Jonah Patel
The lux gene is a bacterial luciferase gene cassette that is unique among bioluminescent bioreporter systems due to its ability to synthesize and/or scavenge all the substrate compounds required for its production of light. The lux operon encodes genes for self-regulation and the production of luminescent proteins. The intensity of light emission in most luminescent marine bacteria and other bacteria harbouring the cloned luxA and luxB genes depends on parameters such as the cAMP system and the density of the cell suspension. The lux genes can be placed under the control of a constitutive promoter, such as the constitutive lactococcal consensus promoter PCP25, which can be optimized for the host strain. LuxR is a constitutively expressed protein that can bind AHL and stimulate transcription from the right-hand lux promoter (pLuxR).
| Characteristics | Values |
|---|---|
| Lux gene products | Luciferase enzyme, substrate |
| Lux genes | luxA-E, luxAB, luxCDEfrp, luxAB, luxCDEfrp:WT, luxCDEfrp:CO |
| Lux operon | Encodes genes for self-regulation and the production of luminescent proteins |
| Lux operon genes | luxA, luxB, luxC, luxD, luxE |
| LuxA and LuxB gene products | α and β subunits |
| LuxC, LuxD, and LuxE gene products | Reductase, transferase, and synthase |
| Lux gene cassette | Bacterial luciferase (lux) gene cassette |
| Lux gene cassette genes | luxCDABE |
| Lux gene cassette substrate compounds | FMNH2, aldehyde co-substrates, molecular oxygen |
| Lux gene expression | Constitutive |
| LuxR | Regulatory protein, constitutively expressed protein |
| LuxR-dependent genes | qsrP, acfA, ribB |
| LuxR-independent gene | nadB |
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What You'll Learn
The Lux Operon
The intensity of light emission in luminescent bacteria depends on several factors, including the density of the cell suspension and the accumulation of an endogenously synthesized autoinducer, N-(3-oxohexanoyl)-l-homoserine lactone for V. fischeri and N-(3-hydroxybutanoyl)-l-homoserine lactone for V. harveyi. The maximum luminescence is attained at an autoinducer concentration of about 5 μM, resulting in a significant increase in light output compared to the basal level.
Additionally, the Lux Operon has been engineered in other organisms, including human cells. While challenging, it is possible to alter the chemistry of human cell cultures to emit light through a two-vector lux expression system. The Lux Operon provides a model for understanding how gene clusters and promoters can create non-traditional products from nucleotide base sequences.
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Quorum sensing
The LuxR protein is a regulatory factor that plays a crucial role in the expression of genes involved in various biological processes. It is one of the most studied members of a family of transcriptional regulator proteins involved in acyl-homoserine lactone-mediated quorum sensing. LuxR can bind to AHL, and this complex can stimulate transcription from the right-hand lux promoter (pLuxR). This positive feedback loop further increases the transcription of LuxI, leading to higher AHL production and enhanced bioluminescence.
In addition to V. fischeri, quorum sensing has been observed in other organisms, such as Pseudomonas aeruginosa, which has over 400 quorum-sensing-controlled genes. The Lux system has unique advantages due to its ability to autonomously produce luminescent signals. It can synthesize and scavenge all the substrate compounds required for light production, allowing it to generate signals continuously or in response to specific triggers across a wide range of hosts. This versatility has led to its application in eukaryotic cells and even human cell lines, where it is used for biomedical detection and tumour visualization in small animal models.
The intensity of light emission in luminescent marine bacteria, such as V. fischeri, depends on parameters like the cAMP system and cell suspension density. Specifically, the accumulation of an endogenously synthesized autoinducer, N-(3-oxohexanoyl)-l-homoserine lactone, influences the maximum luminescence attained by the bacterial culture. This autoinducer concentration results in a 5000-fold stimulation compared to the basal level of luminescence.
The lux genes can be placed under the control of various promoters, including constitutive promoters, to achieve constant expression. For example, the luxA-E genes have been placed under the control of the constitutive E. coli frr promoter, resulting in the successful generation of bioluminescence in Salmonella enterica strains. Additionally, the lux operon encodes genes for self-regulation and the production of luminescent proteins, further highlighting the importance of quorum sensing in the regulation of bioluminescence.
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Luciferase
The bacterial luciferase gene cassette consists of five genes: luxCDABE. The luxA and luxB genes code for the α and β subunits of the luciferase enzyme, which together catalyze the light-producing reaction. The luxC, luxD, and luxE genes encode for the complex components that synthesize and recirculate fatty aldehyde, the substrate for luciferase.
The intensity of light emission in luminescent bacteria depends on various parameters, including cell suspension density and the accumulation of an endogenously synthesized and secreted autoinducer. The autoinducer concentration influences the transcription of the lux operon, with LuxR being the transcriptional activator that responds to the autoinducer.
The lux genes have been transferred to various organisms, generating new luminescent species. The expression of the lux genes in different organisms has led to advancements in understanding the molecular biology of bacterial bioluminescence. The lux operon has been studied in bacteria such as Vibrio fischeri, Escherichia coli, and Vibrio harveyi, as well as in plant and mammalian cell lines.
