Uncovering a hidden functional role of the XRE-cupin protein PsdR as a novel quorum-sensing regulator in Pseudomonas aeruginosa.

XRE-cupin family proteins containing an DNA-binding domain and a cupin signal-sensing domain are widely distributed in bacteria. In Pseudomonas aeruginosa, XRE-cupin transcription factors have long been recognized as regulators exclusively controlling cellular metabolism pathways. However, their potential functional roles beyond metabolism regulation remain unknown. PsdR, a typical XRE-cupin transcriptional regulator, was previously characterized as a local repressor involved solely in dipeptide metabolism. Here, by measuring quorum-sensing (QS) activities and QS-controlled metabolites, we uncover that PsdR is a new QS regulator in P. aeruginosa. Our RNA-seq analysis showed that rather than a local regulator, PsdR controls a large regulon, including genes associated with both the QS circuit and non-QS pathways. To unveil the underlying mechanism of PsdR in modulating QS, we developed a comparative transcriptome approach named "transcriptome profile similarity analysis" (TPSA). Using this TPSA method, we revealed that PsdR expression causes a QS-null-like transcriptome profile, resulting in QS-inactive phenotypes. Based on the results of TPSA, we further demonstrate that PsdR directly binds to the promoter for the gene encoding the QS master transcription factor LasR, thereby negatively regulating its expression and influencing QS activation. Moreover, our results showed that PsdR functions as a negative virulence regulator, as inactivation of PsdR enhanced bacterial cytotoxicity on host cells. In conclusion, we report on a new QS regulation role for PsdR, providing insights into its role in manipulating QS-controlled virulence. Most importantly, our findings open the door for a further discovery of untapped functions for other XRE-Cupin family proteins.

Streptococcal quorum sensing peptide CSP-7 contributes to muscle inflammation and wasting.

Muscle wasting diseases, such as cancer cachexia and age-associated sarcopenia, have a profound and detrimental impact on functional independence, quality of life, and survival. Our understanding of the underlying mechanisms is currently limited, which has significantly hindered the development of targeted therapies. In this study, we explored the possibility that the streptococcal quorum sensing peptide Competence Stimulating Peptide 7 (CSP-7) might be a previously unidentified contributor to clinical muscle wasting. We found that CSP-7 selectively triggers muscle cell inflammation in vitro, specifically the release of IL-6. Furthermore, we demonstrated that CSP-7 can traverse the gastrointestinal barrier in vitro and is present in the systemic circulation in humans in vivo. Importantly, CSP-7 was associated with a muscle wasting phenotype in mice in vivo. Overall, our findings provide new mechanistic insights into the pathophysiology of muscle inflammation and wasting.

AHL-differential quorum sensing regulation of amino acid metabolism in Hafnia alvei H4.

Quorum sensing (QS) regulation of functional metabolites is rarely reported but a common trait of some bacteria. In this study, we found that QS promoted the extracellular accumulation of glycine and serine while inhibiting the extracellular accumulation of methionine in Hafnia alvei H4. The correlation analysis of five QS signals with the above three QS-regulated amino acids suggested that these QS signals may have functional differences in amino acid regulation. The exogenous AHL add-back studies on genes involved in glycine, serine, and methionine metabolic pathway highlighted that N-octanoyl-l-homoserine lactone (C8-HSL) downregulated the expression of sdhC/fumA genes involved in the succinate to malate pathway, thereby reducing the metabolic flux of the tricarboxylic acid (TCA) cycle as an amino acid metabolism platform. Further in-depth research revealed that the QS system promoted the conversion of folate to tetrahydrofolate (THF) by positively regulating the expression of folA and folM, thus impairing the ability of folate to promote methionine accumulation. Moreover, folate positively regulated the expression of the QS signal synthesis gene luxI, promoting the synthesis of QS signals, which may further enhance the influence of the QS system on amino acid metabolism. These findings contribute to the understanding of amino acid metabolism regulated by QS and provide new perspectives for accurate control of metabolic regulation caused by QS.IMPORTANCEAs one of the important regulatory mechanisms of microorganisms, quorum sensing (QS) is involved in the regulation of various physiological activities. However, few studies on the regulation of amino acid metabolism by QS are available. This study demonstrated that the LuxI-type QS system of Hafnia alvei H4 was involved in the regulation of multiple amino acid metabolism, and different types of QS signals exhibited different roles in regulating amino acid metabolism. Additionally, the regulatory effects of the QS system on amino acid metabolism were investigated from two important cycles that influence the conversion of amino acids, including the TCA cycle and the folate cycle. These findings provide new ideas on the role of QS system in the regulation of amino acid metabolism in organisms.

