Biomineralization and AHLs-guided quorum sensing enhanced phosphorus recovery in the alternating aerobic/anaerobic biofilm system under metal ion stress.

The alternating aerobic/anaerobic biofilm system had been applied for phosphorus (P) enrichment and recovery because of the advantage of low energy consumption and high efficiency. The metal ions and N-acyl-L-homoserine lactones (AHLs) in system were studied to better clarify the mechanism of P uptake/release under metal ion stress. The results indicated that the increase of metal ions stimulated the release of AHLs, and AHLs-guided quorum sensing (QS) enhanced P uptake. Moreover, biomineralization could stimulate the increase of P content in biofilm (Pbiofilm). Meanwhile, some ortho-p was converted to short-chain poly-p in extracellular polymer substance (EPS), and others were transferred into cell through EPS to synthesize poly-p. With the Pbiofilm increased, more P could be absorbed/released due to the shift in the metabolic model of polyphosphate accumulating organisms (PAOs). The release of AHLs between microorganisms was also inhibited when PAOs reached the state of P saturation (75.6 ± 2.5 mg/g SS), which meant that the effect of signaling function would tend to stabilize, and the 169.2 ± 2.6 mg/L P concentration in the enriched solution was obtained due to the P release was inhibited. Moreover, P was rapidly transferred to the new enriched solution after the P was recovered, and PAOs restored its capability of P uptake/release. In addition, 31P-NMR analysis demonstrated that EPS played a major role in PAOs compared to cell, and inorganic phosphorus (IP) played an essential role in the uptake/release of P compared to organic phosphorus (OP). Furthermore, the microbiological analysis showed that Candidatus Accumulibacter was positively correlated with AHLs (P < 0.05). This study provided essential support for clarifying the P metabolism mechanism of PAOs.

Influence of glucose on swarming and quorum sensing of Dickeya solani.

Dickeya solani is a pathogen most frequently responsible for infecting potato plants in Europe. As in the case of most plant pathogens, its ability to colonize and invade the host depends on chemotaxis and motility. The coordinated movement of Dickeya over solid surfaces is governed by a quorum sensing mechanism. In D. solani motility is regulated by ExpI-ExpR proteins, homologous to luxI-luxR system from Vibrio fisheri, in which N-acyl-homoserine lactones (AHLs) serve as signaling molecules. Moreover, in many Gram-negative bacteria motility is coupled with central metabolism via carbon catabolite repression. This enables them to reach more nutrient-efficient niches. The aim of this study was to analyze the swarming motility of D. solani depending on the volume of the medium in the cultivation plate and glucose content. We show that the ability of this bacterium to move is strictly dependent on both these factors. Moreover, we analyze the production of AHLs and show that the quorum sensing mechanism in D. solani is also influenced by the availability of glucose in the medium and that the distribution of these signaling molecules are different depending on the volume of the medium in the plate.

N-Heterocycles Scaffolds as Quorum Sensing Inhibitors. Design, Synthesis, Biological and Docking Studies.

Quorum sensing is a communication system among bacteria to sense the proper time to express their virulence factors. Quorum sensing inhibition is a therapeutic strategy to block bacterial mechanisms of virulence. The aim of this study was to synthesize and evaluate new bioisosteres of N-acyl homoserine lactones as Quorum sensing inhibitors in Chromobacterium violaceum CV026 by quantifying the specific production of violacein. Five series of compounds with different heterocyclic scaffolds were synthesized in good yields: thiazoles, 16a-c, thiazolines 17a-c, benzimidazoles 18a-c, pyridines 19a-c and imidazolines 32a-c. All 15 compounds showed activity as Quorum sensing inhibitors except 16a. Compounds 16b, 17a-c, 18a, 18c, 19c and 32b exhibited activity at concentrations of 10 µM and 100 µM, highlighting the activity of benzimidazole 18a (IC50 = 36.67 µM) and 32b (IC50 = 85.03 µM). Pyridine 19c displayed the best quorum sensing inhibition activity (IC50 = 9.66 µM). Molecular docking simulations were conducted for all test compounds on the Chromobacterium violaceum CviR protein to gain insight into the process of quorum sensing inhibition. The in-silico data reveal that all 15 the compounds have higher affinity for the protein than the native AHL ligand (1). A strong correlation was found between the theoretical and experimental results.

