Quorum-sensing Regulator Research Articles

LitR and its quorum-sensing regulators modulate biofilm formation by Vibrio fischeri.

Quorum sensing controls numerous processes ranging from the production of virulence factors to biofilm formation. Biofilms, communities of bacteria that are attached to one another and/or a surface, are common in nature, and when they form, they can produce a quorum of bacteria. One model system to study biofilms is the bacterium Vibrio fischeri, which forms a biofilm that promotes the colonization of its symbiotic host. Many factors promote V. fischeri biofilm formation in vitro, including the symbiosis polysaccharide (SYP) and cellulose, but the role of quorum sensing is currently understudied. Recently, a quorum-sensing-dependent transcription factor, LitR, was shown to negatively influence V. fischeri biofilm formation in the context of a biofilm-overproducing strain. To better understand the importance of LitR, we identified conditions in which the impact of LitR on biofilm formation could be observed in an otherwise wild-type strain and then investigated its role and the roles of upstream quorum regulators in biofilm phenotypes. In static conditions, LitR and its upstream quorum regulators, including autoinducer synthases LuxS and AinS, contributed to control over biofilms that were both SYP and cellulose dependent. In shaking liquid conditions, LitR and AinS contributed to control over biofilms that were primarily cellulose dependent. LitR modestly inhibited cellulose transcription in a manner that depended on the transcription factor VpsR. These findings expand our understanding of LitR and the quorum-sensing pathway in the physiology of V. fischeri and illuminate negative control mechanisms that prevent robust biofilm formation by wild-type V. fischeri under laboratory conditions.IMPORTANCEQuorum sensing is a key regulatory mechanism that controls diverse phenotypes in numerous bacteria, including Vibrio fischeri. In many microbes, quorum sensing has been shown to control biofilm formation, yet in V. fischeri, the link between quorum sensing and biofilm formation has been understudied. This study fills that knowledge gap by identifying roles for the quorum sensing-controlled transcription factor, LitR, and its upstream quorum-sensing regulators, including the autoinducer synthases AinS and LuxS, in inhibiting biofilm formation under specific conditions. It also determined that LitR inhibits the transcription of genes required for cellulose biosynthesis. This work thus expands our understanding of the complex control over biofilm regulation.

Optimized peptide inhibitor Aqs1C targets LasR to disrupt quorum sensing and biofilm formation in Pseudomonas aeruginosa: Insights from MD simulations and in vitro studies.

Pseudomonas aeruginosa (PA) is a critical pathogen, and its antibiotic resistance is largely driven by the quorum-sensing regulator LasR. Herein, we report the design, synthesis, and characterization of Aqs1C, a mutated peptide derivative of Aqs1, optimized to inhibit LasR and its quorum-sensing pathway. By introducing a targeted mutation, Aqs1C exhibited enhanced stability and binding affinity for LasR protein compared to its predecessor, Aqs1B. Using molecular dynamics simulations (MDS), the Aqs1C-LasR complex demonstrated a marked increase in structural stability, reflected in reduced root mean square deviation (RMSD) values and lower binding free energy. Electrostatic complementarity analysis showed stronger and more favorable interactions between Aqs1C and LasR. Further, GaMD experiments were able to reproduce the binding state between Aqs1C and LasR, indicating the binding mechanism between them. These molecular insights correlated with functional in vitro assays. Aqs1C effectively inhibited quorum-sensing-associated virulence factors in PA, involving biofilm formation (77.6 % inhibition), pyocyanin production (75.7 % inhibition), protease secretion (61.1 % inhibition), and rhamnolipid production (74.1 % inhibition), at a 100 μg/mL concentration, in a comparable or superior pattern to azithromycin (AZM). Molecular modelling, MDS, and GaMD insights and in vitro assays established Aqs1C as a promising candidate for therapeutic development to mitigate PA infections through targeted quorum-sensing disruption.

Understanding phage BX-1 resistance in Vibrio alginolyticus AP-1 and the role of quorum-sensing regulation.

