scholarly journals Identification of Extracellular N-Acylhomoserine Lactone Acylase from a Streptomyces sp. and Its Application to Quorum Quenching

2005 ◽  
Vol 71 (5) ◽  
pp. 2632-2641 ◽  
Author(s):  
Sun-Yang Park ◽  
Hye-Ok Kang ◽  
Hak-Sun Jang ◽  
Jung-Kee Lee ◽  
Bon-Tag Koo ◽  
...  
Keyword(s):  
Virulence Factors ◽  
Pathogenic Bacteria ◽  
Quorum Quenching ◽  
Homoserine Lactone ◽  
Amino Acid Sequences ◽  
Amide Bond ◽  
Mass Spectrometry Analysis ◽  
Penicillin G ◽  
Spectrometry Analysis ◽  
Acyl Chains

ABSTRACT N-Acylhomoserine lactones (AHLs) play an important role in regulating virulence factors in pathogenic bacteria. Recently, the enzymatic inactivation of AHLs, which can be used as antibacterial targets, has been identified in several soil bacteria. In this study, strain M664, identified as a Streptomyces sp., was found to secrete an AHL-degrading enzyme into a culture medium. The ahlM gene for AHL degradation from Streptomyces sp. strain M664 was cloned, expressed heterologously in Streptomyces lividans, and purified. The enzyme was found to be a heterodimeric protein with subunits of approximately 60 kDa and 23 kDa. A comparison of AhlM with known AHL-acylases, Ralstonia strain XJ12B AiiD and Pseudomonas aeruginosa PAO1 PvdQ, revealed 35% and 32% identities in the deduced amino acid sequences, respectively. However, AhlM was most similar to the cyclic lipopeptide acylase from Streptomyces sp. strain FERM BP-5809, exhibiting 93% identity. A mass spectrometry analysis demonstrated that AhlM hydrolyzed the amide bond of AHL, releasing homoserine lactone. AhlM exhibited a higher deacylation activity toward AHLs with long acyl chains rather than short acyl chains. Interestingly, AhlM was also found to be capable of degrading penicillin G by deacylation, showing that AhlM has a broad substrate specificity. The addition of AhlM to the growth medium reduced the accumulation of AHLs and decreased the production of virulence factors, including elastase, total protease, and LasA, in P. aeruginosa. Accordingly, these results suggest that AHL-acylase, AhlM could be effectively applied to the control of AHL-mediated pathogenicity.

AhlD, an N-acylhomoserine lactonase in Arthrobacter sp., and predicted homologues in other bacteria

Microbiology ◽  
2003 ◽  
Vol 149 (6) ◽  
pp. 1541-1550 ◽  
Author(s):  
Sun-Yang Park ◽  
Sang Jun Lee ◽  
Tae-Kwang Oh ◽  
Jong-Won Oh ◽  
Bon-Tag Koo ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Pathogenic Bacteria ◽  
Erwinia Carotovora ◽  
Homoserine Lactone ◽  
Amino Acid Sequences ◽  
Mass Spectrometry Analysis ◽  
Signal Molecules ◽  
Genome Database ◽  
Spectrometry Analysis ◽  
A Genome

