Violacein Biosynthesis Research Articles

Regulation of virulence in Chromobacterium violaceum and strategies to combat it.

Chromobacterium is a rod-shaped, Gram-negative, facultatively anaerobic bacteria with a cosmopolitan distribution. Just about 160 Chromobacterium violaceum incidents have been reported globally, but then once infected, it has the ability to cause deadly septicemia, and infections in the lungs, liver, brain, spleen, and lymphatic systems that might lead to death. C. violaceum produces and utilizes violacein to kill bacteria that compete with it in an ecological niche. Violacein is a hydrophobic bisindole that is delivered through an efficient transport route termed outer membrane vesicles (OMVs) through the aqueous environment. OMVs are small, spherical segments detached from the outer membrane of Gram-negative bacteria. C. violaceum OMV secretions are controlled by a mechanism called the quorum sensing system CviI/CviR, which enables cell-to-cell communication between them and regulation of various virulence factors such as biofilm formation, and violacein biosynthesis. Another virulence factor bacterial type 3 secretion system (T3SS) is divided into two types: Cpi-1 and Cpi-2. Cpi-1's needle and rod effector proteins are perhaps recognized by NAIP receptors in humans and mice, activating the NLRC4 inflammasome cascade, effectively clearing spleen infections via pyroptosis, and cytotoxicity mediated by IL-18-driven Natural killer (NK) cells in the liver. In this paper, we attempt to interrelate quorum-controlled biofilm formation, violacein production, violacein delivery by OMVs and T3SS effector protein production and host mediated immunological effects against the Cpi1 of T3SS. We suggest a research path with natural bioactive molecule like palmitic acid that can act as an anti-quorum agent by reducing the expression of virulence factors as well as an immunomodulatory agent that can augment innate immune defense by hyperactivation of NLRC4 inflammasome hence dramatically purge C. violaceum infections.

Infiltration of the entire violacein biosynthesis pathway into a hierarchically etched MIL-101 metal–organic framework

Infiltration of the entire violacein biosynthesis pathway into a hierarchically etched MIL-101 metal–organic framework

Assessment of Morita-Baylis-Hillman adducts as novel modulators of quorum sensing phenotypes in Chromobacterium CV026 by targeting CviR

This work presents Morita-Baylis-Hillman adducts (MBHA) as new modulators of the violacein biosynthesis in the biosensor strain Chromobacterium CV026. Seven adducts displayed violacein reduction higher than 50±1% when tested at concentrations lower than 625 μM. The most active MBHA inhibited hydrolysis of chitin concomitantly with inhibition of violacein production in CV026, suggesting the disruption of its QS machinery. One MBHA induced the violacein biosynthesis in CV026 in the absence of the cognate AI, also supporting its role as a QS modulator of this QS system. Further, RT-qPCR and competition experiments characterize these two molecules as transcriptional modulators of the QS-regulated operon vioABCDE. Blind docking studies for MBHA showed a strong preference for adducts positioned within the CviR autoinducer-binding domain (AIBD). Our results show MBHA as a promising chemical scaffold for the development of novel QS inhibitors affecting LuxR/LuxI-systems.

Assembly of Metabolons in Yeast Using Cas6-Mediated RNA Scaffolding.

Cells often localize pathway enzymes in close proximity to reduce substrate loss via diffusion and to ensure that carbon flux is directed toward the desired product. To emulate this strategy for the biosynthesis of heterologous products in yeast, we have taken advantage of the highly specific Cas6-RNA interaction and the predictability of RNA hybridizations to demonstrate Cas6-mediated RNA-guided protein assembly within the yeast cytosol. The feasibility of this synthetic scaffolding technique for protein localization was first demonstrated using a split luciferase reporter system with each part fused to a different Cas6 protein. In Saccharomyces cerevisiae, the luminescence signal increased 3.6- to 20-fold when the functional RNA scaffold was also expressed. Expression of a trigger RNA, designed to prevent the formation of a functional scaffold by strand displacement, decreased the luminescence signal by nearly 2.3-fold. Temporal control was also possible, with induction of scaffold expression resulting in an up to 11.6-fold increase in luminescence after 23 h. Cas6-mediated assembly was applied to create a two-enzyme metabolon to redirect a branch of the violacein biosynthesis pathway. Localizing VioC and VioE together increased the amount of deoxyviolacein (desired) relative to prodeoxyviolacein (undesired) by 2-fold. To assess the generality of this colocalization method in other yeast systems, the split luciferase reporter system was evaluated in Kluyveromyces marxianus; RNA scaffold expression resulted in an increase in the luminescence signal of up to 1.9-fold. The simplicity and flexibility of the design suggest that this strategy can be used to create metabolons in a wide range of recombinant hosts of interest.

