Hexanoyl Homoserine Lactone Research Articles

Functional characterization of quorum sensing LuxR-type transcriptional regulator, EasR in Enterobacter asburiae strain L1.

Over the past decades, Enterobacter spp. have been identified as challenging and important pathogens. The emergence of multidrug-resistant Enterobacteria especially those that produce Klebsiella pneumoniae carbapenemase has been a very worrying health crisis. Although efforts have been made to unravel the complex mechanisms that contribute to the pathogenicity of different Enterobacter spp., there is very little information associated with AHL-type QS mechanism in Enterobacter spp. Signaling via N-acyl homoserine lactone (AHL) is the most common quorum sensing (QS) mechanism utilized by Proteobacteria. A typical AHL-based QS system involves two key players: a luxI gene homolog to synthesize AHLs and a luxR gene homolog, an AHL-dependent transcriptional regulator. These signaling molecules enable inter-species and intra-species interaction in response to external stimuli according to population density. In our recent study, we reported the genome of AHL-producing bacterium, Enterobacter asburiae strain L1. Whole genome sequencing and in silico analysis revealed the presence of a pair of luxI/R genes responsible for AHL-type QS, designated as easI/R, in strain L1. In a QS system, a LuxR transcriptional protein detects and responds to the concentration of a specific AHL controlling gene expression. In E. asburiae strain L1, EasR protein binds to its cognate AHLs, N-butanoyl homoserine lactone (C4-HSL) and N–hexanoyl homoserine lactone (C6-HSL), modulating the expression of targeted genes. In this current work, we have cloned the 693 bp luxR homolog of strain L1 for further characterization. The functionality and specificity of EasR protein in response to different AHL signaling molecules to activate gene transcription were tested and validated with β-galactosidase assays. Higher β-galactosidase activities were detected for cells harboring EasR, indicating EasR is a functional transcriptional regulator. This is the first report documenting the cloning and characterization of transcriptional regulator, luxR homolog of E. asburiae.

Recombinant N-acyl homoserine lactone-Lactonase AiiAQSI-1 Attenuates Aeromonas hydrophila Virulence Factors, Biofilm Formation and Reduces Mortality in Crucian Carp.

Quorum quenching (QQ) is a promising alternative infection-control strategy to antibiotics that controls quorum-regulated virulence without killing the pathogens. Aeromonas hydrophila is an opportunistic gram-negative pathogen living in freshwater and marine environments. A. hydrophila possesses an N-acyl homoserine lactone (AHL)-based quorum-sensing (QS) system that regulates virulence, so quorum signal-inactivation (i.e., QQ) may represent a new way to combat A. hydrophila infection. In this study, an AHL lactonase gene, aiiA was cloned from Bacillus sp. strain QSI-1 and expressed in Escherichia coli strain BL21(DE3). The A. hydrophila hexanoyl homoserine lactone (C6-HSL) QS signal molecule was degraded by AiiAQSI-1, which resulted in a decrease of bacterial swimming motility, reduction of extracellular protease and hemolysin virulence factors, and inhibited the biofilm formation of A. hydrophila YJ-1 in a microtiter assay. In cell culture studies, AiiAQSI-1 decreased the ability of A. hydrophila adherence to and internalization by Epithelioma papulosum cyprini (EPC) cells. During in vivo studies, oral administration of AiiAQSI-1 via feed supplementation attenuated A. hydrophila infection in Crucian Carp. Results from this work indicate that feed supplementation with AiiAQSI-1 protein has potential to control A. hydrophila aquaculture disease via QQ.

