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Quorum sensing is a well-studied intercellular communication system in bacteria that is triggered by and depends on secreted chemical signaling molecules called autoinducers. 1,2 The process allows a whole population of bacteria to collectively regulate gene expression and therefore their behavior as they reach a certain cell density. In this way, a bacterial population can act like a multicellular organism by synchronizing the actions of all members of a bacterial group.3
Quorum sensing regulates a variety of different processes in bacteria, including bioluminescence, symbiosis, expression of virulence genes, as well as antimicrobial resistance and the formation of biofilms in different bacteria.
In this blog, we look at how microplate readers can be used to advance findings and applications in quorum sensing research. This includes understanding how quorum sensing offers new approaches for drug discovery, microbiology in general, and antimicrobial research.
What triggers quorum sensing?
Quorum sensing is triggered by the accumulation of signaling molecules known as autoinducers in the extracellular environment. The amounts of these autoinducers increase as the bacterial population density increases. Bacteria detect the presence of these molecules through specific receptors or sensor proteins in their cell membrane as soon as the concentration of autoinducers reaches a threshold level. Detection of these molecules leads to a series of signaling cascades that ultimately leads to changes in gene expression and coordinated behaviors among the bacterial population.
The four stages of quorum sensing
In general, quorum sensing occurs in four main stages. These entail autoinducer production, autoinducer accumulation, detection of autoinducers, and the regulation of gene expression. The bacteria continuously produce autoinducers at a basal level as they reach and maintain the threshold concentration. The four stages allow bacteria to monitor their population density and adjust their behavior as needed.
Breakthrough discoveries in quorum sensing
Some of the first hints that quorum sensing was taking place in bacterial populations were reported in the late 1960s and early 1970s.2 First researchers revealed that the genetic competence of Streptococcus pneumoniae required the production of extracellular molecules. Second the luminescence of different marine bacteria was also shown to depend on the action of signaling molecules released from bacterial cells. However, it was not until the 1980s that two breakthrough discoveries started to cement the idea that bacteria could communicate and coordinate their actions based on their population density.
Identification of quorum sensing inducers
One of the first of these breakthroughs was the chemical identification of the quorum sensing signal for the luciferase of Photobacterium fischeri (Photobacterium fischeri was later named Vibrio fischeri; in 2007, Vibrio fischeri was reclassified as Aliivibrio fischeri but Vibrio fischeri continues to be widely used in the literature). 4 The autoinducer excreted by strain MJ-1 of V. fischeri was isolated, extracted and purified and its chemical structure was determined by a combination of nuclear magnetic resonance spectroscopy, infrared spectroscopy, and mass spectroscopy. N-3-Oxohexanoyl-L-homoserine lactone (Fig. 1) was thus identified as the quorum sensing signal of V. fischeri luciferase. 
The LuxR-LuxL System
In another development, the working details of the LuxR-LuxL system in V. fischeri were described for the first time. The system comprises the LuxI enzyme that synthesizes N-3-oxo-hexanoyl-L-homoserine lactone and a LuxR receptor protein that detects the presence of the acyl-homoserine lactone and which is responsible for the activation of gene expression.
The gene cluster responsible for light production consists of eight genes (luxA-E, luxG, luxI and luxR). luxI and luxR code for the main regulator proteins responsible for quorum sensing. When N-3-oxo-hexanoyl-L-homoserine lactone is produced by the LuxI enzyme it specifically interacts with the LuxR transcriptional regulator. When the concentration of N-3-oxo-hexanoyl-L-homoserine lactone reaches concentrations in the nanomolar range, the expression of the luxICDABE operon takes place and bioluminescence ensues (Fig. 2). 4-6 The luminescence reaction occurs in special light organs in nature within certain marine animals like squids and fish where the bacteria participate in a symbiotic relationship.
Today it is known that many other bacterial species control different processes with LuxI-LuxR-like systems. This includes for example activities like gene conjugation, exoenzyme production, and antibiotic synthesis.2
Gram-negative and Gram-positive quorum sensing
Since these breakthrough discoveries, a diverse group of signaling molecules have emerged that can be grouped into two major types of quorum sensing molecules. Gram-negative bacteria use molecules related to the N-acyl homoserine lactones and Gram-positive bacteria use quorum sensing molecules that are either amino acids or short peptides (Fig. 3). 
Research continues in many directions. The first three-dimensional structures of proteins involved in quorum sensing were established in the early 2000s and structures of key transcriptional regulators have also been reported. The expanding portfolio of structural data should prove useful in the development of new inhibitors and drugs that target components of the quorum sensing system.
Examples of quorum sensing phenomena
Virulence
Quorum sensing regulates the expression of a range of virulence factors that are essential for bacterial pathogenicity. In the presence of other bacteria, it allows pathogens to regulate the expression of virulence factors such as specific toxins or control the formation of biofilms (see next section). By interfering with quorum sensing it may prove possible to attenuate virulence, reduce the severity of infections, or decrease the reliance on antibiotics to control infectious bacteria.
