Crystal structure of bacterial ubiquitin ADP-ribosyltransferase CteC reveals a substrate-recruiting insertion.

ADP-ribosylation is a post-translational modification involved in regulation of diverse cellular pathways. Interestingly, many pathogens have been identified to utilize ADP-ribosylation as a way for host manipulation. A recent study found that CteC, an effector from the bacterial pathogen Chromobacterium violaceum, hinders host ubiquitin (Ub) signaling pathways via installing mono-ADP-ribosylation on threonine 66 of Ub. However, the molecular basis of substrate recognition by CteC is not well understood. In this article, we probed the substrate specificity of this effector at protein and residue levels. We also determined the crystal structure of CteC in complex with NAD+, which revealed a canonical mono-ADP-ribosyltransferase fold with an additional insertion domain. The AlphaFold-predicted model differed significantly from the experimentally determined structure, even in regions not used in crystal packing. Biochemical and biophysical studies indicated unique features of the NAD+ binding pocket, while showing selectivity distinction between Ub and structurally close Ub-like modifiers and the role of the insertion domain in substrate recognition. Together, this study provides insights into the enzymatic specificities and the key structural features of a novel bacterial ADP-ribosyltransferase involved in host-pathogen interaction.

Recent Advances in Synthetic, Industrial and Biological Applications of Violacein and Its Heterologous Production.

Violacein, a purple pigment first isolated from a gram-negative coccobacillus Chromobacterium violaceum, has gained extensive research interest in recent years due to its huge potential in the pharmaceutic area and industry. In this review, we summarize the latest research advances concerning this pigment, which include (1) fundamental studies of its biosynthetic pathway, (2) production of violacein by native producers, apart from C. violaceum, (3) metabolic engineering for improved production in heterologous hosts such as Escherichia coli, Citrobacter freundii, Corynebacterium glutamicum, and Yarrowia lipolytica, (4) biological/pharmaceutical and industrial properties, (5) and applications in synthetic biology. Due to the intrinsic properties of violacein and the intermediates during its biosynthesis, the prospective research has huge potential to move this pigment into real clinical and industrial applications.

Feature-Based Molecular Networking for the Targeted Identification of Gq-Inhibiting FR900359 Derivatives.

Both the soil bacterium Chromobacterium vaccinii and the bacterial endosymbiont Candidatus Burkholderia crenata of the plant Ardisia crenata are producers of FR900359 (FR). This cyclic depsipeptide is a potent and selective Gq protein inhibitor used extensively to investigate the intracellular signaling of G protein coupled receptors (GPCRs). In this study, the metabolomes of both FR producers were investigated and compared using feature-based molecular networking (FBMN). As a result, 30 previously unknown FR derivatives were identified, one-third being unique to C. vaccinii. Guided by MS, a novel FR derivative, FR-6 (compound 1), was isolated, and its structure unambiguously established. In a whole-cell biosensing assay based on detection of dynamic mass redistribution (DMR) as readout for Gq inhibition, FR-6 suppressed Gq signaling with micromolar potency (pIC50 = 5.56). This functional activity was confirmed in radioligand binding assays (pKi = 7.50). This work demonstrates the power of molecular networking, guiding the way to a novel Gq-inhibiting FR derivative and underlining the potency of FR as a Gq inhibitor.

The chromodepsins - chemistry, biology and biosynthesis of a selective Gq inhibitor natural product family.

Covering: up to April 2021The bacterial cyclic depsipeptides FR900359 (FR) and YM-254890 (YM) were shown to selectively inhibit Gαq proteins with high potency and selectivity and have recently emerged as valuable pharmacological tools due to their effective mechanism of action. Here, we summarize important aspects of this small and specialized natural product family, for which we propose the name chromodepsins, starting from their discovery, producing organisms and structural variety. We then review biosynthesis, structure-activity relationships and ecological and evolutionary aspects of the chromodepsins. Lastly, we discuss their mechanism of action, potential medicinal applications and future opportunities and challenges for further use and development of these complex inhibitor molecules from nature.

N-Heterocycles Scaffolds as Quorum Sensing Inhibitors. Design, Synthesis, Biological and Docking Studies.

