A single mutation in P450BM-3 enhances acyl homoserine lactone: acyl homoserine substrate binding selectivity nearly 250-fold

Quorum sensing is the process by which bacteria alter gene regulation in response to their population density. The enzymatic inactivation of quorum signals has shown promise for use in genetically modified organisms resistant to pathogens. We recently characterized the ability of a cytochrome P450, P450BM-3, to oxidize the quorum sensing signals known as acyl homoserine lactones. The oxidation of the acyl homoserine lactones reduced their activity as quorum signals. The enzyme also oxidized the inactive lactonolysis products, acyl homoserines. The enzyme showed similar binding affinity for the acyl homoserine lactones and acyl homoserines. The latter reaction may lead to problems when lactonases and the P450-dependent system are used in tandem, as oxidation of the acyl homoserines produced by lactonolysis in vivo may compete with acyl homoserine lactone oxidation by the cytochrome P450. We report here that a single mutation (R47S) in P450BM-3 is capable of increasing the acyl homoserine lactone: acyl homoserine substrate binding selectivity of the enzyme nearly 250-fold, reducing the potential for competition by acyl homoserines and significantly enhancing the potential for use of P450BM-3 as part of a pathogen resistance system in genetically modified crops.     

Identification and characterization of N-acylhomoserine lactone-acylase from the fish intestinal Shewanella sp. strain MIB015

N-Acylhomoserine lactones (AHLs) are used as quorum-sensing signal molecules by many gram-negative bacteria. We have reported that Shewanella sp. strain MIB015 degrades AHLs. In the present study, we cloned the aac gene from MIB015 by PCR with specific primers based on the aac gene in Shewanella oneidensis strain MR-1, which showed high homology with the known AHL-acylases. Escherichia coli expressing Aac showed high degrading activity of AHLs with long acyl chains. HPLC analysis revealed that Aac worked as AHL-acylase, which hydrolyzed the amide bond of AHL. In addition, expression of Aac in fish pathogen Vibrio anguillarum markedly reduced AHL production and biofilm formation. In conclusion, this study indicates that Aac might be effective in quenching quorum sensing of fish pathogens.     

A probable aculeacin A acylase from the Ralstonia solanacearum GMI1000 is N-acyl-homoserine lactone acylase with quorum-quenching activity

Background  

The infection and virulence functions of diverse plant and animal pathogens that possess quorum sensing systems are regulated by N-acylhomoserine lactones (AHLs) acting as signal molecules. AHL-acylase is a quorum quenching enzyme and degrades AHLs by removing the fatty acid side chain from the homoserine lactone ring of AHLs. This blocks AHL accumulation and pathogenic phenotypes in quorum sensing bacteria.     

Properties of the Folate-hydrolyzing Enzyme of Crithidia fasciculate as a Carboxypeptidase

N-Acylhomoserine lactones (AHLs) are used as quorum-sensing signal molecules by many gram-negative bacteria. We have reported that Shewanella sp. strain MIB015 degrades AHLs. In the present study, we cloned the aac gene from MIB015 by PCR with specific primers based on the aac gene in Shewanella oneidensis strain MR-1, which showed high homology with the known AHL-acylases. Escherichia coli expressing Aac showed high degrading activity of AHLs with long acyl chains. HPLC analysis revealed that Aac worked as AHL-acylase, which hydrolyzed the amide bond of AHL. In addition, expression of Aac in fish pathogen Vibrio anguillarum markedly reduced AHL production and biofilm formation. In conclusion, this study indicates that Aac might be effective in quenching quorum sensing of fish pathogens.     

Evidence for acyl homoserine lactone signal production in bacteria associated with marine sponges

We report for the first time the production of acyl homoserine lactones (AHLs) by bacteria associated with marine sponges. Given the involvement of AHLs in bacterial colonization of many higher organisms, we speculate that such quorum sensing signals could play a part in interactions between sponges and the dense bacterial communities living within them.     

