Quorum Quenching Activity Research Articles

Multiple Quorum Quenching Enzymes Are Active in the Nosocomial Pathogen Acinetobacter baumannii ATCC17978.

Acinetobacter baumannii presents a typical luxI/luxR quorum sensing (QS) system (abaI/abaR) but the acyl-homoserine lactone (AHL) signal profile and factors controlling the production of QS signals in this species have not been determined yet. A very complex AHL profile was identified for A. baumannii ATCC17978 as well as for A. nosocomialis M2, but only when cultivated under static conditions, suggesting that surface or cell-to-cell contact is involved in the activation of the QS genes. The analysis of A. baumanni clinical isolates revealed a strain-specific AHL profile that was also affected by nutrient availability. The concentration of OHC12-HSL, the major AHL found in A. baumannii ATCC17978, peaked upon stationary-phase establishment and decreases steeply afterwards. Quorum quenching (QQ) activity was found in the cell extracts of A. baumannii ATCC17978, correlating with the disappearance of the AHLs from the culture media, indicating that AHL concentration may be self-regulated in this pathogen. Since QQ activity was observed in strains in which AidA, a novel α/β-hydrolase recently identified in A. baumannii, is not present, we have searched for additional QQ enzymes in A. baumannii ATCC17978. Seven putative AHL-lactonase sequences could be identified in the genome and the QQ activity of 3 of them could be confirmed. At least six of these lactonase sequences are also present in all clinical isolates as well as in A. nosocomialis M2. Surface-associated motility and biofilm formation could be blocked by the exogenous addition of the wide spectrum QQ enzyme Aii20J. The differential regulation of the QQ enzymes in A. baumannii ATCC17978 and the full dependence of important virulence factors on the QS system provides a strong evidence of the importance of the AHL-mediated QS/QQ network in this species.

Quorum-Quenching Bacteria Isolated From Red Sea Sediments Reduce Biofilm Formation by Pseudomonas aeruginosa.

Quorum sensing (QS) is the process by which bacteria communicate with each other through small signaling molecules such as N-acylhomoserine lactones (AHLs). Certain bacteria can degrade AHL molecules by a process called quorum quenching (QQ); therefore, QQ can be used to control bacterial infections and biofilm formation. In this study, we aimed to identify new species of bacteria with QQ activity. Red Sea sediments were collected either from the close vicinity of seagrass or from areas with no vegetation. We isolated 72 bacterial strains, which were tested for their ability to degrade/inactivate AHL molecules. Chromobacterium violaceum CV026-based bioassay was used for the initial screening of isolates with QQ activity. QQ activity was further quantified using high-performance liquid chromatography-tandem mass spectrometry. We found that these isolates could degrade AHL molecules of different acyl chain lengths as well as modifications. 16S-rRNA sequencing of positive QQ isolates showed that they belonged to three different genera. Specifically, two isolates belonged to the genus Erythrobacter; four, Labrenzia; and one, Bacterioplanes. The genome of one representative isolate from each genus was sequenced, and potential QQ enzymes, namely, lactonases and acylases, were identified. The ability of these isolates to degrade the 3OXOC12-AHLs produced by Pseudomonas aeruginosa PAO1 and hence inhibit biofilm formation was investigated. Our results showed that the isolate VG12 (genus Labrenzia) is better than other isolates at controlling biofilm formation by PAO1 and degradation of different AHL molecules. Time-course experiments to study AHL degradation showed that VG1 (genus Erythrobacter) could degrade AHLs faster than other isolates. Thus, QQ bacteria or enzymes can be used in combination with an antibacterial to overcome antibiotic resistance.

Quorum sensing in rhizobia isolated from the spores of the mycorrhizal symbiont Rhizophagus intraradices.

