Quorum-sensing Receptor Research Articles

Functional Diversity of Quorum Sensing Receptors in Pathogenic Bacteria: Interspecies, Intraspecies and Interkingdom Level.

The formation of biofilm by pathogenic bacteria is considered as one of the most powerful mechanisms/modes of resistance against the action of several antibiotics. Biofilm is formed as a structural adherent over the surfaces of host, food and equipments etc. and is further functionally coordinated by certain chemicals produced itself. These chemicals are known as quorum sensing (QS) signaling molecules and are involved in the cross talk at interspecies, intraspecies and interkingdom levels thus resulting in the production of virulence factors leading to pathogenesis. Bacteria possess receptors to sense these chemicals, which interact with the incoming QS molecules. It is followed by the secretion of virulence molecules, regulation of bioluminescence, biofilm formation, antibiotic resistance development and motility behavioral responses. In the natural environment, different bacterial species (Gram-positive and Gram-negative) produce QS signaling molecules that are structurally and functionally different. Recent and past research shows that various antagonistic molecules (naturally and chemically synthesized) are characterized to inhibit the formation of biofilm and attenuation of bacterial virulence by blocking the QS receptors. This review article describes about the diverse QS receptors at their structural, functional and production levels. Thus, by blocking these receptors with inhibitory molecules can be a potential therapeutic approach to control pathogenesis. Furthermore, these receptors can also be used as a structural platform to screen the most potent inhibitors with the help of bioinformatics approaches.

Quorum Sensing Signal Selectivity and the Potential for Interspecies Cross Talk.

Many species of proteobacteria communicate with kin and coordinate group behaviors through a form of cell-cell signaling called acyl-homoserine lactone (AHL) quorum sensing (QS). Most AHL receptors are thought to be specific for their cognate signal, ensuring that bacteria cooperate and share resources only with closely related kin cells. Although specificity is considered fundamental to QS, there are reports of "promiscuous" receptors that respond broadly to nonself signals. These promiscuous responses expand the function of QS systems to include interspecies interactions and have been implicated in both interspecies competition and cooperation. Because bacteria are frequently members of polymicrobial communities, AHL cross talk between species could have profound impacts. To better understand the prevalence of QS promiscuity, we measured the activity of seven QS receptors in their native host organisms. To facilitate comparison of our results to previous studies, we also measured receptor activity using heterologous expression in Escherichia coli We found that the standard E. coli methods consistently overestimate receptor promiscuity and sensitivity and that overexpression of the receptors is sufficient to account for the discrepancy between native and E. coli reporters. Additionally, receptor overexpression resulted in AHL-independent activity in Pseudomonas aeruginosa Using our activation data, we developed a quantitative score of receptor selectivity. We find that the receptors display a wide range of selectivity and that most receptors respond sensitively and strongly to at least one nonself signal, suggesting a broad potential for cross talk between QS systems.IMPORTANCE Specific recognition of cognate signals is considered fundamental to cell signaling circuits as it creates fidelity in the communication system. In bacterial quorum sensing (QS), receptor specificity ensures that bacteria cooperate only with kin. There are examples, however, of QS receptors that respond promiscuously to multiple signals. "Eavesdropping" by these promiscuous receptors can be beneficial in both interspecies competition and cooperation. Despite their potential significance, we know little about the prevalence of promiscuous QS receptors. Further, many studies rely on methods requiring receptor overexpression, which is known to increase apparent promiscuity. By systematically studying QS receptors in their natural parent strains, we find that the receptors display a wide range of selectivity and that there is potential for significant cross talk between QS systems. Our results provide a basis for hypotheses about the evolution and function of promiscuous signal receptors and for predictions about interspecies interactions in complex microbial communities.

Dihydropyrrolones as bacterial quorum sensing inhibitors

Bacteria regulate their pathogenicity and biofilm formation through quorum sensing (QS), which is an intercellular communication system mediated by the binding of signaling molecules to QS receptors such as LasR. In this study, a range of dihydropyrrolone (DHP) analogues were synthesized via the lactone-lactam conversion of lactone intermediates. The synthesized compounds were tested for their ability to inhibit QS, biofilm formation and bacterial growth of Pseudomonas aeruginosa. The compounds were also docked into a LasR crystal structure to rationalize the observed structure-activity relationships. The most active compound identified in this study was compound 9i, which showed 63.1% QS inhibition of at 31.25 µM and 60% biofilm reduction at 250 µM with only moderate toxicity towards bacterial cell growth.