The marine ecosystem is characterized by a rich diversity of bacterial hosts and their phages. The propagation of phages is primarily limited by their ability to adsorb to host cells and is further challenged by various bacterial defense mechanisms. To fully realize the potential of phage therapy in aquaculture, a comprehensive understanding of phage-host interactions and their regulation is essential. In this study, we isolated a novel Myoviridae phage, BX-1, capable of infecting Vibrio alginolyticus AP-1, and characterized its resistant mutants. We elucidated the essential role of the bacterial cellulose biosynthesis-related gene bcsE, which functions as a cyclic di-GMP-binding protein, in influencing host susceptibility to phage BX-1. Interestingly, Congo Red, Calcofluor White staining, and cellulose content assays indicated that deletion of bcsE in strain AP-1 does not completely abolish cellulose production, suggesting that bcsE is not essential for bacterial cellulose synthesis. Furthermore, investigating the signaling molecules that regulate phage-host interactions, we find that in a high cell density state (ΔluxO), bacterial cells upregulate their susceptibility to phage BX-1, which leads to a rapid development of resistance. Conversely, cells in a low-density state (ΔhapR) exhibit reduced susceptibility to phage BX-1 while still producing comparable phage progenies. This population density-dependent response is primarily enhanced by the predicted quorum-sensing autoinducer CAI-1, synthesized by the gene cqsA. Collectively, our findings reveal the intricate dynamics of phage-host interactions, adding a new layer of complexity to our understanding of phage receptor regulations.IMPORTANCEPhage therapy has garnered significant attention as a promising solution to antibiotic resistance in aquaculture. However, its application is hindered by a limited understanding of the genotypic and phenotypic dynamics governing phage-host interactions. Bacteria have developed various defense mechanisms against phages, such as mutations in phage receptors. In this study, we demonstrate that the bacterial cellulose biosynthesis-related gene bcsE plays a crucial role in determining susceptibility to phage BX-1, while quorum-sensing (QS) systems significantly influence collective phage-related behaviors. By characterizing the mechanisms of phage resistance and the regulatory role of QS in susceptibility, our findings enhance the understanding of phage-host interactions and pave the way for more effective phage therapy applications. Collectively, these insights illuminate the evolutionary complexities of phage-defense systems and the broader strategies that bacteria employ to coexist with phages.

ZnO-Rutin nanostructure as a potent antibiofilm agent against Pseudomonas aeruginosa

Pseudomonas aeruginosa is a common human pathogen that is resistant to multiple antibiotics due to its ability to form biofilms. Developing novel nanoformulations capable of inhibiting and removing biofilms offers a promising solution for controlling biofilm-related infections. In this study, we investigated the anti-biofilm activity of rutin-conjugated ZnO nanoparticles (ZnO-Rutin NPs) in pathogenic strains of P. aeruginosa. The synthesized ZnO-Rutin NPs had amorphous shapes with sizes ranging from 14 to 100 nm. The broth microdilution assay revealed that ZnO-Rutin NPs, with an MIC value of 2 mg/mL, exhibit greater antimicrobial activity than ZnO NPs and rutin alone. Based on crystal violet staining, the biofilm inhibition rate by ½ MIC of the conjugated nanoparticles was recorded at above 90%. The significant reduction in exopolysaccharide (62.75–66.37%) and alginate (38.3–57.61%) levels, as well as the formation of thin biofilms in the ZnO-Rutin NP-treated group, confirmed the anti-biofilm potential of these nanoparticles. Additionally, a significant decrease in the metabolic activity and viable cells of mature biofilms was observed after exposure to the conjugated nanoparticles. Furthermore, ZnO-Rutin NPs considerably attenuated the expression of the Las-Rhl quorum-sensing transcriptional regulator genes (lasR and rhlR) in P. aeruginosa by 0.39–0.40 and 0.25–0.42 folds, respectively. This work demonstrated that ZnO-Rutin NPs are remarkably capable of inhibiting the initial stage of biofilm formation and eradicating mature biofilms, suggesting they could be a useful agent for treating P. aeruginosa biofilm-related infections.

Acyl-Homoserine Lactone Enhances the Resistance of Anammox Consortia under Heavy Metal Stress: Quorum Sensing Regulatory Mechanism.