Structural Basis for Substrate Binding and Catalytic Mechanism of the Key Enzyme VioD in the Violacein Synthesis Pathway.

Violacein is a pigment synthesized by gram-negative bacteria with various biological activities such as antimicrobial, antiviral, and anticancer activities. VioD is a key oxygenase converting protodeoxyviolaceinic acid (PDVA) to protoviolaceinic acid in violacein biosynthesis. To elucidate the catalytic mechanism of VioD, here, we resolved two crystal structures of VioD, a binary complex structure containing VioD and a FAD and a ternary complex structure composed of VioD, a FAD and a 2-ethyl-1-hexanol (EHN). Structural analysis revealed a deep funnel like binding pocket with wide entrance, this pocket is positively charged. The EHN is located at the deep bottom of the binding pocket near isoalloxazine ring. Further docking simulation help us to propose the mechanism of the hydroxylation of the substrate catalyzed by VioD. Bioinformatic analysis suggested and emphasized the importance of the conserved residues involved in substrate binding. Our results provide a structural basis for the catalytic mechanism of VioD. This article is protected by copyright. All rights reserved.

Sensitivity of Janthinobacterium Lividum to Low Concentrations of Hydrogen Peroxide and the Effect of Mild Oxidative Stress on Pigment Yield

In the course of assessing the sensitivity of the Janthinobacterium lividum VKM B-3515 strain to low concentrations of hydrogen peroxide, it was found that at a content of 0.003% H2O2, the growth properties of the bacterium during submerged cultivation without pigmentation differed statistically insignificantly relative to the control variant at 16 hours of incubation and beyond. Whereas in the presence of peroxide at 12 hours the optical density was lower than in the control by 97%. When cultivating by the surface method, the respiration intensity did not significantly differ between the control and experimental variants. However, during the extraction of the pigment, it was found that in the presence of hydrogen peroxide, the optical density of the acetone extract significantly exceeds the control variant by 28%. It can be assumed that, at the same growth parameters of the culture, the biosynthesis of violacein is stimulated and the population can adapt to the peroxide content, and the peroxide concentration itself decreases due to the cost of catalytic reactions. Further studies of the sensitivity of J. lividum VKM B-3515 to various oxidizing agents will allow us to consider the effect of weak oxidative stress on the biosynthesis of violacein.

α-Alkylidene δ-lactones inhibit quorum sensing phenotypes in Chromobacterium strain CV026 showing interaction with the CviR receptor.

Disruption of bacterial quorum sensing (QS) is presented as a promising strategy to overcome clinically relevant and phytopathogenic bacteria. This work presents α-alkylidene δ-lactones as new chemical scaffolds that inhibit the biosynthesis of violacein in the biosensor strain Chromobacterium CV026. Three molecules displayed higher than 50% violacein reduction when tested at concentrations lower than 625 µM. The most active α-alkylidene δ-lactone inhibited the hydrolysis of chitin concomitantly with the inhibition of violacein production in CV026, suggesting the disruption of its QS machinery. Further, RT-qPCR and competition experiments showed this molecule to be a transcriptional inhibitor of the QS-regulated operon vioABCDE. Docking calculations suggested a good correlation between binding affinity energies and inhibition effects, with all molecules positioned within the CviR autoinducer-binding domain (AIBD). The most active lactone yielded the best binding affinity energy, most probably due to its unprecedented binding with the AIBD. Our results show α-alkylidene δ-lactones as promising chemical scaffolds for the development of new QS inhibitors affecting LuxR/LuxI-systems.

Janthinobacterium sp. Strain SLB01 as Pathogenic Bacteria for Sponge Lubomirskia baikalensis

Sponges (phylum Porifera) are ancient, marine and inland water, filter feeding metazoans. In recent years, diseased sponges have been increasingly occurring in marine and freshwater environments. Endemic freshwater sponges of the Lubomirskiidae family are widely distributed in the coastal zone of Lake Baikal. The strain Janthinobacterium sp. SLB01 was isolated previously from the diseased sponge Lubomirskia baikalensis (Pallas, 1776), although its pathogenicity is still unknown. The aim of this study was to confirm whether the Janthinobacterium sp. strain SLB01 is the pathogen found in Baikal sponge. To address this aim, we infected the cell culture of primmorphs of the sponge L. baikalensis with strain SLB01 and subsequently reisolated and sequenced the strain Janthinobacterium sp. PLB02. The results showed that the isolated strain has more than 99% homology with strain SLB01. The genomes of both strains contain genes vioABCDE of violacein biosynthesis and floc formation, for strong biofilm, in addition to the type VI secretion system (T6SS) as the main virulence factor. Based on a comparison of complete genomes, we showed the similarity of the studied bacterial strains of Janthinobacterium spp. with the described strain of Janthinobacterium lividum MTR. This study will help expand our understanding of microbial interactions and determine one of the causes in the development of diseases and death in Baikal sponges.