Cooperation of quorum sensing and central carbon metabolism in the pathogenesis of Gram-positive bacteria.

Quorum sensing (QS), an integral component of bacterial communication, is essential in coordinating the collective response of diverse bacterial pathogens. Central carbon metabolism (CCM), serving as the primary metabolic hub for substances such as sugars, lipids, and amino acids, plays a crucial role in the life cycle of bacteria. Pathogenic bacteria often utilize CCM to regulate population metabolism and enhance the synthesis of specific cellular structures, thereby facilitating in adaptation to the host microecological environment and expediting infection. Research has demonstrated that QS can both directly or indirectly affect the CCM of numerous pathogenic bacteria, thus altering their virulence and pathogenicity. This article reviews the interplay between QS and CCM in Gram-positive pathogenic bacteria, details the molecular mechanisms by which QS modulates CCM, and lays the groundwork for investigating bacterial pathogenicity and developing innovative infection treatment drugs.

Taming Pseudomonas aeruginosa AM26 the barbarian: Targeting the PQS quorum sensing network using crude mandarin extract.

Pseudomonas aeruginosa, one of the most notorious organisms, causes fatal diseases like-, meningitis, pneumonia as well as worsens the prognosis of cystic fibrosis patients. It is also multi-drug resistant and resists a wide range of antibiotics. Attempts have been made to reduce its virulence/pathogenic potential using a number of organic compounds. For this purpose, the Quorum sensing (QS) system of P. aeruginosa was targeted, which regulates its virulence. Pseudomonas Quinolone System (PQS), one of the four quorum sensing systems, producing pyocyanin pigment was chosen. 2-heptyl-3-hydroxy-4-quinolone (HHQ) is a ligand which binds to PQS protein is responsible for pyocyanin pigment production. Attempts were made to find a compound analogous to HHQ which could bind to PQS active site and inhibit the pigment formation. In-silico analysis was performed to estimate possible interactions and to find/predict the possible PQS inhibitors.

Carbon metabolism characteristics of quorum quenching bacteria Rhodococcus sp. BH4 determine the bioaugmentation efficiency under different carbon source conditions.

Carbon sources are critical factors influencing bacterial bioaugmentation, however, the underlying mechanisms, particularly the metabolic characteristics of bioaugmented bacteria remain poorly understood. The bioaugmented bacterium Rhodococcus sp. BH4 secretes the quorum quenching (QQ) enzyme QsdA to disrupt the quorum sensing (QS) in the activated sludge (AS) process, reducing AS yield in-situ. This study investigated the carbon metabolic characteristics of BH4 and explored the effects on bioaugmentation with different influent carbon sources. Because of the absence of glucose-specific phosphoenol phosphotransferase system (PTS), BH4 prefers sodium acetate to glucose. However, the lactones produced during extracellular glucose metabolism enhance BH4 qsdA expression. Moreover, BH4 possess carbon catabolite repression (CCR), acetate inhibits glucose utilization. BH4 microbeads were added to reactors with different carbon sources (R1: sodium acetate; R2: glucose; R3: a mixture of sodium acetate and glucose) for in-situ AS yield reduction. During operation, AS reduction efficiency decreased in the following order: R1 > R3 > R2. R2 and R3 microbeads exhibited similar QQ activity to R1, with less BH4 biomass at 5 d. 13C labeling and Michaelis-Menten equation showed that, due to differences in the competitiveness of carbon sources, R1 BH4 obtained the most carbon, whereas R2 BH4 obtained the least carbon. Moreover, acetate inhibited glucose utilization of R3 BH4. Transcriptome analysis showed that R1 BH4 qsdA expression was the lowest, R2 BH4 was the most serious form of programmed cell death, and the R3 BH4 PTS pathway was inhibited. At 10 d, R1 BH4 biomass and microbead QQ activity were higher than that in R3, and the R2 BH4 lost viability and QQ activity. This study provides new insights into bioaugmentation from the perspectives of carbon source competitiveness, carbon metabolism pathways, and CCR.