Anaerobic ammonium oxidation (anammox) represents an energy-efficient process for the removal of biological nitrogen from ammonium-rich wastewater. However, the susceptibility of anammox bacteria to coexisting heavy metals considerably restricts their use in engineering practices. Here, we report that acyl-homoserine lactone (AHL), a signaling molecule that mediates quorum sensing (QS), significantly enhances the nitrogen removal rate by 24% under Cu2+ stress. A suite of macro-/microanalytical and bioinformatic analyses was exploited to unravel the underlying mechanisms of AHL-induced Cu2+ resistance. Macro-/microanalytical evidence indicated that AHL regulations on the production, spatial distribution, and functional groups of extracellular polymeric substances were not significant, ruling out extracellular partitioning and complexation as a principal mechanism. Meanwhile, molecular biological evidence showed that AHL upregulated the transcriptional levels of resistance genes (sod, kat, cysQ, and czcC responsible for antioxidation defense, Cu2+ sequestration, and transport) to appreciable extents, indicating intracellular resistance as the primary mechanism. This study yielded a mechanistic understanding of the regulatory roles of AHL in extracellular and intracellular resistance of anammox consortia, providing a fundamental basis for utilizing QS regulation for efficient nitrogen removal in wastewaters with heavy metal stress.

Quorum sensing regulating the productivity and stability of cross-feeding cocultivation

Quorum sensing (QS), a dynamic microbial communication mechanism, has been widely applied to synthetic biology for different functions. The incorporation of genome-scale models (GEMs) provides a powerful tool for understanding the metabolic capabilities and interactions within microbial communities. However, the introduction of the relevant QS devices for the stability and productivity of the microbial community lacks systematic analysis, let alone the GEMs-based rational design and optimization of QS-regulated microbial ecosystems. In this study, we integrated different QS regulation strategies with the metabolic division of labor to establish a combinational GEM for systematically designing and optimizing the process of the two-strain cross-feeding cocultivation. Specifically, taking the production of salidroside within two cross-feeding fermentation as an example, the GEMs were developed for the static and QS-based dynamic regulation strategies, and the corresponding simulation and optimization were performed. Then, we further constructed the QS-based self-regulating, cross-regulating, and hybrid-regulating QS communication network (QSCN) by using synthetic biology toolkits to investigate the productivity and stability of cross-feeding cocultivation. Results showed that the hybrid QSCN can better realize the cell density control and the improvement of productivity (562.57 mg/L). This study offers insights for developing more comprehensive model and regulation strategies for QS-based control in diverse applications.

Quorum sensing in Vibrio controls carbon metabolism to optimize growth in changing environmental conditions.

Bacteria sense population density via the cell-cell communication system called quorum sensing (QS). The evolution of QS and its maintenance or loss in mixed bacterial communities is highly relevant to understanding how cell-cell signaling impacts bacterial fitness and competition, particularly under varying environmental conditions such as nutrient availability. We uncovered a phenomenon in which Vibrio cells grown in minimal medium optimize expression of the methionine and tetrahydrofolate (THF) synthesis genes via QS. Strains that are genetically "locked" at high cell density grow slowly in minimal glucose media and suppressor mutants accumulate via inactivating mutations in metF (methylenetetrahydrofolate reductase) and luxR (the master QS transcriptional regulator). In mixed cultures, QS mutant strains initially coexist with wild-type, but as glucose is depleted, wild-type outcompetes the QS mutants. Thus, QS regulation of methionine/THF synthesis is a fitness benefit that links nutrient availability and cell density, preventing accumulation of QS-defective mutants.

Quorum sensing orchestrates parallel cell death pathways in Vibrio cholerae via Type 6 secretion-dependent and -independent mechanisms

Quorum sensing (QS) is a cell-to-cell communication process that enables bacteria to coordinate group behaviors. In Vibrio cholerae colonies, a program of spatial-temporal cell death is among the QS-controlled traits. Cell death occurs in two phases, first along the colony rim, and subsequently, at the colony center. Both cell death phases are driven by the type 6 secretion system (T6SS). Here, we show that HapR, the master QS regulator, does not control t6ss gene expression nor T6SS-mediated killing activity. Nonetheless, a ΔhapR strain displays no cell death at the colony rim. RNA-Sequencing (RNA-Seq) analyses reveal that HapR activates expression of an operon containing four genes of unknown function, vca0646-0649. Epistasis and overexpression studies show that two of the genes, vca0646 and vca0647, are required to drive cell death in both a ΔhapR and a ΔhapR Δt6ss strain. Thus, vca0646-0649 are regulated by HapR but act independently of the T6SS machinery to cause cell death, suggesting that a second, parallel pathway to cell death exists in V. cholerae.

