Quorum Sensing Response Research Articles

Surfactin: A Quorum-Sensing Signal Molecule to Relieve CCR in Bacillus amyloliquefaciens.

Bacillus utilize preferred sugars such as glucose over other carbon sources due to carbon catabolite repression (CCR). Surfactin is a small signal molecule to regulate the quorum-sensing (QS) response such as biofilm formation and sporulation in B. subtilis. Here, the srfA operon for synthesis of surfactin was mutated for disrupting the production of surfactin in B. amyloliquefaciens. The srfA-mutant strain showed a defective biofilm and sporulation but could be restored by addition with surfactin, indicating that surfactin is a QS signal molecule in B. amyloliquefaciens. Unexpectedly, mutation of srfA also led to the cells’ death although nutrients were still enough to support the bacterial growth during this period. Analysis of transcriptomes found that the srfA-mutant strain could not relieve CCR to use non-preferred carbon sources after glucose exhaustion due to deficiency of surfactin. This was further verified by the fact that addition with glucose could dramatically restore the growth, and addition with surfactin could improve the enzymes’ activity (e.g., glucanase and α-amylase) to use non-preferred carbon sources in the srfA-mutant strain. After glucose exhaustion, the cells produce surfactin to relieve CCR for utilizing non-preferred sugars. As a signal molecule to regulate QS, surfactin also directly or indirectly relieves the CcpA-mediated CCR to utilize non-preferred carbon sources countering nutrient limitation (e.g., glucose deprivation) in the environment. In conclusion, our findings provide the first evidence that the QS signal molecule of surfactin is also involved in relieving the CcpA-mediated CCR in B. amyloliquefaciens.

Naringenin Inhibition of the Pseudomonas aeruginosa Quorum Sensing Response Is Based on Its Time-Dependent Competition With N-(3-Oxo-dodecanoyl)-L-homoserine Lactone for LasR Binding.

Bacterial quorum sensing (QS) is a cell-to-cell communication system that governs the expression of a large set of genes involved in bacterial–host interactions, including the production of virulence factors. Conversely, the hosts can produce anti-QS compounds to impair virulence of bacterial pathogens. One of these inhibitors is the plant flavonoid naringenin, which impairs the production of QS-regulated Pseudomonas aeruginosa virulence factors. In the present work, we analyze the molecular basis for such inhibition. Our data indicate that naringenin produces its effect by directly binding the QS regulator LasR, hence competing with its physiological activator, N-(3-oxo-dodecanoyl)-L-homoserine lactone (3OC12-HSL). The in vitro analysis of LasR binding to its cognate target DNA showed that the capacity of naringenin to outcompete 3OC12-HSL, when the latter is previously bound to LasR, is low. By using an E. coli LasR-based biosensor strain, which does not produce 3OC12-HSL, we determined that the inhibition of LasR is more efficient when naringenin binds to nascent LasR than when this regulator is already activated through 3OC12-HSL binding. According to these findings, at early exponential growth phase, when the amount of 3OC12-HSL is low, naringenin should proficiently inhibit the P. aeruginosa QS response, whereas at later stages of growth, once 3OC12-HSL concentration reaches a threshold enough for binding LasR, naringenin would not efficiently inhibit the QS response. To test this hypothesis, we analyze the potential effect of naringenin over the QS response by adding naringenin to P. aeruginosa cultures at either time zero (early inhibition) or at stationary growth phase (late inhibition). In early inhibitory conditions, naringenin inhibited the expression of QS-regulated genes, as well as the production of the QS-regulated virulence factors, pyocyanin and elastase. Nevertheless, in late inhibitory conditions, the P. aeruginosa QS response was not inhibited by naringenin. Therefore, this time-dependent inhibition may compromise the efficiency of this flavonoid, which will be effective just when used against bacterial populations presenting low cellular densities, and highlight the importance of searching for QS inhibitors whose mechanism of action does not depend on the QS status of the population.

Parallel quorum-sensing system in Vibrio cholerae prevents signal interference inside the host.