Soybean-mediated suppression of BjaI/BjaR1 quorum sensing in Bradyrhizobium diazoefficiens impacts symbiotic nitrogen fixation.

The acyl-homoserine lactones (AHLs)-mediated LuxI/LuxR quorum sensing (QS) system orchestrates diverse bacterial behaviors in response to changes in population density. The role of the BjaI/BjaR1 QS system in Bradyrhizobium diazoefficiens USDA 110, which shares homology with LuxI/LuxR, remains elusive during symbiotic interaction with soybean. Here this genetic system in wild-type (WT) bacteria residing inside nodules exhibited significantly reduced activity compared to free-living cells, potentially attributed to soybean-mediated suppression. The deletion mutant strain ΔbjaR1 showed significantly enhanced nodulation induction and nitrogen fixation ability. Nevertheless, its ultimate symbiotic outcome (plant dry weight) in soybeans was compromised. Furthermore, comparative analysis of the transcriptome, proteome, and promoter activity revealed that the inactivation of BjaR1 systematically activated and inhibited genomic modules associated with nodulation and nitrogen metabolism. The former appeared to be linked to a significant decrease in the expression of NodD2, a key cell-density-dependent repressor of nodulation genes, while the latter conferred bacterial growth and nitrogen fixation insensitivity to environmental nitrogen. In addition, BjaR1 exerted a positive influence on the transcription of multiple genes involved in a so-called central intermediate metabolism within the nodule. In conclusion, our findings highlight the crucial role of the BjaI/BjaR1 QS circuit in positively regulating bacterial nitrogen metabolism and emphasize the significance of the soybean-mediated suppression of this genetic system for promoting efficient symbiotic nitrogen fixation by B. diazoefficiens.IMPORTANCEThe present study demonstrates, for the first time, that the BjaI/BjaR1 QS system of Bradyrhizobium diazoefficiens has a significant impact on its nodulation and nitrogen fixation capability in soybean by positively regulating NodD2 expression and bacterial nitrogen metabolism. Moreover, it provides novel insights into the importance of suppressing the activity of this QS circuit by the soybean host plant in establishing an efficient mutual relationship between the two symbiotic partners. This research expands our understanding of legumes' role in modulating symbiotic nitrogen fixation through rhizobial QS-mediated metabolic functioning, thereby deepening our comprehension of symbiotic coevolution theory. In addition, these findings may hold great promise for developing quorum quenching technology in agriculture.

A pore-scale reactive transport modeling study for quorum sensing-driven biofilm dispersal in heterogeneous porous media.

Microorganisms regulate the expression of energetically expensive phenotypes via a collective decision-making mechanism known as quorum sensing (QS). This study investigates the intricate dynamics of biofilm growth and QS-controlled biofilm dispersal in heterogeneous porous media, employing a pore-scale reactive transport modeling approach. Model simulations carried out under various fluid flow conditions and biofilm growth scenarios reveal that QS processes are influenced not only by the biomass density of biofilm colonies but also by a complex interplay between pore architecture, flow velocity, and the rates of biofilm growth and dispersal. This study demonstrates that pore architecture controls the initiation of QS processes and advection gives rise to oscillatory growth of biofilms. Such oscillation is suppressed if biofilm dynamics are in favor of sustaining a sufficiently high signal concentration, such as fast growth or slow dispersal rates. By establishing a mathematical framework, this study contributes to the fundamental understanding of QS-controlled biofilm dynamics in complex environments.

A balancing act: investigations on the impact of altered signal sensitivity in bacterial quorum sensing.

