The Evolution of the Bacterial Luciferase Gene Cassette (lux) as a Real-Time Bioreporter

The bacterial luciferase gene cassette (lux) is unique among bioluminescent bioreporter systems due to its ability to synthesize and/or scavenge all of the substrate compounds required for its production of light. As a result, 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. While originally employed extensively as a bacterial bioreporter system for the detection of specific chemical signals in environmental samples, the use of lux as a bioreporter technology has continuously expanded over the last 30 years to include expression in eukaryotic cells such as Saccharomyces cerevisiae and even human cell lines as well. Under these conditions, the lux system has been developed for use as a biomedical detection tool for toxicity screening and visualization of tumors in small animal models. As the technologies for lux signal detection continue to improve, it is poised to become one of the first fully implantable detection systems for intra-organismal optical detection through direct marriage to an implantable photon-detecting digital chip. This review presents the basic biochemical background that allows the lux system to continuously autobioluminesce and highlights the important milestones in the use of lux-based bioreporters as they have evolved from chemical detection platforms in prokaryotic bacteria to rodent-based tumorigenesis study targets. In addition, the future of lux imaging using integrated circuit microluminometry to image directly within a living host in real-time will be introduced and its role in the development of dose/response therapeutic systems will be highlighted.

insects displaying their luminescent signal after dusk, while in marine environments bioluminescence is most commonly observed in single celled bacteria that are found either living freely or in symbiosis with larger hosts. It is these bioluminescent bacteria that are the most abundant and widely distributed of the light emitting organisms on Earth and they can be found in both aquatic (freshwater and marine) and terrestrial environments. Despite the widespread prevalence of bacterial bioluminescence, however, the majority of these organisms are classified into just three genera: Vibrio, Photobacterium, and Photorhabdus (Xenorhabdus) [1]. Although they are viable as free-living bacteria, these organisms are most commonly observed in symbiosis with a larger host. There is still no consensus as to the evolutionary benefit of bioluminescent production, however, in general it is theorized that the production of light can aid in the consumption of free living bacteria by higher trophic organisms, transferring them to a more controlled, nutrient rich habitat inside the host, or that, likewise, symbiotic bacteria can aid their hosts through the production of light that attracts prey, aids in camouflage, or attracts mates, in return for the shelter and nutrients provided by living within the body of the host organism [8]. Regardless of the reasons, the genetic system employed for the generation of bioluminescence is well conserved across all known bioluminescent bacteria. The luciferase protein is a heterodimer formed by the luxA and luxB gene products. 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. In some species, there is an additional gene, frp, that functions as a flavin reductase to aid in the regeneration of the required FMNH 2 substrate. Together with molecular oxygen, these components are all that are required to produce a bioluminescent signal [9] (Figure 1).

Figure 1.
The luxCDABEfrp genes work synergistically with endogenous myristic acid, FMN, and O 2 to generate a bioluminescent signal. The frp gene is not found in all organisms expressing the remaining lux genes. Originally published in and used with permission from [1].
In addition, some marine species have additional genes that govern the expression of the remainder of the operon. The luxI and luxR genes function as an autoinducer and transcriptional activator (Figure 2), allowing the bioluminescent bacteria to participate in quorum sensing, therefore producing

Development of lux as a Method for Visualizing Gene Expression
The first use of lux as a biomonitoring technology came soon after its transgenic expression in E. coli, when Enbreghet et al. [14] fused the lux cassette to an inducible promoter that could be used to monitor gene expression in vivo. Using this experimental design it became possible to monitor autonomous bioluminescence as an indicator for the transcriptional activity of a promoter of interest. Using this method, the first major targets of study were the E. coli lac and ara promoters and it was discovered that upon IPTG or arabinose induction, light production in hosts expressing lux fusions increased between 600 to 1,000-fold. Following these reports the lux system was used to monitor regulation of the lateral flagella genes in Vibrio parahaemolyticus [14,15], providing its first demonstration in a previously uncharacterized system. These applications represented a significant shift in the way gene expression was investigated because, unlike traditional biochemical assays using enzymatic reporters, the bioluminescent signal from the lux genes could be easily detected and measured with high sensitivity without cell perturbation. This allowed the same sample to be continuously monitored, thus revealing the dynamics of gene expression through changes in bioluminescence over time. This new method was therefore capable of generating data that could not previously be generated.