Most beneficial services provided by arbuscular mycorrhizal fungi (AMF), encompassing improved crop performance and soil resource availability, are mediated by AMF-associated bacteria, showing key-plant growth-promoting (PGP) traits, i.e., the production of indole acetic acid, siderophores and antibiotics, and activities increasing the availability of plant nutrients by nitrogen fixation and phosphate mobilization. Such functions may be affected by the ability of AMF-associated bacteria to communicate through the production and secretion of extracellular small diffusible chemical signals, N-acyl homoserine lactone signal molecules (AHLs), that regulate bacterial behavior at the community level (quorum sensing, QS). This work investigated the occurrence and extent of QS among rhizobia isolated from AMF spores, using two different QS reporter strains, Agrobacterium tumefaciens NTL4 pZRL4 and Chromobacterium violaceum CV026. We also assessed the quorum quenching (QQ) activity among Bacillus isolated from the same AMF spores. Most rhizobia were found to be quorum-signaling positive, including six isolates producing very high levels of AHLs. The results were confirmed by microtiter plate assay, which detected 65% of the tested bacteria as medium/high AHL producers. A 16S rDNA sequence analysis grouped the rhizobia into two clusters, consistent with the QS phenotype. None of the tested bacteria showed QQ activity able to disrupt the QS signaling, suggesting the absence of antagonism among bacteria living in AMF sporosphere. Our results provide the first evidence of the ability of AMF-associated rhizobia to communicate through QS, suggesting further studies on the potential importance of such a behavior in association with key-plant growth-promoting functions.

Identification of N-acyl homoserine lactone-degrading bacteria isolated from rainbow trout (Oncorhynchus mykiss).

A variety of pathogens use quorum sensing (QS) to control the expression of their virulence factors. QS interference has hence been proposed as a promising antivirulence strategy. The specific aim of this study was to isolate bacteria from trout tissue able to degrade N-acyl homoserine lactones (AHL), a QS molecule family. In total 132 isolates were screened for AHL degradation using Chromobacterium violaceum CV026 as a biosensor. Twenty-four quorum-quenching (QQ) isolates were identified biochemically and characterized using 16S rDNA sequencing. They belong to Bacillus, Enterobacter, Citrobacter, Acinetobacter, Agrobacterium, Pseudomonas and Stentrophomonas genera. Four Bacillus spp. showed the highest and fastest QQ activity. AHL degradation proved to be enzymatic in most isolates (except for Stentrophomonas spp. and Pseudomonas sp.) as QQ activity could be destroyed by heat and/or proteinase K treatments. All QQ activity proved to be cell-bound except for Pseudomonas sp., where it could be detected in the supernatant. The results of aiiA gene homology analysis revealed the presence of aiiA gene encoding AHL lactonase in all examined isolates except Pseudomonas syringae and Enterobacter cloacae. The HXHXDH motif conserved in all AHL lactonases and considered to be essential for AHL degradation was detected in all AiiAs after sequence alignment. Some known and novel QQ bacteria were isolated from trouts and characterized in terms of enzymatic or nonenzymatic AHL degradation activity and their extracellular or intracellular location. In addition, an aiiA gene and its HXHXDH motif were detected in most isolates. We could isolate and identify some novel QQ bacteria including Enterobacter hormaechei, Acinetobacter radioresistens and Citrobacter gillenii. The aiiA gene was detected for the first time in these strains as well as in Stenotrophomonas maltophilia. Our QQ isolates could be used for biocontrol of bacterial infections in aquaculture.

Isolation of Marine Bacillus sp. with Antagonistic and Organic-Substances-Degrading Activities and Its Potential Application as a Fish Probiotic.

We report on the isolation and characterization of an acid- and bile-tolerant bacterial strain, Bacillus sp. YB1701 with antibacterial and quorum-quenching activity. Strain YB1701 was isolated from coastal sediment samples and characterized by biochemical tests and 16S rRNA sequencing. In vitro study indicated that strain YB1701 can survive at pH 2.0 for up to 3 h and tolerate bile up to 2.0% concentration even after 12 h of exposure. Strain YB1701 showed antimicrobial activity against fish pathogens Aeromonas hydrophila and Vibrio parahemolyticus using an agar well diffusion assay. The trial test showed dietary supplementation of YB1701 significantly improved the resistance of Carassius auratus gibelio against A. hydrophila challenge. The safety assessment revealed that the isolate Bacillus sp. YB1701 was not cytotoxic to Carassius auratus gibelio or mice and did not exhibit hemolytic activity on rabbit blood agar plate. Disc-diffusion assays using a panel of antibiotics listed by the European Food Safety Authority (EFSA) showed that YB1701 was susceptible to selected antibiotics. Under laboratory conditions, the degradation rate of organic waste (predominately fish excrement) for 14 days by YB1701 was 79.69%. Results from the present study suggest that strain YB1701 is a potential probiotic strain and can be used in aquaculture for degrading organic waste and improving disease resistance of fish against bacterial infection. Further study is needed to assess the utility of strain YB1701 on a commercial scale.

