Quorum Sensing Signal Production Research Articles

Impact of Quorum Sensing on the Virulence and Survival Traits of Burkholderia plantarii.

Quorum sensing (QS) is a mechanism by which bacteria detect and respond to cell density, regulating collective behaviors. Burkholderia plantarii, the causal agent of rice seedling blight, employs the LuxIR-type QS system, common among Gram-negative bacteria, where LuxI-type synthase produces QS signals recognized by LuxR-type regulators to control gene expression. This study aimed to elucidate the QS mechanism in B. plantarii KACC18965. Through whole-genome analysis and autoinducer assays, the plaI gene, responsible for QS signal production, was identified. Motility assays confirmed that C8-homoserine lactone (C8-HSL) serves as the QS signal. Physiological experiments revealed that the QS-defective mutant exhibited reduced virulence, impaired swarming motility, and delayed biofilm formation compared to the wild type. Additionally, the QS mutant demonstrated weakened antibacterial activity against Escherichia coli and decreased phosphate solubilization. These findings indicate that QS in B. plantarii significantly influences various pathogenicity and survival traits, including motility, biofilm formation, antibacterial activity, and nutrient acquisition, highlighting the critical role of QS in pathogen virulence and adaptability.

Phenotypic memory in quorum sensing.

Quorum sensing (QS) is a regulatory mechanism used by bacteria to coordinate group behavior in response to high cell densities. During QS, cells monitor the concentration of external signals, known as autoinducers, as a proxy for cell density. QS often involves positive feedback loops, leading to the upregulation of genes associated with QS signal production and detection. This results in distinct steady-state concentrations of QS-related molecules in QS-ON and QS-OFF states. Due to the slow decay rates of biomolecules such as proteins, even after removal of the initial stimuli, cells can retain elevated levels of QS-associated biomolecules for extended periods of time. This persistence of biomolecules after the removal of the initial stimuli has the potential to impact the response to future stimuli, indicating a memory of past exposure. This phenomenon, which is a consequence of the carry-over of biomolecules rather than genetic inheritance, is known as "phenotypic" memory. This theoretical study aims to investigate the presence of phenotypic memory in QS and the conditions that influence this memory. Numerical simulations based on ordinary differential equations and analytical modeling were used to study gene expression in response to sudden changes in cell density and extracellular signal concentrations. The model examined the effect of various cellular parameters on the strength of QS memory and the impact on gene regulatory dynamics. The findings revealed that QS memory has a transient effect on the expression of QS-responsive genes. These consequences of QS memory depend strongly on how cell density was perturbed, as well as various cellular parameters, including the Fold Change in the expression of QS-regulated genes, the autoinducer synthesis rate, the autoinducer threshold required for activation, and the cell growth rate.

Membrane-enclosed Pseudomonas quinolone signal attenuates bacterial virulence by interfering with quorum sensing.

Outer membrane vesicle (OMV)-delivered Pseudomonas quinolone signal (PQS) plays a critical role in cell-cell communication in Pseudomonas aeruginosa. However, the functions and mechanisms of membrane-enclosed PQS in interspecies communication in microbial communities are not clear. Here, we demonstrate that PQS delivered by both OMVs from P. aeruginosa and liposome reduces the competitiveness of Burkholderia cenocepacia, which usually shares the same niche in the lungs of cystic fibrosis patients, by interfering with quorum sensing (QS) in B. cenocepacia through the LysR-type regulator ShvR. Intriguingly, we found that ShvR regulates the production of the QS signals cis-2-dodecenoic acid (BDSF) and N-acyl homoserine lactone (AHL) by directly binding to the promoters of signal synthase-encoding genes. Perception of PQS influences the regulatory activity of ShvR and thus ultimately reduces QS signal production and virulence in B. cenocepacia. Our findings provide insights into the interspecies communication mediated by the membrane-enclosed QS signal among bacterial species residing in the same microbial community. IMPORTANCE Quorum sensing (QS) is a ubiquitous cell-to-cell communication mechanism. Previous studies showed that Burkholderia cenocepacia mainly employs cis-2-dodecenoic acid (BDSF) and N-acyl homoserine lactone (AHL) QS systems to regulate biological functions and virulence. Here, we demonstrate that Pseudomonas quinolone signal (PQS) delivered by outer membrane vesicles from Pseudomonas aeruginosa or liposome attenuates B. cenocepacia virulence by targeting the LysR-type regulator ShvR, which regulates the production of the QS signals BDSF and AHL in B. cenocepacia. Our results not only suggest the important roles of membrane-enclosed PQS in interspecies and interkingdom communications but also provide a new perspective on the use of functional nanocarriers loaded with QS inhibitors for treating pathogen infections.