An Autoinducer Analogue Reveals an Alternative Mode of Ligand Binding for the LasR Quorum-Sensing Receptor

Bacteria use a cell-cell communication process called quorum sensing to coordinate collective behaviors. Quorum sensing relies on production and group-wide detection of extracellular signal molecules called autoinducers. Here, we probe the activity of the Pseudomonas aeruginosa LasR quorum-sensing receptor using synthetic agonists based on the structure of the native homoserine lactone autoinducer. The synthetic compounds range from low to high potency, and agonist activity tracks with the ability of the agonist to stabilize the LasR protein. Structural analyses of the LasR ligand binding domain complexed with representative synthetic agonists reveal two modes of ligand binding, one mimicking the canonical autoinducer binding arrangement, and the other with the lactone head group rotated approximately 150°. Iterative mutagenesis combined with chemical synthesis reveals the amino acid residues and the chemical moieties, respectively, that are key to enabling each mode of binding. Simultaneous alteration of LasR residues Thr75, Tyr93, and Ala127 converts low-potency compounds into high-potency compounds and converts ligands that are nearly inactive into low-potency compounds. These results show that the LasR binding pocket displays significant flexibility in accommodating different ligands. The ability of LasR to bind ligands in different conformations, and in so doing, alter their potency as agonists, could explain the difficulties that have been encountered in the development of competitive LasR inhibitors.

Synthesis, Structure Elucidation and Biological Activities of Some Novel 4(3H)-Quinazolinones as Anti-Biofilm Agents

Background: Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic infections in immunocompromised patients. The inhibition of Quorum Sensing (QS) system has been recognized as an attractive strategy for the treatment of P. aeruginosa infections. In the present study, a series of novel 2-methyl-3-[4-(substituedaminosulfonyl)phenyl]-4(3H)-quinazolinones (1-8) were synthesized and tested for their biofilm formation and swarming motility inhibitory activities in P. aeruginosa PA01. </P><P> Findings: These compounds were found to reduce biofilm formation by 20-32% and swarming motility by 51-62% in P. aeruginosa PA01 at a concentration of 12.5µM. Molecular docking studies were also performed to elucidate the possible key interactions of these compounds with the active site of the P. aeruginosa QS receptor LasR. Furthermore, some molecular properties related to drug likeness and ADME were predicted. </P><P> Results and Conclusion: Results of this study demonstrated that compounds 1-8 can influence QS-regulated biofilm formation and swarming motility in P. aeruginosa PA01 by binding LasR protein and could be developed as anti-biofilm agents to treat chronic biofilm associated infections caused by P. aeruginosa and other clinically significant pathogens.

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

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

Chlorogenic acid attenuates virulence factors and pathogenicity of Pseudomonas aeruginosa by regulating quorum sensing

Quorum sensing (QS) is a cell-to-cell communication that is used by bacteria to regulate collective behaviors. Quorum sensing controls virulence factor production in many bacterial species and it is regarded as an attractive target to combat bacterial pathogenicity, especially against antibiotic-resistant bacteria. Chlorogenic acid (CA), abundant in fruits, vegetables, and Chinese herbs, processes multiple activities. In this research, we explored its quorum sensing quenching activity. In Pseudomonas aeruginosa, CA significantly inhibited the formation of biofilm, the ability of swarming, and virulence factors including protease and elastase activities and rhamnolipid and pyocyanin production. CA showed similar inhibitory effects in Chromobacterium violaceum on its biofilm formation, swarming motility, chitinolytic activity and violacein production. We examined the expression of QS-related genes inP.aeruginosa and found these genes were all downregulated by CA treatment. Computational modeling revealed that CA can form hydrogen bonds with all three QS receptors. Caenorhabditis elegans and mouse infection models were employed to explore the anti-virulence ability of CA and its effect on pathogenesis process in vivo. CA extended the survival period and reduced the quantity of P. aeruginosa in nematode gut, showing a moderate protective effect on C. elegans. In mice wound model, CA-treated groups showed an accelerating healing rate and the bacteria number in wound area was also decreased by CA treatment. It is suggested by our research that CA has potential to be used as an anti-virulence factor in P. aeruginosa infection.