Cultivable microbiota associated with Aurelia aurita and Mnemiopsis leidyi.

The associated microbiota of marine invertebrates plays an important role to the host in relation to fitness, health, and homeostasis. Cooperative and competitive interactions between bacteria, due to release of, for example, antibacterial substances and quorum sensing (QS)/quorum quenching (QQ) molecules, ultimately affect the establishment and dynamics of the associated microbial community. Aiming to address interspecies competition of cultivable microbes associated with emerging model species of the basal animal phyla Cnidaria (Aurelia aurita) and Ctenophora (Mnemiopsis leidyi), we performed a classical isolation approach. Overall, 84 bacteria were isolated from A. aurita medusae and polyps, 64 bacteria from M. leidyi, and 83 bacteria from ambient seawater, followed by taxonomically classification by 16S rRNA gene analysis. The results show that A. aurita and M. leidyi harbor a cultivable core microbiome consisting of typical marine ubiquitous bacteria also found in the ambient seawater. However, several bacteria were restricted to one host suggesting host-specific microbial community patterns. Interbacterial interactions were assessed by (a) a growth inhibition assay and (b) QS interference screening assay. Out of 231 isolates, 4 bacterial isolates inhibited growth of 17 isolates on agar plates. Moreover, 121 of the 231 isolates showed QS-interfering activities. They interfered with the acyl-homoserine lactone (AHL)-based communication, of which 21 showed simultaneous interference with autoinducer 2. Overall, this study provides insights into the cultivable part of the microbiota associated with two environmentally important marine non-model organisms and into interbacterial interactions, which are most likely considerably involved in shaping a healthy and resilient microbiota.

Growth stimulatory effect of AHL producing Serratia spp. from potato on homologous and non-homologous host plants.

Plant growth promoting rhizobacteria are known to improve plant performance by developing healthy and productive interactions with the host plants. These associations may be symbiotic or asymbiotic depending upon the genetic potential of the resident microbe and promiscuity of the host. Present study describes the potential of two Serratia spp. strains for promotion of plant growth in homologous as well as non-homologous hosts. The strains KPS-10 and KPS-14; native to potato rhizosphere belong to genus Serratia based on 16S rRNA gene sequences (accession no. LN831934 and LN831937 respectively) and contain multiple plant growth promoting properties along-with the production of quorum sensing acyl homoserine lactone (AHL) molecules. Both Serratia spp. strains showed solubilization of inorganic tri-calcium phosphate while KPS-14 also exhibited phytase activity (1.98 10-10 kcat). KPS-10 showed higher P-solubilization activity (128.5 µg/mL), IAA production (8.84 µg/mL), antifungal activity and also showed the production of two organic acids i.e., gluconic acid and lactic acid. Both strains produced three common AHLs: C6-HSL, 3oxo-C10-HSL, 3oxo-C12-HSL while some strain-specific AHLs (3OH-C5-HSL, 3OH-C6-HSL, C10-HSL specific to KPS-10 and 3OH-C6-HSL, C8-HSL, 3oxo-C9-HSL, 3OH-C9-HSL specific to KPS-14). Strains showed roots and rhizosphere colonization of potato and other non-homologous hosts up to one month. In planta AHLs-detection confirmed a likely role of AHLs during seedling growth and development where both extracted AHLs or bacteria inoculated roots showed extensive root hair. A significant increase in root/shoot lengths, root/ shoot fresh weights, root/shoot dry weights was observed by inoculation in different hosts. PGP-characteristics along with the AHLs-production signify the potential of both strains as candidate for the development of bio-inoculum for potato crop in specific and other crops in general. This inoculum will not only reduce the input of chemical fertilizer to the environment but also improve soil quality and plant growth.

Close-up on a bacterial informational war in the geocaulosphere.

