Quantification of Violacein in Chromobacterium violaceum and Its Inhibition by Bioactive Compounds

Summary

The objective of this protocol is to quantify violacein production by Chromobacterium violaceum ATCC 12472 as a proxy for assessing quorum sensing inhibition by bioactive compounds.

Abstract

Bacteria communicate through a system known as quorum sensing (QS), which allows them to coordinate their behavior in response to changes in cell density. This process involves the production, secretion, and detection of small extracellular signaling molecules, usually called autoinducers (AI). QS regulates a wide range of genes and functions, including biofilm formation and dispersal, swarming motility, production of virulence factors, antibiotic resistance, and bioluminescence, among others. QS inhibition has become a promising antivirulence strategy against antibiotic-resistant bacteria as it exerts a lower selective pressure compared to traditional antibiotics. In the bacterium Chromobacterium violaceum ATCC 12472, the QS proteins CviI/CviR, which are homologous to the LuxI/LuxR-type QS proteins of Aliivibrio fisheri, control many genes, including those responsible for producing the purple pigment violacein. C. violaceum has been widely used as a biosensor strain in QS inhibition studies, particularly the inhibition of violacein production. Many compounds have the potential to inhibit QS by binding to QS proteins, either LuxI or LuxR homologues, disrupting QS circuits. This protocol describes the method for quantifying violacein produced by C. violaceum ATCC 12472 and its inhibition by bioactive compounds at concentrations that do not affect bacterial growth. The assay described here demonstrates a significant reduction in violacein production by the tested compounds. This study highlights the potential for using this protocol to screen promising candidates in QS inhibition research.

Introduction

Bacteria can communicate through a system known as QS, which enables coordinated modulation of their behavior in response to changes in cell density. This process involves production, secretion, and detection of small extracellular signaling molecules, also called autoinducers, which allow communication both between different species and within the same species^1,2.

This communication is capable of regulating genes and functions such as biofilm formation and dispersion, motility and adhesion, cell proliferation, synthesis of exoenzymes and siderophores, sporulation, acid resistance, horizontal gene transfer, antibiotic resistance, bioluminescence, production of exopolysaccharides, among others^3,4,5. Given the relevance of QS-regulated phenotypes, there is great interest in developing systems capable of interfering with this cellular communication. The inhibition of QS signaling is also termed quorum quenching (QQ)⁶. QQ can occur in several ways, including inhibitors of the synthesis of autoinducers, enzymatic inactivation of these signals, and molecules with an antagonistic effect on these molecules^4,7.

The growing threat of antibiotic resistance has driven research into alternative therapies, including quorum sensing inhibitors (QSIs), which disrupt bacterial communication without directly killing pathogens. These compounds may show synergistic effects when combined with antibiotics, enhancing bacterial susceptibility and allowing reduced antibiotic doses, which is a key strategy to slow resistance development. For example, resveratrol and kanamycin together delay Aeromonas hydrophila growth⁸, while N-acetylcysteine synergizes with tobramycin to disrupt Pseudomonas aeruginosa biofilms⁵.

With antibiotic overuse and stagnant drug development, antivirulence approaches like QS inhibition offer a paradigm shift⁹. Instead of targeting viability, they target virulence factors, rendering bacteria less harmful and more vulnerable to host immunity or antibiotics⁵. This strategy imposes weaker selective pressure compared to traditional antibiotics, minimizing resistance and preserving beneficial microbiota^10,11. Despite promising lab results, real-world validation through clinical trials remains underexplored¹². QS inhibition represents a promising complement to antibiotics, but its translation to practice requires further evidence of efficacy and safety in complex biological environments.

C. violaceum is a beta proteobacterium that forms violet colonies with a smooth and shiny surface. They are facultative anaerobic bacilli and do not form spores¹³. The mechanism of QS in C. violaceum involves a positive feedback loop based on the gene system analogous to the lux model of Aliivibrio fisheri, which contains proteins CviI (homologous to LuxI) and CviR (homologous to LuxR; Figure 1). Violacein exhibits biocidal activity against various organisms, including fungi, bacteria, nematodes, and viruses, particularly during the microbial stationary growth phase, when population density is extremely high, and nutrients are nearly depleted. Consequently, violacein production can be regarded as a competitive strategy to prolong the survival of the microbial colony¹⁴.