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

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

Structural and Biochemical Studies of Non-native Agonists of the LasR Quorum-Sensing Receptor Reveal an L3 Loop “Out” Conformation for LasR

Chemical strategies to block quorum sensing (QS) could provide a route to attenuate virulence in bacterial pathogens. Considerable research has focused on this approach in Pseudomonas aeruginosa, which uses the LuxR-type receptor LasR to regulate much of its QS network. Non-native ligands that antagonize LasR have been developed, yet we have little understanding of the mode by which these compounds interact with LasR and alter its function, as the receptor is unstable in their presence. Herein, we report an approach to circumvent this challenge through the study of a series of synthetic LasR agonists with varying levels of potency. Structural investigations of these ligands with the LasR ligand-binding domain reveal that certain agonists can enforce a conformation that deviates from that observed for other, often more potent agonists. These results, when combined with cell-based and biophysical analyses, suggest a functional model for LasR that could guide future ligand design.

Anti-quorum sensing and antibiofilm potential of Alternaria alternata, a foliar endophyte of Carica papaya, evidenced by QS assays and in-silico analysis

In the present study, secondary metabolites from an endophytic fungus, Alternaria alternata, colonizing Carica papaya, demonstrated antiquorum sensing properties against Pseudomonas aeruginosa. This study reports the antagonistic effects of fungal crude extract of A. alternata against the various quorum sensing (QS) associated virulent factors such as percentage decrease in production of pyocyanin, alginate, chitinase and rhamnolipid; significant decrease in proteases activity such as LasA protease activity, staphylolytic activity, Las B elastase; and a marked decrease in biofilm formation and associated factors such as exopolysaccharide (EPS) production and cell surface hydrophobicity (CSH). Further, motility pattern i.e., swimming and swarming was also found to be inhibited. This down regulation of QS and associated factors are further supported by in-silico analysis of interaction between QS receptor LasR and bioactive molecules viz., sulfurous acid, 2-propyl tridecyl ester and 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester present in fungal crude extract, found based on GCMS analysis, sketches the modulating ability of QS expression. This is the first report on an endophytic fungus of C. papaya having a role in QS inhibition against P. aeruginosa and lays a platform to explore further the endophytes for potent therapeutic agents in QS.

Eavesdropping and crosstalk between secreted quorum sensing peptide signals that regulate bacteriocin production in Streptococcus pneumoniae.

Quorum sensing (QS), where bacteria secrete and respond to chemical signals to coordinate population-wide behaviors, has revealed that bacteria are highly social. Here, we investigate how diversity in QS signals and receptors can modify social interactions controlled by the QS system regulating bacteriocin secretion in Streptococcus pneumoniae, encoded by the blp operon (bacteriocin-like peptide). Analysis of 4096 pneumococcal genomes detected nine blp QS signals (BlpC) and five QS receptor groups (BlpH). Imperfect concordance between signals and receptors suggested widespread social interactions between cells, specifically eavesdropping (where cells respond to signals that they do not produce) and crosstalk (where cells produce signals that non-clones detect). This was confirmed in vitro by measuring the response of reporter strains containing six different blp QS receptors to cognate and non-cognate peptides. Assays between pneumococcal colonies grown adjacent to one another provided further evidence that crosstalk and eavesdropping occur at endogenous levels of signal secretion. Finally, simulations of QS strains producing bacteriocins revealed that eavesdropping can be evolutionarily beneficial even when the affinity for non-cognate signals is very weak. Our results highlight that social interactions can mediate intraspecific competition among bacteria and reveal that competitive interactions can be modified by polymorphic QS systems.

Role of SdiA on Biofilm Formation by Atypical Enteropathogenic Escherichia coli.

