Long-chain Acyl Homoserine Lactones Research Articles

Roles of transcriptional factor PsrA in the regulation of quorum sensing in Pseudomonas aeruginosa PAO1.

The transcription factor PsrA regulates fatty acid metabolism, the type III secretion system, and quinolone signaling quorum sensing system in Pseudomonas aeruginosa. To explore additional roles of PsrA in P. aeruginosa, this study engineered a P. aeruginosa PAO1 strain to carry a recombinant plasmid with the psrA gene (pMMBpsrA) and examined the impact of elevated psrA expression to the bacterium. Transcriptomic analysis revealed that PsrA significantly downregulated genes encoding the master quorum-sensing regulators, RhlR and LasR, and influenced many quorum-sensing-associated genes. The role of PsrA in quorum sensing was further corroborated by testing autoinducer synthesis in PAO1 [pMMBpsrA] using two reporter bacteria strains Chromobacterium violaceum CV026 and Escherichia coli [pSB1075], which respond to short- and long-chain acyl homoserine lactones, respectively. Phenotypic comparisons of isogenic ΔpsrA, ΔlasR, and ΔpsrAΔlasR mutants revealed that the reduced elastase, caseinase, and swarming activity in PAO1 [pMMBpsrA] were likely mediated through LasR. Additionally, electrophoretic mobility shift assays demonstrated that recombinant PsrA could bind to the lasR promoter at a 5'-AAACGTTTGCTT-3' sequence, which displays moderate similarity to the previously reported consensus PsrA binding motif. Furthermore, the PsrA effector molecule oleic acid inhibited PsrA binding to the lasR promoter and restored several quorum sensing-related phenotypes to wild-type levels. These findings suggest that PsrA regulates certain quorum-sensing phenotypes by negatively regulating lasR expression, with oleic acid acting as a crucial signaling molecule.

Antibacterial and Anti-Quorum Sensing Properties of Silver Nanoparticles Phytosynthesized Using Embelia ruminata.

The rise in antibiotic resistance (AR) poses an imminent threat to human health. Nanotechnology, together with mechanisms such as quorum sensing (QS), which relies on communication between bacterial cells, may decrease the selective pressure for AR. Thus, this study aimed to investigate the effectiveness of silver nanoparticles (AgNPs) synthesized at room temperature (Rt) and 80 °C using Embelia ruminata leaf, stem-bark, and fruit extracts as antibacterial and anti-QS agents. The phytosynthesized AgNPs solutions were subjected to various characterization assays and assessed for their antibacterial activities. Quantitative QS assays were performed using Chromobacterium subtsugae CV017 and Chromobacterium violaceum ATCC 12472. Synthesized AgNPs were spherical-to-near-spherical in shape, poly-dispersed, and crystalline, with a size range of 21.06-32.15 nm. Fruit AgNPs showed stronger antibacterial activity than AgNPs from other plant organs against selected bacterial strains. In the QS assays, fruit 80 °C AgNPs demonstrated the most significant violacein inhibition in an assay performed using the short-chain acyl homoserine lactone CV017 biosensor, while the leaf and fruit Rt AgNPs demonstrated the most violacein inhibition in an assay performed using the long-chain acyl homoserine lactone ATCC 12472 biosensor. The investigations carried out in this study lay the groundwork for future innovative research into antibacterial and anti-QS strategies using E. ruminata.

EVALUATION OF QUORUM SENSING SIGNALS OF STRONG BIOFILM PRODUCING BACTERIA VIA LC-MSMS, HPLC AND BIOSENSORS

This study aimed to show presence of Quorum Sensing (QS) signals of Gram-negative and Gram-positive biofilm producing bacteria isolated from real dairy process lines. Defining the profile and chemical composition of QS-signals is an important factor in control of microbial resistance and biofilm production. We especially focused on unusual behaviour of Gram-positive and Gram-negative isolates. Long-chain acyl-homoserine lactones (AHLs) signals (C14-HSL, C16-HSL and C18-HSL) and DFD (4,5-dihidroksi-2,3-pentanedione)-AI-2 signals of the isolates were studied by High-performance liquid chromatography (HPLC) and Liquid Chromatography with tandem mass spectrometry (LC-MSMS) methods. All Gram-positive isolates were defined as AHL-producers. All Gram-negative isolates, formerly defined as non-AHL producers by both biosensors and HPLC methods, were identified as AHL-producers. DFD signal was only detected from Gram-negative Klebsiella pneumonia, Enterobacter cloacae and Klebsiella oxytoca isolates. The results demonstrated that the QS-system is a complex system and biosensor microorganism may not be the best method for QS-signal identification. The results also provided new insights in defining the profile and chemical composition of QS-signals importance for interrupting the chemical communication completely to reduce biofilm formation and prevent resistance gain of microorganisms.