Quorum sensing on the activated performances of gravity-driven membrane (GDM) system at low temperatures: Ammonia removal and flux stabilization

Gravity-driven membrane (GDM) filtration technology offers a low-energy, low-maintenance solution for water purification due to its powerful bio-cake, but faces predicament in contaminant removal under low temperatures. Quorum sensing (QS), a microbial communication process mediated by signal molecules such as C6-HSL (N-Hexanoyl-L-homoserine lactone), has been shown to influence microbial behavior and enhance biofilm formation, with promising potential for consolidation of GDM operation at low temperature. This study investigates the application of C6-HSL to enhance ammonia nitrogen and organics removal in low-temperature GDM systems. C6-HSL was administered at varying dosages (0/50/250/500 nM/d) at 5 °C, with further experiments conducted at 25 °C to assess the impact of QS regulation on GDM performance under room temperature. The results demonstrated that the optimized presence of C6-HSL (500 nM/d) improved the removal efficiency of dissolved organic carbon, ammonia nitrogen, and raised the stable flux (increase ratios at low vs. room temperature: ∼19 % vs. ∼7 %, ∼38 % vs. ∼9 %, and ∼7.9 % vs. 0.4 %, respectively). Analysis of the biofouling layer’s structural characteristics, composition, and biological activity indicated that the increased C6-HSL effectively promoted microbial growth and migration/movement, increased the production of extracellular polymeric substances (EPS) and soluble microbial products (SMP), and accelerated biofilm formation. The resulting thicker (∼172.98 vs. ∼119.21 μm), rougher (∼62.1 vs. ∼55.0 nm) cake layer, equipped with abundant channels for water penetration, contributed to increased contaminant degradation, but also stabilized the flux with slight growth (∼3.26 vs. ∼3.02 LMH) under low temperature. Notably, the enhancement effect of QS regulation was more pronounced at low temperature than at room temperature, attributing to the optimized release and diffusion of signal molecules and the more substantial stimulation of metabolic and enzymatic activity in nitrifying bacteria. This study provides valuable insights and technical support for decentralized water treatment in winter via reliable GDM process operation in challenging temperature conditions.

New Insights into the Persistent Shock Resistance of Anaerobic Granular Sludge Based on Quorum Sensing Regulation: A Novel Gene Regulatory Mechanism

New Insights into the Persistent Shock Resistance of Anaerobic Granular Sludge Based on Quorum Sensing Regulation: A Novel Gene Regulatory Mechanism

Use of Rgg quorum-sensing machinery to create an innovative recombinant protein expression system in Streptococcus thermophilus.

Streptococcus thermophilus holds promise as a chassis for producing and secreting heterologous proteins. Used for thousands of years to ferment milk, this species has generally recognized as safe (GRAS) status in the USA and qualified presumption of safety (QPS) status in Europe. In addition, it can be easily genetically modified thanks to its natural competence, and it secretes very few endogenous proteins, which means less downstream processing is needed to purify target proteins, reducing costs. Extracellular degradation of heterologous proteins can be eliminated by introducing mutations that inactivate the genes encoding the bacterium's three major surface proteases. Here, we constructed an inducible expression system that utilizes a peptide pheromone (SHP1358) and a transcriptional regulator (Rgg1358) involved in quorum-sensing regulation. We explored the functionality of a complete version of the system, in which the inducer is produced by the bacterium itself, by synthesizing a luciferase reporter protein. This complete version was assessed with bacteria grown in a chemically defined medium but also in vivo, in the faeces of germ-free mice. We also tested an incomplete version, in which the inducer had to be added to the culture medium, by synthesizing luciferase and a secreted form of elafin, a human protein with therapeutic properties. Our results show that, in our system, protein production can be modulated by employing different concentrations of the SHP1358 inducer or other SHPs with closed amino acid sequences. We also constructed a genetic background in which all system leakiness was eliminated. In conclusion, with this new inducible expression system, we have added to the set of tools currently used to produce secreted proteins in S. thermophilus, whose myriad applications include the delivery of therapeutic peptides or proteins.