Effective quorum quenching bacteria dose for anti-fouling strategy in membrane bioreactors utilizing fixed-sheet media

Quorum quenching (QQ) has been recognized as an emerging anti-biofouling strategy for membrane bioreactors (MBRs), but studies on effective QQ bacterial doses are needed. Therefore, in this study, the QQ efficacy for sheet-type media containing different amounts of QQ bacteria (i.e., Rhodococcus sp. BH4) with respect to biofouling control during MBR treatment of synthetic and real municipal wastewaters was investigated. The QQ sheets with well-embedded BH4 bacteria were distinctly effective at alleviating biofouling, but their efficacy was highly dependent on the QQ bacterial dose. The activity of the QQ sheet increased with the QQ bacterial density and its pseudo-first order rate constant reached 3.52 h−1 with 100 mg BH4 per liter of reactor volume. No QQ activity loss occurred with long-term use of the QQ sheets. The relationship between QQ dose and fouling delay had a strong, linear correlation. Fouling retardation was more pronounced with real wastewater because it had less biofouling propensity owing to its inferior organics and nutrients. In fact, the MBRs fed with real wastewater experienced a dramatic decrease in extracellular polymeric substance (EPS) content responsible for biofouling. QQ suppressed the EPS secretion leading to fouling mitigation, but with no impact on biological treatment performances.

Metagenomics-based interpretation of AHLs-mediated quorum sensing in Anammox biofilm reactors for low-strength wastewater treatment

Anammox-based nitrogen removal can advance sustainable wastewater treatment, especially for the main-stream wastewater treatment. Two lab-scale Anammox biofilm reactors fed with influent ammonium concentrations of 110 mg/L (Anammox-H) and 50 mg/L (Anammox-L) were operated, and nitrogen removal and mechanisms of quorum sensing (QS)/quorum quenching (QQ) were examined. The achieved nitrogen removal rates were 0.65 g N/L·d in Anammox-H and 0.25 g N/L·d in Anammox-L. Candidatus Kuenenia was dominant in both reactors. Higher contents of tight extracellular polymeric substance and hydrophobic amino acid confirmed that dense Anammox biofilm developed in Anammox-H. N-hexanoyl-l-homoserine lactone and N-octanoyl-l-homoserine lactone were detected in Anammox-H and Anammox-L. High concentrations of acyl-homoserine lactones (AHLs) in water and biomass phases of Anammox-H were consistent with that more active QS processes existed in Anammox-H. Functional genes including hao, pmoA-amoC, nirK and narGZHY were detected and harbored by Candidatus Kuenenia stuttgartiensis, Nitrosomonas europaea and Lautropia sp. SCN 69-89. These organisms not only involved in nitrogen metabolism, but also in pyruvate, fatty acid and S-Adenosy-l-methionine synthesis (important for AHLs). Influent ammonium concentrations affected the balance between QQ and QS activities, resulting in different Anammox activity and biofilm morphology. This study advanced the Anammox application for low-strength wastewater, especially from the aspect of QQ and QS balance.

Mitigation of membrane biofouling by a quorum quenching bacterium for membrane bioreactors

In this study, a quorum-quenching (QQ) bacterium named HEMM-1 was isolated at a membrane bioreactor (MBR) plant. HEMM-1 has diplococcal morphology and 99% sequence identity to Enterococcus species. The HEMM-1 cell-free supernatant (CFS) showed higher QQ activities than the CFS of other QQ bacteria, mostly by degrading N-acyl homoserine lactones (AHLs) with short acyl chains. Instrumental analyses revealed that HEMM-1 CFS degraded AHLs via lactonase activity. Under static, flow, and shear conditions, the HEMM-1 CFS was effective in reducing bacterial and activated-sludge biofilms formed on membrane surfaces. In conclusion, the HEMM-1 isolate is a QQ bacterium applicable to the control of biofouling in MBRs via inhibition of biofilm formation on membrane surfaces.