Quorum sensing is a communication system among bacteria to sense the proper time to express their virulence factors. Quorum sensing inhibition is a therapeutic strategy to block bacterial mechanisms of virulence. The aim of this study was to synthesize and evaluate new bioisosteres of N-acyl homoserine lactones as Quorum sensing inhibitors in Chromobacterium violaceum CV026 by quantifying the specific production of violacein. Five series of compounds with different heterocyclic scaffolds were synthesized in good yields: thiazoles, 16a-c, thiazolines 17a-c, benzimidazoles 18a-c, pyridines 19a-c and imidazolines 32a-c. All 15 compounds showed activity as Quorum sensing inhibitors except 16a. Compounds 16b, 17a-c, 18a, 18c, 19c and 32b exhibited activity at concentrations of 10 µM and 100 µM, highlighting the activity of benzimidazole 18a (IC50 = 36.67 µM) and 32b (IC50 = 85.03 µM). Pyridine 19c displayed the best quorum sensing inhibition activity (IC50 = 9.66 µM). Molecular docking simulations were conducted for all test compounds on the Chromobacterium violaceum CviR protein to gain insight into the process of quorum sensing inhibition. The in-silico data reveal that all 15 the compounds have higher affinity for the protein than the native AHL ligand (1). A strong correlation was found between the theoretical and experimental results.

Structural analysis of the core and polysaccharide from the lipopolysaccharide produced by Chromobacterium violaceum strain ATCC 12472 (NCTC 9757).

The structure of the polysaccharide O-chain of the lipopolysaccharide isolated from the sequenced strain Chromobacterium violaceum ATCC 12472 (NCTC 9757) was investigated by chemical and NMR analyses, and concluded to be -4-α-Leg5Ac7Ala-4-ß-d-ManNAlaA3OAc-3-α-d-GlcNAc-where Leg5Ac7Ala indicates 5-acetamido-7-alanylamido-3,5,7,9-tetradeoxy-d-glycero-d-galacto-non-2-ulopyranosonic acid and ManNAlaA3OAc 3-O-acetyl-2-alanylamido-2-deoxymannopyranuronic acid. The structure of the core with one repeating unit of the polysaccharide attached was also analyzed, and it was found that the O-chain polysaccharide is linked to the core via ß-GlcpNAc, as opposite to α-GlcpNAc inside the O-chain.

A Nonlive Preparation of Chromobacterium sp. Panama (Csp_P) Is a Highly Effective Larval Mosquito Biopesticide.

Given the continued high prevalence of mosquito-transmitted diseases, there is a clear need to develop novel disease and vector control strategies. Biopesticides of microbial origin represent a promising source of new approaches to target disease-transmitting mosquito populations. Here, we describe the development and characterization of a novel mosquito biopesticide, derived from an air-dried, nonlive preparation of the bacterium Chromobacterium sp. Panama (family: Neisseriaceae). This preparation rapidly and effectively kills the larvae of prominent mosquito vectors, including the dengue and Zika vector Aedes aegypti and the human malaria vector Anopheles gambiae During semi-field trials in Puerto Rico, we observed high efficacy of the biopesticide against field-derived A. aegypti populations, and against A. aegypti and Culex species larvae in natural breeding water, indicating the suitability of the biopesticide for use under more natural conditions. In addition to high efficacy, the nonlive Csp_P biopesticide has a low effective dose, a long shelf life, and high heat stability and can be incorporated into attractive larval baits, all of which are desirable characteristics for a biopesticide.IMPORTANCE We have developed a novel preparation to kill mosquitoes from an abundant soil bacterium, Chromobacterium sp. Panama. This preparation is an air-dried powder containing no live bacteria, and it can be incorporated into an attractive bait and fed directly to mosquito larvae. We demonstrate that the preparation has broad spectrum activity against the larval form of the mosquitoes responsible for the transmission of malaria and the dengue, chikungunya, yellow fever, West Nile, and Zika viruses, as well as mosquito larvae that are already resistant to commonly used mosquitocidal chemicals. Our preparation possesses many favorable traits: it kills at a low dosage, and it does not lose activity when exposed to high temperatures, all of which suggest that this preparation could eventually become an effective new tool for controlling mosquitoes and the diseases they spread.

The Bacterial Product Violacein Exerts an Immunostimulatory Effect Via TLR8.

