• Korean Journal of Microbiology
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• v.50 no.2
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• pp.128-136
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• 2014

Acyl homoserine lactone (AHL) lactonase has been proved to be the AHL-degrading enzyme with the highest substrate specificity for AHL molecules and has shown a considerable potential as low-cost and efficient quorum quenching (QQ) technique. However, few studies focused on its inhibitory effect on biofilm formation which is also a quorum sensing (QS)-regulated phenomenon. In this study, QQ activity of six isolates from biofouled reverse osmosis membranes was studied using Chromobacterium violaceum CV026 and Agrobacterium tumefaciens NTL4 as biosensors under various conditions. All of the isolates belonged to the genus Bacillus and showed QQ activity regardless of the acyl chain length or substitution of AHL molecule. The isolates were capable of significantly inhibiting biofilm formation (46.7-58.3%) by Pseudomonas aeruginosa PAO1 and produced heat-sensitive extracellular QQ substances. The LC-MS analysis of the QQ activity of a selected isolate, RO1S-5, revealed the degradation of N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12 AHL) and the production of corresponding acyl homoserine (3-oxo-C12-HS), which indicated the activity of AHL lactonase. The broad AHL substrate range and high substrate specificity suggested that the isolate would be useful for the control of biofilm-related pathogenesis and biofouling in industrial processes.

• Journal of Microbiology and Biotechnology
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• v.30 no.6
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• pp.937-945
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• 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
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• v.48 no.4
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• pp.183-186
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• 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
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• v.42 no.4
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• pp.169-174
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• 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
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• v.35 no.3
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• pp.193-202
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• 2008

Objective: To evaluate efficacy of assisted hatching by laser (AHL) and acidified Tyrode solution (AHA) in selected groups of IVF-ET patients who have a poor prognosis. Methods: From February 2006 to September 2006, total of 328 IVF-ET cycles with advanced female age (${\geq}38$ years), recurrent implantation failure (${\geq}3$ cycles), thick zona (${\geq}17{\mu}m$), and/or poor quality of embryo were randomly divided into assisted hatching by acidified Tyrode solution (AHA, n=180) and the assisted hatching using the ZILOS-tk laser (AHL, n=148) groups. Clinical outcomes were analyzed and compared between AHA and AHL group based on the patient characteristics. Results: In all AHL and AHA group, there were no significant differences in pregnancy (42.6%, 63/148 vs. 33.3%, 60/180) and implantation rates (17.4%, 82/470 vs. 16.0%, 89/556) However, in advanced female age group (Group 1), pregnancy (37.0%, 20/54 vs. 18.7%, 14/75) and implantation rates (14.4%, 23/160 vs. 7.1%, 15/210) in AHL group were significantly (p<0.05) higher than those of AHA, although there was no difference in patient parameters of both groups. And, the clinical outcome of groups with recurrent implantation failure (Group 2), thick zona pellucida (Group 3) and poor quality embryo (Group 4) were improved in AHL compared to those of AHA: 43.8% (21/48) and 31.6% (25/79) in Group 2, 43.8% (32/73) and 34.1% (28/82) in Group 3, 25.0% (7/28) and 14.6% (6/41) in Group 4, but no significance. Conclusion: The AHL improved the pregnancy and implantation rates in patients with advanced female age and recurrent implantation failure when compared to outcomes achieved from AHA. Therefore, this AHL technique may be a efficient and safe method for patients with poor prognosis.

• Microbiology and Biotechnology Letters
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• v.40 no.2
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• pp.83-91
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• 2012

Quorum sensing (QS) is a 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 differences in cell density by sensing the concentration of signal molecules such as N-acyl-homoserine lactones (AHL) and autoinducer-2 (AI-2). In particular, QS plays a key role in biofilm formation, which is a specific bacterial group behavior. Biofilms are dense aggregates of packed microbial communities that grow on surfaces, and are embedded in a self-produced matrix of extracellular polymeric substances (EPS). QS regulates biofilm dispersal as well as the production of EPS. In some bacteria, biofilm formations are regulated by c-di-GMP-mediated signaling as well as QS, thus the two signaling systems are mutually connected. Biofilms are one of the major virulence factors in pathogenic bacteria. In addition, they cause numerous problems in industrial fields, such as the biofouling of pipes, tanks and membrane bioreactors (MBR). Therefore, the interference of QS, referred to as quorum quenching (QQ) has received a great deal of attention. To inhibit biofilm formation, several strategies to disrupt bacterial QS have been reported, and many enzymes which can degrade or modify the signal molecule AHL have been studied. QQ enzymes, such as AHL-lactonase, AHL-acylase, and oxidoreductases may offer great potential for the effective control of biofilm formation and membrane biofouling in the future. This review describes the process of bacterial QS, biofilm formation, and the close relationship between them. Finally, QQ enzymes and their applications for the reduction of biofouling are also discussed.

• BMB Reports
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• v.44 no.10
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• pp.680-685
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• 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
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• v.21 no.10
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• pp.1001-1011
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• 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
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• v.24 no.11
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• pp.1574-1582
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• 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
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• v.15 no.21
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• pp.9319-9325
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• 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.