The relationship between quorum sensing dynamics and biological performances during anaerobic membrane bioreactor treatment

Anaerobic membrane bioreactors (AnMBRs) enhance carbon neutrality with biomethane recovery from wastewater; however, microbial signaling, which may affect biological performances, was poorly understood. Here, we thus evaluate quorum sensing (QS) dynamics while monitoring acyl-homoserine lactones (AHLs) and autoinducer-2 (AI-2) levels during long-term AnMBR operations after sludge inoculation. Significant organic removal and methane production were achieved with the reactor startup. Signal molecule levels varied with transient organic loading rates, depending on their types. A starving condition may cause an increase in short- and medium-chain AHLs and AI-2. Biopolymers, biosolids, volatile fatty acids, and alkalinity levels had positive correlations with short- and medium-chain AHLs and AI-2, whereas methane production had positive correlations with long-chain AHLs. The principal component analysis of QS signal composition and biological performance data explains their interconnectivity. The findings of this study help to understand that QS signals regulate metabolic pathways in addition to microbial group behaviors.

A novel LuxR-type solo of Sinorhizobium meliloti, NurR, is regulated by the chromosome replication coordinator, DnaA and activates quorum sensing.

The genome of Sinorhizobium meliloti, a model for studying plant-bacteria symbiosis, contains eight genes coding for LuxR-like proteins. Two of these, SinR and ExpR, are essential for quorum sensing (QS). Roles and regulation surrounding the others are mostly unknown. Here, we reveal the DNA recognition sequence and regulon of the LuxR-like protein SMc00877. Unlike ExpR, which uses the long-chain acyl homoserine lactones (AHLs) as inducers, SMc00877 functioned independently of AHLs and was even functional in Escherichia coli. A target of SMc00877 is SinR, the major regulator of AHL production in S. meliloti. Disruption of SMc00877 decreased AHL production. A weaker production of AHLs resulted in smaller microcolonies, starting from single cells, and delayed AHL-dependent regulation. SMc00877 was expressed only in growing cells in the presence of replete nutrients. Therefore, we renamed it NurR (nutrient sensitive LuxR-like regulator). We traced this nutrient-sensitive expression to transcription control by the DNA replication initiation factor, DnaA, which is essential for growth. These results indicate that NurR has a role in modulating the threshold of QS activation according to growth. We propose growth behavior as an additional prerequisite to population density for the activation of QS in S. meliloti.

Isolation and Characterization of N-acyl Homoserine Lactone-Producing Bacteria From Cattle Rumen and Swine Intestines.

Quorum sensing systems regulate gene expression in response to bacterial population density. Acyl-homoserine lactones are a class of quorum sensing molecules found in cattle rumen that are thought to regulate the gene expression of enterohemorrhagic Escherichia coli and thus help this pathogen survive in animal gastrointestinal tracts. However, the specific bacteria that produce these signaling molecules in bovine and porcine gastrointestinal tracts are unknown. Here we developed methods to concentrate gastrointestinal fluids and screen the bacteria that produce acyl-homoserine lactones. We isolated a Pseudomonas aeruginosa strain YZ1 from cattle rumen, and an Aeromonas hydrophila strain YZ2 from pig intestine. Mass spectrometry analysis of culture supernatants indicated at least three specific classes of acyl-homoserine lactones produced by YZ1, and a C4-acyl-homoserine lactone produced by YZ2. Transformation of E. coli with P. aeruginosa or A. hydrophila luxI homologs,which can produce short- or long-chain acyl-homoserine lactones conferred upon E. coli the ability to synthesize acyl-homoserine lactones and affected gene expression, motility, and acid tolerance of E. coli. This is the first study reporting the isolation and characterization of acyl-homoserine lactone synthase-positive bacteria from cattle rumen and swine intestines.

Ratiometric Gas Reporting: A Nondisruptive Approach To Monitor Gene Expression in Soils.