Pseudomonas aeruginosa is a good example of a pathogenic bacterium that produces several virulence factors that are tightly regulated by quorum sensing. This includes proteases that contribute to tissue damage and immune evasion (e.g. LasA, LasB and AprA proteases), endotoxins that inhibit protein synthesis in host cells, and the expression of several secretion systems linked to the invasion and evasion of immune responses. We will see later how the blue-green pigment pyocyanin, another virulence factor, contributes to tissue damage and how researchers are looking for possible interventions to ameliorate their effects.
Biofilm formation
Quorum sensing is also involved in the formation of biofilms. Biofilms are layers of microbial organisms that have aggregated to form a colony. Many bacteria develop resistance to antibiotics when they form biofilms that provide protection against immune responses as well as antimicrobial agents. Biofilm formation can be easily quantified by crystal violet staining and detection on a microplate reader, as shown in the application note Testing novel bacteriophages for antibacterial properties with a crystal violet biofilm quantification assay.
Quorum sensing allows bacteria within a biofilm to communicate and coordinate their behavior to form or dismantle biofilms in response to environmental cues and population density. It does so by helping to regulate the expression of different genes involved in various stages of biofilm development and dispersal. P. aeruginosa for example forms robust biofilms that protect it from antibiotic treatments and host immune responses. Disrupting the quorum sensing that brings bacteria together in these biofilms is therefore a way of improving the action of existing antibiotics and offers new routes to novel treatments.
There are several stages of biofilm development. The first is the initial attachment phase which is often through weaker, reversible interactions mediated by extensions like pili or flagella. This is followed by an irreversible attachment phase that forms stronger interactions with the surface. This step may involve the production of adhesins or extracellular polymeric substances. Next the bacteria proliferate and form microcolonies. Microcolonies provide structural stability and serve as centers for further growth. Maturation proceeds with the further production of extracellular polymeric substances including sugars, proteins, and DNA. This extracellular matrix encases the bacterial cells forming a three-dimensional structure. Channels develop in the biofilm to allow the exchange of nutrients, waste products and signaling molecules and the passage of water. Individual bacterial cells may detach from the biofilm and disperse into the environment. This may be a passive process due to mechanical forces or an active process due to enzymes or signaling processes. Dispersal allows bacteria to colonize new surfaces or environments which contributes to the spread and colonization of the biofilm.
One specific role of quorum sensing in bacterial biofilms is the regulation of extracellular matrix production. As mentioned, bacteria can encase themselves in a matrix composed of sugars, proteins and DNA which provides structural support to the biofilm and offers protection against environmental stress. In P. aeruginosa, quorum sensing regulates the expression of genes involved in the synthesis of polysaccharides like alginate as well as proteins like lectins and adhesins. These ingredients contribute to the formation of a robust and cohesive biofilm structure.
Antimicrobial resistance
One area of particular interest to drug developers is finding viable options to combat antimicrobial resistance (AMR). Antibiotics have been a notable resource in the disease-fighting toolbox but increasingly antimicrobial resistance (AMR) threatens to undo progress. AMR is one of the top ten threats to public health worldwide 7,8 and you can read more about AMR and the challenges it poses in the BMG LABTECH blog Advances in combating antimicrobial resistance.
The inhibition of quorum sensing to suppress bacterial virulence may offer distinct advantages for AMR since it places less selective pressure on pathogens and may avoid the development of resistant bacteria. By targeting quorum sensing rather than directly killing bacteria, this type of approach represents an alternative to the use of antibiotics to fight bacterial disease.
The ideal quorum sensing inhibitor would have a high minimum inhibitory concentration (i.e. low antibacterial activity) but be an effective disruptor of quorum sensing signaling. You can read more about quorum sensing inhibition and possible answers to antibiotic resistance in the BMG LABTECH customer voice interview with Dr. Crystale Lim Siew Ying who works as part of the Malaysian Antimicrobial Stewardship Programme.
In other approaches, new quorum sensing inhibitors could also potentially be used in combination with existing antibiotics to enhance their efficacy. By disrupting bacterial communication and weakening their defenses, lower concentrations of antibiotics may be needed to produce a desired effect. This may in turn reduce the selective pressure for antimicrobial resistance.
Microplate readers and uses for quorum sensing research
Microplate readers are invaluable tools for many uses in the life sciences. In the area of microbiology, they allow researchers to study bacteria in many different ways, perform a wide range of assays, and pursue diverse applications including the search for new antimicrobials. Many of these uses depend on having options to determine how quickly bacteria can grow under different conditions. The growth of bacteria can readily be measured by looking at the optical density at 600 nm or the OD600 as it is otherwise known. Details about measuring bacterial growth and the principles involved are described in the BMG LABTECH blog Measure microbial growth using OD600.