Quorum sensing is a signalling mechanism that controls diverse biological functions, including virulence, via N-acylhomoserine lactone (AHL) signal molecules in Gram-negative bacteria. With the aim of isolating strains or enzymes capable of blocking quorum sensing by inactivating AHL, bacteria were screened for AHL degradation by their ability to utilize N-3-oxohexanoyl-l-homoserine lactone (OHHL) as the sole carbon source. Among four isolates, strain IBN110, identified as Arthrobacter sp., was found to grow rapidly on OHHL, and to degrade various AHLs with different lengths and acyl side-chain substitutions. Co-culture of Arthrobacter sp. IBN110 and the plant pathogen Erwinia carotovora significantly reduced both the AHL amount and pectate lyase activity in co-culture medium, suggesting the possibility of applying Arthrobacter sp. IBN110 in the control of AHL-producing pathogenic bacteria. The ahlD gene from Arthrobacter sp. IBN110 encoding the enzyme catalysing AHL degradation was cloned, and found to encode a protein of 273 amino acids. A mass spectrometry analysis showed that AhlD probably hydrolyses the lactone ring of N-3-hexanoyl-l-homoserine lactone, indicating that AhlD is an N-acylhomoserine lactonase (AHLase). A comparison of AhlD with other known AHL-degrading enzymes, Bacillus sp. 240B1 AiiA, a Bacillus thuringiensis subsp. kyushuensis AiiA homologue and Agrobacterium tumefaciens AttM, revealed 25, 26 and 21 % overall identities, respectively, in the deduced amino acid sequences. Although these identities were relatively low, the HXDH≈H≈D motif was conserved in all the AHLases, suggesting that this motif is essential for AHLase activity. From a genome database search based on the conserved motif, putative AhlD-like lactonase genes were found in several other bacteria, and AHL-degrading activities were observed in Klebsiella pneumoniae and Bacillus stearothermophilus. Furthermore, it was verified that ahlK, an ahlD homologue, encodes an AHL-degrading enzyme in K. pneumoniae. Accordingly, the current results suggest the possibility that AhlD-like AHLases could exist in many other micro-organisms.

scholarly journals Endophytic Actinomycetes: A Novel Source of Potential Acyl Homoserine Lactone Degrading Enzymes

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Surang Chankhamhaengdecha ◽  
Suphatra Hongvijit ◽  
Akkaraphol Srichaisupakit ◽  
Pattra Charnchai ◽  
Watanalai Panbangred
Keyword(s):  
Pathogenic Bacteria ◽  
Sequence Similarity ◽  
Soft Rot ◽  
Quorum Quenching ◽  
Homoserine Lactone ◽  
Amide Bond ◽  
Side Chain ◽  
Signal Molecules ◽  
Endophytic Actinomycetes

Several Gram-negative pathogenic bacteria employN-acyl-L-homoserine lactone (HSL) quorum sensing (QS) system to control their virulence traits. Degradation of acyl-HSL signal molecules by quorum quenching enzyme (QQE) results in a loss of pathogenicity in QS-dependent organisms. The QQE activity of actinomycetes in rhizospheric soil and inside plant tissue was explored in order to obtain novel strains with high HSL-degrading activity. Among 344 rhizospheric and 132 endophytic isolates, 127 (36.9%) and 68 (51.5%) of them, respectively, possessed the QQE activity. The highest HSL-degrading activity was at151.30±3.1 nmole/h/mL from an endophytic actinomycetes isolate, LPC029. The isolate was identified asStreptomycesbased on16S  rRNAgene sequence similarity. The QQE from LPC029 revealed HSL-acylase activity that was able to cleave an amide bond of acyl-side chain in HSL substrate as determined by HPLC. LPC029 HSL-acylase showed broad substrate specificity from C6- to C12-HSL in which C10HSL is the most favorable substrate for this enzyme. In anin vitropathogenicity assay, the partially purified HSL-acylase efficiently suppressed soft rot of potato caused byPectobacterium carotovorumssp.carotovorumas demonstrated. To our knowledge, this is the first report of HSL-acylase activity derived from an endophyticStreptomyces.

scholarly journals AidH, an Alpha/Beta-Hydrolase Fold Family Member from an Ochrobactrum sp. Strain, Is a Novel N-Acylhomoserine Lactonase

2010 ◽  
Vol 76 (15) ◽  
pp. 4933-4942 ◽  
Author(s):  
Gui-Ying Mei ◽  
Xiao-Xue Yan ◽  
Ali Turak ◽  
Zhao-Qing Luo ◽  
Li-Qun Zhang
Keyword(s):  
Pathogenic Bacteria ◽  
Divalent Cations ◽  
Hydrolytic Enzyme ◽  
Quorum Quenching ◽  
Homoserine Lactone ◽  
Great Promise ◽  
Fold Family ◽  
Hydrolase Fold ◽  
Enzymatic Inactivation ◽  
Alpha Beta