IMPORTANCE: Quorum sensing (QS) is a widespread form of cell-cell signaling that regulates group behaviors important for competition and cooperation within bacterial communities. The QS systems from different bacterial species have diverse properties, but the functional consequences of this diversity are largely unknown. Taking advantage of hyper- and hypo-sensitive QS receptor variants in the opportunistic pathogen Pseudomonas aeruginosa, we examine the costs and benefits of altered signal sensitivity. We find that the sensitivity of a model QS receptor, LasR, impacts the timing and level of quorum gene expression, and fitness during intra- and interspecies competition. These findings suggest competition with kin and with other bacterial species work together to tune signal sensitivity.

Crosstalk enables mutual activation of coupled quorum sensing pathways through "jump-start" and "push-start" mechanisms.

Many quorum sensing microbes produce more than one chemical signal and detect them using interconnected pathways that crosstalk with each other. While there are many hypotheses for the advantages of sensing multiple signals, the prevalence and functional significance of crosstalk between pathways are much less understood. We explore the effect of intracellular signal crosstalk using a simple model that captures key features of typical quorum sensing pathways: multiple pathways in a hierarchical configuration, operating with positive feedback, with crosstalk at the receptor and promoter levels. We find that crosstalk enables activation or inhibition of one output by the non-cognate signal, broadens the dynamic range of the outputs, and allows one pathway to modulate the feedback circuit of the other. Our findings show how crosstalk between quorum sensing pathways can be viewed not as a detriment to the processing of information, but as a mechanism that enhances the functional range of the full regulatory system. When positive feedback systems are coupled through crosstalk, several new modes of activation or deactivation become possible.

Biocontrol of biofilm forming Burkholderia cepacia using a quorum quenching crude lactonase enzyme extract from a marine Chromohalobacter sp. strain D23.

Biofilm plays advantageous role in Burkholderia cepacia by exerting multi-drug resistance. As quorum sensing (QS) system regulates biofilm formation and pathogenicity in B. cepacia strains, quorum quenching (QQ) may be a novel strategy to control persistent B. cepacia infections. In these regards, 120 halophilic bacteria were isolated from marine sample and tested using Chromobacterium violaceum and C. violaceum CV026-based bioassays initially, showing reduced violacein synthesis by QQ enzyme by 6 isolates. Among them, Chromohalobacter sp. D23 significantly degraded both C6-homoserine lactone (C6-HSL) and C8-HSL due to potent lactonase activity, which was detected by C. violaceum CV026 biosensor. Further high-performance liquid chromatography (HPLC) study confirmed degradation of N-acyl homoserine lactones (N-AHLs) particularly C6-HSL and C8-HSL by crude lactonase enzyme. Chromohalobacter sp. D23 reduced biofilm formation in terms of decreased total biomass and viability in biofilm-embedded cells in B. cepacia significantly which was also evidenced by fluorescence microscopic images. An increase in antibiotic susceptibility of B. cepacia biofilm was achieved when crude lactonase enzyme of Chromohalobacter sp. strain D23 was combined with chloramphenicol (1-5 × MIC). Chromohalobacter sp. D23 also showed prominent decrease in QS-mediated synthesis of virulence factors such as extracellular polymeric substances (EPS), extracellular protease, and hemolysin in B. cepacia. Again crude lactonase enzyme of Chromohalobacter sp. strain D23 inhibited B. cepacia biofilm formation inside nasal oxygen catheters in vitro. Finally, antibiotic susceptibility test and virulence tests revealed sensitivity of Chromohalobacter sp. strain D23 against a wide range of conventional antibiotics as well as absence of gelatinolytic, hemolytic, and serum coagulating activities. Therefore, the current study shows potential quorum quenching as well as anti-biofilm activity of Chromohalobacter sp. D23 against B. cepacia.

Pharmacokinetics, quorum-sensing signal molecules and tryptophan-related metabolomics of the novel anti-virulence drug Fluorothiazinon in a Pseudomonas aeruginosa-induced pneumonia murine model.