The lux genes can be placed under the control of different promoters, including constitutive promoters, to optimize expression in the host strain. For example, the constitutive lactococcal consensus promoter PCP25 has been modified to create a new promoter, Phelp, which gives a strong signal in Listeria and other genera.
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Bioluminescence
The bacterial luciferase gene cassette (lux) is unique among bioluminescent bioreporter systems due to its ability to synthesise and/or scavenge all of the substrate compounds required for its production of light. The lux system has the unique ability to autonomously produce a luminescent signal, either continuously or in response to the presence of a specific trigger, across a wide array of organismal hosts. The lux operon encodes genes for self-regulation and for the production of luminescent proteins. The luciferase protein is a heterodimer formed by the products of the luxA and luxB genes. The luxC, luxD, and luxE gene products encode for a reductase, transferase, and synthase, respectively, that work together in a single complex to generate an aldehyde substrate for the bioluminescent reaction.
The intensity of light emission in most luminescent marine bacteria and in other bacteria harbouring the cloned luxA and luxB genes depends on a number of parameters, including the cAMP system and, in particular, the density of the cell suspension. The maximum luminescence of a bacterial culture is attained at an autoinducer concentration of ∼5 μM (or 1 μg/ml), resulting in a 5000-fold stimulation compared with the basal level of luminescence.
The lux genes can be placed under the control of a variety of promoters depending on the particular application. For constant expression, the lux genes can be placed under the control of a constitutive promoter which can be optimised for the host strain. For example, Riedel, Casey, et al. (2007) and Riedel, Monk, et al. (2007) altered the constitutive lactococcal consensus promoter PCP25 by introducing the 5′ untranslated region of the Listeria monocytogenes hlyA gene. This new promoter Phelp (highly expressed Listeria promoter) gives a strong signal in Listeria and other genera. Howe et al. (2010) generated a plasmid where luxA-E genes are under the control of the constitutive E. coli frr promoter.
The main benefit of inducible promoters is their ability to act as a "switch", allowing for temporal and spatial regulation of gene expression. Several inducible promoters have been developed and applied to bacterial therapies. To convert constitutive promoters into radio-inducible promoters, the Cheo Box sequence could be added. Radiotherapy activates the recA promoter and, at the same time, it has a cytotoxic effect because of the DNA damage it causes. However, with a constitutive gene expression, off-target effects may increase. Further control can be achieved by inducible promoters, which only drive the expression of a target gene under specific conditions or in the presence of a particular trigger signal.
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LuxR
The LuxR protein is constitutively expressed and can bind to AHL (N-acyl homoserine lactones). When bound to AHL, LuxR stimulates transcription from the right-hand lux promoter (pLuxR). This promoter controls the transcription of the LuxI enzyme, leading to a positive feedback loop that increases transcription from the right-hand lux promoter. This system is known as "quorum sensing" and is based on the ability of the Lux pathway to respond to acyl-homoserine lactones.
The lux operon, which includes the LuxR protein, is involved in the production of luminescent proteins and self-regulation. The most well-studied operon is originally isolated from the bacterium Vibrio fischeri, which produces a yellowish bioluminescence of about 490 nm through its operon-produced luciferase.
The lux operon consists of five genes (luxCDABE) whose protein products work together to generate bioluminescent light signals without the need for external substrate additions or manipulations. This process has been observed in both prokaryotes and, more recently, in a mammalian HEK293 cell line.
The lux genes can be placed under the control of a constitutive promoter, such as the E. coli frr promoter, to achieve constant expression. This approach has been used to successfully confer bioluminescence to Salmonella enterica strains. By placing the lux genes under the control of a constitutive promoter, the recombinant strain can produce both the luciferase enzyme and the necessary substrate for bioluminescence.
In experiments with HEK293 cells, the average radiance of cells producing a visibly detectable bioluminescent signal closely correlated with the number of cells present (R2 = 0.95275). This indicates that the bioluminescent flux of the cell population can be used to approximate the overall population size in a living host, providing a non-invasive method for investigators to quantify cell populations.
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Frequently asked questions
The bacterial luciferase gene cassette (lux) is a bioluminescent bioreporter system that can synthesize and/or scavenge all the substrate compounds required for its production of light.
The Lux gene is used to detect specific chemical signals in environmental samples. It has also been developed for use as a biomedical detection tool for toxicity screening and visualization of tumors in small animal models.
The Lux operon encodes genes for self-regulation and the production of luminescent proteins. It consists of five genes (luxCDABE) whose protein products work together to generate bioluminescent light signals.
LuxR is a transcription factor that binds to signaling molecules to regulate the expression of different genes. It is involved in the LuxI-LuxR quorum-sensing system, where it forms a complex with 3-oxo-hexanoyl-l-homoserine lactone (3-oxo-C6-HSL) to activate transcription of the lux operon.
The Lux genes can be placed under the control of a constitutive promoter, such as the E. coli frr promoter or the lactococcal consensus promoter PCP25, to achieve constant expression.