When studying quorum sensing, bacterial growth is also an essential parameter that needs to be measured. If for example researchers are interested in finding inhibitors of quorum sensing, bacterial growth must be measured alongside quorum sensing. Quorum sensing agents do not destroy or inhibit bacterial growth but instead act by quenching pathogenic activities like toxin production, biofilm formation, swimming, swarming, or motility. In other words, quorum sensing inhibitors inhibit virulence rather than bacterial growth. It is therefore necessary to demonstrate the absence of an impact on growth in parallel to the specific quorum sensing action that is under investigation.
Using bioluminescence as a readout
In the application note Monitoring bacterial cell-to-cell communication quorum sensing using a BMG LABTECH microplate reader the effects of quorum sensing were investigated by measuring bioluminescence and bacterial growth in parallel. Different strains of V. fischeri were grown under different defined environmental conditions to determine the impact of quorum sensing on this Gram-negative bacterium. Figure 5 shows batch growth curves for V. fischeri strains grown in different media. This type of growth curve can be monitored in parallel to the measurement of bioluminescence or other output of quorum sensing.
Finding new antimicrobials
The paper “Paecilomycone inhibits quorum sensing in Gram-negative bacteria” describes the identification of an antimicrobial agent that inhibits quorum sensing in both Chromobacterium violaceum and Pseudomonas aeruginosa. 9 This is a good example of how researchers are trying to target the quorum sensing pathway to find new solutions for antimicrobials.
P. aeruginosa is a Gram-negative pathogen that causes severe infections in immunocompromised patients. It is involved in a variety of acute and chronic infections, including urinary tract infections, infections of burns or wounds, and respiratory diseases. Infections are difficult to treat since P. aeruginosa may form biofilms, has a low permeability outer membrane, and has many efflux pumps to remove drugs from within the cell. Increasingly, P. aeruginosa isolates are multidrug resistant and pose challenges to many classes of antibiotics.
In the study, paecilomycone strongly inhibited the production of virulence factors in P. aeruginosa and disrupted biofilm formation at concentrations that did not affect cell growth.
Quorum sensing inhibition assays in P. aeruginosa were performed on a CLARIOstar® Plus microplate reader by measuring fluorescence. P. aeruginosa PAO1 reporter strains were used that express green fluorescent protein when the quorum sensing pathway is activated, including the lasB-GFP, rhlA-GFP, and pqsA-GFP strains. A wild-type strain that constitutively expressed GFP (WT-GFP) was used as control. GFP expression was normalized to the growth of the bacteria and the IC50 was calculated by plotting the maximum slope of GFP expression/bacterial growth.
Not all of the quorum sensing pathways were inhibited by paecilomycone but a strong effect was observed for the pqsA-GFP reporter strain (Fig. 6).
In separate experiments, it was shown that the toxicity of paecilomycone towards eukaryotic cells and organisms was low, which makes this compound an interesting lead for further clinical research. For example, the viability of HepG2 cells treated with paecilomycone for 24 h was measured using resazurin assays. Fluorescence was measured on a PHERAstar® FSX microplate reader using an excitation wavelength of 540 nm and an emission wavelength of 590 nm. Paecilomycone was toxic to HepG2 cells only at high concentrations with an IC50 of 219 µM (see Fig.7). The researchers concluded that paecilomycone inhibited quorum sensing in P. aeruginosa and is a promising candidate for further research.
Cyclodextrins inhibit the synthesis of pigment virulence
P. aeruginosa is perhaps the most studied quorum sensing pathogen to date. Around 6% of all the genes of this organism are regulated by quorum sensing and have roles in virulence, pigment production and biofilm formation. In the paper “Quorum quenching effect of cyclodextrins on the pyocyanin and pyoverdine production of Pseudomonas aeruginosa” researchers looked at the impact of cyclodextrins on these pigment virulence factors that interfere with multiple cellular functions during infection.
α-Cyclodextrin and β-cyclodextrin were both shown to inhibit the synthesis of pigment virulence factors which suggested the potential value of cyclodextrins as quorum sensing inhibitors that could be used as an antivirulence strategy.
The quorum sensing assays were performed by measuring the fluorescence of pigment formation in the presence or absence of the different inhibitors on an Omega series microplate reader. Cell growth was also monitored throughout the experiments and showed no significant changes upon addition of cyclodextrin inhibitors.10
Emerging areas of quorum sensing research
Researchers are interested in finding new ways to exploit the unique features of quorum sensing. For example, they are looking for synthetic compounds that mimic the natural signals of quorum sensing, looking at new alternatives to disrupt biofilms, and searching for ways to attenuate virulence factors. They are also interested in finding new drug targets and combinations of novel and existing drugs that may offer benefits.