Atypical enteropathogenic Escherichia coli are capable to form biofilm on biotic and abiotic surfaces, regardless of the adherence pattern displayed. Several E. coli mechanisms are regulated by Quorum sensing (QS), including virulence factors and biofilm formation. Quorum sensing is a signaling system that confers bacteria with the ability to respond to chemical molecules known as autoinducers. Suppressor of division inhibitor (SdiA) is a QS receptor present in atypical enteropathogenic E. coli (aEPEC) that detects acyl homoserine lactone (AHL) type autoinducers. However, these bacteria do not encode an AHL synthase, but they are capable of sensing AHL molecules produced by other species, establishing an inter-species bacterial communication. In this study, we performed experiments to evaluate pellicle, ring-like structure and biofilm formation on wild type, sdiA mutants and complemented strains. We also evaluated the transcription of genes involved in different stages of biofilm formation, such as bcsA, csgA, csgD, fliC and fimA. The sdiA mutants were capable of forming thicker biofilm structures and showed increased motility when compared to wild type and complemented strains. Moreover, they also showed denser pellicles and ring-like structures. Quantitative real-time PCR (qRT-PCR) analysis demonstrated increased csgA, csgD and fliC transcription on mutant strains. Biofilm formation, as well as csgD, csgA and fimA transcription decreased on wild type strains by the addition of AHL. These results indicate that SdiA participates on the regulation of these phenotypes in aEPEC and that AHL addition enhances the repressor effect of this receptor on the transcription of biofilm and motility related genes.

A comparative study of non-native N-acyl l-homoserine lactone analogs in two Pseudomonas aeruginosa quorum sensing receptors that share a common native ligand yet inversely regulate virulence

Certain bacteria can coordinate group behaviors via a chemical communication system known as quorum sensing (QS). Gram-negative bacteria typically use N-acyl l-homoserine lactone (AHL) signals and their cognate intracellular LuxR-type receptors for QS. The opportunistic pathogen Pseudomonas aeruginosa has a relatively complex QS circuit in which two of its LuxR-type receptors, LasR and QscR, are activated by the same natural signal, N-(3-oxo)-dodecanoyl l-homoserine lactone. Intriguingly, once active, LasR activates virulence pathways in P. aeruginosa, while activated QscR can inactivate LasR and thus repress virulence. We have a limited understanding of the structural features of AHLs that engender either agonistic activity in both receptors or receptor-selective activity. Compounds with the latter activity profile could prove especially useful tools to tease out the roles of these two receptors in virulence regulation. A small collection of AHL analogs was assembled and screened in cell-based reporter assays for activity in both LasR and QscR. We identified several structural motifs that bias ligand activation towards each of the two receptors. These findings will inform the development of new synthetic ligands for LasR and QscR with improved potencies and selectivities.

Design and Synthesis of Imidazo[1,2‐a]pyridines with Carboxamide Group Substitution andIn silicoEvaluation of their Interaction with a LuxR‐type Quorum Sensing Receptor

Quorum sensing, an important process of bacterial communication, is involved in the development of complex behavior and expression of virulence factors that become important due to its key role in infection process. In this manuscript, docking studies were used as a preliminary screening to identify main interactions between LuxR receptor (CviR) ofChromobacterium violaceumas model andN‐acylL‐homoserine lactones. Thus, following this approach, a library of imidazo[1,2‐a]pyridines bearing carboxamide and urea moieties was designed, evaluated on thisin silicomodel previously validated, and synthesized to generate new potential quorum sensing inhibitors. The present article summarizes survey to develop these new molecules with potential biological activity, based on interaction analysis ligand‐receptor.