Extracellular release of two peptidases dominates generation of the trypanosome quorum-sensing signal

Trypanosomes causing African sleeping sickness use quorum-sensing (QS) to generate transmission-competent stumpy forms in mammalian hosts. This density-dependent process is signalled by oligopeptides that stimulate the signal transduction pathway leading to stumpy formation. Here, using mass spectrometry analysis, we identify peptidases released by trypanosomes and, for 12 peptidases, confirm their extracellular delivery. Thereafter, we determine the contribution of each peptidase to QS signal production using systematic inducible overexpression in vivo, and confirm this activity operates through the physiological QS signalling pathway. Gene knockout of the QS-active peptidases identifies two enzymes, oligopeptidase B and metallocarboxypeptidase 1, that significantly reduce QS when ablated individually. Further, combinatorial gene knockout of both peptidases confirms their dominance in the generation of the QS signal, with peptidase release of oligopeptidase B mediated via an unconventional protein secretion pathway. This work identifies how the QS signal driving trypanosome virulence and transmission is generated in mammalian hosts.

In vitro Activity of Antivirulence Drugs Targeting the las or pqs Quorum Sensing Against Cystic Fibrosis Pseudomonas aeruginosa Isolates.

The chronic lung infection caused by Pseudomonas aeruginosa is a major cause of morbidity and mortality in cystic fibrosis (CF) patients. Antivirulence drugs targeting P. aeruginosa quorum sensing (QS) systems are intensively studied as antibiotics substitutes or adjuvants. Previous studies, carried out in non-CF P. aeruginosa reference strains, showed that the old drugs niclosamide and clofoctol could be successfully repurposed as antivirulence drugs targeting the las and pqs QS systems, respectively. However, frequent emergence of QS-defective mutants in the CF lung undermines the use of QS inhibitors in CF therapy. Here, QS signal production and susceptibility to niclosamide and clofoctol have been investigated in 100 P. aeruginosa CF isolates, with the aim of broadening current knowledge on the potential of anti-QS compounds in CF therapy. Results showed that 85, 78, and 69% of the CF isolates from our collection were proficient for the pqs, rhl, and las QS systems, respectively. The ability of both niclosamide and clofoctol to inhibit QS and virulence in vitro was highly variable and strain-dependent. Niclosamide showed an overall low range of activity and its negative effect on las signal production did not correlate with a decreased production of virulence factors. On the other hand, clofoctol displayed a broader QS inhibitory effect in CF isolates, with consequent reduction of the pqs-controlled virulence factor pyocyanin. Overall, this study highlights the importance of testing new antivirulence drugs against large panels of P. aeruginosa CF clinical isolates before proceeding to further pre-clinical studies and corroborates previous evidence that strains naturally resistant to QS inhibitors occur among CF isolates. However, it is also shown that resistance to pqs inhibitors is less frequent than resistance to las inhibitors, thus supporting the development of pqs inhibitors for antivirulence therapy in CF.

The Impact of an Efflux Pump Inhibitor on the Activity of Free and Liposomal Antibiotics against Pseudomonas aeruginosa.