Violacein, an indole-derived, purple-colored natural pigment isolated from Chromobacterium violaceum has shown multiple biological activities. In this work, we studied the effect of violacein in different immune cell lines, namely THP-1, MonoMac 6, ANA-1, Raw 264.7 cells, as well as in human peripheral blood mononuclear cells (PBMCs). A stimulation of TNF-α production was observed in murine macrophages (ANA-1 and Raw 264.7), and in PBMCs, IL-6 and IL-1ß secretion was detected. We obtained evidence of the molecular mechanism of activation by determining the mRNA expression pattern upon treatment with violacein in Raw 264.7 cells. Incubation with violacein caused activation of pathways related with an immune and inflammatory response. Our data utilizing TLR-transfected HEK-293 cells indicate that violacein activates the human TLR8 (hTLR8) receptor signaling pathway and not human TLR7 (hTLR7). Furthermore, we found that the immunostimulatory effect of violacein in PBMCs could be suppressed by the specific hTLR8 antagonist, CU-CPT9a. Finally, we studied the interaction of hTLR8 with violacein in silico and obtained evidence that violacein could bind to hTLR8 in a similar fashion to imidazoquinoline compounds. Therefore, our results indicate that violacein may have some potential in contributing to future immune therapy strategies.

Tyrosol from marine Fungi, a novel Quorum sensing inhibitor against Chromobacterium violaceum and Pseudomonas aeruginosa.

An ethyl acetate extracts isolated from a marine fungal strain, Penicillium chrysogenum DXY-1, obtained from marine sediments surrounding the East Sea, was found to exhibit anti-quorum sensing (anti-QS) activity. Interestingly, a novel active compound was identified as tyrosol by the purification and structural characterization. At a concentration of 0.5 mg/mL, tyrosol decreased QS-regulated violacein production in Chromobacterium violaceum CV026 by 53.5% and decreased QS-regulated pyocyanin production, elastase activity and proteolytic activity in Pseudomonas aeruginosa PA01 by 63.3%, 57.8% and 9.9%, respectively. SEM images showed that tyrosol inhibited biofilm formation in P. aeruginosa PA01 without having any effect on bacterial growth. Molecular docking results revealed that the natural signal molecule C6HSL and tyrosol bound to different receptor pockets of CviR, and tyrosol inhibited the QS activity of CviR in C. violaceum by binding to the DNA-binding domain and blocking pathogenic gene expression. All the data suggest that tyrosol may act as a potential inhibitor of the QS systems to solve the looming crisis of bacterial resistance. We believe that there are other active compounds with relatively high anti-QS activity or synergistic inhibitory effects on QS in the crude extract, which warrants further research.

A potential probiotic Chromobacterium aquaticum with bacteriocin-like activity enhances the expression of indicator genes associated with nutrient metabolism, growth performance and innate immunity against pathogen infections in zebrafish (Danio rerio).

The use of probiotics as alternatives to antibiotics for disease control is a relatively eco-friendly approach in aquaculture; hence, studies isolating and assessing the benefit of potential probiotics to fish farming are common. The zebrafish is an excellent model system for validating beneficial functions of potential probiotics before their practical application in aquaculture. Here, a potentially probiotic Chromobacterium aquaticum was isolated from lake water samples and characterized by biochemical analysis and 16S rDNA sequencing. The probiotic produced extracellular enzymes (protease and xylanase) and a bacteriocin-like substance, which exhibited tolerance to extreme pH and high-temperature conditions and broad-spectrum bactericidal activity against diverse pathogens, including aquatic, foodborne, clinical and plant pathogens. The effects of C. aquaticum on zebrafish nutrient metabolism, growth performance and innate immunity were evaluated by measuring the expression of indicator genes after C. aquaticum feeding for 8 weeks. Fish administered the probiotic exhibited significantly increased hepatic mRNA expression of carbohydrate metabolism-related genes, including glucokinase (GK), hexokinase (HK), glucose-6-phosphatase (G6Pase), and pyruvate kinase (PK-L), and growth-related genes, including the growth hormone receptor (GHR) and insulin-like growth factor-1 (IGF-1). Innate immune-related genes (IL-1ß, IL-6, TNF-α, IL-10, IL-21, NF-κb, lysozyme and complement C3b) were induced in fish with probiotic supplementation. Probiotic-treated fish exhibited a higher survival rate than control fish after challenge with Aeromonas hydrophila and Streptococcus iniae. Together, these data suggest that C. aquaticum, as a probiotic feed supplement, could enhance nutrient metabolism and growth performance and could modulate innate immunity against A. hydrophila and S. iniae in zebrafish.

Antichagasic effect of violacein from Chromobacterium violaceum.