The modular pYT vector series employed for chromosomal gene integration and expression to produce carbazoles and glycolipids in P. putida.

The expression of biosynthetic genes in bacterial hosts can enable access to high-value compounds, for which appropriate molecular genetic tools are essential. Therefore, we developed a toolbox of modular vectors, which facilitate chromosomal gene integration and expression in Pseudomonas putida KT2440. To this end, we designed an integrative sequence, allowing customisation regarding the modes of integration (random, at attTn7, or into the 16S rRNA gene), promoters, antibiotic resistance markers as well as fluorescent proteins and enzymes as transcription reporters. We thus established a toolbox of vectors carrying integrative sequences, designated as pYT series, of which we present 27 ready-to-use variants along with a set of strains equipped with unique 'landing pads' for directing a pYT interposon into one specific copy of the 16S rRNA gene. We used genes of the well-described violacein biosynthesis as reporter to showcase random Tn5-based chromosomal integration leading to constitutive expression and production of violacein and deoxyviolacein. Deoxyviolacein was likewise produced after gene integration into the 16S rRNA gene of rrn operons. Integration in the attTn7 site was used to characterise the suitability of different inducible promoters and successive strain development for the metabolically challenging production of mono-rhamnolipids. Finally, to establish arcyriaflavin A production in P. putida for the first time, we compared different integration and expression modes, revealing integration at attTn7 and expression with NagR/PnagAa to be most suitable. In summary, the new toolbox can be utilised for the rapid generation of various types of P. putida expression and production strains.

Utilizing RNA origami scaffolds in Saccharomyces cerevisiae for dCas9-mediated transcriptional control.

Designer RNA scaffolds constitute a promising tool for synthetic biology, as they can be genetically expressed to perform specific functions in vivo such as scaffolding enzymatic cascades and regulating gene expression through CRISPR-dCas9 applications. RNA origami is a recently developed RNA design approach that allows construction of large RNA nanostructures that can position aptamer motifs to spatially organize other molecules, including proteins. However, it is still not fully understood how positioning multiple aptamers on a scaffold and the orientation of a scaffold affects functional properties. Here, we investigate fusions of single-guide RNAs and RNA origami scaffolds (termed sgRNAO) capable of recruiting activating domains for control of gene expression in yeast. Using MS2 and PP7 as orthogonal protein-binding aptamers, we observe a gradual increase in transcriptional activation for up to four aptamers. We demonstrate that different aptamer positions on a scaffold and scaffold orientation affect transcriptional activation. Finally, sgRNAOs are used to regulate expression of enzymes of the violacein biosynthesis pathway to control metabolic flux. The integration of RNA origami nanostructures at promoter sites achieved here, can in the future be expanded by the addition of functional motifs such as riboswitches, ribozymes and sensor elements to allow for complex gene regulation.

Functional characterization of a novel violacein biosynthesis operon from Janthinobacterium sp. B9-8.

Violacein is a secondary metabolite mainly produced by Gram-negative bacteria that is formed from tryptophan by five enzymes encoded by a single operon. It is a broad-spectrum antibacterial pigment with various important biological activities such as anti-tumor, antiviral, and antioxidative effects. The newly discovered violacein operon vioABCDE was identified in the genome of the extremophile Janthinobacterium sp. B9-8. The key enzyme-encoding genes were cloned to construct the multigene coexpression plasmids pET-vioAB and pRSF-vioCDE. The violacein biosynthesis pathway was heterologously introduced into engineered Escherichia coli VioABCDE and VioABCDE-SD. The factors affecting violacein production, including temperature, pH, inoculum size, carbon and nitrogen source, precursor, and inducers were investigated. The violacein titer of VioABCDE-SD reached 107mg/L in a two-stage fermentation process, representing a 454.4% increase over the original strain. The violacein operon from B9-8 provides a new microbial gene source for the analysis of the violacein synthesis mechanism, and the constructed engineering E. coli strains lay a foundation for the efficient and rapid synthesis of other natural products.Key points• The newly discovered violacein operon vioABCDE was identified in the genome of the extremophile Janthinobacterium sp. B9-8.• The violacein synthesis pathway was reconstructed in E. coli using two compatible plasmids.• A two-stage fermentation process was optimized for improved violacein accumulation.