The geocaulosphere is home to microbes that establish communication between themselves and others that disrupt them. These cell-to-cell communication systems are based on the synthesis and perception of signaling molecules, of which the best known belong to the N-acyl-homoserine lactone (AHL) family. Among indigenous bacteria, certain Gram-positive actinobacteria can sense AHLs produced by soft-rot Gram-negative phytopathogens and can degrade the quorum-sensing AHL signals to impair the expression of virulence factors. We mimicked this interaction by introducing dual-color reporter strains suitable for monitoring both the location of the cells and their quorum-sensing and -quenching activities, in potato tubers. The exchange of AHL signals within the pathogen's cell quorum was clearly detected by the presence of bright green fluorescence instead of blue in a portion of Pectobacterium-tagged cells. This phenomenon in Rhodococcus cells was accompanied by a change from red fluorescence to orange, showing that the disappearance of signaling molecules is due to rhodococcal AHL degradation rather than the inhibition of AHL production. Rhodococci are victorious in this fight for the control of AHL-based communication, as their jamming activity is powerful enough to prevent the onset of disease symptoms.

Potent modulation of the CepR quorum sensing receptor and virulence in a Burkholderia cepacia complex member using non-native lactone ligands.

The Burkholderia cepacia complex (Bcc) is a family of closely related bacterial pathogens that are the causative agent of deadly human infections. Virulence in Bcc species has been shown to be controlled by the CepI/CepR quorum sensing (QS) system, which is mediated by an N-acyl L-homoserine lactone (AHL) signal (C8-AHL) and its cognate LuxR-type receptor (CepR). Chemical strategies to block QS in Bcc members would represent an approach to intercept this bacterial communication process and further delineate its role in infection. In the current study, we sought to identify non-native AHLs capable of agonizing or antagonizing CepR, and thereby QS, in a Bcc member. We screened a library of AHL analogs in cell-based reporters for CepR, and identified numerous highly potent CepR agonists and antagonists. These compounds remain active in a Bcc member, B. multivorans, with one agonist 250-fold more potent than the native ligand C8-AHL, and can affect QS-controlled motility. Further, the CepR antagonists prolong C. elegans survival in an infection model. These AHL analogs are the first reported non-native molecules that both directly modulate CepR and impact QS-controlled phenotypes in a Bcc member, and represent valuable chemical tools to assess the role of QS in Bcc infections.

Stenotrophomonas maltophilia AHL-Degrading Strains Isolated from Marine Invertebrate Microbiota Attenuate the Virulence of Pectobacterium carotovorum and Vibrio coralliilyticus.

Many Gram-negative aquacultural and agricultural pathogens control virulence factor expression through a quorum-sensing (QS) mechanism involving the production of N-acylhomoserine (AHL) signalling molecules. Thus, the interruption of QS systems by the enzymatic degradation of signalling molecules, known as quorum quenching (QQ), has been proposed as a novel strategy to combat these infections. Given that the symbiotic bacteria of marine invertebrates are considered to be an important source of new bioactive molecules, this study explores the presence of AHL-degrading bacteria among 827 strains previously isolated from the microbiota of anemones and holothurians. Four of these strains (M3-1, M1-14, M3-13 and M9-54-2), belonging to the species Stenotrophomonas maltophilia, were selected on the basis of their ability to degrade a broad range of AHLs, and the enzymes involved in their activity were identified. Strain M9-54-2, which showed the strongest AHL-degrading activity, was selected for further study. High-performance liquid chromatography-mass-spectrometry confirmed that the QQ enzyme is not a lactonase. Strain M9-54-2 degraded AHL accumulation and reduced the production of enzymatic activity in Pectobacterium carotovorum CECT 225T and Vibrio coralliilyticus VibC-Oc-193 in in vitro co-cultivation experiments. The effect of AHL inactivation was confirmed by a reduction in potato tuber maceration and brine shrimp (Artemia salina) mortality caused by P. carotovorum and Vibrio coralliilyticus, respectively. This study strengthens the evidence of marine organisms as an underexplored and promising source of QQ enzymes, useful to prevent infections in aquaculture and agriculture. To our knowledge, this is the first time that anemones and holothurians have been studied for this purpose.

Virtual screening of plant compounds and nonsteroidal anti-inflammatory drugs for inhibition of quorum sensing and biofilm formation in Salmonella.