Conformational and docking studies of acyl homoserine lactones as a robust method to investigate bioactive conformations

A method aiming at investigating possible bioactive conformations of acyl homoserine lactone (AHL) quorum sensing (QS) modulators is established. The method relies on the exhaustive conformational analysis of AHLs by varying torsion angles around the amide group then on the selection of the closest conformation to those known from co-crystallized XRD data of AHL-receptor complexes. These latter are then docked as rigid ligand within the receptor binding site, leading to interactions with binding site residues which are highly consistent as compared with the data arising from XRD studies. The method is first validated using AHLs for which XRD data of their complexes with their cognate receptor are available, then extended to examples for which the binding mode is still unknown.Three compounds were used to validate the method: hexanoyl homoserine lactone (HHL) as an example of autoinducer, 3-oxo-butanoyl homoserine lactone (OBHL), as a representative model of 3-oxo-AHLs, and 4-(4-chlorophenoxy)butanoyl homoserine lactone (CPOBHL) as an example of a QS inhibitor. The conformational analysis of these three compounds to their cognate protein (TraR, SdiA, LasR and CviR) provides the data which enable the next rigid docking step. Further rigid docking of the closest conformations compared to the known bioactive ones within the binding sites allows to recover the expected binding mode with high precision (atomic RMSD < 2 Å). This “conformational analysis/torsion angle filter/rigid ligand docking” method was then used for investigating three non-natural AHL-type QS inhibitors without known co-crystallized XRD structures, namely was 2-hexenoyl homoserine lactone (HenHL), 3-oxo-4-phenylbutanoyl homoserine lactone (OPBHL) and 3-(4-bromophenyl)propanoyl homoserine lactone (BPPHL). Results provide insights into their possible binding mode by identifying specific interactions with some key residues within the receptor binding site, allowing discussion of their biological activity.

Overexpressed recombinant quorum quenching lactonase reduces the virulence, motility and biofilm formation of multidrug-resistant Pseudomonas aeruginosa clinical isolates.

The increasing occurrence of resistance among Pseudomonas aeruginosa clinical isolates necessitates finding alternatives to antibiotics for controlling the infection of such pathogenic bacteria. In this study, lactonase gene ahl-1 from Bacillus weihenstephanensis isolate-P65 was successfully cloned and expressed in Escherichia coli BL21 (DE3) under the control of T7 promoter for utilizing its quorum quenching activity against three multidrug-resistant (MDR) P. aeruginosa clinical isolates. The biological activity of the overexpressed lactonase enzyme (Ahl-1), tested using a synthetic signal and Chromobacterium violaceum CV026 as a biosensor, displayed good catalytic activity using hexanoyl homoserine lactone (HHL) as a substrate and Chromobacterium violaceum (CV026) as a biosensor (77.2 and 133nmmin-1 for the crude and the purified Ahl-lactonase enzymes, respectively). Upon challenging its ability to inhibit the virulence of three MDR P. aeruginosa clinical isolates, recombinant Ahl-1 successfully prevented the accumulation of acylhomoserine lactone signals resulting in a significant reduction in the investigated virulence determinants; protease (from 40 up to 75.5%), pyocyanin (48-75.9%), and rhamnolipids (52.7-63.4%) (P value < 0.05). Ahl-1 also displayed significant inhibitory activities on the swarming motility and biofilm formation of the three tested MDR P. aeruginosa clinical isolates (P value < 0.05). Consequently, Ahl-1 lactonase enzyme in this study is considered a promising therapeutic agent to inhibit P. aeruginosa pathogenicity with no fear of emergence of resistance.

Cloning and characterization of short-chain N-acyl homoserine lactone-producing Enterobacter asburiae strain L1 from lettuce leaves.

In gram‐negative bacteria, bacterial communication or quorum sensing (QS) is achieved using common signaling molecules known as N‐acyl homoserine lactones (AHL). We have previously reported the genome of AHL‐producing bacterium, Enterobacter asburiae strain L1. In silico analysis of the strain L1 genome revealed the presence of a pair of luxI/R genes responsible for AHL‐type QS, designated as easIR. In this work, the 639 bp luxI homolog, encoding 212 amino acids, have been cloned and overexpressed in Escherichia coli BL21 (DE3)pLysS. The purified protein (~25 kDa) shares high similarity to several members of the LuxI family among different E asburiae strains. Our findings showed that the heterologously expressed EasI protein has activated violacein production by AHL biosensor Chromobacterium violaceum CV026 as the wild‐type E. asburiae. The mass spectrometry analysis showed the production of N‐butanoyl homoserine lactone and N–hexanoyl homoserine lactone from induced E. coli harboring the recombinant EasI, suggesting that EasI is a functional AHL synthase. E. asburiae strain L1 was also shown to possess biofilm‐forming characteristic activity using crystal violet binding assay. This is the first report on cloning and characterization of the luxI homolog from E. asburiae.