Development of Methylorubrum extorquens AM1 as a promising platform strain for enhanced violacein production from co-utilization of methanol and acetate

Violacein, a blue-violet compound with a wide range of beneficial bioactivities, is an attractive product for microbial production. Currently, violacein production has been demonstrated in several sugar heterotrophs through metabolic engineering; however, the cost of production remains an obstacle for business ventures. To address this issue, the development of host strains that can utilize inexpensive alternative substrates to reduce production costs would enable the commercialization of violacein. In this study, we engineered a facultative methylotroph, Methylorubrum extorquens AM1, to develop a methanol-based platform for violacein production. By optimizing expression vectors as well as inducer concentrations, 11.7 mg/L violacein production was first demonstrated using methanol as the sole substrate. Considering that unidentified bottlenecks for violacein biosynthesis in the shikimate pathway of M. extorquens AM1 would be difficult to address using generic metabolic engineering approaches, random mutagenesis and site-directed mutagenesis were implemented, and a 2-fold improvement in violacein production was achieved. Finally, by co-utilization of methanol and acetate, a remarkable enhancement of violacein production to 118 mg/L was achieved. Our results establish a platform strain for violacein production from non-sugar feedstocks, which may contribute to the development of an economically efficient large-scale fermentation system for violacein production.

Comparative Genomics Reveals Insights into Induction of Violacein Biosynthesis and Adaptive Evolution in Janthinobacterium.

ABSTRACTViolacein has different bioactive properties conferring distinct selective advantages, such as defense from predation and interspecific competition. Adaptation of Janthinobacterium to diverse habitats likely leads to variation in violacein production among phylogenetically closely related species inhabiting different environments, yet genomic mechanisms and the influence of adaptive evolution underpinning violacein biosynthesis in Janthinobacterium are not clear. In this study, we performed genome sequencing, comparative genomic analysis, and phenotypic characterization to investigate genomic factors regulating violacein production in nine Janthinobacterium strains, including a type strain from soil and eight strains we isolated from terrestrial subsurface sediment and groundwater. Results show that although all nine Janthinobacterium strains are phylogenetically closely related and contain genes essential for violacein biosynthesis, they vary in carbon usage and violacein production. Sediment and groundwater strains are weak violacein producers and possess far fewer secondary metabolite biosynthesis genes, indicating genome adaptation compared to soil strains. Further examination suggests that quorum sensing (QS) may play an important role in regulating violacein in Janthinobacterium: the strains exhibiting strong potential in violacein production possess both N-acyl-homoserine lactone (AHL) QS and Janthinobacterium QS (JQS) systems in their genomes, while weaker violacein-producing strains harbor only the JQS system. Preliminary tests of spent media of two Janthinobacterium strains possessing both AHL QS and JQS systems support the potential role of AHLs in inducing violacein production in Janthinobacterium. Overall, results from this study reveal potential genomic mechanisms involved in violacein biosynthesis in Janthinobacterium and provide insights into evolution of Janthinobacterium for adaptation to oligotrophic terrestrial subsurface environment.IMPORTANCE Phylogenetically closely related bacteria can thrive in diverse environmental habitats due to adaptive evolution. Genomic changes resulting from adaptive evolution lead to variations in cellular function, metabolism, and secondary metabolite biosynthesis. The most well-known secondary metabolite produced by Janthinobacterium is the purple-violet pigment violacein. To date, the mechanisms of induction of violacein biosynthesis in Janthinobacterium is not clear. Comparative genome analysis of closely related Janthinobacterium strains isolated from different environmental habitats not only reveals potential mechanisms involved in induction of violacein production by Janthinobacterium but also provides insights into the survival strategy of Janthinobacterium for adaptation to oligotrophic terrestrial subsurface environment.

GenoChemetic Strategy for Derivatization of the Violacein Natural Product Scaffold.