Salmonella belongs to the Enterobacteriaceae family which is widely distributed in the environment due to its adaptive capacity to stress conditions. In addition, Salmonella is able to perform a type of cell-to-cell communication called quorum sensing, which leads to differential gene expression. The quorum sensing system mediated by AI-1, acyl homoserine lactones (AHLs), is incomplete in Salmonella because the luxI homolog gene, which encodes for AI-1 synthase, is missing in the genome. However, a homologue of LuxR, known as SdiA, is present and allows the detection of signaling molecules produced by other species of bacteria, leading to regulation of gene expression, mainly related to virulence and biofilm formation. Thus, in view of the importance of quorum sensing on the physiology regulation of microorganisms, the aim of the present study was to perform a virtual screening of plant compounds and nonsteroidal anti-inflammatory drugs (NASIDs) for inhibition of quorum sensing by molecular docking and biofilm formation in Salmonella. In general, most plant compounds and all NSAIDs bound in, at least, one of the three modeled structures of SdiA proteins of Salmonella Enteritidis PT4 578. In addition, many tested compounds had higher binding affinities than the AHLs and the furanones which are inducers and inhibitors of quorum sensing, respectively. The Z-phytol and lonazolac molecules were good candidates for the in vitro inhibition tests of quorum sensing mediated by AI-1 and biofilm formation in Salmonella. Thus, this study directs future prospecting of plant extracts for inhibition of quorum sensing mechanism depending on AHL and biofilm formation. In addition, the use of inhibitors of quorum sensing and biofilm formation can be combined with antibiotics for better treatment efficacy, as well as the use of these compounds to design new drugs.

Purification and characterisation of a quorum quenching AHL-lactonase from the endophytic bacterium Enterobacter sp. CS66.

The quorum quenching (QQ) activity of endophytic bacteria associated with medicinal plants was explored. Extracts of the Gram-negative Enterobacter sp. CS66 possessed potent N-acylhomoserine lactone (AHL) hydrolytic activity in vitro. Using degenerate primers, we PCR-amplified an open reading frame (denoted aiiE) from CS66 that was 96% identical to the well-characterised AHL-lactonase AiiA from Bacillus thuringiensis, but only 30% was identical to AHL-lactonases from other Gram-negative species. This confirms that close AiiA homologs can be found in both Gram-positive and Gram-negative bacteria. Purified AiiE exhibited potent AHL-lactonase activity against a broad range of AHLs. Furthermore, aiiE was able to reduce the production of secreted plant cell wall-degrading hydrolytic enzymes when expressed in trans in the economically important plant pathogen, Pectobacterium atrosepticum. Our results indicate the presence of a novel AHL-lactonase in Enterobacter sp. CS66 with significant potential as a biocontrol agent.

Attenuation and quantitation of virulence gene expression in quorum-quenched Dickeya chrysanthemi.

N-Acyl-homoserine lactones (AHLs)-dependent quorum sensing (QS) system(s) is recruited by the soft rot bacterium Dickeya chrysanthemi for coordinating its social activities such as secretion of plant cell wall-degrading enzymes, while the main signal molecule and quantity dependence of virulence to QS in this bacterium have not been clarified. To do this end, the involvement of AHLs in African violet leaves and potato tuber maceration; swarming motility; pectate lyase and polygalacturonase enzymes production and in planta expression of virulence genes including pelE, pehX and pemA by electroporating two quorum-quenching vectors. The expression of two types of AHL-lactonase expressing vector caused dramatic decrease in swarming motility, production of pectinolytic enzymes and macerating of plant tissues. The maximum ability of quenching of QS in repression of D. chrysanthemi virulence was assessed quantitatively by q-RT-PCR, as expression of pelE, pehX and pemA genes were decreased 90.5-92.18 % in quenched cells. We also showed that virulence and pathogenicity of this bacterium was under the control of DHL-dependent QS system and that the existence of second DHL operating system is probable for this bacterium. Thus, this signal molecule would be the key point for future research to design DHL-specific lactonase enzymes using bioinformatics methods.

Diarylheptanoids from Alnus viridis ssp. viridis and Alnus glutinosa: Modulation of Quorum Sensing Activity in Pseudomonas aeruginosa.