Many bacteria use quorum sensing (QS) to regulate virulence factor production in response to changes in population density. QS is mediated through the production, secretion, and detection of signaling molecules called autoinducers (AIs) to modulate population-wide behavioral changes. Four histidine kinases, LuxPQ, CqsS, CqsR and VpsS, have been identified in Vibrio cholerae as QS receptors to activate virulence gene expression at low cell density. Detection of AIs by these receptors leads to virulence gene repression at high cell density. The redundancy among these receptors is puzzling since any one of the four receptors is sufficient to support colonization of V. cholerae in the host small intestine. It is believed that one of the functions of such circuit architecture is to prevent interference on any single QS receptor. However, it is unclear what natural molecules can interfere V. cholerae QS and in what environment interference is detrimental. We show here mutants expressing only CqsR without the other three QS receptors are defective in colonizing the host large intestine. We identified ethanolamine, a common intestinal metabolite that can function as a chemical source of QS interference. Ethanolamine specifically interacts with the ligand-binding CACHE domain of CqsR and induces a premature QS response in V. cholerae mutants expressing only CqsR without the other three QS receptors. The effect of ethanolamine on QS gene expression and host colonization is abolished by mutations that disrupt CqsR signal sensing. V. cholerae defective in producing ethanolamine is still proficient in QS, therefore, ethanolamine functions only as an external cue for CqsR. Our findings suggest the inhibitory effect of ethanolamine on CqsR could be a possible source of QS interference but is masked by the presence of the other parallel QS pathways, allowing V. cholerae to robustly colonize the host.

Surface association sensitizes Pseudomonas aeruginosa to quorum sensing

In the pathogen Pseudomonas aeruginosa, LasR is a quorum sensing (QS) master regulator that senses the concentration of secreted autoinducers as a proxy for bacterial cell density. Counterintuitively, previous studies showed that saturating amounts of the LasR ligand, 3OC12-HSL, fail to induce the full LasR regulon in low-density liquid cultures. Here we demonstrate that surface association, which is necessary for many of the same group behaviors as QS, promotes stronger QS responses. We show that lasR is upregulated upon surface association, and that surface-associated bacteria induce LasR targets more strongly in response to autoinducer than planktonic cultures. This increased sensitivity may be due to surface-dependent lasR induction initiating a positive feedback loop through the small RNA, Lrs1. The increased sensitivity of surface-associated cells to QS is affected by the type IV pilus (TFP) retraction motors and the minor pilins. The coupling of physical surface responses and chemical QS responses could enable these bacteria to trigger community behaviors more robustly when they are more beneficial.

Integrated Transcriptional Regulatory Network of Quorum Sensing, Replication Control, and SOS Response in Dinoroseobacter shibae.

Quorum sensing (QS) coordinates population wide gene expression of bacterial species. Highly adaptive traits like gene transfer agents (GTA), morphological heterogeneity, type 4 secretion systems (T4SS), and flagella are QS controlled in Dinoroseobacter shibae, a Roseobacter model organism. Its QS regulatory network is integrated with the CtrA phosphorelay that controls cell division in alphaproteobacteria. To elucidate the network topology, we analyzed the transcriptional response of the QS-negative D. shibae strain ΔluxI1 toward externally added autoinducer (AI) over a time period of 3 h. The signaling cascade is initiated by the CtrA phosphorelay, followed by the QS genes and other target genes, including the second messenger c-di-GMP, competence, flagella and pili. Identification of transcription factor binding sites in promoters of QS induced genes revealed the integration of QS, CtrA phosphorelay and the SOS stress response mediated by LexA. The concentration of regulatory genes located close to the origin or terminus of replication suggests that gene regulation and replication are tightly coupled. Indeed, addition of AI first stimulates and then represses replication. The restart of replication comes along with increased c-di-GMP levels. We propose a model in which QS induces replication followed by differentiation into GTA producing and non-producing cells. CtrA-activity is controlled by the c-di-GMP level, allowing some of the daughter cells to replicate again. The size of the GTA producing subpopulation is tightly controlled by QS via the AI Synthase LuxI2. Finally, induction of the SOS response allows for integration of GTA DNA into the host chromosome.