Natural products and their analogues are often challenging to synthesize due to their complex scaffolds and embedded functional groups. Solely relying on engineering the biosynthesis of natural products may lead to limited compound diversity. Integrating synthetic biology with synthetic chemistry allows rapid access to much more diverse portfolios of xenobiotic compounds, which may accelerate the discovery of new therapeutics. As a proof-of-concept, by supplementing an Escherichia coli strain expressing the violacein biosynthesis pathway with 5-bromo-tryptophan in vitro or tryptophan 7-halogenase RebH in vivo, six halogenated analogues of violacein or deoxyviolacein were generated, demonstrating the promiscuity of the violacein biosynthesis pathway. Furthermore, 20 new derivatives were generated from 5-brominated violacein analogues via the Suzuki–Miyaura cross-coupling reaction directly using the crude extract without prior purification. Herein we demonstrate a flexible and rapid approach to access a diverse chemical space that can be applied to a wide range of natural product scaffolds.

Development of a bioavailable Hg(II) sensing system based on MerR-regulated visual pigment biosynthesis

Engineered microorganisms have proven to be a highly effective and robust tool to specifically detect heavy metals in the environment. In this study, a highly specific pigment-based whole-cell biosensor has been investigated for the detection of bioavailable Hg(II) based on an artificial heavy metal resistance operon. The basic working principle of biosensors is based on the violacein biosynthesis under the control of mercury resistance (mer) promoter and mercury resistance regulator (MerR). Engineered biosensor cells have been demonstrated to selectively respond to Hg(II), and the specific response was not influenced by interfering metal ions. The response of violacein could be recognized by the naked eye, and the time required for the maximum response of violacein (5 h) was less than that of enhanced green fluorescence protein (eGFP) (8 h) in the single-signal output constructs. The response of violacein was almost unaffected by the eGFP in a double-promoter controlled dual-signals output construct. However, the response strength of eGFP was significantly decreased in this genetic construct. Exponentially growing violacein-based biosensor detected concentrations as low as 0.39 μM Hg(II) in a colorimetric method, and the linear relationship was observed in the concentration range of 0.78–12.5 μM. Non-growing biosensor cells responded to concentrations as low as 0.006 μM Hg(II) in a colorimetric method and in a Hg(II) containing plate sensitive assay, and the linear relationship was demonstrated in a very narrow concentration range. The developed biosensor was finally validated for the detection of spiked bioavailable Hg(II) in environmental water samples.

Quorum sensing inhibitory effect of bergamot oil and aspidosperma extract against Chromobacterium violaceum and Pseudomonas aeruginosa.

Quorum sensing (QS) represents a major target for reducing bacterial pathogenicity and antibiotic resistance. This study identifies bergamot and aspidosperma as new potential sources of anti-QS agents. We investigated the anti-QS activity of plant materials on both Chromobacterium violaceum and Pseudomonas aeruginosa. Initially, we determined the minimum inhibitory concentrations (MICs) of plant materials using a broth microdilution method. Subsequently, we tested the effect of sub-MIC concentrations on QS-regulated traits and virulence factors production in test bacteria. Results revealed that bergamot and aspidosperma inhibited the ability of C. violaceum to produce violacein. Other QS-controlled phenotypes of C. violaceum, namely chitinolytic activity, motility, and biofilm formation, were also reduced by both plant materials. Moreover, QS-linked traits of P. aeruginosa were also reduced. Bergamot inhibited swarming but not swimming motility, while aspidosperma diminished both motility types in P. aeruginosa. Both plant materials also demonstrated antibiofilm activity and inhibited the production of protease and pyocyanin in P. aeruginosa. Furthermore, we tested the anti-QS effect of plant materials on the transcriptional level using RT-qPCR. Bergamot dramatically downregulated the C. violaceum autoinducer synthase gene cviI and the vioB gene involved in violacein biosynthesis, confirming the phenotypic observation on its anti-QS activity. Aspidosperma also reduced the expression of cviI and vioB but less drastically than bergamot. In P. aeruginosa, downregulation in the transcripts of the QS genes lasI, lasR, rhlI, and rhlR was also achieved by bergamot and aspidosperma. Therefore, data in the present study suggest the usefulness of bergamot and aspidosperma as sources of antivirulence agents.

Interplay between two quorum sensing-regulated pathways, violacein biosynthesis and VacJ/Yrb, dictates outer membrane vesicle biogenesis in Chromobacterium violaceum.