Emerging areas of quorum sensing research include applications in synthetic biology, microbiome modulation, the study of ecological interactions, and environmental monitoring.
It should be noted that applications in areas like environmental testing are not restricted to the direct impairment of the quorum sensing process. For example, researchers are finding innovative ways to use the properties of quorum sensing organisms as biosensors for the overall health of bacterial communities. One example is in wastewater testing. The gold standard for testing water toxicity for many years uses measurements of the bioluminescence produced by V. fischeri. Luminescence is a sensitive indicator of cellular stress and can be readily measured in test samples.
In the application note “Wastewater testing of biotoxicity using Aliivibrio fischeri luminescence assays” robust measurements were successfully performed on a BMG LABTECH Omega Series microplate reader with significant savings in time and cost versus alternative cuvette-based methods. In addition, the assays could be performed with flash kinetic measurements of luminescence which avoid the interference caused by colored samples that arise on the initial mixing of samples (Fig. 8). This type of assay therefore eliminates the need for additional absorbance measurements and offers significant savings in time and resources.
Overall, research into quorum sensing is closely linked to human health, environmental sustainability and innovation in biotechnology.
BMG LABTECH solutions
What is the preferred BMG LABTECH microplate reader for specific needs and applications related to quorum sensing? BMG LABTECH offers a range of detection devices for sensitive absorbance and fluorescence measurements.
The PHERAstar FSX was specifically conceived for screening campaigns and is your go-to reader for high-performance high-throughput screening.
Both the VANTAstar® and CLARIOstar Plus allow for wavelength flexibility and include Enhanced Dynamic Range technology for superior performance in a single run. They also offer increased light transmission and sensitivity courtesy of Linear Variable Filter MonochromatorsTM and different filter options.
All BMG LABTECH microplate readers have exceptionally fast reading capabilities. In addition, the Omega series, CLARIOstar Plus, and PHERAstar FSX microplate readers come with on-board injectors that can offer the very best options for detection at the time of injection.
Collectively, BMG LABTECH multi-mode readers combine high-quality measurements with miniaturised assays, short measurement times, and offer considerable savings on materials and other resources.
Configure your microplate reader and get an initial recommendation!
References
- Bassler BL, Losick R. Bacterially speaking. Cell. 2006 Apr 21;125(2):237-46. doi: 10.1016/j.cell.2006.04.001. PMID: 16630813.
- Whiteley M, Diggle SP, Greenberg EP. Progress in and promise of bacterial quorum sensing research. Nature. 2017 Nov 15;551(7680):313-320. doi: 10.1038/nature24624.
- Waters CM, Bassler BL. Quorum sensing: cell-to-cell communication in bacteria. Annu Rev Cell Dev Biol. 2005;21:319-46. doi: 10.1146/annurev.cellbio.21.012704.131001.
- Eberhard A, Burlingame AL, Eberhard C, Kenyon GL, Nealson KH, Oppenheimer NJ. Structural identification of autoinducer of Photobacterium fischeri luciferase. Biochemistry. 1981 Apr 28;20(9):2444-9. doi: 10.1021/bi00512a013.
- Engebrecht J, Nealson K, Silverman M. Bacterial bioluminescence: isolation and genetic analysis of functions from Vibrio fischeri. Cell. 1983 Mar;32(3):773-81. doi: 10.1016/0092-8674(83)90063-6.
- Engebrecht J, Silverman M. Identification of genes and gene products necessary for bacterial bioluminescence. Proc Natl Acad Sci U S A. 1984 Jul;81(13):4154-8. doi: 10.1073/pnas.81.13.4154.
- World Health Organization, Fact sheet, Antimicrobial resistance, https://www.who.int/news-room/fact-sheets/detail/antimicrobial-resistance
- Cook MA, Wright GD. The past, present, and future of antibiotics. Science Translational Medicine 2022 Aug 10;14(657):eabo7793. doi: 10.1126/scitranslmed.abo7793.
- Beenker WAG, Hoeksma J, Bannier-Hélaouët M, Clevers H, den Hertog J. Paecilomycone inhibits quorum sensing in Gram-negative bacteria. Microbiol Spectr. 2023 Mar 15;11(2):e0509722. doi: 10.1128/spectrum.05097-22. Fig.6 in this blog adapted from Fig.3 from this reference under license CC-BY 4.0. Fig.7 in this blog adapted from Fig.8A from this reference under license CC-BY 4.0.
- Fekete-Kertész I, Berkl Z, Buda K, Fenyvesi É, Szente L, Molnár M. Quorum quenching effect of cyclodextrins on the pyocyanin and pyoverdine production of Pseudomonas aeruginosa. Appl Microbiol Biotechnol. 2024 Mar 22;108(1):271. doi: 10.1007/s00253-024-13104-7.