lux-Based Bioluminescence as a Tool for Cellular Population Monitoring
While the Lux proteins do not require exogenous substrate addition, their function does require continued access to the molecular oxygen, FMNH 2 , and aldehyde co-substrates. For this reason, their bioluminescence can only be detected in actively growing cells. This knowledge, combined with the discovery that lux bioluminescent output is proportionally correlated to the number of cells present, has therefore been used as a simple, sensitive, and non-destructive means for in situ bacterial monitoring. This was first demonstrated by Shaw et al. [16] in 1986 when constitutively expressed V. fischeri luxCDABE genes were introduced into the phytopathogen Xanthomonas campestris, and their subsequent invasion of a cauliflower leaf was visualized. Similarly, de Weger and colleagues [17] were successfully able to detect luxCDABE-labeled Pseudomonas fluorescens in the rhizosphere of soybean roots using the same technique. Additionally, through the use of a lux-based system rather than an enzymatic reporter, it was possible for these researchers to achieve detection limits three orders of magnitude lower than what was previously possible, leading to improved signal detection. These early examples highlighted the application of lux-based bioluminescence as a rapid, simple and sensitive tool for in situ detection of living bacteria and established the foundation for future research using lux to monitor genetically engineered microorganisms. In perhaps the most notable use of the lux genes for tracking a cellular population, a P. fluorescens strain was transformed with the lux genes and used for the first bioremediation-related environmental field release of a genetically engineered microorganism in 1996.
This release was approved by the Environmental Protection Agency to determine the efficiency of bioremediation process monitoring through inoculation of the bioluminescent strain directly into contaminated soil and to determine its ability to monitor the bioremediation of polycyclic aromatic hydrocarbons [18]. By placing the lux genes under the control of promoters in the naphthalene degradation pathway, it was possible to monitor their bioluminescent output as a measure of naphthalene contamination in the soil [19]. Using a combination of bioluminescent and traditional culture based detection methods, the release area was monitored for two years after the release of bioluminescent P. fluorescens. Over this time, regular sampling was performed to track the amount of bacteria present in the soil, as well as the amount of bioluminescence produced, which were indicative of organism presence and naphthalene degradation, respectively ( Figure 3). Based on culture detection methods, the bioluminescent P. fluorescens persisted in both contaminated and non-contaminated soils, decaying at similar rates and producing similar colony counts [18]. The long term nature and difficulty in remote monitoring of bacterial populations presented in this study illustrates how the unique properties of the lux operon can provide it with an advantage over its substrate requiring bioluminescent or UV stimulation requiring fluorescent counterparts. Because of its autonomous nature, the lux-tagged P. fluorescens could be continually surveyed for bioluminescent production, without the need for repeated stimulation to induce a reporter signal.