Synergistic activity of cosecreted natural products from amoebae-associated bacteria

Investigating microbial interactions from an ecological perspective is a particularly fruitful approach to unveil both new chemistry and bioactivity. Microbial predator-prey interactions in particular rely on natural products as signal or defense molecules. In this context, we identified a grazing-resistant Pseudomonas strain, isolated from the bacterivorous amoeba Dictyostelium discoideum. Genome analysis of this bacterium revealed the presence of two biosynthetic gene clusters that were found adjacent to each other on a contiguous stretch of the bacterial genome. Although one cluster codes for the polyketide synthase producing the known antibiotic mupirocin, the other cluster encodes a nonribosomal peptide synthetase leading to the unreported cyclic lipopeptide jessenipeptin. We describe its complete structure elucidation, as well as its synergistic activity against methicillin-resistant Staphylococcus aureus, when in combination with mupirocin. Both biosynthetic gene clusters are regulated by quorum-sensing systems, with 3-oxo-decanoyl homoserine lactone (3-oxo-C10-AHL) and hexanoyl homoserine lactone (C6-AHL) being the respective signal molecules. This study highlights the regulation, richness, and complex interplay of bacterial natural products that emerge in the context of microbial competition.

Draft Genome Perspective of Staphylococcus saprophyticus Strain SU8, an N-Acyl Homoserine Lactone-Degrading Bacterium.

Staphylococcus saprophyticus strain SU8 was isolated from a pristine water source in Malaysia and it exhibited degradation of N-hexanoylhomoserine lactone. Here we report the draft genome sequence of S. saprophyticus strain SU8 to further understand its quorum quenching abilities.

English

By playing a major role in the virulence of several pathogenic bacteria, N-Acyl homoserine-lactone (AHL) -dependent quorum sensing, is now considered a useful target for antimicrobial therapy. In the present study a total number of 63 Streptomyces isolates recovered from different soil samples collected from different localities in Egypt were screened for quorum quenching activity using a fast, reliable, simple screening method and Chromobacterium violaceum mutant strain (CV026) as a biosensor. Primary screening against synthetic hexanoyl homoserine lactone (HHL) revealed that 8 isolates showed quorum quenching activity while secondary screening against the extracted naturally produced acyl homoserine lactone (AHL) signals of seven Pseudomonas aeruginosa clinical isolates showed a variable profile of activity. Characterization of activity showed that isolate that coded St62, showing highest activity among other Streptomyces isolates, could degrade 2 &micro;M HHL in 4 h. The crude enzyme of this isolate was found to have high thermal stability retaining &gt;83% of its activity when pre-incubated at 80&deg;C for 60 min. It also retained &gt;90% of the activity when pre-incubated at pH 8, pH 9 and pH 10 and &gt;80% when pre-incubated at pH 6. All the tested metal ions and EDTA had no effect on activity except for Cu++ which caused partial reduction in activity. Enzyme extract was found to have acylase activity equal to 5.2 U/mg total protein.&nbsp; Vmax and Km were found to be equal to 19.92 nM.min-1 per mg protein and 23.05 nM, respectively. Maximum catalytic activity was observed at pH 8 over a temperature range of 20-50&deg;C. The enzyme also showed preference in hydrolyzing AHLs with long acyl chains than short acyl chains but yet digested all the tested AHL standards. Finally, identification of the selected isolate using 16S ribosomal RNA gene analysis and phylogenetic analysis showed it to be a Streptomyces minutiscleroticus which is, to the best of our knowledge, the first identified quorum quenching bacterial species of its type. Key words: Quorum sensing, Streptomyces minutiscleroticus, Antipathogenic drugs, Acyl homoserine lactones.