Fluorescent proteins are ubiquitous tools that are used to monitor the dynamic functions of natural and synthetic genetic circuits. However, these visual reporters can only be used in transparent settings, a limitation that complicates nondisruptive measurements of gene expression within many matrices, such as soils and sediments. We describe a new ratiometric gas reporting method for nondisruptively monitoring gene expression within hard-to-image environmental matrices. With this approach, C2H4 is continuously synthesized by ethylene forming enzyme to provide information on viable cell number, and CH3Br is conditionally synthesized by placing a methyl halide transferase gene under the control of a conditional promoter. We show that ratiometric gas reporting enables the creation of Escherichia coli biosensors that report on acylhomoserine lactone (AHL) autoinducers used for quorum sensing by Gram-negative bacteria. Using these biosensors, we find that an agricultural soil decreases the bioavailable concentration of a long-chain AHL up to 100-fold. We also demonstrate that these biosensors can be used in soil to nondisruptively monitor AHLs synthesized by Rhizobium leguminosarum and degraded by Bacillus thuringiensis. Finally, we show that this new reporting approach can be used in Shewanella oneidensis, a bacterium that lives in sediments.

Improvement of bioelectrochemical property and energy recovery by acylhomoserine lactones (AHLs) in microbial electrolysis cells (MECs)

Quorum sensing (QS) has been extensively studied as a cell–cell communication system, where small chemical signal molecules (acylhomoserine lactones, AHLs) can regulate the bacterial communications in bioelectrochemical systems via chemical signaling and electric signaling. In this study, electrochemical activity of bio-anode is substantially promoted by adding two kinds of AHLs with different chain length at the stage of community formation in microbial electrolysis cells (MECs). Hydrogen yield increase is observed by adding of two chain length AHLs, 3-oxo-hexanoyl-homoserine lactone (3OC6-HSL) and 3-oxo-dodecanoyl homoserine lactone (3OC12-HSL). A higher MEC current is acquired with addition of 3OC6-HSL than 3OC12-HSL at a fixed voltage of 0.8 V (vs. SHE). The highest yield is up to 3.8 ± 0.2 mol H2 mol−1 acetate at 10 μM 3OC6-HSL, which is increased 29% over control MECs. Evaluated on applied voltage, energy efficiency is increased to 171.6 ± 21.3% with short chain AHL, however, no significant improvement is performed on energy efficiency and coulombic efficiency with long-chain AHL. The study shows that bioelectrochemical characteristics of MECs varied on the chain length of AHL signal molecules and short-chain AHLs have a more positive effect on electron transfer and energy recovery in MECs.

Applications of thin-layer chromatography in extraction and characterisation of ajoene from garlic bulbs

Novel and inexpensive methods of thin-layer chromatography (TLC) were employed for the extraction, characterisation and mechanism of quorum sensing inhibition by ajoene, a component from toluene garlic bulb (Allium sativum L.) extract (TGE). TLC profiling of TGE was carried out using ethyl acetate as solvent. Out of total spots extracted from TLC, four spots exhibited quorum sensing inhibitory (QSI) potential. Among those, spot 5 was identified as Z-ajoene by TLC and confirmed by NMR and MS. HPLC analysis indicated 97.7% purity for purified ajoene. TLC densitometric analysis quantified 221.08 μmol/g of ajoene in TGE and indicated that ajoene is stable at 4°C and at acidic pH. HPTLC profiling showed that ajoene exhibits QSI effect by inhibiting the production of both long-chain acyl homoserine lactones and Pseudomonas quinolone signal (PQS) by P. aeruginosa and also by inactivating PQS molecules.

Rhizobial homologs of the fatty acid transporter FadL facilitate perception of long-chain acyl-homoserine lactone signals.