Modulation of Pseudomonas aeruginosa Quorum Sensing by Glutathione.

Pseudomonas aeruginosa uses quorum sensing (QS) to regulate the production of a battery of secreted products. At least some of these products are shared among the population and serve as public goods. When P. aeruginosa is grown on casein as the sole carbon and energy source, the QS-induced extracellular protease elastase is required for growth. We isolated a P. aeruginosa variant, which showed increased production of QS-induced factors after repeated transfers in casein broth. This variant, P. aeruginosa QS*, had a mutation in the glutathione synthesis gene gshA We describe several experiments that show a gshA coding variant and glutathione affect the QS response. The P. aeruginosa QS transcription factor LasR has a redox-sensitive cysteine (C79). We report that GshA variant cells with a LasR C79S substitution show a similar QS response to that of wild-type P. aeruginosa Surprisingly, it is not LasR but the QS transcription factor RhlR that is more active in bacteria containing the variant gshA Our results demonstrate that QS integrates information about cell density and the cellular redox state via glutathione levels.IMPORTANCEPseudomonas aeruginosa and other bacteria coordinate group behaviors using a chemical communication system called quorum sensing (QS). The QS system of P. aeruginosa is complex, with several regulators and signals. We show that decreased levels of glutathione lead to increased gene activation in P. aeruginosa, which did not occur in a strain carrying the redox-insensitive variant of a transcription factor. The ability of P. aeruginosa QS transcription factors to integrate information about cell density and cellular redox state shows these transcription factors can fine-tune levels of the gene products they control in response to at least two types of signals or cues.

Hybrid centralized/decentralized control of a network of bacteria-based bio-hybrid microrobots

Engineering microrobotic systems using a bio-hybrid approach that couples synthetic micro/nanoscale components with live cells is a powerful approach to address key shortcomings of micro/nanotechnology such as providing an on-board power source, communication, and decentralized control. In the last decade, a number of centralized control strategies based on native biological mechanisms have been demonstrated; however, decentralized cooperative control of a swarm of bio-hybrid microrobots has rarely been explored. In this work, we use our previously developed agent-based computational model to explore the utility of integrating synthetic biology with bio-hybrid microrobotics with the goal of developing strategies for robust decentralized control of bio-hybrid microrobotic systems. To this end, our Nanoscale Bacteria-Enabled Autonomous Delivery System (NanoBEADS), wherein each agent is comprised of an Escherichia coli bacterium conjugated with an ensemble of nanoparticles, was used as a model system. We imparted bacteria with engineered biological circuits to facilitate agent-agent communication and enable predictable and robust cooperative control of a network of NanoBEADS agents. We developed a hybrid control strategy wherein a centralized chemotaxis-based control scheme is used to direct migration, and a decentralized quorum sensing (QS)-based control scheme enables the agents to independently coordinate a cooperative behavior (fluorescent protein expression). We then computationally analyzed the role of bacterial subsystem properties and spatiotemporal changes in population organization in the emergent behavior (i.e., the activation time of the QS response) of the NanoBEADS. We show that the spatial changes in NanoBEADS agents’ distribution, in response to the centralized control input, and the mass transport boundary conditions are critical factors in determining the dynamics as well as the sensitivity and robustness of the QS response. We analyze the stark differences in feasible QS parameter domains under dissimilar chemoattractant gradients and show that the flow boundary conditions may greatly influence system function. Altogether, we show that a bacteria-based bio-hybrid system must be designed with careful consideration of growth rate, chemotaxis properties, QS properties, and the environment initial and boundary conditions in the context of its target application. This modeling framework can serve as an insightful tool for the predictive design of bio-hybrid microrobotic swarms with a tunable and robust response.