Pseudomonas aeruginosa (PA) infection is commonly associated with hospital-acquired infections in patients with immune deficiency and/or severe lung diseases. Managing this bacterium is complex due to drug resistance and high adaptability. Fluorothiazinon (FT) is an anti-virulence drug developed to suppress the virulence of bacteria as opposed to bacterial death increasing host's immune response to infection and improving treatment to inhibit drug resistant bacteria. We aimed to evaluate FT pharmacokinetics, quorum sensing signal molecules profiling and tryptophan-related metabolomics in blood, liver, kidneys, and lungs of mice. Study comprised three groups: a group infected with PA that was treated with 400 mg/kg FT ("infected treated group"); a non-infected group, but also treated with the same single drug dose ("non-infected treated group"); and an infected group that received a vehicle ("infected non-treated group"). PA-mediated infection blood pharmacokinetics profiling was indicative of increased drug concentrations as shown by increased Cmax and AUCs. Tissue distribution in liver, kidneys, and lungs, showed that liver presented the most consistently higher concentrations of FT in the infected versus non-infected mice. FT showed that HHQ levels were decreased at 1 h after dosing in lungs while PQS levels were lower across time in lungs of infected treated mice in comparison to infected non-treated mice. Metabolomics profiling performed in lungs and blood of infected treated versus infected non-treated mice revealed drug-associated metabolite alterations, especially in the kynurenic and indole pathways.

Joining the bacterial conversation: increasing the cultivation efficiency of soil bacteria with acyl-homoserine lactones and cAMP.

IMPORTANCE: Microorganisms are a repository of interesting metabolites and functions. Therefore, accessing them is an important exercise for advancing not only basic questions about their physiology but also to advance technological applications. In this sense, increasing the culturability of environmental microorganisms remains an important endeavor for modern microbiology. Because microorganisms do not live in isolation in their environments, molecules can be added to the cultivation strategies to "inform them" that they are present in growth-permissive environmental conditions. Signaling molecules such as acyl-homoserine lactones and 3',5'-cyclic adenosine monophosphate belong to the plethora of molecules used by bacteria to communicate with each other in a phenomenon called quorum sensing. Therefore, including quorum sensing molecules can be an incentive for microorganisms, specifically soil bacteria, to increase their numbers on solid media.

Engineering a Pseudomonas putida as living quorum quencher for biofilm formation inhibition, benzenes degradation, and environmental risk evaluation.

Bacterial communication interruption based on quorum quenching (QQ) has been proven its potential in biofilm formation inhibition and biofouling control. However, it would be more satisfying if QQ could be combined with the efficient degradation of contaminants in environmental engineering. In this study, we engineered a biofilm of Pseudomonas putida through introducing a QQ synthetic gene, which achieved both biofilm formation inhibition and efficient degradation of benzene series in wastewater. The aiiO gene introduced into the P. putida by heat shock method was highly expressed to produce QQ enzyme to degrade AHL-based signal molecules. The addition of this engineered P. putida reduced the AHLs concentration, quorum sensing gene expression, and connections of the microbial community network in activated sludge and therefore inhibited the biofilm formation. Meanwhile, the sodium benzoate degradation assay indicated an enhanced benzene series removal ability of the engineering bacteria on activated sludge. Besides, we also demonstrated a controllable environmental risk of this engineered bacteria through monitoring its abundance and horizontal gene transfer test. Overall, the results of this study suggest an alternative strategy to solve multiple environmental problems through genetic engineering means and provide support for the application of engineered bacteria in environmental biotechnology.

Activation of a Vibrio cholerae CBASS anti-phage system by quorum sensing and folate depletion.

IMPORTANCE: To counteract infection with phage, bacteria have evolved a myriad of molecular defense systems. Some of these systems initiate a process called abortive infection, in which the infected cell kills itself to prevent phage propagation. However, such systems must be inhibited in the absence of phage infection to prevent spurious death of the host. Here, we show that the cyclic oligonucleotide based anti-phage signaling system (CBASS) accomplishes this by sensing intracellular folate molecules and only expressing this system in a group. These results enhance our understanding of the evolution of the seventh Vibrio cholerae pandemic and more broadly how bacteria defend themselves against phage infection.