High Prevalence of Quorum-Sensing and Quorum-Quenching Activity among Cultivable Bacteria and Metagenomic Sequences in the Mediterranean Sea.

There is increasing evidence being accumulated regarding the importance of N-acyl homoserine lactones (AHL)-mediated quorum-sensing (QS) and quorum-quenching (QQ) processes in the marine environment, but in most cases, data has been obtained from specific microhabitats, and subsequently little is known regarding these activities in free-living marine bacteria. The QS and QQ activities among 605 bacterial isolates obtained at 90 and 2000 m depths in the Mediterranean Sea were analyzed. Additionally, putative QS and QQ sequences were searched in metagenomic data obtained at different depths (15–2000 m) at the same sampling site. The number of AHL producers was higher in the 90 m sample (37.66%) than in the 2000 m sample (4.01%). However, the presence of QQ enzymatic activity was 1.63-fold higher in the 2000 m sample. The analysis of putative QQ enzymes in the metagenomes supports the relevance of QQ processes in the deepest samples, found in cultivable bacteria. Despite the unavoidable biases in the cultivation methods and biosensor assays and the possible promiscuous activity of the QQ enzymes retrieved in the metagenomic analysis, the results indicate that AHL-related QS and QQ processes could be common activity in the marine environment.

Quorum quenching activity of indigenous quorum quenching bacteria and its potential application in mitigation of membrane biofouling

AbstractBACKGROUNDQuorum sensing (QS) could regulate gene expression so as to mediate some bacterial behaviors such as the production of extracellular polymeric substances (EPS) and biofilm formation. In this study, a quorum quenching (QQ) bacterium was isolated from an indigenous lab‐scale membrane bioreactor (MBR) and encapsulated in a dumpling‐shaped microbial bag for biofouling control.RESULTSThis QQ bacteria has a higher genetic homology with Acinetobacter bereziniae strain. The effect of time and temperature on the QQ activity of the microbial bag was tested by degrading N‐acyl homoserine lactone (AHL). The results showed the degradation of C8‐HSL (N‐Octanoyl‐DL‐homoserine lactone) and C6‐HSL (N‐Hexanoyl‐L‐homoserine lactone) could reach equilibrium within 2 h. In addition, a suitable temperature for AHL degradation was 20 °C, at which the rate of degradation of C6‐HSL and C8‐HSL by the microbial bag was 88% and 69%, respectively. The antifouling efficiency of the microbial bag was investigated by a constant pressure filtration system. With the microbial bag added, the decrease of permeability was obviously improved. After 14 days, the permeability with QQ decreased to 18% while the control experiment exhibited almost no permeability.CONCLUSIONThe excellent AHL degradation rate and the filtration test showed that this QQ bacteria has an excellent antifouling potential in membrane filtration systems. © 2017 Society of Chemical Industry

Oxygenated N-Acyl Alanine Methyl Esters (NAMEs) from the Marine Bacterium Roseovarius tolerans EL-164.

The marine bacterium Roseovarius tolerans EL-164 (Rhodobacteraceae) can produce unique N-acylalanine methyl esters (NAMEs) besides strucutrally related N-acylhomoserine lactones (AHLs), bacterial signaling compounds widespread in the Rhodobacteraceae. The structures of two unprecedented NAMEs carrying a rare terminally oxidized acyl chain are reported here. The compounds (Z)-N-16-hydroxyhexadec-9-enoyl-l-alanine methyl ester (Z9-16-OH-C16:1-NAME, 3) and (Z)-N-15-carboxypentadec-9-enoyl-l-alanine methyl ester (16COOH-C16:1-NAME, 4) were isolated, and the structures were determined by NMR and MS experiments. Both compounds were synthesized to prove assignments and to test their biological activity. Finally, non-natural, structurally related Z9-3-OH-C16:1-NAME (18) was synthesized to investigate the mass spectroscopy of structurally related NAMEs. Compound 3 showed moderate antibacterial activity against microorganisms such as Bacillus, Streptococcus, Micrococcus, or Mucor strains. In contrast to AHLs, quorum-sensing or quorum-quenching activity was not observed.