Production of cross-domain signalling molecules by Halophilic archaea

Cell-cell communication through the production of autoinducer molecules has been widely studied in bacteria and found to play a pivotal role in biofilm formation and gene regulation. Quorum Sensing (QS) within the domain Archaea is understudied compared with their bacterial counterparts. The aim of this study was to determine whether archaea are capable of cross-kingdom signalling through the production of QS inducing compounds and QS inhibitory activities. A combination of culture-dependent (crude extracts from archaeal isolates) and culture-independent (genomic mining) techniques were employed to investigate the production of compounds capable of modulating bacterial QS. Crude archaeal extracts were screened for activity using the bioreporter strains Agrobacterium tumefaciens ATCC BAA-2240, Escherichia coli JM109 pSB536, pSB401 and pSB1142, Chromobacterium violaceum CV026 and Pseudomonas aeruginosa MW-1. Active strains were further characterised using initial bio-assay guided fractionation. Preliminary results revealed different strains were capable of eliciting a QS response in the bacterial bio-reporters. Initial characterisation using LC-MS, TLC-overlays, and other biochemical tests, suggests the possible production of Butyryl Homoserine Lactones or homologs of this molecule by at least one of the strains. Conversely, other halophilic archaea were capable of inhibiting the production of QS-controlled pathways, as demonstrated by the reduction in virulence factor production by P. aeruginosa. Further characterisation of these compounds will prove to be an invaluable insight into the poorly understood mechanisms behind archaeal QS and equally will reveal the role of these compounds in cross-kingdom signalling.

Inactivation of the quorum-sensing transcriptional regulators LasR or RhlR does not suppress the expression of virulence factors and the virulence of Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa is an environmental bacterium but is also an opportunistic pathogen. The aim of this work is to evaluate the contribution of P. aeruginosa LasR and RhlR transcriptional regulators of the quorum-sensing response (QSR) to the production of virulence factors, and to its virulence in a mouse abscess model. The QSR is a complex regulatory network that modulates the expression of several virulence factors, including elastase, pyocyanin and rhamnolipids. LasR, when complexed with the auto-inducer 3-oxo-dodecanoyl lactone (3O-C12-HSL), produced by LasI, is at the top of the QSR regulatory cascade since it activates transcription of some genes encoding virulence factors (such as the gene coding for elastase, lasB) and also transcription of both rhlR and rhlI, encoding the synthase of the auto-inducer butanoyl-homoserine lactone (C4-HSL). In turn RhlR, coupled with C4-HSL, activates the transcription of genes encoding for the enzymes involved in pyocyanin and rhamnolipid production. Several efforts have been made to obtain inhibitors of LasR activity that would suppress the QSR. However, these attempts have used chemical compounds that might not be specific for LasR inactivation. In this work we show that individual inactivation of either lasR or rhlR did not block the QSR, nor did it impair P. aeruginosa virulence, and that even a lasR rhlR double mutant still presented residual virulence, even lacking the production of virulence factors. These results show that the inhibition of either lasR or rhlR is not a straightforward approach to blocking P. aeruginosa virulence, due to the great complexity of the QSR.

Modeling the Role of Feedback in the Adaptive Response of Bacterial Quorum Sensing.

Bacterial quorum sensing (QS) is a form of intercellular communication that relies on the production and detection of diffusive signaling molecules called autoinducers. Such a mechanism allows the bacteria to track their cell density in order to regulate group behavior, such as biofilm formation and bioluminescence. In a number of bacterial QS systems, including V. harveyi, multiple signaling pathways are integrated into a single phosphorylation-dephosphorylation cycle. In this paper, we propose a weight control mechanism, in which QS uses feedback loops to 'decode' the integrated signals by actively changing the sensitivity in different pathways. We first use a slow/fast analysis to reduce a single-cell model to a planar dynamical system involving the concentrations of phosphorylated signaling protein LuxU and a small non-coding RNA. In addition to identifying the weight control mechanism, we show that adding a feedback loop can lead to a bistable QS response in certain parameter regimes. We then combine the slow/fast analysis with a contraction mapping theorem in order to reduce a population model to an effective single-cell model, and show how the weight control mechanism allows bacteria to have a finer discrimination of their social and physical environment.

