The quorum-quenching lactonase from Bacillus thuringiensis is a metalloprotein

Lactonases from Bacillus species hydrolyze the N-acylhomoserine lactone (AHL) signaling molecules used in quorum-sensing pathways of many Gram-negative bacteria, including Pseudomonas aeruginosa and Erwinia carotovora, both significant pathogens. Because of sequence similarity, these AHL lactonases have been assigned to the metallo-beta-lactamase superfamily of proteins, which includes metalloenzymes of diverse activity, mechanism, and metal content. However, a recent study claims that AHL lactonase from Bacillus sp. 240B1 is not a metalloprotein [Wang, L. H., et al. (2004) J. Biol. Chem. 279, 13645]. Here, the gene for an AHL lactonase from Bacillus thuringiensis is cloned, and the protein is expressed, purified, and found to bind 2 equiv of zinc. The metal-bound form of AHL lactonase catalyzes the hydrolysis of N-hexanoyl-(S)-homoserine lactone but not the (R) enantiomer. Removal of both zinc ions results in loss of activity, and reconstitution with zinc restores activity, indicating the importance of metal ions for catalytic activity. Metal content, sequence alignments, and X-ray absorption spectroscopy of the zinc-containing lactonase all support a proposed dinuclear zinc binding site similar to that found in glyoxalase II.     

Mechanism of the quorum-quenching lactonase (AiiA) from Bacillus thuringiensis. 2. Substrate modeling and active site mutations

The N-acyl- l-homoserine lactone hydrolases (AHL lactonases) have attracted considerable attention because of their ability to quench AHL-mediated quorum-sensing pathways in Gram-negative bacteria and because of their relation to other enzymes in the metallo-beta-lactamase superfamily. To elucidate the detailed catalytic mechanism of AHL lactonase, mutations are made on residues that presumably contribute to substrate binding and catalysis. Steady-state kinetic studies are carried out on both the wild-type and mutant enzymes using a spectrum of substrates. Two mutations, Y194F and D108N, present significant effects on the overall catalysis. On the basis of a high-resolution structural model of the enzyme-product complex, a hybrid quantum mechanical/molecular mechanical method is used to model the substrate binding orientation and to probe the effect of the Y194F mutation. Combining all experimental and computational results, we propose a detailed mechanism for the ring-opening hydrolysis of AHL substrates as catalyzed by the AHL lactonase from Bacillus thuringiensis. Several features of the mechanism that are also found in related enzymes are discussed and may help to define an evolutionary thread that connects the hydrolytic enzymes of this mechanistically diverse superfamily.     

Structure and specificity of a quorum-quenching lactonase (AiiB) from Agrobacterium tumefaciens

N-Acyl-l-homoserine lactone (AHL) mediated quorum-sensing regulates virulence factor production in a variety of Gram-negative bacteria. Proteins capable of degrading these autoinducers have been called "quorum-quenching" enzymes, can block many quorum-sensing dependent phenotypes, and represent potentially useful reagents for clinical, agricultural, and industrial applications. The most characterized quorum-quenching enzymes to date are the AHL lactonases, which are metalloproteins that belong to the metallo-beta-lactamase superfamily. Here, we report the cloning, heterologous expression, purification, metal content, substrate specificity, and three-dimensional structure of AiiB, an AHL lactonase from Agrobacterium tumefaciens. Much like a homologous AHL lactonase from Bacillus thuringiensis, AiiB appears to be a metal-dependent AHL lactonase with broad specificity. A phosphate dianion is bound to the dinuclear zinc site and the active-site structure suggests specific mechanistic roles for an active site tyrosine and aspartate. To our knowledge, this is the second representative structure of an AHL lactonase and the first of an AHL lactonase from a microorganism that also produces AHL autoinducers. This work should help elucidate the hydrolytic ring-opening mechanism of this family of enzymes and also facilitate the design of more effective quorum-quenching catalysts.     