Screening and preliminary characterization of quenching activities of soil Bacillus isolates against acyl homoserine lactones of clinically isolated Pseudomonas aeruginosa

Aim: Quorum quenching of Pseudomonas aeruginosa homoserine lactone signal molecules represents a new approach for control of infection of this pathogenic highly resistant microorganism. The present study aimed at screening of soil for quorum sensing inhibitory bacteria active against clinically isolated P. aeruginosa together with the characterization of their activity and finally identification of promising isolates. Methodology and results: Using a fast, reliable and simple screening method, 161 bacterial isolates collected from soil from different places in Egypt were screened for their quorum quenching activity against synthetic hexanoyl homoserine lactone using Chromobacterium violaceum mutant strain (CV026) as a biosensor. All 32 positive isolates were found to belong to Bacillus species. Secondary screening against the signals extracted from seven Pseudomonas isolates, analyzed by thin layer chromatography, was done. The activity of all the positive isolates was found to be intracellular. Activity against different concentrations of synthetic hexanoyl homoserine lactone showed that some isolates could degrade more than 20 µM even when diluted 100 fold. Selected isolates were found to have broad spectrum activity against other synthetic homoserine lactone standards. Maximum activity for most of the selected isolates was found to occur between 25-60 °C. Crude enzyme extracts of the promising isolates were collected by sonication, protein concentrations of the obtained extracts were measured and their activities were compared by well diffusion method. Finally, the isolates with promising quorum quenching activities were identified using 16S ribosomal RNA sequencing. Conclusion, significance and impact of study: Having high activity against homoserine lactone autoinducers, the enzyme produced by Bacillus isolates represents a new promising antipathogenic drug suppressing Pseudomonas virulence.

Quorum sensing activity of Enterobacter asburiae isolated from lettuce leaves.

Bacterial communication or quorum sensing (QS) is achieved via sensing of QS signaling molecules consisting of oligopeptides in Gram-positive bacteria and N-acyl homoserine lactones (AHL) in most Gram-negative bacteria. In this study, Enterobacteriaceae isolates from Batavia lettuce were screened for AHL production. Enterobacter asburiae, identified by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) was found to produce short chain AHLs. High resolution triple quadrupole liquid chromatography mass spectrometry (LC/MS) analysis of the E. asburiae spent supernatant confirmed the production of N-butanoyl homoserine lactone (C4-HSL) and N–hexanoyl homoserine lactone (C6-HSL). To the best of our knowledge, this is the first report of AHL production by E. asburiae.

English

Vibrio harveyi&nbsp;strains were isolated from the water samples of low salinity.&nbsp; Isolates were confirmed by both the presence of&nbsp;vhh&nbsp;gene using polymerase chain reaction and bio-chemical tests.&nbsp;&nbsp;V. harveyi&nbsp;was grown in LB medium and its spent culture was treated with ethyl acetate. The organic layer was pooled and dried by Roto - Vapour with vacuum at 30ËšC. The dried residues were dissolved with 50 % acetonitrile and water. The presence of N-acyl homoserine lactones (AHL) compounds from the residues of crude extract and the standard &ldquo;3 - Oxo hexanoyl homoserine lactone&rdquo; were analyzed separately using Liquid chromatography and mass spectrometry (LC-MS). Different peaks were detected and peaks corresponding to 214.4 m/z were identified as N- (3-oxohexanoyl)-L-homoserine lactones in the sample. Different unknown peaks were also identified.&nbsp; Aqueous extracts of 47 terrestrial plants were evaluated against luminescence disease causing&nbsp;V. harveyi,&nbsp;through &ldquo;Agar well diffusion assay&rdquo;. Ten plant extracts showed zone of inhibition (5 to 7 mm) on&nbsp;V. harveyi. Extracts from&nbsp;Phyllanthus niruri&nbsp;and&nbsp;Cynodon dactylon&nbsp;showed the highest zone of inhibition (8.3 mm) against&nbsp;V. harveyi&nbsp;followed byCalotropis gigantean&nbsp;and&nbsp;Costus speciosus&nbsp;(7.3 mm). Plant extracts were reported for the presence of alkanes, alkenes, aromatics, primary and secondary amines etc, byFourier transform infra red spectroscopy&nbsp;(FT-IR). The antagonistic activity may be due to by the presence of these functional compounds in the extracts. Therefore, terrestrial plants could be used to control aquatic bacterial pathogens, which can also be extended to control human bacterial pathogens. &nbsp; Key words:&nbsp;V. harveyi,&nbsp;extraction of N-acyl homoserine lactones (AHL), analysis by Liquid chromatography and mass spectrometry (LC-MS), terrestrial plants, antagonism,Fourier transform infra red spectroscopy.