Quorum sensing (QS) using N-acyl homoserine lactones (AHLs) as signal molecules is a common strategy used by diverse Gram-negative bacteria. A widespread mechanism of AHL sensing involves binding of these molecules by cytosolic LuxR-type transcriptional regulators, which requires uptake of external AHLs. The outer membrane is supposed to be an efficient barrier for diffusion of long-chain AHLs. Here we report evidence that in Sinorhizobium meliloti, sensing of AHLs with acyl chains composed of 14 or more carbons is facilitated by the outer membrane protein FadLSm, a homolog of the Escherichia coli FadLEc long-chain fatty acid transporter. The effect of fadLSm on AHL sensing was more prominent for longer and more hydrophobic signal molecules. Using reporter gene fusions to QS target genes, we found that fadLSm increased AHL sensitivity and accelerated the course of QS. In contrast to FadLEc, FadLSm did not support uptake of oleic acid, but did contribute to growth on palmitoleic acid. FadLSm homologs from related symbiotic α-rhizobia and the plant pathogen Agrobacterium tumefaciens differed in their ability to facilitate long-chain AHL sensing or to support growth on oleic acid. FadLAt was found to be ineffective toward long-chain AHLs. We obtained evidence that the predicted extracellular loop 5 of FadLSm and further α-rhizobial FadL proteins contains determinants of specificity to long-chain AHLs. Replacement of a part of loop 5 by the corresponding region from α-rhizobial FadL proteins transferred sensitivity for long-chain AHLs to FadLAt.

Quorum Sensing Signalling and Biofilm Formation of Brewery-Derived Bacteria, and Inhibition of Signalling by Natural Compounds

Bacteria use quorum sensing signalling in various functions, e.g. while forming biofilms, and inhibition of this signalling could be one way to control biofilm formation. The aim of this study was to evaluate the production of signalling molecules and its correlation with the biofilm formation capability of bacteria isolated from brewery filling process. A further aim was to study berry extracts and wood-derived terpenes for their possible quorum sensing inhibitory effects. Out of the twenty bacteria studied, five produced short-chain and five long-chain AHL (acyl homoserine lactone) signalling molecules when tested with the Chromobacterium violaceum CV026 reporter bacterium. Production of AI-2 (autoinducer-2) signalling molecules was detected from nine strains with the Vibrio harveyi BB170 bioassay. Over half of the strains produced biofilm in the microtitre plate assay, but the production of AHL and AI-2 signalling molecules and biofilm formation capability did not directly correlate with each other. Out of the 13 berry extracts and wood-derived terpenes screened, four compounds decreased AHL signalling without effect on growth. These were betulin, raspberry extract and two cloudberry extracts. The effect of these compounds on biofilm formation of the selected six bacterial strains varied. The phenolic extract of freeze-dried cloudberry fruit caused a statistically significant reduction of biofilm formation of Obesumbacterium proteus strain. Further experiments should aim at identifying the active compounds and revealing whether quorum sensing inhibition causes structural changes in the biofilms formed.

Long-chain acylhomoserine lactones increase the anoxic ammonium oxidation rate in an OLAND biofilm

The oxygen-limited autotrophic nitrification/denitrification (OLAND) process comprises one-stage partial nitritation and anammox, catalyzed by aerobic and anoxic ammonium-oxidizing bacteria (AerAOB and AnAOB), respectively. The goal of this study was to investigate whether quorum sensing influences anoxic ammonium oxidation in an OLAND biofilm, with AnAOB colonizing 13% of the biofilm, as determined with fluorescent in situ hybridization (FISH). At high biomass concentrations, the specific anoxic ammonium oxidation rate of the OLAND biofilm significantly increased with a factor of 1.5 ± 0.2 compared to low biomass concentrations. Supernatant obtained from the biofilm showed no ammonium-oxidizing activity on itself, but its addition to low OLAND biomass concentrations resulted in a significant activity increase of the biomass. In the biofilm supernatant, the presence of long-chain acylhomoserine lactones (AHLs) was shown using the reporter strain Chromobacterium violaceum CV026, and one specific AHL, N-dodecanoyl homoserine lactone (C(12)-HSL), was identified via LC-MS/MS. Furthermore, C(12)-HSL was detected in an AnAOB-enriched community, but not in an AerAOB-enriched community. Addition of C(12)-HSL to low OLAND biomass concentrations resulted in a significantly higher ammonium oxidation rate (p < 0.05). To our knowledge, this is the first report demonstrating that AHLs enhance the anoxic ammonium oxidation process. Future work should confirm which species are responsible for the in situ production of C(12)-HSL in AnAOB-based applications.