Evaluation of a novel anti-biofouling microorganism (Bacillus sp. T5) for control of membrane biofouling and its effect on bacterial community structure in membrane bioreactors.

The effects of a newly isolated quorum quenching (QQ) bacteria (Bacillus sp. T5) on the microbial community has been evaluated via the Illumina sequencing method. Membrane bioreactors (MBRs) operated with this novel QQ bacterium to evaluate the improvement in the performance of MBR. Anti-biofouling effect of T5 was enhanced as 71% compared to the control reactor. Also, QQ bacteria did not have any negative effect on the removal of organics during the process. Gram-negative bacteria were found to be dominant over Gram-positive bacteria. Proteobacteria, Actinobacteria, Bacteroidetes, Acidobacteria, Firmicutes, and Chloroflexi were dominant phyla in the control and QQ reactors. The proportion of Alphaproteobacteria was most significant among Proteobacteria. The relative abundances of Actinobacteria, Acidobacteria, and Firmicutes were significantly affected by Quorum quenching mechanism. On the other hand, QQ activity of Bacillus sp. T5 significantly influenced the relative abundance of Proteobacteria, Bacteroidetes, and Chloroflexi. The QQ process appeared to generate variations in the structure of the microbial community. According to the results of the molecular analyses, the syntrophic interaction of Bacillus sp. T5 and indigenous Gram-negative and Gram-positive bacterial community is critical to the performance of MBRs.

Quorum quenching activity of rhizosphere bacteria against Ralstonia solanacearum

Many Gram-negative bacteria control the expression of virulence factors through quorum sensing (QS) mechanism. Enzymatic degradation of these signalling molecules is an important possible way to interfere with this QS mechanism. Such quorum quenching (QQ) enzymes are widespread in the bacterial world. The Pseudomonas aeruginosa 2apa, Pseudomonas fluorescens, Serratia marcescens and Azospirillum amazonense which are used as biocontrol agent were tested for QQ activity using Chromobacterium violaceum CV026 biosensor strain. Cell-free lysate of P. aeruginosa 2apa exhibited effective N-acyl homoserine lactone (AHL) degrading ability by inhibiting violacein production in biosensor strain. Further when the cell-free lysate was applied to Ralstonia solanacearum, it resulted in inhibition of biofilm formation. The confocal laser scanning microscopic analysis showed decrease in total biomass formation in treated slides. Our study shows the potential of AHL degradation by AHL acylase present in cell-free lysate of P. aeruginosa 2apa and inhibition of biofilm formation in R. solanacearum.

Quorum sensing network in clinical strains of A. baumannii: AidA is a new quorum quenching enzyme.

Acinetobacter baumannii is an important pathogen that causes nosocomial infections generally associated with high mortality and morbidity in Intensive Care Units (ICUs). Currently, little is known about the Quorum Sensing (QS)/Quorum Quenching (QQ) systems of this pathogen. We analyzed these mechanisms in seven clinical isolates of A. baumannii. Microarray analysis of one of these clinical isolates, Ab1 (A. baumannii ST-2_clon_2010), previously cultured in the presence of 3-oxo-C12-HSL (a QS signalling molecule) revealed a putative QQ enzyme (α/ß hydrolase gene, AidA). This QQ enzyme was present in all non-motile clinical isolates (67% of which were isolated from the respiratory tract) cultured in nutrient depleted LB medium. Interestingly, this gene was not located in the genome of the only motile clinical strain growing in this medium (A. baumannii strain Ab421_GEIH-2010 [Ab7], isolated from a blood sample). The AidA protein expressed in E. coli showed QQ activity. Finally, we observed downregulation of the AidA protein (QQ system attenuation) in the presence of H2O2 (ROS stress). In conclusion, most of the A. baumannii clinical strains were not surface motile (84%) and were of respiratory origin (67%). Only the pilT gene was involved in surface motility and related to the QS system. Finally, a new QQ enzyme (α/ß hydrolase gene, AidA protein) was detected in these strains.