Characterization and Complete Sequence of Lactonase Enzyme fromBacillus weihenstephanensisIsolate P65 with Potential Activity against Acyl Homoserine Lactone Signal Molecules

Acyl homoserine lactones (AHLs) are the most common class of quorum sensing signal molecules (autoinducers) that have been reported to be essential for virulence of many relevant pathogenic bacteria such as Pseudomonas aeruginosa. New approach for controlling infections of such bacteria is through quorum quenching. In this study, the acyl homoserine lactone inhibitory activity of the crude enzyme from a Bacillus weihenstephanensis-isolate P65 was characterized. The crude enzyme was found to have relatively high thermal stability and was stable in pH range 6 to 9. The crude enzyme extract was found to have lactonase activity of 36.3 U/mg total protein. Maximum enzyme activity was achieved within a range of 28–50°C and pH 6–9. None of the metals used enhanced the activity neither did EDTA inhibit it. However, a concentration of 10 mM Fe+2 reduced the activity to 73.8%. Catalytic activity and kinetic constants were determined using hexanoyl homoserine lactone as a substrate. Studying enzyme substrate specificity using synthetic standard signals displayed broad spectrum of activity. The enzyme was found to be constitutive. Isolation and complete nucleotide sequence of the respective lactonase gene were done and submitted to the Genbank database under accession code KC823046.

β-Cyclodextrin Interaction with &lt;i&gt;N&lt;/i&gt;-Hexanoyl Homoserine Lactone as Quorum Sensing Signal Produced in Gram-Negative Bacteria

β-Cyclodextrin Interaction with &lt;i&gt;N&lt;/i&gt;-Hexanoyl Homoserine Lactone as Quorum Sensing Signal Produced in Gram-Negative Bacteria

Infrared Study of the Bacterial Autoinducer N-Hexanoyl-Homoserine Lactone (C6-HSL) in the Gas-Phase, Water, and Octanol Solutions

The N-hexanoyl-homoserine lactone (C6-HSL) molecule has been investigated by means of infrared multiphoton dissociation (IRMPD) and Fourier-transform infrared spectroscopy (FT-IR) under different conditions in an attempt to mimic biological situations encountered in communication between bacteria for quorum sensing. The protonated molecular ion was studied in the gas-phase that corresponds to a solvent-free situation somewhat analogous to that encountered in the receptor. The simulation of the IRMPD spectrum of the isolated ion was then conducted by means of quantum chemistry calculations in vacuum. In the case of the neutral species, the FT-IR spectra were recorded in D(2)O, mimicking the cytosolic and extracellular media as well as in 1-octanol that is often used for simulation of cell membranes. The interpretation was conducted by considering a C6-HSL molecule in its endo or exo conformation hydrogen-bonded to, respectively, six D(2)O and four 1-octanol molecules. A satisfying agreement with the experimental FT-IR studies conducted in solution at room temperature was obtained as long as a continuum IEFPCM model was added to the explicit solvent environment.

Interception of quorum sensing signal molecule by furanone to enhance shelf life of fermented milk

Fermented milk prepared by culturing the probiotic culture Leuconostoc mesenteroides, is a nutritious drink but at the same time a large number of spoilage microorganisms can grow in it when stored for a long time. The spoilage bacterial cultures are known to communicate with each other by release of signaling molecule which is described as quorum sensing. In the present work, the predominant bacterial culture, Pseudomonas was found to be responsible for spoiling the fermented milk. The signal molecule was identified as hexanoyl homoserine lactone by TLC, GC, and GCMS method. Two furanones namely 2(5H)-furanone and bromofuranone were used to reduce the virulence of Pseudomonas sp. Results showed that 2(5H)-furanone has inhibitory activity against Pseudomonas. This was supported with data of reduced motility, reduced acyl homoserine lactone (HHSL and BHSL), low rhamnolipid content and reduced exoprotease activity of Pseudomonas sp. when furanone was supplemented. With addition of 2(5H)-furanones the shelf life was increased up to 9 days by reducing the spoilage bacterial cultures.