Diarylheptanoids from the barks of Alnus viridis ssp. viridis (green alder) and Alnus glutinosa (black alder) were explored for anti-quorum sensing activity. Chemicals with anti-quorum sensing activity have recently been examined for antimicrobial applications. The anti-quorum sensing activity of the selected diarylheptanoids was determined using two biosensors, namely Pseudomonas aeruginosa PAO1 and Chromobacterium violaceum CV026. Although all of the investigated compounds negatively influenced the motility of P. aeruginosa PAO1, four were able to inhibit biofilm formation of this human opportunistic pathogen for 40-70 %. Three of the diarylheptanoids (3, 4, and 5) negatively influenced the biosynthesis of pyocyanin, which is under the control of quorum sensing. Platyphyllenone (7) and hirsutenone (5) were able to inhibit the biosynthesis of violacein in C. violaceum CV026, with 5 being able to inhibit the synthesis of both biopigments. Only one of the tested diarylheptanoids (1) was shown to significantly decrease the production of acyl homoserine lactones (AHL) in P. aeruginosa PAO1, more specifically, production of the long chain N-(3-oxododecanoyl)-l-HSL. On the other side, four diarylheptanoids (2-5) significantly reduced the synthesis of 2-alkyl-4-quinolones, part of the P. aeruginosa quinolone-mediated signaling system. To properly assess therapeutic potential of these compounds, their in vitro antiproliferative effect on normal human lung fibroblasts was determined, with doses affecting cell proliferation between 10 and 100 µg/mL. This study confirms that the barks of green and black alders are rich source of phytochemicals with a wide range of biological activities that could further be exploited as natural agents against bacterial contaminations and infections.

Cranberry-derived proanthocyanidins impair virulence and inhibit quorum sensing of Pseudomonas aeruginosa.

Bacteria have evolved multiple strategies for causing infections that include producing virulence factors, undertaking motility, developing biofilms, and invading host cells. N-acylhomoserine lactone (AHL)-mediated quorum sensing (QS) tightly regulates the expression of multiple virulence factors in the opportunistic pathogenic bacterium Pseudomonas aeruginosa. Thus, inhibiting QS could lead to health benefits. In this study, we demonstrate an anti-virulence activity of a cranberry extract rich in proanthocyanidins (cerPAC) against P. aeruginosa in the model host Drosophila melanogaster and show this is mediated by QS interference. cerPAC reduced the production of QS-regulated virulence determinants and protected D. melanogaster from fatal infection by P. aeruginosa PA14. Quantification of AHL production using liquid chromatography-mass spectrometry confirmed that cerPAC effectively reduced the level of AHLs produced by the bacteria. Furthermore, monitoring QS signaling gene expression revealed that AHL synthases LasI/RhlI and QS transcriptional regulators LasR/RhlR genes were inhibited and antagonized, respectively, by cerPAC. Molecular docking studies suggest that cranberry-derived proanthocyanidin binds to QS transcriptional regulators, mainly interacting with their ligand binding sites. These findings provide insights into the underlying mechanisms of action of a cerPAC to restrict the virulence of P. aeruginosa and can have implications in the development of alternative approaches to control infections.

Pseudomonas aeruginosa Biofilm Formation and Persistence, along with the Production of Quorum Sensing-Dependent Virulence Factors, Are Disrupted by a Triterpenoid Coumarate Ester Isolated from Dalbergia trichocarpa, a Tropical Legume.

Recently, extracts of Dalbergia trichocarpa bark have been shown to disrupt P. aeruginosa PAO1 quorum sensing (QS) mechanisms, which are key regulators of virulence factor expression and implicated in biofilm formation. One of the active compounds has been isolated and identified as oleanolic aldehyde coumarate (OALC), a novel bioactive compound that inhibits the formation of P. aeruginosa PAO1 biofilm and its maintenance as well as the expression of the las and rhl QS systems. Consequently, the production of QS-controlled virulence factors including, rhamnolipids, pyocyanin, elastase and extracellular polysaccharides as well as twitching and swarming motilities is reduced. Native acylhomoserine lactones (AHLs) production is inhibited by OALC but exogenous supply of AHLs does not restore the production of virulence factors by OALC-treated cultures, indicating that OALC exerts its effect beyond AHLs synthesis in the QS pathways. Further experiments provided a significant inhibition of the global virulence factor activator gacA by OALC. OALC disorganizes established biofilm structure and improves the bactericidal activity of tobramycin against biofilm-encapsulated PAO1 cells. Finally, a significant reduction of Caenorhabditis elegans paralysis was recorded when the worms were infected with OALC-pre-treated P. aeruginosa. Taken together, these results show that triterpenoid coumarate esters are suitable chemical backbones to target P. aeruginosa virulence mechanisms.