An in vitro and in silico identification of antibiofilm small molecules from seawater metaclone SWMC166 against Vibrio cholerae O1

This study aimed to determine the antibiofilm activity of seawater microbes against Vibrio cholerae (VCO1) through functional metagenomics approach. A metagenomic library was constructed from Palk Bay seawater and the library was screened to identify the biofilm inhibitory metaclone. Metaclone SWMC166 (harbouring ∼30 kb metagenomic insert) was found to exhibit antibiofilm activity against VCO1. The biofilm inhibitory potential of partially purified ethyl acetate extract of SWMC166 (EA166) was further evaluated through microscopic studies and biochemical assays. Further, EA166 treated VCO1 divulged up-regulation of genes involved in high cell density-mediated quorum sensing (QS) pathway which was analysed by real-time PCR. In order to identify the genes of interest (within ∼30 kb insert), subcloning was performed through shotgun approach. Small molecules from positive subclones SC5 and SC8 were identified through HRLC-MS analysis. Resulted small molecules were docked against QS receptors of V. cholerae to identify the bioactive metabolites. Docking studies revealed that totally seven metabolites were able to interact with QS receptors that can possibly trigger the QS cascade and sequentially inhibit the biofilm formation and virulence factors of VCO1.

Attenuation of quorum sensing controlled virulence factors and biofilm formation in Pseudomonas aeruginosa by pentacyclic triterpenes, betulin and betulinic acid

The production of virulence determinants and biofilm formation in numerous pathogens is regulated by the cell-density-dependent phenomenon, Quorum sensing (QS). The QS system in multidrug resistant opportunistic pathogen, P. aeruginosa constitutes of three main regulatory circuits namely Las, Rhl, and Pqs which are closely linked to its pathogenicity and establishment of chronic infections. In spite intensive antibiotic therapy, P. aeruginosa continue to be an important cause of nosocomial infections and also the major cause of mortality in Cystic Fibrosis patients with 80% of the adults suffering from chronic P. aeruginosa infection. Hence, targeting QS circuit offers an effective intervention to the ever increasing problem of drug resistant pathogens. In the present study, the pentacyclic triterpenes i.e. Betulin (BT) and Betulinic acid (BA) exhibited significant attenuation in production of QS-regulated virulence factors and biofilm formation in P. aeruginosa, at the sub-lethal concentration. The test compound remarkably interfered in initial stages of biofilm development by decreasing the exopolysaccharide production and cell surface hydrophobicity. Based on the in vivo studies, the test compounds notably enhanced the survival of Caenorhabditis elegans infected with P. aeruginosa. Furthermore, molecular docking analysis revealed that BT and BA can act as a strong competitive inhibitor for QS receptors, LasR and RhlR. The findings suggest that BT and BA can serve as potential anti-infectives in the controlling chronic infection of P. aeruginosa.

Association of furanone C-30 with biofilm formation & antibiotic resistance in Pseudomonas aeruginosa.

Background & objectives:Pseudomonas aeruginosa is an opportunistic pathogen that can cause nosocomial bloodstream infections in humans. This study was aimed to explore the association of furanone C-30 with biofilm formation, quorum sensing (QS) system and antibiotic resistance in P. aeruginosa.Methods:An in vitro model of P. aeruginosa bacterial biofilm was established using the standard P. aeruginosa strain (PAO-1). After treatment with 2.5 and 5 μg/ml of furanone C-30, the change of biofilm morphology of PAO-1 was observed, and the expression levels of QS-regulated virulence genes (lasB, rhlA and phzA2), QS receptor genes (lasR, rhlR and pqsR) as well as QS signal molecule synthase genes (lasI, rhlI, pqsE and pqsH) were determined. Besides, the AmpC expression was quantified in planktonic and mature biofilm induced by antibiotics.Results:Furanone C-30 treatment significantly inhibited biofilm formation in a dose-dependent manner. With the increase of furanone C-30 concentration, the expression levels of lasB, rhlA, phzA2, pqsR, lasI, rhlI, pqsE and pqsH significantly decreased in mature biofilm bacteria while the expression levels of lasR and rhlR markedly increased. The AmpC expression was significantly decreased in both planktonic and biofilm bacteria induced by imipenem and ceftazidime.Interpretation & conclusions:Furanone C-30 may inhibit biofilm formation and antibiotic resistance in P. aeruginosa through regulating QS genes. The inhibitory effect of furanone C-30 on las system appeared to be stronger than that on rhl system. Further studies need to be done with different strains of P. aeruginosa to confirm our findings.

Production of Bacterial Quorum Sensing Antagonists, Caffeoyl- and Feruloyl-HSL, by an Artificial Biosynthetic Pathway.