Regulation of Bacterial Biofilm Formation by Ultrasound: Role of Autoinducer-2 and Finite-Element Analysis of Acoustic Streaming.

OBJECTIVE: The formation of bacterial biofilm regulated by quorum sensing (QS) is a critical factor that contributes to infections of indwelling medical devices. Autoinducer-2 (AI-2), as a signal molecule in QS, plays a crucial role in mediating bacterial signaling and regulating their biological behavior. This study investigated the impact of ultrasonic vibration at varying frequencies on biofilm formation in a mixture of Staphylococcus aureus and Escherichia coli. METHODS: By exciting ultrasound at different frequencies (20, 100 and 200 kHz), a vibration with an amplitude of 100 nm was generated on the material surface located at the bottom of the petri dish containing mixed bacteria. We measured the content of AI-2 and bacteria in the mixed bacterial solution and bioburden on material surfaces at different time points during culture. In addition, the relationships among AI-2 content, bacterial concentration and distribution were assessed through finite-element analysis of acoustic streaming under ultrasonic vibration. RESULTS: The AI-2 gradient is influenced by the diversity of acoustic streaming patterns on the material surface and in the mixed bacterial solution caused by ultrasonic vibration at different frequencies, thereby regulating biofilm formation. The experimental results showed that the optimal inhibition effect on AI-2 and minimal bacterial adhesion degree was achieved when applying an ultrasonic frequency of 100 kHz with a power intensity of 46.1 mW/cm2 under an amplitude of 100 nm. CONCLUSION: Ultrasound can affect the QS system of bacteria, leading to alterations in their biological behavior. Different species of bacteria exhibit varying degrees of chemotaxis toward different frequencies.

Revealing the roles of chemical communication in restoring the formation and electroactivity of electrogenic biofilm under electrical signaling disruption.

Electrogenic biofilms in microbial electrochemical systems have played significant roles in simultaneous wastewater treatment and energy recovery owing to their unique extracellular electron transfer. Their formation has been shown to be regulated by electrical and chemical communication, but the interaction between these signal communication pathways has not been studied. This research investigated the coordination between intracellular c-di-GMP signaling and reinforced quorum sensing with or without exogenous HSL (a common quorum sensing molecule), on the formation of mixed-cultured electrogenic biofilm under electrical signaling disruption by tetraethylammonium (TEA, a broad-range potassium channel blocker). Intracellular c-di-GMP was spontaneously reinforced in response to TEA stress, and metagenomic analysis revealed that the dominant DGC (the genes for producing c-di-GMP) induced the eventual biofilm formation by mediating exopolysaccharide synthesis. Meanwhile, reinforced quorum sensing by exogenous HSL could also benefit the biofilm restoration, however, it alleviated the TEA-induced communication stress, resulting in the weakening of c-di-GMP dominance. Interestingly, suppressing electrical communication with or without HSL addition both induced selective enrichment of Geobacter of 85.5% or 30.1% respectively. Functional contribution analysis revealed the significant roles of Geobacter and Thauera in c-di-GMP signaling, especially Thauera in resistance to TEA stress. This study proposed a potential strategy for electrogenic biofilm regulation from the perspectives of cell-to-cell communication.

A Streptomyces tendae Specialized Metabolite Inhibits Quorum Sensing in Group A Streptococcus.

Quorum sensing (QS) is a means of bacterial communication accomplished by microbe-produced signals and sensory systems. QS systems regulate important population-wide behaviors in bacteria, including secondary metabolite production, swarming motility, and bioluminescence. The human pathogen Streptococcus pyogenes (group A Streptococcus [GAS]) utilizes Rgg-SHP QS systems to regulate biofilm formation, protease production, and activation of cryptic competence pathways. Given their reliance on small-molecule signals, QS systems are attractive targets for small-molecule modulators that would then affect gene expression. In this study, a high-throughput luciferase assay was employed to screen an Actinobacteria-derived secondary metabolite (SM) fraction library to identify small molecule inhibitors of Rgg regulation. A metabolite produced by Streptomyces tendae D051 was found to be a general inhibitor of GAS Rgg-mediated QS. Herein, we describe the biological activity of this metabolite as a QS inhibitor. IMPORTANCE Streptococcus pyogenes, a human pathogen known for causing infections such as pharyngitis and necrotizing fasciitis, uses quorum sensing (QS) to regulate social responses in its environment. Previous studies have focused on disrupting QS as a means to control specific bacterial signaling outcomes. In this work, we identified and described the activity of a naturally derived S. pyogenes QS inhibitor. This study demonstrates that the inhibitor affects three separate but similar QS signaling pathways.