The Use of lux for Exogenous Target Detection
Following the work that demonstrated how the lux cassette could be used as a tool for visualizing gene expression, it soon became clear that these genes could be adapted for use as a traditional bioreporter target through activation under specific, predetermined conditions as well. By expressing the lux cassette under the control of a promoter with a known inducer, the resultant bioluminescent emission could be used as an indicator for the presence of the given stimulus, and fluctuation of the bioluminescent signal could be interpreted as changes in the bioavailable concentration of the inducer compound. Building upon these ideas, the first use of bioluminescence for monitoring metabolic activity was demonstrated in Pseudomonas putida by Burlage et al. in 1990 [20]. Here, naphthalene degradation was monitored using a transcriptional fusion of the salicylate inducible nah promoter and the luxCDABE genes. Salicylate is an intermediate metabolite of naphthalene, which is eventually degraded to acetaldehyde and pyruvate in Pseudomonas. Therefore, naphthalene degradation could be correlated to the light emission upon induction with naphthalene-derived salicylate. The nondestructive nature of the lux system allowed for this analysis to occur in real time in a growing culture, providing continuous monitoring of naphthalene metabolism across various stages of growth. It was later determined by King et al. [19] that the bioluminescent signal was controlled in a dose/response fashion (Figure 4), therefore demonstrating its usefulness in determining contaminant levels in mixed environmental samples. This opened the door for a multitude of environmental bioreporters featuring lux, such as that developed by Applegate and colleagues that was used to monitor for water soluble benzene, toluene, ethylbenzene, and xylene (BTEX) compounds indicative of petroleum spills. This reporter, constructed by linking expression of the lux cassette to the toluene dioxygenase promoter, was capable of detecting as little as 30 µg of toluene/L in as quickly as 2 h and maintained its detection ability for over 100 generations without antibiotic selection [21]. Adapted and used with permission from [19].
Another common target for lux-based environmental sensing has been phenol. Notably, Abd-El-Haleem et al. [22] constructed one of the first lux-based phenol biosensors by inserting a mopR-like promoter fused to the V. fischeri lux cassette genes into Acinetobacter sp DF4. This reporter was capable of demonstrating a lower detection limit of 2.5 ppm in 4 h when exposed to phenol, and was only responsive to three of the ten phenol derivatives tested, suggesting that it was relatively specific as well. This is, however, not by any means the only lux-based phenol reporter to be developed. Davidov et al. [23] made extensive use of recA promoters fused to lux cassettes, with each of the reporters containing a slight variation in its promoter sequence, that were expressed either in E. coli or Salmonella typhimurium and using lux genes from either V. fischeri or P. luminescens. The most sensitive of these reporters was that expressing the V. fischeri lux genes in E. coli, which was capable of detecting 0.008 mg phenol/L in 2 h. This same construct, when expressed in S. typhimurium was also capable of detecting phenol in 2 h but required a minimum concentration of 16 mg phenol/L, demonstrating the differences in host phenol bioavailability.

Further Uses of lux as a Bacterial Bioreporter
As the popularity of the lux system has grown over the years, an increasing number of bacterial reporters have been leveraged for the detection of a wide variety of contaminants. While this review focuses only on the seminal examples of lux's growth as a reporter system, a larger list of target compounds and detection limits of various bioreporters can be found in recent reviews [24,25] and Table 1. Heat shock 20 min Various, depending on chemical inducer used [47,48] Heavy metals A multi-bioreporter panel for detecting and identifying multiple heavy metal contaminants in a single sample [49]

Eukaryotic Expression of the lux Cassette
Despite its success as a bacterial bioreporter, widespread use of the lux system faced a major hurdle in that it was initially believed to be capable of expression only in prokaryotes. Although several attempts were made to express the lux genes in eukaryotic hosts, none of these made significant headway [74][75][76]. It would not be until 2003 that the first major achievement was documented with the demonstration of autonomous bioluminescence from the yeast Saccharomyces cerevisiae [5]. Following this breakthrough, the lux genes continued to be modified and improved for eukaryotic expression, later being developed into a reliable yeast-based bioassay tool and, eventually, becoming capable of expression in a human cell line [7], opening the door for continued development in the future.