ABSTRACT N-Acylhomoserine lactones (AHLs) are signaling molecules in many quorum-sensing (QS) systems that regulate interactions between various pathogenic bacteria and their hosts. Quorum quenching by the enzymatic inactivation of AHLs holds great promise in preventing and treating infections, and several such enzymes have been reported. In this study, we report the characterization of a novel AHL-degrading protein from the soil bacterium Ochrobactrum sp. strain T63. This protein, termed AidH, shares no similarity with any of the known AHL degradases but is highly homologous with a hydrolytic enzyme from Ochrobactrum anthropi ATCC 49188 that contains the alpha/beta-hydrolase fold. By liquid chromatography-mass spectrometry (MS) analysis, we demonstrate that AidH functions as an AHL-lactonase that hydrolyzes the ester bond of the homoserine lactone ring of AHLs. Mutational analyses indicate that the G-X-Nuc-X-G motif or the histidine residue conserved among alpha/beta-hydrolases is critical for the activity of AidH. Furthermore, the AHL-inactivating activity of AidH requires Mn2+ but not several other tested divalent cations. We also showed that AidH significantly reduces biofilm formation by Pseudomonas fluorescens 2P24 and the pathogenicity of Pectobacterium carotovorum, indicating that this enzyme is able to effectively quench QS-dependent functions in these bacteria by degrading AHLs.

scholarly journals Quorum Quenching Properties and Probiotic Potentials of Intestinal Associated Bacteria in Asian Sea Bass Lates calcarifer

Marine Drugs ◽  
2019 ◽  
Vol 18 (1) ◽  
pp. 23 ◽  
Author(s):  
Reza Ghanei-Motlagh ◽  
Takavar Mohammadian ◽  
Darioush Gharibi ◽  
Simon Menanteau-Ledouble ◽  
Esmaeil Mahmoudi ◽  
...  
Keyword(s):  
High Capacity ◽  
Quorum Quenching ◽  
Homoserine Lactone ◽  
Amino Acid Sequences ◽  
Diffusion Method ◽  
Asian Sea Bass ◽  
Pathogenic Vibrio ◽  
Rdna Sequencing ◽  
Degrading Bacteria

Quorum quenching (QQ), the enzymatic degradation of N-acyl homoserine lactones (AHLs), has been suggested as a promising strategy to control bacterial diseases. In this study, 10 AHL-degrading bacteria isolated from the intestine of barramundi were identified by 16S rDNA sequencing. They were able to degrade both short and long-chain AHLs associated with several pathogenic Vibrio species (spp.) in fish, including N-[(RS)-3-Hydroxybutyryl]-l-homoserine lactone (3-oh-C4-HSL), N-Hexanoyl-l-homoserine lactone (C6-HSL), N-(β-Ketocaproyl)-l-homoserine lactone (3-oxo-C6-HSL), N-(3-Oxodecanoyl)-l-homoserine lactone (3-oxo-C10-HSL), N-(3-Oxotetradecanoyl)-l-homoserine lactone (3-oxo-C14-HSL). Five QQ isolates (QQIs) belonging to the Bacillus and Shewanella genera, showed high capacity to degrade both synthetic AHLs as well as natural AHLs produced by Vibrio harveyi and Vibrio alginolyticus using the well-diffusion method and thin-layer chromatography (TLC). The genes responsible for QQ activity, including aiiA, ytnP, and aaC were also detected. Analysis of the amino acid sequences from the predicted lactonases revealed the presence of the conserved motif HxHxDH. The selected isolates were further characterized in terms of their probiotic potentials in vitro. Based on our scoring system, Bacillus thuringiensis QQ1 and Bacillus cereus QQ2 exhibited suitable probiotic characteristics, including the production of spore and exoenzymes, resistance to bile salts and pH, high potential to adhere on mucus, appropriate growth abilities, safety to barramundi, and sensitivity to antibiotics. These isolates, therefore, constitute new QQ probiotics that could be used to control vibriosis in Lates calcalifer.