Colostrum hexasaccharide, a novel Staphylococcus aureus quorum-sensing inhibitor.

The discovery of quorum-sensing (QS) systems regulating antibiotic resistance and virulence factors (VFs) has afforded a novel opportunity to prevent bacterial pathogenicity. Dietary molecules have been demonstrated to attenuate QS circuits of bacteria. But, to our knowledge, no study exploring the potential of colostrum hexasaccharide (CHS) in regulating QS systems has been published. In this study, we analyzed CHS for inhibiting QS signaling in Staphylococcus aureus. We isolated and characterized CHS from mare colostrum by high-performance thin-layer chromatography (HPTLC), reverse-phase high-performance liquid chromatography evaporative light-scattering detection (RP-HPLC-ELSD), (1)H and (13)C nuclear magnetic resonance (NMR), and electrospray ionization mass spectrometry (ESI-MS). Antibiofilm activity of CHS against S. aureus and its possible interference with bacterial QS systems were determined. The inhibition and eradication potentials of the biofilms were studied by microscopic analyses and quantified by 96-well-microtiter-plate assays. Also, the ability of CHS to interfere in bacterial QS by degrading acyl-homoserine lactones (AHLs), one of the most studied signal molecules for Gram-negative bacteria, was evaluated. The results revealed that CHS exhibited promising inhibitory activities against QS-regulated secretion of VFs, including spreading ability, hemolysis, protease, and lipase activities, when applied at a rate of 5 mg/ml. The results of biofilm experiments indicated that CHS is a strong inhibitor of biofilm formation and also has the ability to eradicate it. The potential of CHS to interfere with bacterial QS systems was also examined by degradation of AHLs. Furthermore, it was documented that CHS decreased antibiotic resistance in S. aureus. The results thus give a lead that mare colostrum can be a promising source for isolating a next-generation antibacterial.

Transcriptome of the quorum-sensing signal-degrading Rhodococcus erythropolis responds differentially to virulent and avirulent Pectobacterium atrosepticum.

Social bacteria use chemical communication to coordinate and synchronize gene expression via the quorum-sensing (QS) regulatory pathway. In Pectobacterium, a causative agent of the blackleg and soft-rot diseases on potato plants and tubers, expression of the virulence factors is collectively controlled by the QS-signals N-acylhomoserine lactones (NAHLs). Several soil bacteria, such as the actinobacterium Rhodococcus erythropolis, are able to degrade NAHLs, hence quench the chemical communication and virulence of Pectobacterium. Here, next-generation sequencing was used to investigate structural and functional genomics of the NAHL-degrading R. erythropolis strain R138. The R. erythropolis R138 genome (6.7 Mbp) contained a single circular chromosome, one linear (250 kbp) and one circular (84 kbp) plasmid. Growth of R. erythropolis and P. atrosepticum was not altered in mixed-cultures as compared with monocultures on potato tuber slices. HiSeq-transcriptomics revealed that no R. erythropolis genes were differentially expressed when R. erythropolis was cultivated in the presence vs absence of the avirulent P. atrosepticum mutant expI, which is defective for QS-signal synthesis. By contrast 50 genes (<1% of the R. erythropolis genome) were differentially expressed when R. erythropolis was cultivated in the presence vs absence of the NAHL-producing virulent P. atrosepticum. Among them, quantitative real-time reverse-transcriptase-PCR confirmed that the expression of some alkyl-sulfatase genes decreased in the presence of a virulent P. atrosepticum, as well as deprivation of organic sulfur such as methionine, which is a key precursor in the synthesis of NAHL by P. atrosepticum.

Evidence of autoinducer-dependent and -independent heterogeneous gene expression in Sinorhizobium fredii NGR234.