食源假单胞菌群体感应信号分子的研究

从市售鲜鱼中分离的3株革兰氏阴性菌,经16S rDNA鉴定为假单胞菌属,该菌是一种导致食品腐败的重要腐败细菌。N-酰基-高丝氨酸内酯(AHLs)是革兰氏阴性菌群体感应(QS)系统中一类重要的信号分子,以密度依赖的方式调控某些生理性状的表达。利用AHLs检测菌株对3株假单胞菌进行检测发现,均产生AHLs类信号分子,且FML05-1和FML05-2至少产生两种AHLs,主要的信号分子是N-3-氧代-辛酰基-高丝氨酸内酯(N- 3-oxo-C_8-HSL)。同时对菌株FML05-2在生长过程中所产生的AHLs的活性变化进行研究,发现AHLs活性在菌体生长至12h时达到最大。首次对食源假单胞菌所产生的AHLs进行了研究,为以干扰腐败细菌群体感应为靶点的食品防腐保鲜策略提供研究基础。     

Structural basis and specificity of acyl-homoserine lactone signal production in bacterial quorum sensing

Synthesis and detection of acyl-homoserine lactones (AHLs) enables many gram-negative bacteria to engage in quorum sensing, an intercellular signaling mechanism that activates differentiation to virulent and biofilm lifestyles. The AHL synthases catalyze acylation of S-adenosyl-L-methionine by acyl-acyl carrier protein and lactonization of the methionine moiety to give AHLs. The crystal structure of the AHL synthase, EsaI, determined at 1.8 A resolution, reveals a remarkable structural similarity to the N-acetyltransferases and defines a common phosphopantetheine binding fold as the catalytic core. Critical residues responsible for catalysis and acyl chain specificity have been identified from a modeled substrate complex and verified through functional analysis in vivo. A mechanism for the N-acylation of S-adenosyl-L-methionine by 3-oxo-hexanoyl-acyl carrier protein is proposed.     

AmiE,a Novel N-Acylhomoserine Lactone Acylase Belonging to the Amidase Family,from the Activated-Sludge Isolate Acinetobacter sp. Strain Ooi24

Many Gram-negative bacteria use N-acyl-l-homoserine lactones (AHLs) as quorum-sensing signal molecules. We have reported that Acinetobacter strains isolated from activated sludge have AHL-degrading activity. In this study, we cloned the amiE gene as an AHL-degradative gene from the genomic library of Acinetobacter sp. strain Ooi24. High-performance liquid chromatography analysis revealed that AmiE functions as an AHL acylase, which hydrolyzes the amide bond of AHL. AmiE showed a high level of degrading activity against AHLs with long acyl chains but no activity against AHLs with acyl chains shorter than eight carbons. AmiE showed homology with a member of the amidases (EC 3.5.1.4) but not with any known AHL acylase enzymes. An amino acid sequence of AmiE from Ooi24 showed greater than 99% identities with uncharacterized proteins from Acinetobacter ursingii CIP 107286 and Acinetobacter sp. strain CIP 102129, but it was not found in the draft or complete genome sequences of other Acinetobacter strains. The presence of transposase-like genes around the amiE genes of these three Acinetobacter strains suggests that amiE is transferred by a putative transposon. Furthermore, the expression of AmiE in Pseudomonas aeruginosa PAO1 reduced AHL accumulation and elastase activity, which were regulated by AHL-mediated quorum sensing.     

Specificity of acyl-homoserine lactone synthases examined by mass spectrometry

Many gram-negative bacteria produce a specific set of N-acyl-L-homoserine-lactone (AHL) signaling molecules for the purpose of quorum sensing, which is a means of regulating coordinated gene expression in a cell-density-dependent manner. AHLs are produced from acylated acyl-carrier protein (acyl-ACP) and S-adenosyl-L-methionine by the AHL synthase enzyme. The appearance of specific AHLs is due in large part to the intrinsic specificity of the enzyme for subsets of acyl-ACP substrates. Structural studies of the Pantoea stewartii enzyme EsaI and AHL-sensitive bioassays revealed that threonine 140 in the acyl chain binding pocket directs the enzyme toward production of 3-oxo-homoserine lactones. Mass spectrometry was used to examine the range of AHL molecular species produced by AHL synthases under a variety of conditions. An AHL selective normal-phase chromatographic purification with addition of a deuterated AHL internal standard was followed by reverse-phase liquid chromatography-tandem mass spectrometry in order to obtain estimates of the relative amounts of different AHLs from biological samples. The AHLs produced by wild-type and engineered EsaI and LasI AHL synthases show that intrinsic specificity and different cellular conditions influence the production of AHLs. The threonine at position 140 in EsaI is important for the preference for 3-oxo-acyl-ACPs, but the role of the equivalent threonine in LasI is less clear. In addition, LasI expressed in Escherichia coli produces a high proportion of unusual AHLs with acyl chains consisting of an odd number of carbons. Furthermore, these studies offer additional methods that will be useful for surveying and quantitating AHLs from different sources.     