AIMS: Violacein (VIO), a bacterial pigment produced by Chromobacterium violaceum, was examined to evaluate the antichagasic activity and its action mechanism against Trypanosoma cruzi Y strain. METHODS AND RESULTS: Violacein was tested against the epimastigote, trypomastigote and amastigote forms of T. cruzi Y strain (benznidazole-resistant strain). VIO inhibited all T. cruzi developmental forms, including amastigotes, which is implicated in the burden of infection in the chronic phase of Chagas disease (CD). VIO induced cell death in T. cruzi through apoptosis, as determined by flow cytometry analyses with specific molecular probes and morphological alterations, such as involvement of reactive oxygen species and changes in mitochondrial membrane potential and cell shrinkage. CONCLUSION: The results suggest antichagasic activity of VIO against T. cruzi Y strain with apoptotic involvement. SIGNIFICANCE AND IMPACT OF THE STUDY: The treatment of CD has limited efficacy and side effects that restrict patient tolerability and compliance. The VIO molecule could be used as a model for therapeutic alternatives for this disease.

Structure-function characterization of an insecticidal protein GNIP1Aa, a member of an MACPF and ß-tripod families.

The crystal structure of the Gram-negative insecticidal protein, GNIP1Aa, has been solved at 2.5-Å resolution. The protein consists of two structurally distinct domains, a MACPF (membrane attack complex/PerForin) and a previously uncharacterized type of domain. GNIP1Aa is unique in being a prokaryotic MACPF member to have both its structure and function identified. It was isolated from a Chromobacterium piscinae strain and is specifically toxic to Diabrotica virgifera virgifera larvae upon feeding. In members of the MACPF family, the MACPF domain has been shown to be important for protein oligomerization and formation of transmembrane pores, while accompanying domains define the specificity of the target of the toxicity. In GNIP1Aa the accompanying C-terminal domain has a unique fold composed of three pseudosymmetric subdomains with shared sequence similarity, a feature not obvious from the initial sequence examination. Our analysis places this domain into a protein family, named here ß-tripod. Using mutagenesis, we identified functionally important regions in the ß-tripod domain, which may be involved in target recognition.

Anti-hypertensive mechanisms of cyclic depsipeptide inhibitor ligands for Gq/11 class G proteins.

Augmented vasoconstriction is a hallmark of hypertension and is mediated partly by hyper-stimulation of G protein couple receptors (GPCRs) and downstream signaling components. Although GPCR blockade is a key component of current anti-hypertensive strategies, whether hypertension is better managed by directly targeting G proteins has not been thoroughly investigated. Here, we tested whether inhibiting Gq/11 proteins in vivo and ex vivo using natural cyclic depsipeptide, FR900359 (FR) from the ornamental plant, Ardisia crenata, and YM-254890 (YM) from Chromobacterium sp. QS3666, or it's synthetic analog, WU-07047 (WU), was sufficient to reverse hypertension in mice. All three inhibitors blocked G protein-dependent vasoconstriction, but to our surprise YM and WU and not FR inhibited K+-induced Ca2+ transients and vasoconstriction of intact vessels. However, each inhibitor blocked whole-cell L-type Ca2+ channel current in vascular smooth muscle cells. Subcutaneous injection of FR or YM (0.3 mg/kg, s.c.) in normotensive and hypertensive mice elicited bradycardia and marked blood pressure decrease, which was more severe and long lasting after the injection of FR relative to YM (FRt1/2 ≅ 12 h vs. YMt1/2 ≅ 4 h). In deoxycorticosterone acetate (DOCA)-salt hypertension mice, chronic injection of FR (0.3 mg/kg, s.c., daily for seven days) reversed hypertension (vehicle SBP: 149 ± 5 vs. FR SBP: 117 ± 7 mmHg), without any effect on heart rate. Our results together support the hypothesis that increased LTCC and Gq/11 activity is involved in the pathogenesis of hypertension, and that dual targeting of both proteins can reverse hypertension and associated cardiovascular disorders.

Impact of violacein from Chromobacterium violaceum on the mammalian gut microbiome.