scholarly journals Novel Bifunctional α-l-Arabinofuranosidase/Xylobiohydrolase (ABF3) from Penicillium purpurogenum

2010 ◽  
Vol 76 (15) ◽  
pp. 5247-5253 ◽  
Author(s):  
Mar�a Cristina Ravanal ◽  
Eduardo Callegari ◽  
Jaime Eyzaguirre
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Glycosyl Hydrolase ◽  
Biochemical Properties ◽  
Amino Acid Sequences ◽  
Mass Spectrometry Analysis ◽  
Carbohydrate Binding ◽  
Penicillium Purpurogenum ◽  
Spectrometry Analysis ◽  
Amino Terminal

ABSTRACT The soft rot fungus Penicillium purpurogenum grows on a variety of natural substrates and secretes various isoforms of xylanolytic enzymes, including three arabinofuranosidases. This work describes the biochemical properties as well as the nucleotide and amino acid sequences of arabinofuranosidase 3 (ABF3). This enzyme has been purified to homogeneity. It is a glycosylated monomer with a molecular weight of 50,700 and can bind cellulose. The enzyme is active with p-nitrophenyl α-l-arabinofuranoside and p-nitrophenyl β-d-xylopyranoside with a Km of 0.65 mM and 12 mM, respectively. The enzyme is active on xylooligosaccharides, yielding products of shorter length, including xylose. However, it does not hydrolyze arabinooligosaccharides. When assayed with polymeric substrates, little arabinose is liberated from arabinan and debranched arabinan; however, it hydrolyzes arabinose and releases xylooligosaccharides from arabinoxylan. Sequencing both ABF3 cDNA and genomic DNA reveals that this gene does not contain introns and that the open reading frame is 1,380 nucleotides in length. The deduced mature protein is composed of 433 amino acids residues and has a calculated molecular weight of 47,305. The deduced amino acid sequence has been validated by mass spectrometry analysis of peptides from purified ABF3. A total of 482 bp of the promoter were sequenced; putative binding sites for transcription factors such as CreA (four), XlnR (one), and AreA (three) and two CCAAT boxes were found. The enzyme has two domains, one similar to proteins of glycosyl hydrolase family 43 at the amino-terminal end and a family 6 carbohydrate binding module at the carboxyl end. ABF3 is the first described modular family 43 enzyme from a fungal source, having both α-l-arabinofuranosidase and xylobiohydrolase functionalities.

scholarly journals Chemical characterization and mode of action of Ligustrum lucidum flower essential oil against food-borne pathogenic bacteria

2016 ◽  
Vol 11 (1) ◽  
pp. 269 ◽  
Author(s):  
Vivek K. Bajpai ◽  
Siddhartha Singh ◽  
Archana Mehta
Keyword(s):  
Essential Oil ◽  
Mode Of Action ◽  
Pathogenic Bacteria ◽  
Membrane Integrity ◽  
Chemical Characterization ◽  
Mass Spectrometry Analysis ◽  
Gas Chromatography Mass Spectrometry ◽  
Spectrometry Analysis ◽  
Ligustrum Lucidum ◽  
Food Borne

<p class="Abstract">Current research analyzes the chemical composition of <em>Ligustrum lucidum</em> flower essential oil obtained by the hydrodistillation, and examines its anti-microbial mode of action against food-borne pathogenic bacteria. Gas chromatography-mass spectrometry analysis of the oil resulted in the determination of 44 different compounds, representing 85.2% of the total oil. The oil (1 mg/disc) showed significant antibacterial effect as diameters of inhibition zones (14.6 ± 0.2 – 19.7 ± 0.3 mm), as well as minimum inhibitory and minimum bactericidal concentrations values (250–1000 and 250–2000 µg/mL), respectively. Based on the susceptibility, <em>L. lucidum</em> flower oil revealed its mode of action on membrane integrity as confirmed by increased release of extracellular ATP (2.5 and 2.2 pg/mL), leakage of potassium ions (950 and 900 mM/L), loss of 260-nm absorbing materials (4.2 and 3.9 optical density), and increase in relative electrical conductivity (10.6 and 9.8%) against <em>Staphylococcus aureus</em> KCTC-1621 (Gram-positive) and <em>Salmonella enterica</em> ATCC-4731 (Gram-negative), respectively.</p><p> </p>

scholarly journals Exploration of the Quorum-Quenching Mechanism in Pseudomonas nitroreducens W-7 and Its Potential to Attenuate the Virulence of Dickeya zeae EC1