Characterization of quorum regulatory small RNAs in an emerging pathogen Vibrio fluvialis and their roles toward type VI secretion system VflT6SS2 modulation

ABSTRACT The type VI secretion system (T6SS) is essential for Gram-negative bacteria to antagonize a wide variety of prokaryotic and eukaryotic competitors and thus gain survival advantages. Two sets of T6SS have been found in Vibrio fluvialis, namely VflT6SS1 and VflT6SS2, among which VflT6SS2 is functionally expressed. The CqsA/LuxS-HapR quorum sensing (QS) system with CAI-1 and AI-2 as signal molecules can regulate VflT6SS2 by regulators LuxO and HapR, with LuxO repressing while HapR activating VflT6SS2. Quorum regulatory small RNAs (Qrr sRNAs) are Hfq-dependent trans-encoded sRNAs that control Vibrio quorum sensing. In V. fluvialis, Qrr sRNAs have not been characterized and their regulatory function is unknown. In this study, we first identified four Qrr sRNAs in V. fluvialis and demonstrated that these Qrr sRNAs are regulated by LuxO and involved in the modulation of VflT6SS2 function. On the one hand, Qrr sRNAs act on HapR, the activator of both the major and the auxiliary clusters of VflT6SS2, and then indirectly repress VflT6SS2. On the other hand, they directly repress VflT6SS2 by acting on tssB2 and tssD2_a, the first gene of the major cluster and the highly transcriptional one among the two units of the first auxiliary cluster, respectively. Our results give insights into the role of Qrr sRNAs in CAI-1/AI-2 based QS and VflT6SS2 modulation in V. fluvialis and further enhance understandings of the network between QS and T6SS regulation in Vibrio species.

Combinatorial control of Pseudomonas aeruginosa biofilm development by quorum-sensing and nutrient-sensing regulators.

The human pathogen Pseudomonas aeruginosa, a leading cause of hospital-acquired infections, inhabits and forms sessile antibiotic-resistant communities called biofilms in a wide range of biotic and abiotic environments. In this study, we examined how two global sensory signaling pathways-the RhlR quorum-sensing system and the CbrA/CbrB nutritional adaptation system-intersect to control biofilm development. Previous work has shown that individually these two systems repress biofilm formation. Here, we used biofilm analyses, RNA-seq, and reporter assays to explore the combined effect of information flow through RhlR and CbrA on biofilm development. We find that the ΔrhlRΔcbrA double mutant exhibits a biofilm morphology and an associated transcriptional response distinct from wildtype and the parent ΔrhlR and ΔcbrA mutants indicating codominance of each signaling pathway. The ΔrhlRΔcbrA mutant gains suppressor mutations that allow biofilm expansion; these mutations map to the crc gene resulting in loss of function of the carbon catabolite repression protein Crc. Furthermore, the combined absence of RhlR and CbrA leads to a drastic reduction in the abundance of the Crc antagonist small RNA CrcZ. Thus, CrcZ acts as the molecular convergence point for quorum- and nutrient-sensing cues. We find that in the absence of antagonism by CrcZ, Crc promotes the expression of biofilm matrix components-Pel exopolysaccharide, and CupB and CupC fimbriae. Therefore, this study uncovers a regulatory link between nutritional adaption and quorum sensing with potential implications for anti-biofilm targeting strategies.IMPORTANCEBacteria often form multicellular communities encased in an extracytoplasmic matrix called biofilms. Biofilm development is controlled by various environmental stimuli that are decoded and converted into appropriate cellular responses. To understand how information from two distinct stimuli is integrated, we used biofilm formation in the human pathogen Pseudomonas aeruginosa as a model and studied the intersection of two global sensory signaling pathways-quorum sensing and nutritional adaptation. Global transcriptomics on biofilm cells and reporter assays suggest parallel regulation of biofilms by each pathway that converges on the abundance of a small RNA antagonist of the carbon catabolite repression protein, Crc. We find a new role of Crc as it modulates the expression of biofilm matrix components in response to the environment. These results expand our understanding of the genetic regulatory strategies that allow P. aeruginosa to successfully develop biofilm communities.

Extracellular proteolysis of tandemly duplicated pheromone propeptides affords additional complexity to bacterial quorum sensing.