Violacein is a violet pigment produced by Chromobacterium violaceum that possesses several functions such as antibacterial, antiviral, antifungal, and antioxidant activities. The search for potential compounds and therapies that may interfere with and modulate the gut microbial consortia without causing severe damage and increased resistance is important for the treatment of inflammatory, allergic, and metabolic diseases. The aim of the present work was to evaluate the ability of violacein to change microbial patterns in the mammalian gut by favoring certain groups over the others in order to be used as a therapy for diseases associated with changes in the intestinal microflora. To do this, we used male Wistar rats, and administered violacein orally, in low (50 µg/ml) and high (500 µg/ml) doses for a month. Initially, the changes in the microbial diversity were observed by DGGE analyses that showed that the violacein significantly affects the gut microbiota of the rats. Pyrosequencing of 16S rDNA was then employed using a 454 GS Titanium platform, and the results demonstrated that higher taxonomic richness was observed with the low violacein treatment group, followed by the control group and high violacein treatment group. Modulation of the microbiota at the class level was observed in the low violacein dose, where Bacilli and Clostridia (Firmicutes) were found as dominant. For the high violacein dose, Bacilli followed by Clostridia and Actinobacteria were present as the major components. Further analyses are crucial for a better understanding of how violacein affects the gut microbiome and whether this change would be beneficial to the host, providing a framework for the development of alternative treatment strategies for intestinal diseases using this compound.

Anti-Quorum Sensing Activity of 12 Essential Oils on chromobacterium violaceum and Specific Action of cis-cis-p-Menthenolide from Corsican Mentha suaveolens ssp. Insularis.

Quorum sensing (QS) is a bacterial communication mechanism used to express various survival or virulence traits leading to enhanced resistance. Chromobacterium violaceum is a commonly used strain that highlights anti-QS action of bioactive substances. Here, we wanted to see if 12 selected essential oils (EO) could exert anti-QS activity. We measured the sublethal minimal QS inhibitory concentration (MQSIC) by assessing violacein production of C. violaceum along with bacterial growth. To confirm the QS disruption, we also proceed to surface bacterial observations using scanning electron microscopy (SEM). We showed that cis-cis-p-menthenolide extracted and isolated from a plant endemic to occidental Mediterranean Sea islands, Mentha suaveolens ssp. insularis, acts as an inhibitor of violacein production and biofilm formation. Measured MQSIC was much lower than the minimal inhibitory concentration (MIC): 0.10 mg·mL-1 vs. 3.00 mg·mL-1. Moreover, disturbance of QS-related traits was confirmed by the degradation of C. violaceum biofilm matrix. There is a clear structure⁻activity relationship between cis-cis-p-menthenolide and anti-QS activity. Indeed, its isomer molecule (mintlactone) exerts a poor anti-QS action. These results indicate that inhibition of violacein production and biofilm formation by cis-cis-p-menthenolide might be related to a disruption in the QS mechanism.

Production of N-acyl homoserine lactones by Chromobacterium haemolyticum KM2 isolated from the river water in Malaysia.

Quorum sensing (QS) is a term used to describe cell-to-cell communication that enables bacteria to orchestrate group behaviours according to density of bacterial cells. In Gram-negative bacteria, this signalling system is widely known to regulate a variety of different phenotypes such as antibiotic production and biofilm formation. In this study, we report the production of N-acyl homoserine lactones produced by Chromobacterium haemolyticum strain KM2, a bacterium isolated from a river water of a reserved tropical national park. Preliminary screening of QS activity using biosensor reporter assays indicated that C. haemolyticum strain KM2 produces both short- and long-chain AHLs. Analysis with high-resolution liquid chromatography-mass spectrometry (LC-MS/MS) analysis revealed the production of three AHLs by strain KM2: N-octanoyl-L-homoserine lactone (C8-HSL), N-dodecanoyl-L-homoserine lactone (C12-HSL), and N-3-oxo-dodecanoyl-L-homoserine lactone (OC12-HSL). This bacterial isolate also exhibited strong ß-haemolytic activity. To the best of our knowledge, this is the first documentation of QS activity and multiple AHLs production by C. haemolyticum strain KM2.

Antibiofilm effect of Nocardiopsis sp. GRG 1 (KT235640) compound against biofilm forming Gram negative bacteria on UTIs.