N-acylhomoserine lactonase producing Rhodococcus spp. with different AHL-degrading activities

N-acylhomoserine lactones (AHLs) are conserved signal molecules that control diverse biological activities in quorum sensing system of Gram-negative bacteria. Recently, several soil bacteria were found to degrade AHLs, thereby interfering with the quorum sensing system. Previously, Rhodococcus erythropolis W2 was reported to degrade AHLs by both oxido-reductase and AHL-acylase. In the present study, two AHL-utilizing bacteria, strains LS31 and PI33, were isolated and identified as the genus Rhodococcus. They exhibited different AHL-utilization abilities: Rhodococcus sp. strain LS31 rapidly degraded a wide range of AHLs, including N-3-oxo-hexanoyl-l-homoserine lactone (OHHL), whereas Rhodococcus sp. strain PI33 showed relatively less activity towards 3-oxo substituents. Coculture of strain LS31 with Erwinia carotovora effectively reduced the amount of OHHL and pectate lyase activity, compared with coculture of strain PI33 with E. carotovora. A mass spectrometry analysis indicated that both strains hydrolyzed the lactone ring of AHL to generate acylhomoserine, suggesting that AHL-lactonases (AHLases) from the two Rhodococcus strains are involved in the degradation of AHL, in contrast to R. erythropolis W2. To the best of our knowledge, this is the first report on AHLases of Rhodococcus spp.     

Acyl-homoserine lactone acylase from Ralstonia strain XJ12B represents a novel and potent class of quorum-quenching enzymes

N-acylhomoserine lactones (AHLs) are used as signal molecules by many quorum-sensing Proteobacteria. Diverse plant and animal pathogens use AHLs to regulate infection and virulence functions. These signals are subject to biological inactivation by AHL-lactonases and AHL-acylases. Previously, little was known about the molecular details underlying the latter mechanism. An AHL signal-inactivating bacterium, identified as a Ralstonia sp., was isolated from a mixed-species biofilm. The signal inactivation encoding gene from this organism, which we call aiiD, was cloned and successfully expressed in Escherichia coli and inactivated three AHLs tested. The predicted 794-amino-acid polypeptide was most similar to the aculeacin A acylase (AAC) from Actinoplanes utahensis and also shared significant similarities with cephalosporin acylases and other N-terminal (Ntn) hydrolases. However, the most similar homologues of AiiD are deduced proteins of undemonstrated function from available Ralstonia, Deinococcus and Pseudomonas genomes. LC-MS analyses demonstrated that AiiD hydrolyses the AHL amide, releasing homoserine lactone and the corresponding fatty acid. Expression of AiiD in Pseudomonas aeruginosa PAO1 quenched quorum sensing by this bacterium, decreasing its ability to swarm, produce elastase and pyocyanin and to paralyze nematodes. Thus, AHL-acylases have fundamental implications and hold biotechnological promise in quenching quorum sensing.     

Acyl-homoserine lactones modulate the settlement rate of zoospores of the marine alga Ulva intestinalis via a novel chemokinetic mechanism

Bacteria utilize quorum sensing to regulate the expression of cell density-dependant phenotypes such as biofilm formation and virulence. Zoospores of the marine alga Ulva intestinalis exploit the acyl-homoserine lactone (AHL) quorum sensing system to identify bacterial biofilms for preferential settlement. Here, we demonstrate that AHLs act as strong chemoattractants for Ulva zoospores. Chemoattraction does not involve a chemotactic orientation towards the AHL source. Instead, it occurs through a chemokinesis in which zoospore swimming speed is rapidly decreased in the presence of AHLs. The chemoresponse to AHLs was dependant on the nature of the acyl side chain, with N-(3-oxododecanoyl)-homoserine lactone (30-C12-HSL) being the most effective signal molecule. Mean zoospore swimming speed decreased more rapidly over wild-type biofilms of the marine bacteria Vibrio anguillarum relative to biofilms of the vanM mutant, in which AHL synthesis is disrupted. These data implicate a role for AHL-mediated chemokinesis in the location and preferential settlement of Ulva zoospores on marine bacterial assemblages. Exposure to AHLs did not inhibit the negative phototaxis of Ulva zoospores, indicating that chemoattraction to bacterial biofilms does not preclude the response to a light stimulus in substrate location.     