AidP, a novel N-Acyl homoserine lactonase gene from Antarctic Planococcus sp.

Planococcus is a Gram-positive halotolerant bacterial genus in the phylum Firmicutes, commonly found in various habitats in Antarctica. Quorum quenching (QQ) is the disruption of bacterial cell-to-cell communication (known as quorum sensing), which has previously been described in mesophilic bacteria. This study demonstrated the QQ activity of a psychrotolerant strain, Planococcus versutus strain L10.15T, isolated from a soil sample obtained near an elephant seal wallow in Antarctica. Whole genome analysis of this bacterial strain revealed the presence of an N-acyl homoserine lactonase, an enzyme that hydrolyzes the ester bond of the homoserine lactone of N-acyl homoserine lactone (AHLs). Heterologous gene expression in E. coli confirmed its functions for hydrolysis of AHLs, and the gene was designated as aidP (autoinducer degrading gene from Planococcus sp.). The low temperature activity of this enzyme suggested that it is a novel and uncharacterized class of AHL lactonase. This study is the first report on QQ activity of bacteria isolated from the polar regions.

Application of quorum quenching bacteria entrapping sheets to enhance biofouling control in a membrane bioreactor with a hollow fiber module

Quorum quenching (QQ) has been recognized as an innovative approach for biofouling control in membrane bioreactors (MBRs). Recently, QQ bacteria entrapping beads (QQ-beads) have been developed and verified to have excellent anti-biofouling potential in a pilot-scale MBR with flat-sheet membrane modules. In this study, considering the dense structure of hollow fiber (HF) bundles into which QQ-beads can hardly penetrate, QQ bacteria entrapping sheets (QQ-sheets) were developed as a new shape of QQ-media suitable for MBRs with HF modules. In a lab-scale MBR, QQ-sheets with a thickness of 0.5mm exhibited a greater physical washing effect than did QQ-beads with a diameter of 3.5mm because the former collided with membrane surfaces at the inner as well as the outer part of HF bundles, whereas the latter only made contact with the outer part. Moreover, QQ-sheets showed 2.5-fold greater biological QQ activity than did QQ-beads due to their greater total surface area at a fixed volume of QQ-media. These results suggest high potential for QQ-sheets to be used in MBRs with HF modules.

Enhancing the Physical Properties and Lifespan of Bacterial Quorum Quenching Media through Combination of Ionic Cross-Linking and Dehydration.

Quorum quenching (QQ) bacteria entrapped in a polymeric composite hydrogel (QQ medium) have been successfully applied in membrane bioreactors (MBRs) for effective biofouling control. However, in order to bring QQ technology closer to practice, the physical strength and lifetime of QQ media should be improved. In this study, enforcement of physical strength, as well as an extension of the lifetime of a previously reported QQ bacteria entrapping hollow cylinder (QQ-HC), was sought by adding a dehydration procedure following the cross-linking of the polymeric hydrogel by inorganic compounds like Ca2+ and boric acid. Such prepared medium demonstrated enhanced physical strength possibly through an increased degree of physical cross-linking. As a result, a longer lifetime of QQ-HCs was confirmed, which led to improved biofouling mitigation performance of QQ-HC in an MBR. Furthermore, QQ-HCs stored under dehydrated condition showed higher QQ activity when the storage time lasted more than 90 days owing to enhanced cell viability. In addition, the dormant QQ activity after the dehydration step could be easily restored through reactivation with real wastewater, and the reduced weight of the dehydrated media is expected to make handling and transportation of QQ media highly convenient and economical in practice.

Attenuation of Quorum Sensing Regulated Virulence of Pectobacterium carotovorum subsp. carotovorum through an AHL Lactonase Produced by Lysinibacillus sp. Gs50.