Bacterial interactions are vital for adapting to changing environments, with quorum sensing (QS) systems playing a central role in coordinating behaviors through small signaling molecules. The RRNPPA family is the prevalent QS systems in Bacillota and mediating communication through secreted oligopeptides, which are processed into active pheromones by extracellular proteases. Notably, in several cases the propeptides show the presence of multiple putative pheromones within their sequences, which has been proposed as a mechanism to diversify peptide-receptor specificity and potentially facilitate new functions. However, neither the processes governing the maturation of propeptides containing multiple pheromones, nor their functional significance has been evaluated. Here, using 2 Rap systems from bacteriophages infecting Bacillus subtilis that exhibit different types of pheromone duplication in their propeptides, we investigate the maturation process and the molecular and functional activities of the produced pheromones. Our results reveal that distinct maturation processes generate multiple mature pheromones, which bind to receptors with varying affinities but produce identical structural and biological responses. These findings add additional layers in the complexity of QS communication and regulation, opening new possibilities for microbial social behaviors, highlighting the intricate nature of bacterial interactions and adaptation.

Rapid Revealing of Quorum Sensing (QS)-Regulated PLA, Biofilm and Lysine Targets of Lactiplantibacillus plantarum L3.

Quorum sensing (QS) can regulate the production of multiple functional factors in bacteria, but the process of identifying its regulatory targets is very complex and labor-intensive. In this study, an efficient and rapid method to find QS targets through prediction was used. The genome of Lactiplantibacillus plantarum (L. plantarum) L3 was sequenced and characterized, and then linked the L. plantarum L3 genome to the STRING database for QS system regulatory target prediction. A total of 3,167,484 base pairs (bps) were examined from the genome of L. plantarum L3, and 30 QS-related genes were discovered (including luxS). The STRING database prediction indicated that the 30 QS-related genes are mainly involved in the regulation of nine metabolic pathways. Furthermore, metE, metK, aroB, cysE, and birA1 were predicted to be regulatory targets of the LuxS/AI-2 QS system, and these five targets were validated based on quantitative real-time PCR and content determination. Successful elucidation of the LuxS/AI-2 QS system's key targets and regulation mechanism in L. plantarum L3 demonstrated the effectiveness of the new approach for predicting QS targets and provides a scientific basis for future work on improving regulation of functional factor production.

The LuxO-OpaR quorum-sensing cascade differentially controls Vibriophage VP882 lysis-lysogeny decision making in liquid and on surfaces.

Quorum sensing (QS) is a process of cell-to-cell communication that bacteria use to synchronize collective behaviors. QS relies on the production, release, and group-wide detection of extracellular signaling molecules called autoinducers. Vibrios use two QS systems: the LuxO-OpaR circuit and the VqmA-VqmR circuit. Both QS circuits control group behaviors including biofilm formation and surface motility. The Vibrio parahaemolyticus temperate phage φVP882 encodes a VqmA homolog (called VqmAφ). When VqmAφ is produced by φVP882 lysogens, it binds to the host-produced autoinducer called DPO and launches the φVP882 lytic cascade. This activity times induction of lysis with high host cell density and presumably promotes maximal phage transmission to new cells. Here, we explore whether, in addition to induction from lysogeny, QS controls the initial establishment of lysogeny by φVP882 in naïve host cells. Using mutagenesis, phage infection assays, and phenotypic analyses, we show that φVP882 connects its initial lysis-lysogeny decision to both host cell density and whether the host resides in liquid or on a surface. Host cells in the low-cell-density QS state primarily undergo lysogenic conversion. The QS regulator LuxO~P promotes φVP882 lysogenic conversion of low-cell-density planktonic host cells. By contrast, the ScrABC surface-sensing system regulates lysogenic conversion of low-cell-density surface-associated host cells. ScrABC controls the abundance of the second messenger molecule cyclic diguanylate, which in turn, modulates motility. The scrABC operon is only expressed when its QS repressor, OpaR, is absent. Thus, at low cell density, QS-dependent derepression of scrABC drives lysogenic conversion in surface-associated host cells. These results demonstrate that φVP882 integrates cues from multiple sensory pathways into its lifestyle decision making upon infection of a new host cell.