Structural determinants driving homoserine lactone ligand selection in the Pseudomonas aeruginosa LasR quorum-sensing receptor

Quorum sensing is a cell-cell communication process that bacteria use to orchestrate group behaviors. Quorum sensing is mediated by signal molecules called autoinducers. Autoinducers are often structurally similar, raising questions concerning how bacteria distinguish among them. Here, we use the Pseudomonas aeruginosa LasR quorum-sensing receptor to explore signal discrimination. The cognate autoinducer, 3OC12 homoserine lactone (3OC12HSL), is a more potent activator of LasR than other homoserine lactones. However, other homoserine lactones can elicit LasR-dependent quorum-sensing responses, showing that LasR displays ligand promiscuity. We identify mutants that alter which homoserine lactones LasR detects. Substitution at residue S129 decreases the LasR response to 3OC12HSL, while enhancing discrimination against noncognate autoinducers. Conversely, the LasR L130F mutation increases the potency of 3OC12HSL and other homoserine lactones. We solve crystal structures of LasR ligand-binding domains complexed with noncognate autoinducers. Comparison with existing structures reveals that ligand selectivity/sensitivity is mediated by a flexible loop near the ligand-binding site. We show that LasR variants with modified ligand preferences exhibit altered quorum-sensing responses to autoinducers in vivo. We suggest that possessing some ligand promiscuity endows LasR with the ability to optimally regulate quorum-sensing traits.

Role of the Multidrug Resistance Efflux Pump MexCD-OprJ in the Pseudomonas aeruginosa Quorum Sensing Response.

Multidrug efflux pumps constitute a category of antibiotic resistance determinants that are a part of the core bacterial genomes. Given their conservation, it is conceivable that they present functions beyond the extrusion of antibiotics currently used for therapy. Pseudomonas aeruginosa stands as a relevant respiratory pathogen, with a high prevalence at hospitals and in cystic fibrosis patients. Part of its success relies on its low susceptibility to antibiotics and on the production of virulence factors, whose expression is regulated in several cases by quorum sensing (QS). We found that overexpression of the MexCD-OprJ multidrug efflux pump shuts down the P. aeruginosa QS response. Our results support that MexCD-OprJ extrudes kynurenine, a precursor of the alkyl-quinolone signal (AQS) molecules. Anthranilate and octanoate, also AQS precursors, do not seem to be extruded by MexCD-OprJ. Kynurenine extrusion is not sufficient to reduce the QS response in a mutant overexpressing this efflux pump. Impaired QS response is mainly due to the extrusion of 4-hydroxy-2-heptylquinoline (HHQ), the precursor of the Pseudomonas Quinolone Signal (PQS), leading to low PQS intracellular levels and reduced production of QS signal molecules. As the consequence, the expression of QS-regulated genes is impaired and the production of QS-regulated virulence factors strongly decreases in a MexCD-OprN P. aeruginosa overexpressing mutant. Previous work showed that MexEF-OprJ, another P. aeruginosa efflux pump, is also able of extruding kynurenine and HHQ. However, opposite to our findings, the QS defect in a MexEF-OprN overproducer is due to kynurenine extrusion. These results indicate that, although efflux pumps can share some substrates, the affinity for each of them can be different. Although the QS response is triggered by population density, information on additional elements able of modulating such response is still scarce. This is particularly important in the case of P. aeruginosa lung chronic infections, a situation in which QS-defective mutants are accumulated. If MexCD-OprJ overexpression alleviates the cost associated to triggering the QS response when un-needed, it could be possible that MexCD-OprJ antibiotic resistant overproducer strains might be selected even in the absence of antibiotic selective pressure, acting as antibiotic resistant cheaters in heterogeneous P. aeruginosa populations.

The plant compound rosmarinic acid induces a broad quorum sensing response in Pseudomonas aeruginosa PAO1.