The eradication of Pseudomonas aeruginosa in cystic fibrosis patients has become continuously difficult due to its increased resistance to treatments. This study assessed the efficacy of free and liposomal gentamicin and erythromycin, combined with Phenylalanine arginine beta-naphthylamide (PABN), a broad-spectrum efflux pump inhibitor, against P. aeruginosa isolates. Liposomes were prepared and characterized for their sizes and encapsulation efficiencies. The antimicrobial activities of formulations were determined by the microbroth dilution method. Their activity on P. aeruginosa biofilms was assessed, and the effect of sub-inhibitory concentrations on bacterial virulence factors, quorum sensing (QS) signals and bacterial motility was also evaluated. The average diameters of liposomes were 562.67 ± 33.74 nm for gentamicin and 3086.35 ± 553.95 nm for erythromycin, with encapsulation efficiencies of 13.89 ± 1.54% and 51.58 ± 2.84%, respectively. Liposomes and PABN combinations potentiated antibiotics by reducing minimum inhibitory and bactericidal concentrations by 4–32 fold overall. The formulations significantly inhibited biofilm formation and differentially attenuated virulence factor production as well as motility. Unexpectedly, QS signal production was not affected by treatments. Taken together, the results indicate that PABN shows potential as an adjuvant of liposomal macrolides and aminoglycosides in the management of lung infections in cystic fibrosis patients.

Identification potential inhibitors against the Streptococcus quorum-sensing signal pathway

Streptococcal infections are common in human and antibiotics are frequently prescribed in clinical practice. However, infections caused by drug-resistant strains are particularly difficult to treat using common antibiotics. Hence, there is an urgent need for new antibiotics. Quorum sensing is a regulatory mechanism involving cell communication that is thought to play an important role in various bacterial infections, including those caused by Streptococcus. The ATP-binding cassette transporter ComA of Streptococcus is essential for quorum-sensing signal production. The inhibition of the ComA peptidase domain (ComA PEP) suppresses the quorum-sensing pathway and resulting changes in phenotype and/or behavior. Using virtual screening and molecular dynamics simulations, two promising candidate compounds, ZINC32918029 and ZINC6751571, were found. These compounds had similar binding modes and interactions to the experimentally determined reference inhibitor 6CH. However, a significantly stronger negative binding energy was achieved (−113.501 ± 15.312 KJ/mol and −103.153 ± 11.912 KJ/mol for ZINC32918029 and ZINC6751571, respectively). Molecular dynamics simulations also revealed that ZINC32918029 and ZINC6751571 had a strong affinity for ComA PEP. These results indicate that ZINC32918029 and ZINC6751571 are promising candidate inhibitors of the Streptococcus quorum-sensing pathway. Communicated by Ramaswamy H. Sarma

Anthranilic acid from Ralstonia solanacearum plays dual roles in intraspecies signalling and inter-kingdom communication

Quorum sensing (QS) signals are widely utilized by bacteria to regulate biological functions in response to cell population density. Previous studies have demonstrated that Ralstonia solanacearum employs two different types of QS systems. We report here that anthranilic acid controls important biological functions and the production of QS signals in R. solanacearum. It was demonstrated that the biosynthesis of anthranilic acid is mainly performed by TrpEG. The accumulation of anthranilic acid and the transcription of trpEG occur in a cell density-dependent manner in R. solanacearum. Both the anthranilic acid and TrpEG homologues are conserved in various bacterial species. Moreover, we show that Sporisorium scitamineum sexual mating and hypha formation are strongly inhibited by the addition of exogenous anthranilic acid. Our results suggest that anthranilic acid is important for the physiology of bacteria in addition to its role in inter-kingdom communication.

Sodium salicylate interferes with quorum-sensing-regulated virulence in chronic wound isolates of Pseudomonas aeruginosa in simulated wound fluid.