Quorum sensing (QS) is a mechanism in which Gram negative bacterial pathogens sense their population density through acyl homoserine lactones (AHLs) and regulate the expression of virulence factors. Enzymatic degradation of AHLs by lactonases, known as quorum quenching (QQ), is thus a potential strategy for attenuating QS regulated bacterial infections. We characterised the QQ activity of soil isolate Lysinibacillus sp. Gs50 and explored its potential for controlling bacterial soft rot of crop plants. Lysinibacillus sp. Gs50 inactivated AHL, which could be restored upon acidification, suggested that inactivation was due to the lactone ring hydrolysis of AHL. Heterologous expression of cloned gene for putative hydrolase (792 bp) designated adeH from Lysinibacillus sp. Gs50 produced a ~29 kDa protein which degraded AHLs of varying chain length. Mass spectrometry analysis of AdeH enzymatic reaction product revealed that AdeH hydrolyses the lactone ring of AHL and hence is an AHL lactonase. Multiple sequence alignment of the amino acid sequence of AdeH showed that it belongs to the metallo- β- lactamase superfamily, has a conserved “HXHXDH” motif typical of AHL lactonases. KM for AdeH for C6HSL was found to be 3.089 μM and the specific activity was 0.8 picomol min-1μg-1. AdeH has not so far been reported from any Lysinibacillus sp. and has less than 40% identity with known AHL lactonases. Finally we found that Lysinibacillus sp. Gs50 can degrade AHL produced by Pectobacterium carotovorum subsp. carotovorum (Pcc), a common cause of soft rot. This QQ activity causes a decrease in production of plant cell wall degrading enzymes of Pcc and attenuates symptoms of soft rot in experimental infection of potato, carrot and cucumber. Our results demonstrate the potential of Lysinibacillus sp. Gs50 as a preventive and curative biocontrol agent.

Engineering of a thermostable esterase Est816 to improve its quorum-quenching activity and the underlying structural basis.

N-acyl-homoserine lactones (AHLs) are small diffusible molecules called autoinducers that mediate cell-to-cell communications. Enzymatic degradation of AHLs is a promising bio-control strategy known as quorum-quenching. To improve the quorum-quenching activity of a thermostable esterase Est816, which had been previously cloned, we have engineered the enzyme by random mutagenesis. One of the mutants M2 with double amino acid substitutions (A216V/K238N) showed 3-fold improvement on catalytic efficiency. Based on the crystal structure determined at 2.64 Å, rational design of M2 was conducted, giving rise to the mutant M3 (A216V/K238N/L122A). The kcat/KM value of the mutant M3 is 21.6-fold higher than that of Est816. Furthermore, activity assays demonstrated that M3 reached 99% conversion of 10-μM N-octanoyl-DL-homoserine lactone (C8-HSL) to N-octanoyl- DL-homoserine (C8-Hse) in 20 min, in contrast to the 8 h required by wild type Est816. The dramatic activity enhancement may be attributed to the increased hydrophobic interactions with the lactone ring by the mutation A216V, and the reduced steric clashes between the long side chain of L122 and the aliphatic tail of HSL by the mutation L122A, according to the crystal structure. This study sheds lights on the activity-structure relationship of AHL-lactonases, and may provide useful information in engineering AHL-degrading enzymes.

Effect of the Shape and Size of Quorum-Quenching Media on Biofouling Control in Membrane Bioreactors for Wastewater Treatment.

Recently, spherical beads entrapping quorum quenching (QQ) bacteria have been reported as effective moving QQ-media for biofouling control in MBRs for wastewater treatment owing to their combined effects of biological (i.e., quorum quenching) and physical washing. Taking into account both the mass transfer of signal molecules through the QQ-medium and collision efficiencies of the QQ-medium against the filtration membranes in a bioreactor, a cylindrical medium (QQ-cylinder) was developed as a new shape of moving QQ-medium. The QQ-cylinders were compared with previous QQ-beads in terms of the QQ activity and the physical washing effect under identical loading volumes of each medium in batch tests. It was found that the QQ activity of a QQ-medium was highly dependent on its specific surface area, regardless of the shape of the medium. In contrast, the physical washing effect of a QQ-medium was greatly affected by its geometric structure. The enhanced anti-biofouling property of the QQ-cylinders relative to QQ-beads was confirmed in a continuous laboratory-scale MBR with a flat-sheet membrane module.