Quorum Sensing-Dependent Invasion of Ralstonia solanacearum into Fusarium oxysporum Chlamydospores.

Strains of the Ralstonia solanacearum species complex (RSSC), although known as the causative agent of bacterial wilt disease in plants, induce the chlamydospores of many fungal species and invade them through the spores. The lipopeptide ralstonins are the chlamydospore inducers produced by RSSC and are essential for this invasion. However, no mechanistic investigation of this interaction has been conducted. In this study, we report that quorum sensing (QS), which is a bacterial cell-cell communication, is important for RSSC to invade the fungus Fusarium oxysporum (Fo). ΔphcB, a deletion mutant of QS signal synthase, lost the ability to both produce ralstonins and invade Fo chlamydospores. The QS signal methyl 3-hydroxymyristate rescued these disabilities. In contrast, exogenous ralstonin A, while inducing Fo chlamydospores, failed to rescue the invasive ability. Gene-deletion and -complementation experiments revealed that the QS-dependent production of extracellular polysaccharide I (EPS I) is essential for this invasion. The RSSC cells adhered to Fo hyphae and formed biofilms there before inducing chlamydospores. This biofilm formation was not observed in the EPS I- or ralstonin-deficient mutant. Microscopic analysis showed that RSSC infection resulted in the death of Fo chlamydospores. Altogether, we report that the RSSC QS system is important for this lethal endoparasitism. Among the factors regulated by the QS system, ralstonins, EPS I, and biofilm are important parasitic factors. IMPORTANCE Ralstonia solanacearum species complex (RSSC) strains infect both plants and fungi. The phc quorum-sensing (QS) system of RSSC is important for parasitism on plants, because it allows them to invade and proliferate within the hosts by causing appropriate activation of the system at each infection step. In this study, we confirm that ralstonin A is important not only for Fusarium oxysporum (Fo) chlamydospore induction but also for RSSC biofilm formation on Fo hyphae. Extracellular polysaccharide I (EPS I) is also essential for biofilm formation, while the phc QS system controls these factors in terms of production. The present results advocate a new QS-dependent mechanism for the process by which a bacterium invades a fungus.

Lactonase-mediated inhibition of quorum sensing largely alters phenotypes, proteome, and antimicrobial activities in Burkholderia thailandensis E264.

Introduction: Burkholderia thailandensis is a study model for Burkholderia pseudomallei, a highly virulent pathogen, known to be the causative agent of melioidosis and a potential bioterrorism agent. These two bacteria use an (acyl-homoserine lactone) AHL-mediated quorum sensing (QS) system to regulate different behaviors including biofilm formation, secondary metabolite productions, and motility. Methods: Using an enzyme-based quorum quenching (QQ) strategy, with the lactonase SsoPox having the best activity on B. thailandensis AHLs, we evaluated the importance of QS in B. thailandensis by combining proteomic and phenotypic analyses. Results: We demonstrated that QS disruption largely affects overall bacterial behavior including motility, proteolytic activity, and antimicrobial molecule production. We further showed that QQ treatment drastically decreases B. thailandensis bactericidal activity against two bacteria (Chromobacterium violaceum and Staphylococcus aureus), while a spectacular increase in antifungal activity was observed against fungi and yeast (Aspergillus niger, Fusarium graminearum and Saccharomyces cerevisiae). Discussion: This study provides evidence that QS is of prime interest when it comes to understanding the virulence of Burkholderia species and developing alternative treatments.