Introduction. An important factor for delayed healing of chronic wounds is the presence of bacteria. Quorum sensing (QS), a cell density-dependent signalling system, controls the production of many virulence factors and biofilm formation in Pseudomonas aeruginosa .Aim. Inhibition by sodium salicylate (NaSa) of QS-regulated virulence expression was evaluated in QS-characterized clinical wound isolates of P. aeruginosa, cultured in serum-containing medium.Methodology. Fourteen clinical P. aeruginosa strains from chronic wounds were evaluated for the production of QS signals and virulence factors. Inhibition of QS by NaSa in P. aeruginosa clinical strains, wild-type PAO1 and QS reporter strains was evaluated using in vitro assays for the production of biofilm, pyocyanin, siderophores, alkaline protease, elastase and stapholytic protease.Results. Six clinical strains secreted several QS-associated virulence factors and signal molecules and two were negative for all factors. Sub-inhibitory concentrations of NaSa downregulated the expression of the QS-related genes lasB, rhlA and pqsA and reduced the secretion of several virulence factors in PAO1 and clinical strains cultured in serum. Compared to serum-free media, the presence of serum increased the expression of QS genes and production of siderophores and pyocyanin but decreased biofilm formation.Conclusions. Pseudomonas aeruginosa from chronic wound infections showed different virulence properties. While very few strains showed no QS activity, approximately half were highly virulent and produced QS signals, suggesting that the targeting of QS is a viable and relevant strategy for infection control. NaSa showed activity as a QS-inhibitor by lowering the virulence phenotypes and QS signals at both transcriptional and extracellular levels.

Signal Production and Response Specificity in the phc Quorum Sensing Systems of Ralstonia solanacearum Species Complex.

Ralstonia solanacearum strains are devastating plant pathogens with global distribution, a wide host range, and genetic diversity, and they are now also referred to as the R. solanacearum species complex (RSSC). RSSC strains employ the quorum sensing (QS) system composed of the phcBSR operon to regulate their virulence on plants. The RSSC strains previously examined produce either (R)-methyl 3-hydroxymyristate (3-OH MAME) or (R)-methyl 3-hydroxypalmitate (3-OH PAME) as their QS signals. Analogously, the phylogenetic analyses of the signal synthase PhcB and the signal receptor PhcS from 15 RSSC strains revealed that these proteins have two clades dependent on their QS signal types. However, the biochemical mechanism underlying this selectivity of QS signal production remains to be elucidated. We demonstrated that the PhcB methyltransferases synthesize QS signals from the cognate fatty acids (R)-3-hydroxymyristic acid or (R)-3-hydroxypalmitic acid. The RSSC strains used here produced both fatty acids, and thus the selectivity of QS signal production depends on the activity of PhcB enzymes. On the other hand, the enantioselective supply of the precursors functioned in the production of enantiopure QS signals. The opposite QS signals weakly induced the production of virulence factors in the RSSC strains. Furthermore, the complementation of the phcB gene encoding the 3-OH PAME-type synthase to the phcB-deletion mutant of the 3-OH MAME-producing strain did not rescue its virulence on tomato plants. Taken together, we propose that the specific production of 3-OH MAME/3-OH PAME ensures full virulence of the RSSC strains.

Interference With Quorum-Sensing Signal Biosynthesis as a Promising Therapeutic Strategy Against Multidrug-Resistant Pathogens.

Faced with the global health threat of increasing resistance to antibiotics, researchers are exploring interventions that target bacterial virulence factors. Quorum sensing is a particularly attractive target because several bacterial virulence factors are controlled by this mechanism. Furthermore, attacking the quorum-sensing signaling network is less likely to select for resistant strains than using conventional antibiotics. Strategies that focus on the inhibition of quorum-sensing signal production are especially attractive because the enzymes involved are expressed in bacterial cells but are not present in their mammalian counterparts. We review here various approaches that are being taken to interfere with quorum-sensing signal production via the inhibition of autoinducer-2 synthesis, PQS synthesis, peptide autoinducer synthesis, and N-acyl-homoserine lactone synthesis. We expect these approaches will lead to the discovery of new quorum-sensing inhibitors that can help to stem the tide of antibiotic resistance.

Multiple Quorum Quenching Enzymes Are Active in the Nosocomial Pathogen Acinetobacter baumannii ATCC17978.