The interference of plant compounds with bacterial quorum sensing (QS) is a major mechanism through which plants and bacteria communicate. However, little is known about the modes of action and effects on signal integrity during this type of communication. We have recently shown that the plant compound rosmarinic acid (RA) specifically binds to the Pseudomonas aeruginosa RhlR QS receptor. To determine the effect of RA on expression patterns, we carried out global RNA-seq analysis. The results show that RA induces the expression of 128 genes, amongst which many virulence factor genes. RA triggers a broad QS response because 88% of the induced genes are known to be controlled by QS, and because RA stimulated genes were found to be involved in all four QS signalling systems within P. aeruginosa. This finding was confirmed through the analysis of transcriptional fusions transferred to wt and a rhlI/lasI double mutant. RA did not induce gene expression in the rhlI/lasI/rhlR triple mutant indicating that the effects observed are due to the RA-RhlR interaction. Furthermore, RA induced seven sRNAs that were all encoded in regions close to QS and/or RA induced genes. This work significantly enhances our understanding of plant bacteria interaction.

Focusing quorum sensing signalling by nano-magnetic assembly.

Quorum sensing (QS) exists widely among bacteria, enabling a transition to multicellular behaviour after bacterial populations reach a particular density. The coordination of multicellularity enables biotechnological application, dissolution of biofilms, coordination of virulence, and so forth. Here, a method to elicit and subsequently disperse multicellular behaviour among QS-negative cells is developed using magnetic nanoparticle assembly. We fabricated magnetic nanoparticles (MNPs, ∼5 nm) that electrostatically collect wild-type (WT) Escherichia coli BL21 cells and brings them into proximity of bioengineered E. coli [CT104 (W3110 lsrFG- luxS- pCT6 + pET-DsRed)] reporter cells that exhibit a QS response after receiving autoinducer-2 (AI-2). By shortening the distance between WT and reporter cells (e.g., increasing local available AI-2 concentrations), the QS response signalling was amplified four-fold compared to that in native conditions without assembly. This study suggests potential applications in facilitating intercellular communication and modulating multicellular behaviours based on user-specified designs.

VqsA, a Novel LysR-Type Transcriptional Regulator, Coordinates Quorum Sensing (QS) and Is Controlled by QS To Regulate Virulence in the Pathogen Vibrio alginolyticus.

The quorum sensing (QS) system controls bacterial group behaviors in response to cell density. In vibrios, LuxR and AphA are two master QS regulators (MQSRs) controlling gene expression in response to high or low cell density. Other regulators involved in the regulation of these two MQSRs and QS pathways remain to be determined. Here, we performed bacterial one-hybrid (B1H)-assay-based screens of transcriptional factors (TFs) to identify TFs that can directly regulate the expression of luxR and aphA from a library of 285 TFs encoded by the fish pathogen Vibrio alginolyticus A total of 7 TFs were identified to bind to the promoters of both luxR and aphA Among these TFs, the novel LysR-type transcriptional regulator (LTTR) VqsA could activate LuxR and repress AphA transcription. Meanwhile, LuxR and AphA exerted feedback inhibition and activation of vqsA expression, respectively, indicating that VqsA coordinates QS and is also regulated by QS. In addition, VqsA inhibited its own expression by directly binding to its own promoter region. The VqsA-binding sites in the promoter regions of luxR and aphA as well as the binding sites of LuxR, AphA, and VqsA in the vqsA gene were uncovered by electrophoretic mobility shift assays (EMSAs) and DNase I footprinting analysis. Finally, VqsA was verified to play essential roles in QS-regulated phenotypes, i.e., type VI secretion system 2 (T6SS2)-dependent interbacterial competition, biofilm formation, exotoxin production, and in vivo virulence of V. alginolyticus Collectively, our data showed that VqsA is an important QS regulator in V. alginolyticusIMPORTANCE Investigation of the mechanism of regulation of quorum sensing (QS) systems will facilitate an understanding of bacterial pathogenesis and the identification of effective QS interference (QSI) targets. Here, we systematically screened transcriptional factors (TFs) that modulate the expression of the master QS regulators (MQSRs) LuxR and AphA, and a novel LysR-type transcriptional regulator, VqsA, was identified. Our data illuminated the mechanisms mediating the interaction among LuxR, AphA, and VqsA as well as the effects of these regulators on the expression and output of QS. The impaired expression of virulence genes as a result of vqsA disruption demonstrated that VqsA is an important player in QS regulation and pathogenesis and may be the third MQSR involved in sensing environmental signals by vibrios to coordinate QS responses. This study will facilitate the development of strategies to interfere with QS and effectively control this pathogen that plagues the aquaculture industry.

