suggest that cysQ protein has PAP-specific phosphatase activity in addition to DPNPase activity. Microbial complementation procedure described in this paper is useful for structure-activity studies of PAP-specific phosphatases identified from microbes and plants.

• Journal of Applied Biological Chemistry
• /
• 제42권4호
• /
• pp.169-174
• /
• 1999

Arabidopsis AHL gene contains 4 exons encoding a putative protein highly homologous to the yeast salt-sensitive enzyme HAL2, a 3'(2'),5'-bisphosphate nucleotidase involving in reductive sulfate assimilation. AHL cDNA complemented yeast met22 (hal2) mutant. AHL fusion protein expressed in E. coli exhibited $Mg^{2+}$-dependent, 3'-phosphoadenosine 5'-phosphate (PAP)-specific phosphatase activity. $Li^+,\;Na^+,\;K^+$ and $Ca^{2+}$ ions inhibit the enzyme activity by competing with $Mg^{2+}$ for the active site of the enzyme. The enzyme activity was also sensitive to ${\mu}M$ concentrations of toxic heavy metal ions such as $Cd^{2+},\;Cu^{2+}$ and $Zn^{2+}$, but was not recovered by addition of more $Mg^{2+}$ ions, suggesting that these ions inactivate the enzyme with a mechanism other than competition with $Mg^{2+}$ ions. Inhibition of the AHL enzyme activity may result in accumulation of PAP, which is highly toxic to the cell. Thus, the AHL enzyme could be one of the intial targets of heavy metal toxicity in plants.

• Clinical and Experimental Reproductive Medicine
• /
• 제35권3호
• /
• pp.193-202
• /
• 2008

목 적: 보조부화술이 적용되는 좋지 않은 예후를 보여주는 선별된 환자군을 대상으로 산성 용액을 이용한 AHA 방법과 레이저를 이용한 AHL 방법의 효용성을 비교하여, 보다 효과적으로 임신율과 착상률을 높일 수 있는 보조부화술 방법을 찾고자 하였다. 연구방법: 2006년 2월부터 9월까지 체외수정 시술을 시행한 환자 중 보조부화술이 필요한 328주기를 대상으로 산성용액을 이용한 AHA 방법 (180주기)과 ZILOS-tk 레이저를 이용한 AHL 방법 (148주기)으로 나누어 시행하였다. 보조부화술을 시행한 환자군은 환자의 나이가 38세 이상이거나 투명대의 두께가 $18{\mu}m$ 이상, 기저 FSH 농도가 15 mIU/ml이상, 체외수정 시술을 3번 이상 실패한 환자, 이식하는 배아의 상태가 양호하지 않은 환자들로 이상에 적용요인이 있으면 시행하여 무작위로 보조부화술 방법간에 환자들의 임상적 특징과 임신율과 착상률을 분석하였다. 결 과: 전체 보조부화술을 시행한 환자군에 AHL 방법과 AHA 방법간에 임신율 (42.6%, 63/148 vs. 33.3%, 60/180)과 착상률 (17.4%, 82/470 vs. 16.0%, 89/556)에 유의적 차이는 나타나지 않았다. 그러나 나이가 많은 환자군인 Group 1은 임신율 (37.0%, 20/54 vs. 18.7%, 14/75)과 착상률 (14.4%, 23/160 vs. 7.1%, 15/210)이 AHL 방법이 AHA 방법보다 유의적 (p<0.05)으로 높게 나타났다. 전체 환자군이나 선별된 각 군내에 보조부화술 방법간에 환자의 임상적 특징은 유의적 차이가 나타나지 않았다. 3번 이상 체외시술에 실패한 환자군 [Group 2: 43.8% (21/48)과 31.6% (25/79)], 투명대의 두께가 $18{\mu}m$ 이상인 환자군 [Group 3: 43.8% (32/73)과 34.1% (28/82)], 이식한 배아의 질이 양호하지 않은 환자군[Group 4: 25.0% (7/28)과 14.6% (6/41)]에서는 AHL 방법이 임상결과는 좋았으나 유의적 차이는 없었다. 결 론: 레이저를 이용한 AHL 방법이 나이가 많은 환자군과 3번 이상 체외수정 시술에 실패한 환자군에서 AHA방법에 비해 높은 임신율과 착상률을 나타내었다. 결론적으로, AHL을 이용한 보조부화술이 임상적으로 보다 효과적이고 안전한 방법이라고 사료된다.