Thiolactone modulators of quorum sensing revealed through library design and screening

Quorum sensing (QS) is a process by which bacteria use small molecules or peptidic signals to assess their local population densities. At sufficiently high density, bacteria can alter gene expression levels to regulate group behaviors involved in a range of important and diverse phenotypes, including virulence factor production, biofilm formation, root nodulation, and bioluminescence. Gram-negative bacteria most commonly use N-acylated l-homoserine lactones (AHLs) as their QS signals. The AHL lactone ring is hydrolyzed relatively rapidly at biological pH, and the ring-opened product is QS inactive. We seek to identify AHL analogues with heightened hydrolytic stability, and thereby potentially heightened activity, for use as non-native modulators of bacterial QS. As part of this effort, we probed the utility of thiolactone analogues in the current study as QS agonists and antagonists in Gram-negative bacteria. A focused library of thiolactone analogs was designed and rapidly synthesized in solution. We examined the activity of the library as agonists and antagonists of LuxR-type QS receptors in Pseudomonas aeruginosa (LasR), Vibrio fischeri (LuxR), and Agrobacterium tumefaciens (TraR) using bacterial reporter strains. The thiolactone library contained several highly active compounds, including some of the most active LuxR inhibitors and the most active synthetic TraR agonist reported to date. Analysis of a representative thiolactone analog revealed that its hydrolysis half-life was almost double that of its parent AHL in bacterial growth medium.     

Orally administered thermostable N-acyl homoserine lactonase from Bacillus sp. strain AI96 attenuates Aeromonas hydrophila infection in zebrafish

N-Acylated homoserine lactone (AHL) lactonases are capable of degrading signal molecules involved in bacterial quorum sensing and therefore represent a new approach to control bacterial infection. Here a gene responsible for the AHL lactonase activity of Bacillus sp. strain AI96, 753 bp in length, was cloned and then expressed in Escherichia coli. The deduced amino acid sequence of Bacillus sp. AI96 AiiA (AiiA(AI96)) is most similar to those of other Bacillus sp. AHL lactonases (~80% sequence identity) and was consequently categorized as a member of the metallo-β-lactamase superfamily. AiiA(AI96) maintains ~100% of its activity at 10°C to 40°C at pH 8.0, and it is very stable at 70°C at pH 8.0 for at least 1 h; no other Bacillus AHL lactonase has been found to be stable under these conditions. AiiA(AI96) resists digestion by proteases and carp intestinal juice, and it has broad-spectrum substrate specificity. The supplementation of AiiA(AI96) into fish feed by oral administration significantly attenuated Aeromonas hydrophila infection in zebrafish. This is the first report of the oral administration of an AHL lactonase for the efficient control of A. hydrophila.     

Isolation and characterization of N-acylhomoserine lactonase from the thermophilic bacterium, Geobacillus caldoxylosilyticus YS-8

Geobacillus caldoxylosilyticus YS-8, which was isolated from volcanic soil in Indonesia, was found to degrade various N-acylhomoserine lactones (AHLs) with different lengths and acyl side-chain substitutions over a wide temperature range of 30-70 °C. The purified AHL-degrading enzyme showed a single band of 32 kDa, and its N-terminal amino acid sequence was determined to be ANVIKARPKLYVMDN, tentatively suggesting that the AHL-degrading enzyme was AHL lactonase. The AHL-degrading activity of the purified enzyme was maximized at pH 7.5 and 50 °C, and it retained about 50% of its activity even after a heat treatment at 60 °C for 3 h, exhibiting properties consistent with a thermostable enzyme. The mass spectrometric analysis demonstrated that the AHL-degrading enzyme catalyzed lactone ring opening of N-3-oxohexanoyl-L-homoserine lactone and N-hexanoyl-L-homoserine lactone by hydrolyzing the lactones and working as an AHL lactonase.     