Stopping Autoinducer-2 Chatter by Means of an Indigenous Bacterium ( Acinetobacter sp. DKY-1): A New Antibiofouling Strategy in a Membrane Bioreactor for Wastewater Treatment.

Bacterial quorum quenching (QQ) by means of degrading signaling molecules has been applied to antibiofouling strategies in a membrane bioreactor (MBR) for wastewater treatment. However, the target signaling molecules have been limited to N-acyl homoserine lactones participating in intraspecies quorum sensing. Here, an approach to disrupting autoinducer-2 (AI-2) signaling molecules participating in interspecies quorum sensing was pursued as a next-generation antibiofouling strategy in an MBR for wastewater treatment. We isolated an indigenous QQ bacterium ( Acinetobacter sp. DKY-1) that can attenuate the expression of the quorum-sensing (QS) response through the inactivation of an autoinducer-2 signaling molecule, 4,5-dihydroxy-2,3-pentanedione (DPD), among four kinds of autoinducer-2 QS bacteria. DKY-1 released AI-2 QQ compounds, which were verified to be hydrophilic with a molecular weight of <400 Da. The addition of DKY-1 entrapping beads into an MBR significantly decreased DPD concentration and remarkably reduced membrane biofouling. This new approach, combining molecular biology with wastewater engineering, could enlarge the range of QQ-MBR for antibiofouling and energy savings in the field of wastewater treatment.

Quantitative Investigation of the Role of Intra-/Intercellular Dynamics in Bacterial Quorum Sensing.

Bacteria utilize diffusible signals to regulate population density-dependent coordinated gene expression in a process called quorum sensing (QS). While the intracellular regulatory mechanisms of QS are well-understood, the effect of spatiotemporal changes in the population configuration on the sensitivity and robustness of the QS response remains largely unexplored. Using a microfluidic device, we quantitatively characterized the emergent behavior of a population of swimming E.coli bacteria engineered with the lux QS system and a GFP reporter. We show that the QS activation time follows a power law with respect to bacterial population density, but this trend is disrupted significantly by microscale variations in population configuration and genetic circuit noise. We then developed a computational model that integrates population dynamics with genetic circuit dynamics to enable accurate (less than 7% error) quantitation of the bacterial QS activation time. Through modeling and experimental analyses, we show that changes in spatial configuration of swimming bacteria can drastically alter the QS activation time, by up to 22%. The integrative model developed herein also enables examination of the performance robustness of synthetic circuits with respect to growth rate, circuit sensitivity, and the population's initial size and spatial structure. Our framework facilitates quantitative tuning of microbial systems performance through rational engineering of synthetic ribosomal binding sites. We have demonstrated this through modulation of QS activation time over an order of magnitude. Altogether, we conclude that predictive engineering of QS-based bacterial systems requires not only the precise temporal modulation of gene expression (intracellular dynamics) but also accounting for the spatiotemporal changes in population configuration (intercellular dynamics).

Chitosan nanoencapsulation of flavonoids enhances their quorum sensing and biofilm formation inhibitory activities against an E.coli Top 10 biosensor.