Plants produce compounds originating from secondary metabolism, which play a crucial role in increasing their ability to adapt to unfavorable environments. These metabolites, such as alkaloids, phenols, flavonoids, quinones, tannins, terpenes, and lectins, are widely recognized as defense mechanisms against herbivores and microorganisms¹⁵. The potential of plant-derived compounds as QSI has attracted special attention due to the dynamic interactions between plants and microorganisms. Studies on plant-pathogen interactions in the rhizosphere have demonstrated that plants can respond to bacterial autoinducers by secreting bioactive compounds capable of acting as inhibitors against pathogens¹⁶.

In this study, we describe a practical method aimed at quantifying violacein production by C. violaceum, focusing on its inhibition by bioactive compounds. This method serves as an initial screening tool for selecting compounds with potential QSI activities. It provides a rapid and practical approach for future therapeutic development against Gram-negative pathogenic bacteria. This standardized protocol will be useful because it provides a detailed description that has not been done in previous studies and will facilitate comparison among different studies in the future^17,18,19.

Protocol

1. Culture preparation (Figure 2)

1. Grow in a test tube a 5 mL culture of C. violaceum ATCC 12472 in Luria Bertani broth (LB: tryptone 10 g/L, yeast extract 5 g/L, 5g/L NaCl) at 30 °C in a shaking incubator (220 rpm) for 24 h.
2. Adjust the culture to an OD₅₉₅ of 0.1 in LB broth, corresponding to approximately 1 x 10⁸ CFU/mL.

2. Compound preparation

1. In a flat-bottom 96-well plate, add LB broth and the bioactive compound according to a serial dilution calculation, following the description below. Depending on their solubility, bioactive compounds are typically diluted in pure DMSO or a DMSO: water (1:1) solution.
   NOTE: In methodologies that assess QSI, it is crucial to ensure that the concentrations used are below the minimum inhibitory concentration (sub-MIC), meaning that they do not affect bacterial growth. Therefore, it is recommended to perform growth curve assays at different concentrations to confirm that the compound specifically inhibits cell-to-cell communication rather than microbial viability. Please see Supplementary Figure 1 for growth curve assays of the tested bioactive compounds used in this protocol.
2. To calculate the bioactive compound concentration in the well, considering a stock solution of farnesol and linalool at 20 mg/mL and a target concentration of 200 µg/mL in the well, as well as resveratrol at 2.5 mg/mL in the stock solution and 25 µg/mL in the well, perform the calculation as follows:
   C₁V₁ = C₂V₂
   20,000 µg/mL x V₁ = 200 µg/mL x 200 µL
   V₁ = 40,000/20,000 µL
   V₁ = 2 µL
   where C₁ = Stock solution concentration (20 mg/mL, which is equivalent to 20,000 µg/mL); V₁ = volume of stock solution to be added; C₂ = desired final concentration (200 µg/mL); V₂ = final volume in the well (200 µL).

3. Serial dilution²⁰ and plate preparation (Figure 3)

NOTE: Serial dilution is used for quickly evaluating a range of concentrations of a tested compound. In this process, the compound is successively diluted at a constant ratio, in this case 1:2. Each dilution reduces the concentration of the compound by half compared to the previous concentration, resulting in a range of decreasing concentrations²⁰.