• 한국미생물·생명공학회지
• /
• 제40권2호
• /
• pp.83-91
• /
• 2012

본 총설은 N-acyl-homoserine lactone (AHL)에 기반한 quorum sensing(QS)을 비롯한 다양한 QS 시스템 및 생물막 형성과의 관련성에 대한 연구 동향을 정리하였다. 또한 anti-QS으로서 quorum quenching 전략을 이용한 생물막 억제 연구 동향에 대해 중점적으로 서술하였다. 세균의 독특한 신호전달 체계인 QS는 AHL과 같은 특정한 신호분자의 농도에 의해 세균의 집단적 행동 양식이 결정되는 세포밀도-의존성 유전자 발현 조절 메커니즘이다. QS 시스템은 미생물의 부착 및 생물막 형성에 있어 중요한 역할을 한다. AI-1이나 AI-2에 의한 QS는 생물막 형성 과정에 필요한 세포외 다당류, 단백질, 세포 외 DNA 등 주요한 구성 성분 등의 생산뿐만 아니라, 세균의 운동성 조절, 부착, 생물막 해체 과정까지도 조절하는 기능을 한다. 일부 세균의 경우 QS시스템 이외에도 second messenger로 알려진 c-di-GMP에 의한 signaling이 QS와 서로 연결되어 생물막 형성이나 병독성과 같은 타깃들을 함께 조절한다. 생물막은 병원성 세균에 의한 감염 시 여러 가지 병독성 가운데 가장 중요한 요소 중 하나이기 때문에, 생물막 형성을 조절하는 QS를 차단하기 위한 다양한 anti-quorum sensing 전략이 연구되고 있다. Anti-QS 접근 방식은 의학적 이용뿐만 아니라 물에 노출되어있는 MBR을 비롯한 많은 산업적 장치 등에서 생물막 형성으로 인한 손상 및 오염을 방지하기 위해 쓰일 수 있다. Anti-QS 전략 중 신호분자인 AHL을 무력화 시키는 quorum quenching 효소(AHL-lactonase, AHL-acylase, oxidoreductas)를 이용하여 생물막 형성을 억제할 수 있으며, 막을 이용한 수처리 공정에서 막에 발생하는 biofouling을 완화시킬 수 있는 새로운 anti-fouling 처리 기술로서 이러한 QQ 효소의적용 가능성을 보여 주고 있다.

• BMB Reports
• /
• 제44권10호
• /
• pp.680-685
• /
• 2011

The AT-hook motif is a small DNA-binding protein motif that has been found in the high mobility group of non-histone chromosomal proteins. The Arabidopsis genome contains 29 genes encoding the AT-hook motif DNA-binding protein (AHL). Recent studies of Arabidopsis genes (AtAHLs) have revealed that they might play diverse functional roles during plant growth and development. In this report, we mined 20 AHL genes (OsAHLs) from the rice genome database using AtAHL genes as queries and characterized their molecular features. A phylogenetic tree revealed that OsAHL proteins can be classified into 2 evolutionary clades. Tissue expression pattern analysis revealed that all of the OsAHL genes might be functionally expressed genes with 3 distinct expression patterns. Nuclear localization analysis using transgenic Arabidopsis showed that several OsAHL proteins are exclusively localized in the nucleus, indicating that they may act as architectural transcription factors to regulate expression of their target genes during plant growth and development.