Quorum sensing regulating the heterogeneous transformation of antibiotic resistance genes in microplastic biofilms

Microplastics (MPs) provide a specific habitat for bacterial adhesion and the adsorption of extracellular DNA (eDNA), making them hotspots for the transformation of antibiotic resistance genes (ARGs) in the environment. However, the heterogeneous mechanism of ARG regulation by quorum sensing (QS) in MP biofilms remains unclear. This study demonstrated that QS signals, including C8-HSL, 3-oxo-C10-HSL, and C12-HSL, were detected at ng/L levels in Bacillus subtilis biofilms colonized on MPs. The addition of these three AHLs enhanced natural transformation by 1–2 orders of magnitude in both MP and natural substrate (NS) biofilms, while QS inhibitors decreased transformation frequencies by 2 orders of magnitude. Moreover, transformation frequencies in MP biofilms increased 6.0-fold under QS regulation mediated by AHLs, compared to a 2.5-fold increase in NS biofilms. QS-regulated biofilm formation and EPS secretion were found to be responsible for ARG dissemination in MP biofilms, as indicated by the positive correlation between transformation frequencies and EPS variation. Additionally, mRNA expression analysis revealed that activation and inhibition of the QS system in MP biofilms regulated the expression of natural transformation-related genes, including epsG (regulating EPS secretion) and recO (regulating eDNA transformation). These results highlight the significant influence of the QS system on ARG dissemination through heterogeneous biofilm formation and EPS secretion in MP biofilms. Understanding the effect and mechanism of QS on ARG natural transformation provides new insights into controlling ARG spread by targeting QS in host bacteria within MP biofilms.

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.

Disrupting quorum sensing as a strategy to inhibit bacterial virulence in human, animal, and plant pathogens.

The development of sustainable alternatives to conventional antimicrobials is needed to address bacterial virulence while avoiding selecting resistant strains in a variety of fields, including human, animal, and plant health. Quorum sensing (QS), a bacterial communication system involved in noxious bacterial phenotypes such as virulence, motility, and biofilm formation, is of utmost interest. In this study, we harnessed the potential of the lactonase SsoPox to disrupt QS of human, fish, and plant pathogens. Lactonase treatment significantly alters phenotypes including biofilm formation, motility, and infection capacity. In plant pathogens, SsoPox decreased the production of plant cell wall degrading enzymes in Pectobacterium carotovorum and reduced the maceration of onions infected by Burkholderia glumae. In human pathogens, lactonase treatment significantly reduced biofilm formation in Acinetobacter baumannii, Burkholderia cepacia, and Pseudomonas aeruginosa, with the cytotoxicity of the latter being reduced by SsoPox treatment. In fish pathogens, lactonase treatment inhibited biofilm formation and bioluminescence in Vibrio harveyi and affected QS regulation in Aeromonas salmonicida. QS inhibition can thus be used to largely impact the virulence of bacterial pathogens and would constitute a global and sustainable approach for public, crop, and livestock health in line with the expectations of the One Health initiative.

Lighting the way: how the Vibrio fischeri model microbe reveals the complexity of Earth's "simplest" life forms.

Vibrio (Aliivibrio) fischeri's initial rise to fame derived from its alluring production of blue-green light. Subsequent studies to probe the mechanisms underlying this bioluminescence helped the field discover the phenomenon now known as quorum sensing. Orthologs of quorum-sensing regulators (i.e., LuxR and LuxI) originally identified in V. fischeri were subsequently uncovered in a plethora of bacterial species, and analogous pathways were found in yet others. Over the past three decades, the study of this microbe has greatly expanded to probe the unique role of V. fischeri as the exclusive symbiont of the light organ of the Hawaiian bobtail squid, Euprymna scolopes. Buoyed by this optically amenable host and by persistent and insightful researchers who have applied novel and cross-disciplinary approaches, V. fischeri has developed into a robust model for microbe-host associations. It has contributed to our understanding of how bacteria experience and respond to specific, often fluxing environmental conditions and the mechanisms by which bacteria impact the development of their host. It has also deepened our understanding of numerous microbial processes such as motility and chemotaxis, biofilm formation and dispersal, and bacterial competition, and of the relevance of specific bacterial genes in the context of colonizing an animal host. Parallels in these processes between this symbiont and bacteria studied as pathogens are readily apparent, demonstrating functional conservation across diverse associations and permitting a reinterpretation of "pathogenesis." Collectively, these advances built a foundation for microbiome studies and have positioned V. fischeri to continue to expand the frontiers of our understanding of the microbial world inside animals.