A new series comprising phenylacetyl-homoserine lactones (HSLs), caffeoyl-HSL and feruloyl-HSL, was biologically synthesized using an artificial de novo biosynthetic pathway. We developed an Escherichia coli system containing artificial biosynthetic pathways that yield phenylacetyl-HSLs from simple carbon sources. These artificial biosynthetic pathways contained the LuxI-type synthase gene (rpaI) in addition to caffeoyl-CoA and feruloyl-CoA biosynthetic genes, respectively. Finally, the yields for caffeoyl-HSL and feruloyl-HSL were 97.1 ± 10.3 and 65.2 ± 5.7 mg/l, respectively, by tyrosine-overproducing E. coli with a L-methionine feeding strategy. In a quorum sensing (QS) competition assay, feruloyl-HSL and p-coumaroyl-HSL antagonized the QS receptor TraR in Agrobacterium tumefaciens NT1, whereas caffeoyl-HSL did not.

Mechanistic understanding of Phenyllactic acid mediated inhibition of quorum sensing and biofilm development in Pseudomonas aeruginosa.

Pseudomonas aeruginosa depends on its quorum sensing (QS) system for its virulence factors' production and biofilm formation. Biofilms of P. aeruginosa on the surface of indwelling catheters are often resistant to antibiotic therapy. Alternative approaches that employ QS inhibitors alone or in combination with antibiotics are being developed to tackle P. aeruginosa infections. Here, we have studied the mechanism of action of 3-Phenyllactic acid (PLA), a QS inhibitory compound produced by Lactobacillus species, against P. aeruginosa PAO1. Our study revealed that PLA inhibited the expression of virulence factors such as pyocyanin, protease, and rhamnolipids that are involved in the biofilm formation of P. aeruginosa PAO1. Swarming motility, another important criterion for biofilm formation of P. aeruginosa PAO1, was also inhibited by PLA. Gene expression, mass spectrometric, functional complementation assays, and in silico data indicated that the quorum quenching and biofilm inhibitory activities of PLA are attributed to its ability to interact with P. aeruginosa QS receptors. PLA antagonistically binds to QS receptors RhlR and PqsR with a higher affinity than its cognate ligands N-butyryl-L-homoserine lactone (C4-HSL) and 2-heptyl-3,4-dihydroxyquinoline (PQS; Pseudomonas quinolone signal). Using an in vivo intraperitoneal catheter-associated medaka fish infection model, we proved that PLA inhibited the initial attachment of P. aeruginosa PAO1 on implanted catheter tubes. Our in vitro and in vivo results revealed the potential of PLA as anti-biofilm compound against P. aeruginosa.

Broad-Host-Range Expression Reveals Native and Host Regulatory Elements That Influence Heterologous Antibiotic Production in Gram-Negative Bacteria.

ABSTRACTHeterologous expression has become a powerful tool for studying microbial biosynthetic gene clusters (BGCs). Here, we extend the transformation-associated recombination cloning and heterologous expression platform for microbial BGCs to include Gram-negative proteobacterial expression hosts. Using a broad-host-range expression platform, we test the implicit assumption that biosynthetic pathways are more successfully expressed in more closely related heterologous hosts. Cloning and expression of the violacein BGC from Pseudoalteromonas luteoviolacea 2ta16 revealed robust production in two proteobacterial hosts, Pseudomonas putida KT2440 and Agrobacterium tumefaciens LBA4404, but very little production of the antibiotic in various laboratory strains of Escherichia coli, despite their closer phylogenetic relationship. We identified a nonclustered LuxR-type quorum-sensing receptor from P. luteoviolacea 2ta16, PviR, that increases pathway transcription and violacein production in E. coli by ∼60-fold independently of acyl-homoserine lactone autoinducers. Although E. coli harbors the most similar homolog of PviR identified from all of the hosts tested, overexpression of various E. coli transcription factors did not result in a statistically significant increase in violacein production, while overexpression of two A. tumefaciens PviR homologs significantly increased production. Thus, this work not only introduces a new genetic platform for the heterologous expression of microbial BGCs, it also challenges the assumption that host phylogeny is an accurate predictor of host compatibility.