Populations of genetically identical Sinorhizobium fredii NGR234 cells differ significantly in their expression profiles of autoinducer (AI)-dependent and AI-independent genes. Promoter fusions of the NGR234 AI synthase genes traI and ngrI showed high levels of phenotypic heterogeneity during growth in TY medium on a single-cell level. However, adding very high concentrations of N-(3-oxooctanoyl-)-l-homoserine lactone resulted in a more homogeneous expression profile. Similarly, the lack of internally synthesized AIs in the background of the NGR234-ΔtraI or the NGR234-ΔngrI mutant resulted in a highly homogenous expression of the corresponding promoter fusions in the population. Expression studies with reporter fusions of the promoter regions of the quorum-quenching genes dlhR and qsdR1 and the type IV pilus gene cluster located on pNGR234b suggested that factors other than AI molecules affect NGR234 phenotypic heterogeneity. Further studies with root exudates and developing Arabidopsis thaliana seedlings provide the first evidence that plant root exudates have strong effects on the heterogeneity of AI synthase and quorum-quenching genes in NGR234. Therefore, plant-released octopine appears to play a key role in modulation of heterogeneous gene expression.

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.

Elastase LasB of Pseudomonas aeruginosa promotes biofilm formation partly through rhamnolipid-mediated regulation.

Elastase LasB, an important extracellular virulence factor, is shown to play an important role in the pathogenicity of Pseudomonas aeruginosa during host infection. However, the role of LasB in the life cycle of P. aeruginosa is not completely understood. This report focuses on the impact of LasB on biofilm formation of P. aeruginosa PAO1. Here, we reported that the lasB deletion mutant (ΔlasB) displayed significantly decreased bacterial attachment, microcolony formation, and extracellular matrix linkage in biofilm associated with decreased biosynthesis of rhamnolipids compared with PAO1 and lasB complementary strain (ΔlasB(+)). Nevertheless, the ΔlasB developed restored biofilm formation with supplementation of exogenous rhamnolipids. Further gene expression analysis revealed that the mutant of lasB could result in the downregulation of rhamnolipid synthesis at the transcriptional level. Taken together, these results indicated that LasB could promote biofilm formation partly through the rhamnolipid-mediated regulation.

Anti-quorum sensing activity of Psidium guajava L. flavonoids against Chromobacterium violaceum and Pseudomonas aeruginosa PAO1.

Psidium guajava L., which has been used traditionally as a medicinal plant, was explored for anti-quorum sensing (QS) activity. The anti-QS activity of the flavonoid (FL) fraction of P. guajava leaves was determined using a biosensor bioassay with Chromobacterium violaceum CV026. Detailed investigation of the effects of the FL-fraction on QS-regulated violacein production in C. violaceum ATCC12472 and pyocyanin production, proteolytic, elastolytic activities, swarming motility and biofilm formation in Pseudomonas aeruginosa PAO1 was performed using standard methods. Possible mechanisms of QS-inhibition were studied by assessing violacein production in response to N-acyl homoserine lactone (AHL) synthesis in the presence of the FL-fraction in C. violaceum ATCC31532 and by evaluating the induction of violacein in the mutant C. violaceum CV026 by AHL extracted from the culture supernatants of C. violaceum 31532. Active compounds in the FL-fraction were identified by liquid chromatography-mass spectrometry (LC-MS). Inhibition of violacein production by the FL-fraction in a C. violaceum CV026 biosensor bioassay indicated possible anti-QS activity. The FL-fraction showed concentration-dependent decreases in violacein production in C. violaceum 12472 and inhibited pyocyanin production, proteolytic and elastolytic activities, swarming motility and biofilm formation in P. aeruginosa PAO1. Interestingly, the FL-fraction did not inhibit AHL synthesis; AHL extracted from cultures of C. violaceum 31532 grown in the presence of the FL-fraction induced violacein in the mutant C. violaceum CV026. LC-MS analysis revealed the presence of quercetin and quercetin-3-O-arabinoside in the FL-fraction. Both quercetin and quercetin-3-O-arabinoside inhibited violacein production in C. violaceum 12472, at 50 and 100 µg/mL, respectively. Results of this study provide scope for further research to exploit these active molecules as anti-QS agents.