2021 ◽  
Vol 12 ◽  
Author(s):  
Wenping Zhang ◽  
Xinghui Fan ◽  
Jiayi Li ◽  
Tian Ye ◽  
Sandhya Mishra ◽  
...  
Keyword(s):  
Host Plants ◽  
Pathogenic Bacteria ◽  
Bacterial Pathogens ◽  
Disease Incidence ◽  
Quorum Quenching ◽  
Homoserine Lactone ◽  
Economic Losses ◽  
Highly Efficient ◽  
Pseudomonas Nitroreducens ◽  
Wide Range

Quorum quenching (QQ) is a novel, promising strategy that opens up a new perspective for controlling quorum-sensing (QS)-mediated bacterial pathogens. QQ is performed by interfering with population-sensing systems, such as by the inhibition of signal synthesis, catalysis of degrading enzymes, and modification of signals. In many Gram-negative pathogenic bacteria, a class of chemically conserved signaling molecules named N-acyl homoserine lactones (AHLs) have been widely studied. AHLs are involved in the modulation of virulence factors in various bacterial pathogens including Dickeya zeae. Dickeya zeae is the causal agent of plant-rot disease of bananas, rice, maize, potatoes, etc., causing enormous economic losses of crops. In this study, a highly efficient AHL-degrading bacterial strain W-7 was isolated from activated-sludge samples and identified as Pseudomonas nitroreducens. Strain W-7 revealed a superior ability to degrade N-(3-oxododecanoyl)-l-homoserine lactone (OdDHL) and completely degraded 0.2 mmol/L of OdDHL within 48 h. Gas chromatography-mass spectrometry (GC-MS) identified N-cyclohexyl-propanamide as the main intermediate metabolite during AHL biodegradation. A metabolic pathway for AHL in strain W-7 was proposed based on the chemical structure of AHL and intermediate products. In addition to the degradation of OdDHL, this strain was also found to be capable of degrading a wide range of AHLs including N-(3-oxohexanoyl)-l-homoserine lactone (OHHL), N-(3-oxooctanoyl)-l-homoserine lactone (OOHL), and N-hexanoyl-l-homoserine lactone (HHL). Moreover, the application of strain W-7 as a biocontrol agent could substantially attenuate the soft rot caused by D. zeae EC1 to suppress tissue maceration in various host plants. Similarly, the application of crude enzymes of strain W-7 significantly reduced the disease incidence and severity in host plants. These original findings unveil the biochemical aspects of a highly efficient AHL-degrading bacterial isolate and provide useful agents that exhibit great potential for the control of infectious diseases caused by AHL-dependent bacterial pathogens.

scholarly journals Enterobacter cancerogenus produces short chain N-3-oxo-acylhomoserine lactones quorum sensing molecules

2015 ◽  
Author(s):  
Kok-Gan Chan ◽  
Wen-Si Tan
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
High Resolution ◽  
Quorum Sensing ◽  
Homoserine Lactone ◽  
Mass Spectrometry Analysis ◽  
Liquid Chromatography Mass Spectrometry ◽  
Spectrometry Analysis ◽  
Synthesis Activity

Enterobacter cancerogenus strain M004 genome size is 5.67 Mb. Here, its luxI homologue, designated as ecnI which is ecnI gene (633 bp) was cloned and overexpressed. Its AHL synthesis activity was verified using the high-resolution liquid chromatography-mass spectrometry analysis revealed the production of N-(3-oxo-hexanoyl)-L-homoserine lactone (3-oxo-C6-HSL) and N-(3-oxo-hexanoyl)-L-homoserine lactone (3-oxo-C8-HSL). The cloning and characterization of luxI homologue of E. cancerogenus strain M004 was firstly reported here.