Outer membrane vesicles (OMVs) are lipid nanoparticles released by Gram-negative bacteria, which play multiple roles in bacterial physiology and adaptation to diverse environments. In this work, we demonstrate that OMVs released by the environmental pathogen Chromobacterium violaceum deliver the antimicrobial compound violacein to competitor bacteria, mediating its toxicity in vivo at a long distance. OMVs purified by ultracentrifugation from the wild-type strain, but not from a violacein-abrogated mutant ΔvioABCDE, contained violacein and inhibited several Gram-positive bacteria. Competition tests using co-culture and transwell assays indicated that the C. violaceum wild-type strain killed Staphylococcus aureus better than the ΔvioABCDE mutant strain. We found that C. violaceum achieves growth phase-dependent OMV release by the concerted expression of two quorum sensing (QS)-regulated pathways, namely violacein biosynthesis and VacJ/Yrb system. Although both pathways were activated at high cell density in a QS-dependent manner, the effect on vesiculation was the opposite. While the ΔvioABCDE mutant produced twofold fewer vesicles than the wild-type strain, indicating that violacein induces OMV biogenesis for its own delivery, the ΔvacJ and ΔyrbE mutants were hypervesiculating strains. Our findings uncovered QS-regulated pathways involved in OMV biogenesis used by C. violaceum to package violacein into OMVs for interbacterial competition.

Genetic control of violacein biosynthesis to enable a pigment-based whole-cell lead biosensor.

Environmental risks continue to grow due to heavy metal contamination caused by anthropogenic activities. Accumulation of harmful quantities of lead poses a threat to aquatic organisms, plants, and human beings. Whole-cell biosensors, which can proliferate independently, can detect the bioavailable fraction to assess the effect of target heavy metal on the environmental ecosystem. In this study, the biosynthesis pathway of violacein was heterogeneously constructed under the control of the T7 lac promoter in E. coli. A dose–response relationship existed between the inducer and the production of violacein. The biosynthesis pathway of violacein was finally engineered under the regulation of Pb(ii)-dependent metalloregulator PbrR to assemble Pb(ii)-inducible whole-cell biosensor. It permitted specific biosensing of Pb(ii) with extraordinary selectivity, and could resist the interferences from various metal ions. Color change by the intracellular accumulation of violacein could be recognized with the naked eye directly with high concentration of lead exposure, and quantified by determining the absorbance at 490 nm after butanol extraction. A good linear range for Pb(ii) concentrations of 0.1875–1.5 μM was obtained. The novel pigment-based whole-cell biosensor could detect concentrations as low as 0.1875 μM Pb(ii) based on in vitro quantification of violacein extracted by butanol, which is significantly lower than reported fluorescent protein-based PbrR-regulated biosensors based on direct measurement of whole cell fluorescence. These results indicate that genetically controlled violacein biosynthesis can enable a sensitive, visual, and qualitative biosensor for monitoring the presence of bioavailable Pb(ii) in lead-contaminated water.

Comparative biological and antiquorum activity of extracts of lamiaceae plants, grown in the Russian Federation

The purpose of this study was to assess the biological activity and antiquorum activity of extracts of Lamiaceae plants. Material and methods. Extracts of 4 medicinal plants of the Lamiáceae were used as test substances. Bioluminescent analysis were used on the model Escherichia coli MG1655 and antiquorum activity of medicinal plant extracts - on the model Chromobacterium violaceum ATCC 31532. Result. An analysis of the bioluminescent testing of Mentha piperita leaf aqueous extract showed no toxic effect throughout the entire record period over the entire concentration range. Low concentrations of analyte (0.625 mg/ml - 0.0097 mg/ml) cause a slight induction of lux-operon. Sālvia officinālis extract showed a slight antibacterial effect on the tested strain (EC50 = 10 mg/ml). The effect is achieved after 50-60 minutes of contact of microbial cells with components of Sālvia officinālis extract and persists until the end of the analyzed period of time (180 minutes). Extract of Oríganum vulgáre showed a decrease in the level of luminescence by 30% when the analyte was exposed to a concentration of 10 mg/ml. The extract of Thýmus serpýllum showed a pronounced antibacterial effect on the test organism, with a maximum concentration of 10 mg/ml, a 50% inhibition of luminescence is observed. All the tested compounds in subinhibitory concentrations suppressed the quorum-dependent biosynthesis of violacein.

CRISPR/Cas-directed programmable assembly of multi-enzyme complexes.

We describe a versatile CRISPR/Cas-based strategy to construct multi-enzyme complexes scaffolded on a DNA template in programmable patterns. Catalytically inactive dCas9 nuclease was used in combination with SpyCatcher-SpyTag chemistry to assemble enzymes in a highly modular fashion. Five enzymes comprising the violacein biosynthesis pathway were precisely organized in nanometer proximity; a notable increase in violacein production demonstrated the benefits of scaffolding.