MomL,a Novel Marine-Derived N-Acyl Homoserine Lactonase from Muricauda olearia

Gram-negative bacteria use N-acyl homoserine lactones (AHLs) as quorum sensing (QS) signaling molecules for interspecies communication, and AHL-dependent QS is related with virulence factor production in many bacterial pathogens. Quorum quenching, the enzymatic degradation of the signaling molecule, would attenuate virulence rather than kill the pathogens, and thereby reduce the potential for evolution of drug resistance. In a previous study, we showed that Muricauda olearia Th120, belonging to the class Flavobacteriia, has strong AHL degradative activity. In this study, an AHL lactonase (designated MomL), which could degrade both short- and long-chain AHLs with or without a substitution of oxo-group at the C-3 position, was identified from Th120. Liquid chromatography-mass spectrometry analysis demonstrated that MomL functions as an AHL lactonase catalyzing AHL degradation through lactone hydrolysis. MomL is an AHL lactonase belonging to the metallo-β-lactamase superfamily that harbors an N-terminal signal peptide. The overall catalytic efficiency of MomL for C₆-HSL is ∼2.9 × 10⁵ s⁻¹ M⁻¹. Metal analysis and site-directed mutagenesis showed that, compared to AiiA, MomL has a different metal-binding capability and requires the histidine and aspartic acid residues for activity, while it shares the “HXHXDH” motif with other AHL lactonases belonging to the metallo-β-lactamase superfamily. This suggests that MomL is a representative of a novel type of secretory AHL lactonase. Furthermore, MomL significantly attenuated the virulence of Pseudomonas aeruginosa in a Caenorhabditis elegans infection model, which suggests that MomL has the potential to be used as a therapeutic agent.     

Diversity and polymorphism in AHL-lactonase gene (aiiA) of Bacillus

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.     

Directed evolution of a thermostable quorum-quenching lactonase from the amidohydrolase superfamily

A thermostable quorum-quenching lactonase from Geobacillus kaustophilus HTA426 (GI: 56420041) was used as an initial template for in vitro directed evolution experiments. This enzyme belongs to the phosphotriesterase-like lactonase (PLL) group of enzymes within the amidohydrolase superfamily that hydrolyze N-acylhomoserine lactones (AHLs) that are involved in virulence pathways of quorum-sensing pathogenic bacteria. Here we have determined the N-butyryl-l-homoserine lactone-liganded structure of the catalytically inactive D266N mutant of this enzyme to a resolution of 1.6 Å. Using a tunable, bioluminescence-based quorum-quenching molecular circuit, the catalytic efficiency was enhanced, and the AHL substrate range increased through two point mutations on the loops at the C-terminal ends of the third and seventh β-strands. This E101N/R230I mutant had an increased value of k_cat/K_m of 72-fold toward 3-oxo-N-dodecanoyl-l-homoserine lactone. The evolved mutant also exhibited lactonase activity toward N-butyryl-l-homoserine lactone, an AHL that was previously not hydrolyzed by the wild-type enzyme. Both the purified wild-type and mutant enzymes contain a mixture of zinc and iron and are colored purple and brown, respectively, at high concentrations. The origin of this coloration is suggested to be because of a charge transfer complex involving the β-cation and Tyr-99 within the enzyme active site. Modulation of the charge transfer complex alters the lactonase activity of the mutant enzymes and is reflected in enzyme coloration changes. We attribute the observed enhancement in catalytic reactivity of the evolved enzyme to favorable modulations of the active site architecture toward productive geometries required for chemical catalysis.     

The quorum-quenching metallo-gamma-lactonase from Bacillus thuringiensis exhibits a leaving group thio effect

Lactone-hydrolyzing enzymes derived from some Bacillus species are capable of disrupting quorum sensing in bacteria that use N-acyl-l-homoserine lactones (AHLs) as intercellular signaling molecules. Despite the promise of these quorum-quenching enzymes as therapeutic and anti-biofouling agents, the ring opening mechanism and the role of metal ions in catalysis have not been elucidated. Labeling studies using (18)O, (2)H, and the AHL lactonase from Bacillus thuringiensis implicate an addition-elimination pathway for ring opening in which a solvent-derived oxygen is incorporated into the product carboxylate, identifying the alcohol as the leaving group. (1)H NMR is used to show that metal binding is required to maintain proper folding. A thio effect is measured for hydrolysis of N-hexanoyl-l-homoserine lactone and the corresponding thiolactone by AHL lactonase disubstituted with alternative metal ions, including Mn(2+), Co(2+), Zn(2+), and Cd(2+). The magnitude of the thio effect on k(cat) values and the thiophilicity of the metal ion substitutions vary in parallel and are consistent with a kinetically significant interaction between the leaving group and the active site metal center during turnover. X-ray absorption spectroscopy confirms that dicobalt substitution does not result in large structural perturbations at the active site. Finally, substitution of the dinuclear metal site with Cd(2+) results in a greatly enhanced catalyst that can hydrolyze AHLs 1600-24000-fold faster than other reported quorum-quenching enzymes.     