Acinetobacter baumannii presents a typical luxI/luxR quorum sensing (QS) system (abaI/abaR) but the acyl-homoserine lactone (AHL) signal profile and factors controlling the production of QS signals in this species have not been determined yet. A very complex AHL profile was identified for A. baumannii ATCC17978 as well as for A. nosocomialis M2, but only when cultivated under static conditions, suggesting that surface or cell-to-cell contact is involved in the activation of the QS genes. The analysis of A. baumanni clinical isolates revealed a strain-specific AHL profile that was also affected by nutrient availability. The concentration of OHC12-HSL, the major AHL found in A. baumannii ATCC17978, peaked upon stationary-phase establishment and decreases steeply afterwards. Quorum quenching (QQ) activity was found in the cell extracts of A. baumannii ATCC17978, correlating with the disappearance of the AHLs from the culture media, indicating that AHL concentration may be self-regulated in this pathogen. Since QQ activity was observed in strains in which AidA, a novel α/β-hydrolase recently identified in A. baumannii, is not present, we have searched for additional QQ enzymes in A. baumannii ATCC17978. Seven putative AHL-lactonase sequences could be identified in the genome and the QQ activity of 3 of them could be confirmed. At least six of these lactonase sequences are also present in all clinical isolates as well as in A. nosocomialis M2. Surface-associated motility and biofilm formation could be blocked by the exogenous addition of the wide spectrum QQ enzyme Aii20J. The differential regulation of the QQ enzymes in A. baumannii ATCC17978 and the full dependence of important virulence factors on the QS system provides a strong evidence of the importance of the AHL-mediated QS/QQ network in this species.

Production of quorum-sensing signals by bacteria in the coral mucus layer

Quorum sensing is an integral part of bacterial communication and interaction, but has not been well characterized in coral mucus microbiota. In this study, of 61 coral mucus isolates, five alphaproteobacteria and one Vibrio species were found to produce N-acyl homoserine lactone (AHL), a quorum-sensing signal in bacteria. Eight gammaproteobacteria isolates were found to produce autoinducer-2 (AI-2) quorum-sensing signals along with two actinobacteria of the genus Rothia. Coral mucus is rich in the antioxidant dimethylsulfoniopropionate (DMSP), the concentration of which has been found to increase under heat stress. Neither AHL nor AI-2 activity was induced by DMSP in those coral mucus isolates that did not initially produce quorum-sensing signals. However, the AI-2 activities of one Rothia isolate (SCSIO 13017) from coral mucus and of Vibrio shilonii (DSM 13774 isolated from a bleached coral) were found to increase in response to 5 μM DMSP but decreased in response to 50 μM DMSP for the first time. These findings suggest that the production of quorum-sensing signals in the coral mucus microbiota may play a role in structuring the surface microbial community as they respond to environmental stress.

High-throughput Screening of Small Molecule Inhibitors of the Streptococcus Quorum-sensing Signal Pathway

The main components of the quorum-sensing system are expected to be favorable targets for drug development to combat various chronic infectious diseases. ComA of Streptococcus is an ATP-binding cassette transporter containing a peptidase domain (PEP), which is essential for the quorum-sensing signal production. Using high-throughput screening, we found a potent small molecule that suppressed the S. mutans quorum-sensing pathway through inhibition of PEP activity. The compound effectively attenuated the biofilm formation and competence development of S. mutans without inhibiting cell growth. The kinetic and structural studies with this molecule and a related compound unexpectedly revealed an allosteric site of PEP. This relatively hydrophobic site is thought to undergo large structural changes during the catalytic process. These compounds inhibit PEP activity by binding to and suppressing the structural changes of this site. These results showed that PEP is a good target for inhibitors of the Streptococcus quorum-sensing system.

Regulation Involved in Colonization of Intercellular Spaces of Host Plants in Ralstonia solanacearum.