1. In the first column of the serial dilution, add 196 µL of LB broth (in our example, columns 3, 5, and 7). On the following wells of the serial dilution, add 100 µL of LB broth (in our case, columns 4, 6, and 8).
2. Then, add 2x the amount of bioactive compound calculated in the previous step (2.2) to perform serial dilution. The calculation resulted in 2 µL of each tested compound to achieve the final concentration in the first well, so add 4 µL of the compound (columns 3, 5, and 7).
3. Transfer half of the well's total volume (100 µL) to the next column (in our case, columns 4, 6, and 8), and so on until the last desired concentration, if doing a serial dilution. Remember to thoroughly mix the contents before the transfer.
   NOTE: In this experimental setup, we have only used two concentrations for simplicity and all compounds fit into just one plate.
4. Perform the experiment in at least triplicate per concentration of each compound. Three biological replicates are also recommended. Change the tip and repeat the procedure until the last desired concentration. In the last column (in our case, 4, 6, and 8), take 100 µL from it and discard.
5. Add 80 µL of LB broth to all the wells, totaling 180 µL (rows B to D). Then, add 20 µL of the C. violaceum culture standardized in step 1.2, ensuring a final volume of 200 µL per well (rows B to D). Make sure to change pipette tips every time when changing wells to avoid carryover of higher concentrations between the wells or contamination.
6. If the compound is dissolved in any kind of solvent, make sure to prepare the control with that solvent. We used DMSO. In this case, prepare a solvent control, which will be the untreated control - UC (without bioactive compound; column 9). Add 2 µL of DMSO per control well. This gives a maximum concentration of 1% DMSO in the test.
   NOTE: DMSO at 1% does not affect bacterial growth^21,22.
7. Prepare the blank, or color control if using pigmented compounds, without bacteria. After adding 80 µL of LB broth to all the wells, totaling 180 µL (rows B to D, as previously described), add 20 µL of LB broth to the corresponding blank wells, totaling 200 µL (rows E to G). This is done because there is some background staining from crystal violet that needs to be subtracted from the OD readings, as will be described in step 5 of this protocol.
8. If any column remains empty, fill it with 200 µL of LB broth or sterile distilled water (columns 1, 2, 10, 11, and 12). This step is important to prevent excessive drying of the medium in the incubator.

4. Incubation and extraction of violacein

1. Cover the plate with a lid or a sealing film (as specified in the Table of Materials) to prevent evaporation, depending on the spectrophotometer used.
2. Incubate the plate at 30 °C with agitation at 130 rpm for 24 h.Then, remove the plate from the incubator, place it in a drying incubator at 60 °C, remove the lid, and allow the plate to dry completely (approximately 8 h).
3. After the plate has dried, remove it from the incubator and add 200 µL of pure DMSO to each well, then cover it. Place the plate in an incubator at 25°C while shaking at 130 rpm for 30 min to dissolve the violacein pigment.
4. After this time, remove the plate from the incubator and transfer 100 µL to another microplate, avoiding contact between the tip and the walls of the well.

5. Quantification of violacein production

1. Quantitate the amount of violacein production by measuring the absorbance at 595 nm using an appropriate plate spectrophotometer (Table of Materials). The inhibition of violacein pigment is proportional to lower absorbance readings.
2. Quantify violacein production by using a negative control (untreated control) as the reference for 100% violacein production (Table 1). First, subtract the absorbance value of the blank from each treatment replicate to obtain the absorbance corresponding solely to the violacein pigment (excluding interference from the broth or compound color). Then, calculate the percentage of violacein production for each treatment, using the following equation.
   %violacein production = (OD of the bioactive compound / OD of untreated control) x 100
3. Calculate the statistical analyses in three replicates. Results can be submitted to ANOVA, followed by Tukey's test using the GraphPad Prism 8.0 software. The value of p <0.05 was considered statistically significant.

Results

For the results shown here, three bioactive compounds previously studied by our research group⁸, resveratrol, farnesol, and linalool, were used. Each compound was tested for its ability to inhibit violacein production in C. violaceum ATCC 12472, a phenotype regulated by the QS system over the vioABCDE operon²³.

The results validate the efficacy of our protocol in quantifying violacein production and its inhibition by bioactive c...

Discussion

The first method for extracting and characterizing violacein from C. violaceum emerged in the 1990s, focusing on identifying suitable carbon sources for pigment synthesis. In this process, violacein -- along with the cells and culture medium -- was dried, resuspended in ethanol, and then separated by supernatant extraction. The violacein, now dissolved in ethanol, was subsequently dried again, yielding a pure, dry form of the pigment²⁷. In 1998, the protocol was modified^...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMSO	Synth
Farnesol	Sigma-Aldrich	F203	95%
Linalool	Sigma-Aldrich	L2602	97%
Luria-Bertani Medium	Difco	244620
Optical Adhesive Film	Thermo Fisher	4311971	MicroAmp
Resveratrol	Sigma-Aldrich	R5010	≥99% (HPLC)
Synergy HTX multi-mode reader	Biotek
Tissue Culture Plates 96 well	Kasvi	K12-096