Degradation of N-acyl homoserine lactone quorum sensing signal molecules by forest root-associated fungi

A collection of mycorrhizal and nonmycorrhizal root-associated fungi coming from forest environments was screened for their ability to degrade N-acyl homoserine lactones (AHL) or to prevent AHL recognition by producing quorum sensing inhibitors (QSI). No production of QS-inhibitors or -activators was detected using the two biosensors Chromobacterium violaceum CV026 and Agrobacterium tumefaciens in the culture supernatant of these fungi. However, the ability to degrade C6- and 3O,C6-HSL was detected for three fungal isolates. Acidification assay revealed that the AHL were degraded by a lactonase activity for two of these isolates. These results demonstrated for the first time that the forest root-associated fungi are capable of degrading the AHL signal molecules.     

Identification of quorum-quenching N-acyl homoserine lactonases from Bacillus species

A range of gram-negative bacterial species use N-acyl homoserine lactone (AHL) molecules as quorum-sensing signals to regulate different biological functions, including production of virulence factors. AHL is also known as an autoinducer. An autoinducer inactivation gene, aiiA, coding for an AHL lactonase, was cloned from a bacterial isolate, Bacillus sp. strain 240B1. Here we report identification of more than 20 bacterial isolates capable of enzymatic inactivation of AHLs from different sources. Eight isolates showing strong AHL-inactivating enzyme activity were selected for a preliminary taxonomic analysis. Morphological phenotypes and 16S ribosomal DNA sequence analysis indicated that these isolates probably belong to the species Bacillus thuringiensis. Enzymatic analysis with known Bacillus strains confirmed that all of the strains of B. thuringiensis and the closely related species B. cereus and B. mycoides tested produced AHL-inactivating enzymes but B. fusiformis and B. sphaericus strains did not. Nine genes coding for AHL inactivation were cloned either by functional cloning or by a PCR procedure from selected bacterial isolates and strains. Sequence comparison of the gene products and motif analysis showed that the gene products belong to the same family of AHL lactonases.     

Oryza sativa rice plants contain molecules that activate different quorum-sensing N-acyl homoserine lactone biosensors and are sensitive to the specific AiiA lactonase

Gram-negative bacteria most often use N-acyl homoserine lactones (AHLs) as intercellular quorum-sensing signal molecules. In this study, it was demonstrated that rice plants contain AHL mimic molecules that are very sensitive to the highly specific AiiA lactonase enzyme and can activate three different AHL bacterial biosensors, indicating that the compounds have a homoserine lactone structure and could be AHLs. The possible source and biological significance of this finding are discussed.     

AidC,a Novel N-Acylhomoserine Lactonase from the Potato Root-Associated Cytophaga-Flavobacteria-Bacteroides (CFB) Group Bacterium Chryseobacterium sp. Strain StRB126

N-Acylhomoserine lactones (AHLs) are used as quorum-sensing (QS) signal molecules by many Gram-negative bacteria. We have reported that Chryseobacterium sp. strain StRB126, which was isolated from the root surface of potato, has AHL-degrading activity. In this study, we cloned and characterized the aidC gene from the genomic library of StRB126. AidC has AHL-degrading activity and shows homology to several metallo-β-lactamase proteins from Bacteroidetes, although not to any known AHL-degrading enzymes. Purified AidC, as a maltose-binding fusion protein, showed high degrading activity against all tested AHLs, whether short- or long-chain forms, with or without substitution at carbon 3. High-performance liquid chromatography (HPLC) analysis revealed that AidC functions as an AHL lactonase catalyzing AHL ring opening by hydrolyzing lactones. An assay to determine the effects of covalent and ionic bonding showed that Zn²⁺ is important to AidC activity both in vitro and in vivo. In addition, the aidC gene could also be PCR amplified from several other Chryseobacterium strains. In conclusion, this study indicated that the aidC gene, encoding a novel AHL lactonase, may be widespread throughout the genus Chryseobacterium. Our results extend the diversity and known bacterial hosts of AHL-degrading enzymes.     