Inhibition of Quorum Sensing Mechanism andAeromonas hydrophilaBiofilm Formation by Vanillin

High-quality drinking water is obtained using membrane technology. Biofilm development lowers filtration efficacy and eventually membranes must be replaced. In natural environments, a micro-organism produces different kinds of quorum sensing (QS) molecules resulting in biofilm formation and biofouling. Biological fouling is a major problem in desalination and water purification plants. Biofilm formation can be reduced using eco-friendly substances in the drinking water system based on quorum sensing inhibition (QSI). Vanillin (4-hydroxy-3-methoxybenzaldehyde), a well-known food flavoring agent, was studied for its QSI property against different individual acyl homoserine lactone (AHL) molecules using bio indicator strains. Vanillin showed significant inhibition in short-chain [C4-HSL (69%) and 3-Oxo-C8-HSL (59.8%)] and long-chain AHL molecules. QSI activity was tested on a reverse osmosis (RO) membrane biofilm bacteria Aeromonas hydrophila. Biofilm formation was inhibited up to 46.3% on polystyrene surface. Results suggested that Vanillin could be used as a potential QSI compound that reduces the biofilm formation on RO membranes.

Molecular insights into quorum sensing in Acidithiobacillus ferrooxidans bacteria via molecular modelling of the transcriptional regulator AfeR and of the binding mode of long-chain acyl homoserine lactones

Amino acid sequence alignments of the transcriptional regulator AfeR, which is involved in type 1 quorum sensing (QS) in Acidithiobacillus ferrooxidans bacteria, with other acyl homoserine lactone (AHL)-dependent QS regulators, revealed the presence of strictly or highly conserved residues located in the active site of these proteins. As a consequence, a model of AfeR was constructed to study the binding mode of long-chain AHLs using molecular dynamics and subsequent rigid ligand docking. This study, performed on the tetradecanoyl homoserine lactone C14-AHL, showed that the binding mode involved a curved conformation. Based on these results, the binding mode of tetradec-7-Z enoyl homoserine lactone, an AHL that is conformationally constrained due to the presence of the cis double bond, was investigated. This mono-unsaturated AHL with its preferential curved shape conformation was found to be particularly well adapted to the active site of AfeR. These results should be helpful in the rational design of QS modulators with potential biotechnological applications and especially in the improvement of industrial bioleaching from ores.

Direct binding of the quorum sensing regulator CepR of Burkholderia cenocepacia to two target promoters in vitro

Burkholderia cenocepacia is an opportunistic human pathogen that can aggressively colonize the cystic fibrosis lung. This organism has a LuxR/LuxI-type quorum sensing system that enables cell-cell communication via exchange of acyl homoserine lactones (AHLs). The CepR and CepI proteins constitute a global regulatory system, controlling expression of at least 40 genes, including those controlling swarming motility and biofilm formation. In this study, we isolated seven lacZ fusions in a clinical isolate of B. cenocepacia that are inducible by octanoyl-HSL. Induction of all of these genes requires CepR. The cepI promoter was tested for induction by a set of 33 synthetic autoinducers and analogues, and was most strongly induced by long-chain AHLs lacking 3-oxo substitutions. Expression of this promoter was inhibited by high concentrations of three different autoinducers, each having six-carbon acyl chains. When CepR protein was overproduced in Escherichia coli, it accumulated in a soluble form in the presence of octanoyl-HSL, but accumulated only as insoluble inclusion bodies in its absence. Purified CepR-OHL complexes bound to specific DNA sequences at the cepI and aidA promoters with high specificity. These binding sites included a 16-nucleotide imperfect dyad symmetry. Both CepR binding sites are centred approximately 44 nucleotides upstream of the respective transcription start sites.

Long-chain acyl-homoserine lactone quorum-sensing regulation of Rhodobacter capsulatus gene transfer agent production.

Many proteobacteria use acyl-homoserine lactones as quorum-sensing signals. Traditionally, biological detection systems have been used to identify bacteria that produce acyl-homoserine lactones, although the specificities of these detection systems can limit discovery. We used a sensitive approach that did not require a bioassay to detect production of long-acyl-chain homoserine lactone production by Rhodobacter capsulatus and Paracoccus denitrificans. These long-chain acyl-homoserine lactones are not readily detected by standard bioassays. The most abundant acyl-homoserine lactone was N-hexadecanoyl-homoserine lactone. The long-chain acyl-homoserine lactones were concentrated in cells but were also found in the culture fluid. An R. capsulatus gene responsible for long-chain acyl-homoserine lactone synthesis was identified. A mutation in this gene, which we named gtaI, resulted in decreased production of the R. capsulatus gene transfer agent, and gene transfer agent production was restored by exogenous addition of N-hexadecanoyl-homoserine lactone. Thus, long-chain acyl-homoserine lactones serve as quorum-sensing signals to enhance genetic exchange in R. capsulatus.