Characterization of a Phenazine and Hexanoyl Homoserine Lactone Producing Pseudomonas aurantiaca Strain PB-St2, Isolated from Sugarcane Stem

A novel strain of fluorescent pseudomonad (PB-St2) was isolated from surface-sterilized stems of sugarcane grown in Pakistan. The bacterium was identified as Pseudomonas aurantiaca on the basis of 16S rRNA gene sequence analysis and results from physiological and biochemical characteristics carried out with API50 CH and QTS 24 bacterial identification kits. Assays using substrate specific media for enzymes revealed lipase and protease activities but cellulase, chitinase, or pectinase were not detected. The bacterium was unable to solubilize phosphate or produce indole acetic acid. However, it did produce HCN, siderophores, and homoserine lactones. In dual culture assays on agar, the bacterium showed antifungal activity against an important pathogen of sugarcane in Pakistan, namely Colletotrichum falcatum, as well as for pathogenic isolates of Fusarium oxysporium, F. lateritium but not against F. solani. The antifungal metabolites were identified using thin-layer chromatography, UV-spectra, and MALDI-TOFF spectra and shown to be phenazine-1-carboxylic acid (PCA), 2-hydroxyphenazine (2-OH-PHZ), and N-hexanoyl homoserine lactone (HHL) (assessed using only TLC data). The capacity of this bacterium to produce HCN and 2-OH-PHZ, as well as to inhibit the growth of C. falcatum, has not been previously reported.

Directed evolution of LuxI for enhanced OHHL production

Quorum sensing is a common mechanism used by bacteria to coordinate population behavior, and is involved in a variety of biological processes, such as bioluminescence, virulence factor synthesis, antibiotic production, and biofilm formation. To engineer the LuxI enzyme of the LuxI-LuxR quorum-sensing system, we developed a high throughput genetic selection to identify LuxI mutants with improved OHHL (3-oxo-hexanoyl homoserine lactone) synthesis in E. coli. Using this genetic selection, we created LuxI mutants with improved OHHL synthesis rates and yields through directed evolution, identifying three LuxI mutants after two generations. An in vivo semi-quantitative method allowed for verification of the genetic screen and OHHL yields were quantified using HPLC-MS/MS, revealing an 80-fold increase in a mutant culture compared to the wildtype culture. In addition to OHHL, the yields of C6HSL (hexanoyl homoserine lactone) and C8HSL (octanoyl homoserine lactone) were also improved, and a slight change in substrate specificity towards C6HSL production was observed. Based on alignment with the crystal structure of EsaI, a homolog of LuxI, two mutations are most likely involved in enhancing the interactions between the enzyme and the substrates. The high throughput genetic selection and the semi-quantitative method can be conveniently modified for the directed evolution of LuxI homologs. The identification of these LuxI mutants has implications in synthetic biology, where they can be used for the construction of artificial genetic circuits. In addition, development of drugs that specifically target quorum sensing to attenuate the pathogenesis of gram-negative infectious bacteria might also benefit from the insights into the molecular mechanism of quorum sensing revealed by the amino acid substitutions.