A comparative study of non-native N-acyl l-homoserine lactone analogs in two Pseudomonas aeruginosa quorum sensing receptors that share a common native ligand yet inversely regulate virulence

Certain bacteria can coordinate group behaviors via a chemical communication system known as quorum sensing (QS). Gram-negative bacteria typically use N-acyl l-homoserine lactone (AHL) signals and their cognate intracellular LuxR-type receptors for QS. The opportunistic pathogen Pseudomonas aeruginosa has a relatively complex QS circuit in which two of its LuxR-type receptors, LasR and QscR, are activated by the same natural signal, N-(3-oxo)-dodecanoyl l-homoserine lactone. Intriguingly, once active, LasR activates virulence pathways in P. aeruginosa, while activated QscR can inactivate LasR and thus repress virulence. We have a limited understanding of the structural features of AHLs that engender either agonistic activity in both receptors or receptor-selective activity. Compounds with the latter activity profile could prove especially useful tools to tease out the roles of these two receptors in virulence regulation. A small collection of AHL analogs was assembled and screened in cell-based reporter assays for activity in both LasR and QscR. We identified several structural motifs that bias ligand activation towards each of the two receptors. These findings will inform the development of new synthetic ligands for LasR and QscR with improved potencies and selectivities.     

Restraining Erwinia virulence by expression of N-acyl homoserine lactonase gene pro3A-aiiA in Bacillus thuringiensis subsp leesis

To widen the biological control function of a genetically modified Bacillus thuringiensis subsp leesis strain BMB-005, an acyl homoserine lactonase (AHL lactonase) gene aiiA transcribed by the promoter of insecticidal crystal protein coding gene cry3A, was transformed into strain BMB-005. The amount of AHL lactonase protein produced by transformant BMB821A was 2.4-fold more than that produced by BMB-005. AHL-degradation assay showed that transformant BMB821A could degrade more AHLs molecules than the original strain BMB-005. The result of Erwinia carotovora pathogenicity test showed that the parental strain BMB-005 had no restraint of Erwinia infection, but the transformants exhibited strong restraint of E. carotovora infection on potato slices and cactus stems. Insecticidal bioassay against lepidopteran Spodoptera exigua showed that both strain BMB-005 and transformant BMB821A were toxic to S. exigua. The toxicity of transformant BMB821A (LC(50) was 3.8) was a little attenuated comparing with the toxicity of the original strain BMB-005 (LC(50) was 2.9). The B. thuringiensis strain BMB-005 has high toxicity against Helicoverpa armigera, Plutella xylostella, and S. exigua. This work provided new strategy for developing genetically engineered multi-functional B. thuringiensis strain that possesses insecticidal activity together with restraint of bacterial pathogenicity.     

Biofouling inhibition in MBR by Rhodococcus sp. BH4 isolated from real MBR plant

It has been reported that an indigenous quorum quenching bacterium, Rhodococcus sp. BH4, which was isolated from a real plant of membrane bioreactor (MBR) has promising potential to control biofouling in MBR. However, little is known about quorum quenching mechanisms by the strain BH4. In this study, various characteristics of strain BH4 were investigated to elucidate its behavior in more detail in the mixed liquor of MBR. The N-acyl homoserine lactone hydrolase (AHL–lactonase) gene of strain BH4 showed a high degree of identity to qsdA in Rhodococcus erythropolis W2. The LC-ESI-MS analysis of the degradation product by strain BH4 confirmed that it inactivated AHL activity by hydrolyzing the lactone bond of AHL. It degraded a wide range of N-acyl homoserine lactones (AHLs), but there was a large difference in the degradation rate of each AHL compared to other reported AHL–lactonase-producing strains belonging to Rhodococcus genus. Its quorum quenching activity was confirmed not only in the Luria-Bertani medium, but also in the synthetic wastewater. Furthermore, the amount of strain BH4 encapsulated in the vessel as well as the material of the vessel substantially affected the quorum quenching activity of strain BH4, which provides useful information, particularly for the biofouling control in a real MBR plant from an engineering point of view.     