Urinary tract infections (UTIs) are diverse public health complication and caused by range of pathogens, however mostly Gram negative bacteria cause significant life threatening risks to different populations. The prevalence rate and antimicrobial resistance among the Gram negative uropathogens alarmed significantly heighten the economic burden of these infections. In this study, we investigated the antibiofilm efficiency of Pyrrolo [1,2-a] pyrazine-1,4-dione,hexahydro-3-(2-methylpropyl) extracted from endophytic actinomycetes Nocardiopsis sp. GRG 1 (KT235640) against P. mirabilis and E. coli. The extracted compound was characterized through TLC, HPLC, GC-MS, LC-MS and confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM). The compound, Pyrrolo [1,2-a] pyrazine-1, 4-dione, hexahydro-3-(2-methylpropyl) inhibits both bacterial biofilm formation as well as reduces the viability of preformed biofilms. Furthermore, CLSM image shows cell shrinkage, disorganized cell membrane and loss of viability. The SEM result also confirms the cell wall degradation in treated cells of the bacteria. Hence, the Pyrrolo [1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl) is active against P. mirabilis and E. coli.

Antibacterial mode of action of violacein from Chromobacterium violaceum UTM5 against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA).

Violacein, violet pigment produced by Chromobacterium violaceum, has attracted much attention recently due to its pharmacological properties including antibacterial activity. The present study investigated possible antibacterial mode of action of violacein from C. violaceum UTM5 against Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) strains. Violet fraction was obtained by cultivating C. violaceum UTM5 in liquid pineapple waste medium, extracted, and fractionated using ethyl acetate and vacuum liquid chromatography technique. Violacein was quantified as major compound in violet fraction using HPLC analysis. Violet fraction displayed bacteriostatic activity against S. aureus ATCC 29213 and methicillin-resistant S. aureus ATCC 43300 with minimum inhibitory concentration (MIC) of 3.9 µg/mL. Fluorescence dyes for membrane damage and scanning electron microscopic analysis confirmed the inhibitory effect by disruption on membrane integrity, morphological alternations, and rupture of the cell membranes of both strains. Transmission electron microscopic analysis showed membrane damage, mesosome formation, and leakage of intracellular constituents of both bacterial strains. Mode of action of violet fraction on the cell membrane integrity of both strains was shown by release of protein, K+, and extracellular adenosine 5'-triphosphate (ATP) with 110.5 µg/mL, 2.34 µg/mL, and 87.24 ng/µL, respectively, at 48 h of incubation. Violet fraction was toxic to human embryonic kidney (HEK293) and human fetal lung fibroblast (IMR90) cell lines with LC50 value of 0.998 ± 0.058 and 0.387 ± 0.002 µg/mL, respectively. Thus, violet fraction showed a strong antibacterial property by disrupting the membrane integrity of S. aureus and MRSA strains. This is the first report on the possible mode of antibacterial action of violet fraction from C. violaceum UTM5 on S. aureus and MRSA strains.

Comparison of seven structurally related coumarins on the inhibition of Quorum sensing of Pseudomonas aeruginosa and Chromobacterium violaceum.

Quorum sensing (QS) is a cell-to-cell signaling communication system that controls the virulence behavior of a broad spectrum of bacterial pathogens, participating also in the development of biofilms, responsible of the antibiotic ineffectiveness in many infections. Therefore, QS system is an attractive target for antimicrobial therapy. In this study, we compare the effect of seven structurally related coumarins against bacterial growth, biofilm formation and elastase activity of Pseudomonas aeruginosa. In addition, the anti-pathogenic capacity of the seven coumarins was evaluated on the wild type and the biosensor strain of Chromobacterium violaceum. The comparative study of coumarins showed that molecules with hydroxyl groups on the aromatic ring displayed higher activity on the inhibition of biofilm formation of P. aeruginosa over coumarins with substituents in positions 3 and 4 or without the double 3,4-bond. These 3 or 4-hydroxylated positions caused a decrease in the anti-biofilm activity obtained for coumarin. However, the hydroxyl group in position 3 of the pyrone ring was important for the inhibition of C. violaceum QS and elastolytic activity of P. aeruginosa. The effects observed were active independently of any effect on growth. According to our results, coumarin and its hydroxylated derivatives represent an interesting group of compounds to use as anti-virulence agents against the human pathogen P. aeruginosa.

Micellar Enzymology for Thermal, pH, and Solvent Stability.

This chapter describes methods for enzyme stabilization using micellar solutions. Micellar solutions have been shown to increase the thermal stability, as well as the pH and solvent tolerance of enzymes. This field is traditionally referred to as micellar enzymology. This chapter details the use of ionic and nonionic micelles for the stabilization of polyphenol oxidase, lipase, and catalase, although this method could be used with any enzymatic system or enzyme cascade system.