A soil-borne bacterium Ralstonia solanacearum invading plant roots first colonizes the intercellular spaces of the root, and eventually enters xylem vessels, where it replicates at high levels leading to wilting symptoms. After invasion into intercellular spaces, R. solanacearum strain OE1-1 attaches to host cells and expression of the hrp genes encoding components of the type III secretion system (T3SS). OE1-1 then constructs T3SS and secrets effectors into host cells, inducing expression of the host gene encoding phosphatidic acid phosphatase. This leads to suppressing plant innate immunity. Then, OE1-1 grows on host cells, inducing quorum sensing (QS). The QS contributes to regulation of OE1-1 colonization of intercellular spaces including mushroom-type biofilm formation on host cells, leading to its virulence. R. solanacearum strains AW1 and K60 produce methyl 3-hydroxypalmitate (3-OH PAME) as a QS signal. The methyltransferase PhcB synthesizes 3-OH PAME. When 3-OH PAME reaches a threshold level, it increases the ability of the histidine kinase PhcS to phosphorylate the response regulator PhcR. This results in elevated levels of functional PhcA, the global virulence regulator. On the other hand, strains OE1-1 and GMI1000 produce methyl 3-hydroxymyristate (3-OH MAME) as a QS signal. Among R. solanacearum strains, the deduced PhcB and PhcS amino acid sequences are related to the production of QS signals. R. solanacearum produces aryl-furanone secondary metabolites, ralfuranones, which are extracellularly secreted and required for its virulence, dependent on the QS. Interestingly, ralfuranones affect the QS feedback loop. Taken together, integrated signaling via ralfuranones influences the QS, contributing to pathogen virulence.

Arachis hypogaea L. produces mimic and inhibitory quorum sensing like molecules

A wide variety of plant-associated soil bacteria (rhizobacteria) communicate with each other by quorum sensing (QS). Plants are able to detect and produce mimics and inhibitor molecules of the QS bacterial communicative process. Arachis hypogaea L. (peanut) establishes a nitrogen-fixing symbiosis with rhizobia belonging to the genus Bradyrhizobium. These bacteria use a QS mechanism dependent on the synthesis of N-acyl homoserine lactones (AHLs). Given the relevance that plant-rhizobacteria interactions have at the ecological level, this work addresses the involvement of peanut in taking part in the QS mechanism. By using biosensor bacterial strains capable of detecting AHLs, a series of standard and original bioassays were performed in order to determine both (i) the production of QS-like molecules in vegetal materials and (ii) the expression of the QS mechanism throughout plant-bacteria interaction. Mimic QS-like molecules (mQS) linked to AHLs with long acyl chains (lac-AHL), and inhibitor QS-like molecules (iQS) linked to AHLs with short acyl chains (sac-AHL) were detected in seed and root exudates. The results revealed that synthesis of specific signaling molecules by the plant (such as mQS and iQS) probably modulates the function and composition of the bacterial community established in its rhizosphere. Novel bioassays of QS detection during peanut-Bradyrhizobium interaction showed an intense production of QS signals in the contact zone between root and bacteria. It is demonstrated that root exudates stimulate the root colonization and synthesis of lac-AHL by Bradyrhizobium strains in the plant rhizosphere, which leads to the early stages of the development of beneficial plant-bacteria interactions.

Crystal structure of Pseudomonas aeruginosa RsaL bound to promoter DNA reaffirms its role as a global regulator involved in quorum-sensing.

Pseudomonas aeruginosa possesses at least three well-defined quorum-sensing (QS) (las, rhl and pqs) systems that control a variety of important functions including virulence. RsaL is a QS repressor that reduces QS signal production and ensures homeostasis by functioning in opposition to LasR. However, its regulatory role in signal homeostasis remains elusive. Here, we conducted a ChIP-seq assay and revealed that RsaL bound to two new targets, the intergenic regions of PA2228/PA2229 and pqsH/cdpR, which are required for PQS synthesis. Deletion of rsaL reduced transcription of pqsH and cdpR, thus decreasing PQS signal production. The ΔrsaL strain exhibited increased pyocyanin production and reduced biofilm formation, which are dependent on CdpR or PqsH activity. In addition, we solved the structure of the RsaL–DNA complex at a 2.4 Å resolution. Although the overall sequence similarity is quite low, RsaL folds into a HTH-like structure, which is conserved among many transcriptional regulators. Complementation results of the rsaL knockout cells with different rsaL mutants further confirmed the critical role of the DNA-binding residues (including Arg20, Gln27, Gln38, Gly35, Ser37 and Ser42) that are essential for DNA binding. Our findings reveal new targets of RsaL and provide insight into the detailed characterization of the RsaL–DNA interaction.

Genome sequencing-assisted identification and the first functional validation of N-acyl-homoserine-lactone synthases from the Sphingomonadaceae family.