Phytochemicals have been found to be promising alternatives to conventional antibiotic therapies for the control of bacterial infections, as they may entail less selective pressure and hence reduce the development of resistance. This study involved examining the inhibition of biofilm formation and of quorum sensing (QS), and the cytotoxicity on mammalian cells of two flavonoids, quercetin and baicalein, in free form and associated into chitosan-based nanocapsules. This was done by use of a transformed E. coli Top 10 biosensor strain, while the cytotoxicity was evaluated on MDCK-C7 cells. In free form, application both flavonoids exhibited slight inhibitory activity on the QS response and biofilm formation, a scenario that was improved positively upon encapsulation with chitosan (Mw ∼115,000 g/mol and DA ∼42%). The association efficiency of 99% (quercetin) and 87% (baicalein) was determined, and each formulation had an average diameter of 190 ± 4 and 187 ± 2 nm, and zeta (ζ) potential of +48.1 ± 2.03 and +48.4 ± 3.46 mV, respectively. Both types of systems were stable against aggregation in M9 and MEM media. The in vitro release kinetics data of both flavonoids seemed to be similar with only ∼20% released over the first 5 h, or ∼10% over the first 4 h, respectively, with subsequent sudden release increase up to ∼40% in both cases. The free phytochemicals seemed to be cytotoxic to MDCK-C7 cells at higher doses, however, upon nanoencapsulation, a cytoprotective effect was evidenced. We have gained proof-of-principle of the advantages of encapsulation of two bioactive flavonoids.

Early Detection of Biofouling on Water Purification Membranes by Ambient Ionization Mass Spectrometry Imaging.

By direct analysis of water purification membranes using ambient ionization mass spectrometry, an attempt has been made to understand the molecular signatures of bacterial fouling. Membrane based purification methods are used extensively in water treatment, and a major challenge for them is biofouling. The buildup of microbes and their extracellular polymeric matrix clog the purification membranes and reduce their efficiency. To understand the early stages of bacterial fouling on water purification membranes, we have used desorption electrospray ionization mass spectrometry (DESI MS), where ion formation occurs in ambient conditions and the ionization event is surface sensitive. Biosurfactants at the air-water interface generated by microorganisms as a result of quorum sensing, influence the water-membrane interface and are important for the bacterial attachment. We show that these biosurfactants produced by bacteria can be indicator molecular species signifying initiation of biofilms on membrane surfaces, demonstrated by specific DESI MS signatures. In Pseudomonas aeruginosa, one of the best studied models for biofilm formation, this process is mediated by rhamnolipids forewarning bacterial fouling. Species dependent variation of such molecules can be used for the precise identification of the microorganisms, as revealed by studies on P. aeroginosa (ATCC 25619). The production of biosurfactants is tightly regulated at the transcriptional level by the quorum-sensing (QS) response. Thus, secretion of these extracellular molecules across the membrane surface allows rapid screening of the biofilm community. We show that, the ambient ionization mass spectrometry can detect certain toxic heavy metals present in water, using surfactant-metal complexes as analytes. We believe that such studies conducted on membranes in various input water streams will help design suitable membrane processes specific to the input streams.

Self-sensing in Bacillus subtilis quorum-sensing systems.

Bacterial cell-cell signaling, or quorum sensing, is characterized by the secretion and group-wide detection of small diffusible signal molecules called autoinducers. This mechanism allows cells to coordinate their behavior in a density-dependent manner. A quorum-sensing cell may directly respond to the autoinducers it produces in a cell-autonomous and quorum-independent manner, but the strength of such self-sensing effect and its impact on bacterial physiology are unclear. Here, we explored the existence and impact of self-sensing in the Bacillus subtilis ComQXP and Rap-Phr quorum-sensing systems. By comparing the quorum-sensing response of autoinducer-secreting and non-secreting cells in co-culture, we found that secreting cells consistently showed a stronger response than non-secreting cells. Combining genetic and quantitative analyses, we demonstrated this effect to be a direct result of self-sensing and ruled out an indirect regulatory effect of the autoinducer production genes on response sensitivity. In addition, self-sensing in the ComQXP system affected persistence to antibiotic treatment. Together, these findings indicate the existence of self-sensing in the two most common designs of quorum-sensing systems of Gram-positive bacteria.