Presence of Quorum-sensing Systems Associated with Multidrug Resistance and Biofilm Formation in Bacteroides fragilis

Bacteroides fragilis constitutes 1-2% of the natural microbiota of the human digestive tract and is the predominant anaerobic opportunistic pathogen in gastrointestinal infections. Most bacteria use quorum sensing (QS) to monitor cell density in relation to other cells and their environment. In Gram-negative bacteria, the LuxRI system is common. The luxR gene encodes a transcriptional activator inducible by type I acyl-homoserine lactone autoinducers (e.g., N-[3-oxohexanoyl] homoserine lactone and hexanoyl homoserine lactone [C6-HSL]). This study investigated the presence of QS system(s) in B. fragilis. The genome of American-type culture collection strain no. ATCC25285 was searched for QS genes. The strain was grown to late exponential phase in the presence or absence of synthetic C6-HSL and C8-HSL or natural homoserine lactones from cell-free supernatants from spent growth cultures of other bacteria. Growth, susceptibility to antimicrobial agents, efflux pump gene (bmeB) expression, and biofilm formation were measured. Nine luxR and no luxI orthologues were found. C6-HSL and supernatants from Yersinia enterocolitica, Vibrio cholerae, and Pseudomonas aeruginosa caused a significant (1) reduction in cellular density and (2) increases in expression of four putative luxR genes, bmeB3, bmeB6, bmeB7, and bmeB10, resistance to various antibiotics, which was reduced by carbonyl cyanide-m-chlorophenyl hydrazone (CCCP, an uncoupler that dissipates the transmembrane proton gradient, which is also the driving force of resistance nodulation division efflux pumps) and (3) increase in biofilm formation. Susceptibility of ATCC25285 to C6-HSL was also reduced by CCCP. These data suggest that (1) B. fragilis contains putative luxR orthologues, which could respond to exogenous homoserine lactones and modulate biofilm formation, bmeB efflux pump expression, and susceptibility to antibiotics, and (2) BmeB efflux pumps could transport homoserine lactones.

Expression of the bviIR and cepIR Quorum-Sensing Systems of Burkholderia vietnamiensis

Burkholderia vietnamiensis has both the cepIR quorum-sensing system that is widely distributed among the Burkholderia cepacia complex (BCC) and the bviIR system. Comparison of the expression of cepI, cepR, bviI, and bviR-luxCDABE fusions in B. vietnamiensis G4 and the G4 cepR and bviR mutants determined that the expression of bviI requires both a functional cognate regulator, BviR, and functional CepR. The cepIR system, however, is not regulated by BviR. Unlike the cepIR genes in other BCC species, the cepIR genes are not autoregulated in G4. N-Acyl-homoserine lactone (AHL) production profiles in G4 cepI, cepR, bviI, and bviR mutants confirmed the regulatory organization of the G4 quorum-sensing systems. The regulatory network in strain PC259 is similar to that in G4, except that CepR positively regulates cepI and negatively regulates cepR. AHL production and the bviI expression levels in seven B. vietnamiensis isolates were compared. All strains produced N-octanoyl-homoserine lactone and N-hexanoyl-homoserine lactone; however, only one of four clinical strains but all three environmental strains produced the BviI synthase product, N-decanoyl-homoserine lactone (DHL). The three strains that did not produce DHL expressed bviR but not bviI. Heterologous expression of bviR restored DHL production in these strains. The bviIR loci of the non-DHL-producing strains were sequenced to confirm that bviR encodes a functional transcriptional regulator. Lack of expression of G4 bviI in these three strains indicated that an additional regulatory element may be involved in the regulation of bviIR expression in certain strains of B. vietnamiensis.

Relevance of N-acyl-L-homoserine lactone production by Yersinia enterocolitica in fresh foods

Determination of the food matrix impact on the potential for N-acyl-L-homoserine lactones (AHLs) production by Yersinia enterocolitica. Induction and inhibition of a sensor strain and a fluorescent assay were used to investigate Y. enterocolitica AHL production in artificial media, as well as in different food extracts. All Y. enterocolitica strains tested produced AHLs in artificial media. Thin Layer Chromatography analysis of Y. enterocolitica strains indicated the production of 3-oxo-hexanoyl homoserine lactone and hexanoyl homoserine lactone. Yersinia enterocolitica produced AHL principally in fish and meat extracts. AHL production by Y. enterocolitica was observed in products of animal origin, but were inhibited by some vegetables extracts. This study suggests that quorum sensing systems in Y. enterocolitica is significant in foods but depends upon the type of food. Determination of physiological functions in Y. enterocolitica which are regulated by quorum sensing and their relation to the production of AHLs in foods need to be further assessed.