scholarly journals The Komagataeibacter europaeus GqqA is the prototype of a novel bifunctional N-Acyl-homoserine lactone acylase with prephenate dehydratase activity

2021 ◽  
Author(s):  
Manuel Ferrer ◽  
Laura Fernandes-Lopez ◽  
Sven Falke ◽  
Markus Perbandt ◽  
Winfried Hinrichs ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Lactone Ring ◽  
Phylogenetic Analyses ◽  
Quorum Quenching ◽  
Homoserine Lactone ◽  
Docking Studies ◽  
Protein Domain ◽  
Amide Bond ◽  
Binding Motif ◽  
Acyl Homoserine Lactone

Abstract Previously, we reported the isolation of a quorum quenching protein (QQ), designated GqqA, from Komagataeibacter europaeus CECT 8546 that is highly homologous to prephenate dehydratases (PDT) 1. GqqA strongly interfered with N-acyl-homoserine lactone (AHL) quorum sensing signals from Gram-negative bacteria and affected biofilm formation in its native host strain Komagataeibacter europaeus. Here we present and discuss data identifying GqqA as a novel acylase. ESI-MS-MS data showed unambiguously that GqqA hydrolyzes the amide bond of the acyl side-chain of AHL molecules, but not the lactone ring. Consistent with this observation the protein sequence does not carry a conserved Zn2+ binding motif, known to be essential for metal-dependent lactonases, but in fact harboring the typical periplasmatic binding protein domain (PBP domain), acting as catalytic domain. We report structural details for the native structure at 2.5 Å resolution and for a truncated GqqA structure at 1.7 Å. The structures obtained highlight that GqqA acts as a dimer and complementary docking studies indicate that the lactone ring of the substrate binds within a cleft of the PBP domain and interacts with polar residues Y16, S17 and T174. The biochemical and phylogenetic analyses imply that GqqA represents the first member of a novel type of QQ family enzymes.

scholarly journals Primary Structure and Antibacterial Activity of Chicken Bone Marrow-Derived β-Defensins

2009 ◽  
Vol 53 (11) ◽  
pp. 4647-4655 ◽  
Author(s):  
Chrystelle Derache ◽  
Valérie Labas ◽  
Vincent Aucagne ◽  
Hervé Meudal ◽  
Céline Landon ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Bone Marrow ◽  
Posttranslational Modifications ◽  
Amino Acid Sequences ◽  
Biologically Active ◽  
Mass Spectrometry Analysis ◽  
Gram Positive ◽  
Intact Cells ◽  
Spectrometry Analysis ◽  
Chicken Bone

ABSTRACTThree biologically active β-defensins were purified by chromatography from chicken bone marrow extract: avian β-defensin 1 (AvBD1), AvBD2, and the newly isolated β-defensin AvBD7. Mass spectrometry analyses showed that bone marrow-derived AvBD1, -2, and -7 peptides were present as mature peptides and revealed posttranslational modifications for AvBD1 and AvBD7 in comparison to their in silico-predicted amino acid sequences. Tandem mass spectrometry analysis using the nanoelectrospray-quadrupole time of flight method showed N-terminal glutaminyl cyclization of mature AvBD7 and C-terminal amidation of mature AvBD1 peptide, while posttranslational modifications were absent in bone marrow-derived mature AvBD2 peptide. Furthermore, mass spectrometry analysis performed on intact cells confirmed the presence of these three peptides in mature heterophils. In addition, the antibacterial activities of the three β-defensins against a large panel of gram-positive and -negative bacteria were assessed. While the three defensins displayed similar antibacterial spectra of activity against gram-positive strains, AvBD1 and AvBD7 exhibited the strongest activity against gram-negative strains in comparison to AvBD2.

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