BackgroundMembers of the genus Novosphingobium have been isolated from a variety of environmental niches. Although genomics analyses have suggested the presence of genes associated with quorum sensing signal production e.g., the N-acyl-homoserine lactone (AHL) synthase (luxI) homologs in various Novosphingobium species, to date, no luxI homologs have been experimentally validated.MethodsIn this study, we report the draft genome of the N-(AHL)-producing bacterium Novosphingobium subterraneum DSM 12447 and validate the functions of predicted luxI homologs from the bacterium through inducible heterologous expression in Agrobacterium tumefaciens strain NTL4. We developed a two-dimensional thin layer chromatography bioassay and used LC-ESI MS/MS analyses to separate, detect and identify the AHL signals produced by the N. subterraneum DSM 12447 strain.ResultsThree predicted luxI homologs were annotated to the locus tags NJ75_2841 (NovINsub1), NJ75_2498 (NovINsub2), and NJ75_4146 (NovINsub3). Inducible heterologous expression of each luxI homologs followed by LC-ESI MS/MS and two-dimensional reverse phase thin layer chromatography bioassays followed by bioluminescent ccd camera imaging indicate that the three LuxI homologs are able to produce a variety of medium-length AHL compounds. New insights into the LuxI phylogeny was also gleemed as inferred by Bayesian inference.DiscussionThis study significantly adds to our current understanding of quorum sensing in the genus Novosphingobium and provide the framework for future characterization of the phylogenetically interesting LuxI homologs from members of the genus Novosphingobium and more generally the family Sphingomonadaceae.

Quorum sensing systems differentially regulate the production of phenazine-1-carboxylic acid in the rhizobacterium Pseudomonas aeruginosa PA1201

Pseudomonas aeruginosa strain PA1201 is a newly identified rhizobacterium that produces high levels of the secondary metabolite phenazine-1-carboxylic acid (PCA), the newly registered biopesticide Shenqinmycin. PCA production in liquid batch cultures utilizing a specialized PCA-promoting medium (PPM) typically occurs after the period of most rapid growth, and production is regulated in a quorum sensing (QS)-dependent manner. PA1201 contains two PCA biosynthetic gene clusters phz1 and phz2; both clusters contribute to PCA production, with phz2 making a greater contribution. PA1201 also contains a complete set of genes for four QS systems (LasI/LasR, RhlI/RhlR, PQS/MvfR, and IQS). By using several methods including gene deletion, the construction of promoter-lacZ fusion reporter strains, and RNA-Seq analysis, this study investigated the effects of the four QS systems on bacterial growth, QS signal production, the expression of phz1 and phz2, and PCA production. The possible mechanisms for the strain- and condition-dependent expression of phz1 and phz2 were discussed, and a schematic model was proposed. These findings provide a basis for further genetic engineering of the QS systems to improve PCA production.

Mechanistic analysis of a synthetic inhibitor of the Pseudomonas aeruginosa LasI quorum-sensing signal synthase.

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen responsible for many human infections. LasI is an acyl-homoserine lactone synthase that produces a quorum-sensing (QS) signal that positively regulates numerous P. aeruginosa virulence determinants. The inhibition of the LasI protein is therefore an attractive drug target. In this study, a novel in silico to in vitro complementation was applied to screen thiazolidinedione-type compounds for their ability to inhibit biofilm formation at concentrations not affecting bacterial growth. The compound (z)-5-octylidenethiazolidine-2, 4-dione (TZD-C8) was a strong inhibitor of biofilm formation and chosen for further study. Structural exploration of in silico docking predicted that the compound had high affinity for the LasI activity pocket. The TZD-C8 compound was also predicted to create hydrogen bonds with residues Arg30 and Ile107. Site-directed mutagenesis (SDM) of these two sites demonstrated that TZD-C8 inhibition was abolished in the lasI double mutant PAO-R30D, I107S. In addition, in vitro swarming motility and quorum sensing signal production were affected by TZD-C 8, confirming this compound alters the cell to cell signalling circuitry. Overall, this novel inhibitor of P. aeruginosa quorum sensing shows great promise and validates our mechanistic approach to discovering inhibitors of LuxI-type acyl-homoserine lactone synthases.