• Journal of Microbiology and Biotechnology
• /
• 제21권10호
• /
• pp.1001-1011
• /
• 2011

To explore bacterial diversity for elucidating genetic variability in acylhomoserine lactone (AHL) lactonase structure, we screened 800 bacterial strains. It revealed the presence of a quorum quenching (QQ) AHL-lactonase gene (aiiA) in 42 strains. These 42 strains were identified using rrs (16S rDNA) sequencing as Bacillus strains, predominantly B. cereus. An in silico restriction endonuclease (RE) digestion of 22 AHL lactonase gene (aiiA) sequences (from NCBI database) belonging to 9 different genera, along with 42 aiiA gene sequences from different Bacillus spp. (isolated here) with 14 type II REs, revealed distinct patterns of fragments (nucleotide length and order) with four REs; AluI, DpnII, RsaI, and Tru9I. Our study reflects on the biodiversity of aiiA among Bacillus species. Bacillus sp. strain MBG11 with polymorphism (115Alanine > Valine) may confer increased stability to AHL lactonase, and can be a potential candidate for heterologous expression and mass production. Microbes with ability to produce AHL-lactonases degrade quorum sensing signals such as AHL by opening of the lactone ring. The naturally occurring diversity of QQ molecules provides opportunities to use them for preventing bacterial infections, spoilage of food, and bioremediation.

• Journal of Microbiology and Biotechnology
• /
• 제24권11호
• /
• pp.1574-1582
• /
• 2014

Bacteria recognize changes in their population density by sensing the concentration of signal molecules, N-acyl-homoserine lactones (AHLs). AHL-mediated quorum sensing (QS) plays a key role in biofilm formation, so the interference of QS, referred to as quorum quenching (QQ), has received a great deal of attention. A QQ strategy can be applied to membrane bioreactors (MBRs) for advanced wastewater treatment to control biofouling. To isolate QQ bacteria that can inhibit biofilm formation, we isolated diverse AHL-degrading bacteria from a laboratory-scale MBR and sludge from real wastewater treatment plants. A total of 225 AHL-degrading bacteria were isolated from the sludge sample by enrichment culture. Afipia sp., Acinetobacter sp. and Streptococcus sp. strains produced the intracellular QQ enzyme, whereas Pseudomonas sp., Micrococcus sp. and Staphylococcus sp. produced the extracellular QQ enzyme. In case of Microbacterium sp. and Rhodococcus sp., AHL-degrading activities were detected in the whole-cell assay and Rhodococcus sp. showed AHL-degrading activity in cell-free lysate as well. There has been no report for AHL-degrading capability in the case of Streptococcus sp. and Afipia sp. strains. Finally, inhibition of biofilm formation by isolated QQ bacteria or enzymes was observed on glass slides and 96-well microtiter plates using crystal violet staining. QQ strains or enzymes not only inhibited initial biofilm development but also reduced established biofilms.

• Asian Pacific Journal of Cancer Prevention
• /
• 제15권21호
• /
• pp.9319-9325
• /
• 2014

Alkaloids are the most extensively featured compounds of natural anti-tumor herbs, which have attracted much attention in pharmaceutical research. In our previous studies, a mixture of major three alkaloid components (5, 6-dihydrobicolorine, 7-deoxy-trans-dihydronarciclasine, littoraline) from Hymenocallis littoralis were extracted, analyzed and designated as AHL. In this paper, AHL extracts were added to human liver hepatocellular cells HepG-2, human gastric cancer cell SGC-7901, human breast adenocarcinoma cell MCF-7 and human umbilical vein endothelial cell EVC-304, to screen one or more AHL-sensitive tumor cell. Among these cells, HepG-2 was the most sensitive to AHL treatment, a very low dose ($0.8{\mu}g/ml$) significantly inhibiting proliferation. The non-tumor cell EVC-304, however, was not apparently affected. Effect of AHL on HepG-2 cells was then explored. We found that the AHL could cause HepG-2 cycle arrest at G2/M checkpoint, induce apoptosis, and interrupt polymerization of microtubules. In addition, expression of two cell cycle-regulated proteins, CyclinB1 and CDK1, was up-regulated upon AHL treatment. Up-regulation of the Fas, Fas ligand, Caspase-8 and Caspase-3 was observed as well, which might imply roles for the Fas/FsaL signaling pathway in the AHL-induced apoptosis of HepG-2 cells.