Turnover of quorum sensing signal molecules modulates cross-kingdom signalling

N -acylhomoserine lactone (AHL) quorum-sensing molecules modulate the swimming behaviour of zoospores of the macroalga Ulva to facilitate the location of bacterial biofilms. Here we show that the intertidal surfaces colonized by Ulva are dominated by Alphaproteobacteria , particularly the Rhodobacteraceae family, and the Bacteroidetes family Flavobacteriaceae , and that this diverse assemblage both produces and degrades AHLs. N -acylhomoserine lactones could also be extracted from the surfaces of pebbles recovered from intertidal rock-pools. Bacteria representative of this assemblage were isolated and tested for the production and degradation of AHLs, and for their ability to modulate zoospore settlement at different biofilm densities. Of particular interest was a Shewanella sp. This strain produced three major AHLs (OC4, OC10 and OC12) in the late exponential phase, but the longer-chain AHLs were rapidly degraded in the stationary phase. Degradation occurred via both lactonase and amidase activity. A close relationship was found between AHL synthesis and Ulva zoospore settlement. The Shewanella isolate also interfered with AHL production by a Sulfitobacter isolate and its ability to enhance zoospore settlement in a polymicrobial biofilm. This influence on the attachment of Ulva zoospores suggests that AHL-degrading strains can affect bacterial community behaviour by interfering with quorum sensing between neighbouring bacteria. More importantly, these interactions may exert wider ecological effects across different kingdoms.     

Utilization of acyl-homoserine lactone quorum signals for growth by a soil pseudomonad and Pseudomonas aeruginosa PAO1

Acyl-homoserine lactones (AHLs) are employed by several Proteobacteria as quorum-sensing signals. Past studies have established that these compounds are subject to biochemical decay and can be used as growth nutrients. Here we describe the isolation of a soil bacterium, Pseudomonas strain PAI-A, that degrades 3-oxododecanoyl-homoserine lactone (3OC12HSL) and other long-acyl, but not short-acyl, AHLs as sole energy sources for growth. The small-subunit rRNA gene from strain PAI-A was 98.4% identical to that of Pseudomonas aeruginosa, but the soil isolate did not produce obvious pigments or AHLs or grow under denitrifying conditions or at 42 degrees C. The quorum-sensing bacterium P. aeruginosa, which produces both 3OC12HSL and C4HSL, was examined for the ability to utilize AHLs for growth. It did so with a specificity similar to that of strain PAI-A, i.e., degrading long-acyl but not short-acyl AHLs. In contrast to the growth observed with strain PAI-A, P. aeruginosa strain PAO1 growth on AHLs commenced only after extremely long lag phases. Liquid-chromatography-atmospheric pressure chemical ionization-mass spectrometry analyses indicate that strain PAO1 degrades long-acyl AHLs via an AHL acylase and a homoserine-generating HSL lactonase. A P. aeruginosa gene, pvdQ (PA2385), has previously been identified as being a homologue of the AHL acylase described as occurring in a Ralstonia species. Escherichia coli expressing pvdQ catalyzed the rapid inactivation of long-acyl AHLs and the release of HSL. P. aeruginosa engineered to constitutively express pvdQ did not accumulate its 3OC12HSL quorum signal when grown in rich media. However, pvdQ knockout mutants of P. aeruginosa were still able to grow by utilizing 3OC12HSL. To our knowledge, this is the first report of the degradation of AHLs by pseudomonads or other gamma-Proteobacteria, of AHL acylase activity in a quorum-sensing bacterium, of HSL lactonase activity in any bacterium, and of AHL degradation with specificity only towards AHLs with long side chains.