碳源及温度对食源假单胞菌群体感应信号分子产生的影响

许多革兰氏阴性菌通过产生N-酰基-高丝氨酸内酯（AHLs）类信号分子来调控某些性状的表达,即群体感应（quorum sensing）。假单胞菌是一种导致食品腐败的重要腐败细菌,也产生AHLs。本文研究了不同温度及碳源对食源假单胞菌AHLs产生的影响。结果表明,该假单胞菌在25℃条件下,产生两种AHL信号分子,而在4℃条件下,所产生的短链AHL分子消失,主要产生长链AHL分子。而且在不同碳源（葡萄糖,果糖,木糖,麦芽糖等）的培养基中生长,所产生的AHLs分子种类也不同。同时发现当pH>7.5时,AHLs的稳定性下降。由此得出,在不同的环境条件(碳源及温度)下假单胞菌所产生的AHLs种类不同。为进一步研究群体感应现象在食品腐败中的作用以及开发基于干扰腐败菌群体感应的新型食品防腐技术提供研究基础。     

Effector-stimulated single molecule protein-DNA interactions of a quorum-sensing system in Sinorhizobium meliloti

Intercellular communication by means of small signal molecules coordinates gene expression among bacteria. This population density-dependent regulation is known as quorum sensing. The symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti Rm1021 possesses the Sin quorum sensing system based on N-acyl homoserine lactones (AHL) as signal molecules. Here, we demonstrate that the LuxR-type regulator ExpR binds specifically to a target sequence in the sinRI locus in the presence of different AHLs with acyl side chains from 8 to 20 carbons. Dynamic force spectroscopy based on the atomic force microscope provided detailed information about the molecular mechanism of binding upon activation by six different AHLs. These single molecule experiments revealed that the mean lifetime of the bound protein-DNA complex varies depending on the specific effector molecule. The small differences between individual AHLs also had a pronounced influence on the structure of protein-DNA interaction: The reaction length of dissociation varied from 2.6 to 5.8 A. In addition, dynamic force spectroscopy experiments indicate that N-heptanoyl-DL-homoserine lactone binds to ExpR but is not able to stimulate protein-DNA interaction.     

Development of a sensitive bioassay method for quorum sensing inhibitor screening using a recombinantAgrobacterium tumefaciens

Acylhomoserine lactones (AHLs) are known to be the triggering molecules in the quorum sensing mechanism of many gram-negative bacteria. In order to detect AHL inhibitors that are potential biofilm inhibitors, a convenient and sensitive bioassay was developed based on the β-galactosidase activity (β-GAL) of a recombinantAgrobacterium tumefaciens strain. A series of commercially available AHLs were tested for inducing β-GAL at varying concentrations in agar-plate and liquid cultures of the reporter strain. All AHLs tested exhibited a concentration-dependent induction, and octanoyl homoserine lactone (OHL) showed the highest sensitivity with a detection limit of 0.1 nM in the liquid culture assay. When fimbrolide, a known quorum sensing inhibitor, was added, induction of β-GAL by OHL was repressed. The repression at a constant OHL concentration was dependent on the fimbrolide concentration with the detection limit below 1 ppm, indicating that this assay is a sensitive method for screening AHL inhibitors.     

Evidence for Acyl Homoserine Lactone Signal Production in Bacteria Associated with Marine Sponges

We report for the first time the production of acyl homoserine lactones (AHLs) by bacteria associated with marine sponges. Given the involvement of AHLs in bacterial colonization of many higher organisms, we speculate that such quorum sensing signals could play a part in interactions between sponges and the dense bacterial communities living within them.     

Pseudomonas corrugata contains a conserved N-acyl homoserine lactone quorum sensing system; its role in tomato pathogenicity and tobacco hypersensitivity response

Pseudomonas corrugata is a phytopathogenic bacterium, causal agent of tomato pith necrosis, yet it is an ubiquitous bacterium that is part of the microbial community in the soil and in the rhizosphere of different plant species. Although it is a very heterogeneous species, all the strains tested were able to produce short chain acyl homoserine lactone (AHL) quorum sensing signal molecules. The main AHL produced was N-hexanoyl-L-homoserine lactone (C(6)-AHL). An AHL quorum sensing system, designated PcoI/PcoR, was identified and characterized. The role of the quorum sensing system in the expression of a variety of traits was evaluated. Inactivation of pcoI abolished the production of AHLs. The pcoR mutant, but not the pcoI mutant, was impaired in swarming, unable to cause a hypersensitivity response on tobacco and resulted in a reduced tomato pith necrosis phenotype. The pcoI mutant showed a reduced antimicrobial activity against various fungi and bacteria when assayed on King's B medium. These results demonstrate that the AHL quorum sensing in Ps. corrugata regulates traits that contribute to virulence, antimicrobial activity and fitness. This is the first report of genes of Ps. corrugata involved in the disease development and biological control activity.