lux Expression in Yeast
It was the demonstration of lux function in S. cerevisiae by Gupta et al. [5] in 2003 that marked the first time a eukaryotic organism successfully produced levels of bioluminescence comparable to prokaryotic lux-based bioreporters ( Figure 5). To achieve this, Gupta and colleagues chose to express the lux genes from the terrestrial bacterium P. luminescens rather than those from the traditional marine organisms V. harveyi or V. fischeri. This was done because the resulting luciferase proteins from P. luminescens exhibit a higher thermal stability than those of their marine counterparts. To mimic the organization and expression of the lux genes found in prokaryotic organisms, the luxA and luxB genes were expressed from a single promoter and linked by an internal ribosomal entry site (IRES). Under this expression strategy it was determined that bioluminescence was 20 times greater than that reported for fused luciferases upon exposure to an n-decanal substrate. Building upon these findings, the remainder of the lux genes were incorporated using the same strategy, with a pair of genes linked by an IRES element and driven by a unique promoter. When expressed concurrently this design was capable of producing an easily detectable bioluminescent signal. Using the lessons learned from creation of the bioluminescent yeast strain, work was then begun to develop the eukaryotic lux system into a functional bioreporter for the detection of estrogenic compounds-a task that was not possible using prokaryotic hosts. Sanseverino et al. [45] built upon the lux plasmids developed by Gupta, creating a second set that constitutively expressed the luxC, luxD, luxE, and frp genes while regulating expression of the remaining luxA and luxB genes through insertion of human estrogen response elements ( Figure 6). Upon exposure to estrogenic compounds, yeast expressing these regulated lux genes would produce a bioluminescent signal in as quickly as 1 h. This improved significantly over the colorimetric yeast estrogen screen, which could take as long as five days to produce results under identical conditions. Within two years of this successful demonstration of lux-based bioluminescent yeast as estrogen reporters, the same group had expanded   It is possible that this two plasmid expression system could itself contribute to the production of bioluminescence in human cells since Yagur-Kroll and Belkin [80] have recently reported that splitting the five lux genes into two smaller units (luxAB and luxCDE) resulted in improved bioreporter performance in E. coli. This increased bioluminescent production is hypothesized to be due to the associated enhanced transcriptional and/or translational efficiency achieved through the expression of smaller open reading frames, which theoretically could serve the same function in eukaryotic cells as well.
Using this expression strategy, it was demonstrated that these changes were both necessary and sufficient for autonomous production of a bioluminescent signal when expressed in a human kidney cell line (Figure 8) [7]. It should be noted that bioluminescent production from the human-optimized lux cassette was demonstrated to be several orders of magnitude lower than that of the more common firefly luciferase reporter and therefore greater numbers of bioluminescent cells were required to produce a significantly detectable signal in both cell culture (15,000 lux-expressing cells vs. 50 firefly luciferase-expressing cells) and small animal imaging experiments (25,000 lux-expressing cells vs. 2,500 firefly luciferase cells). However, due to the autonomous nature of the lux system, bioluminescent production was maintained over a longer period and produced less variability than did the firefly luciferase system [78]. In one interesting experiment that took advantage of the autonomous nature of lux bioluminescence, constitutively bioluminescent human cells expressing the lux genes were used to evaluate the cytotoxicity of the aldehyde n-decanal [77]-the same aldehyde that was used by Patterson et al. [6] to stimulate bioluminescent production in cell extracts containing optimized luxA and luxB genes. By monitoring the changes in bioluminescent production following aldehyde treatment, it was possible to evaluate not only which concentrations were cytotoxic to the cells, but also at what time following exposure the effects began to take place, how long the cells were able to continue functioning under a diminished capacity following introduction of the aldehyde, and at what point cells succumbed to treatment and died. It was demonstrated that treatment with 0.00001%, 0.0001%, and 0.001% volumes of aldehyde did not show any changes in bioluminescence [77], despite the fact that this compound has been shown previously to function as a substrate for the lux reaction [6]. However, while treatment with a 0.1% volume of aldehyde quickly diminished bioluminescent production, treatment with 0.01%  Figure 10). Figure 10. Despite its small size, the BBIC chip contains all of the necessary circuitry for the detection and reporting of bioluminescent cells. By imaging directly on the chip prior to photons passing through host tissue, signal collection will be greatly simplified. Used with permission from [84].
When bioluminescent bacterial cells are interfaced to the BBIC, as few as 5,000 can be detected and distinguished from background [81] and it is sensitive enough to differentiate bioluminescent output levels stemming from changes in the concentration of the exposed stimulating analyte [81,82]. These devices could be paired with lux-expressing eukaryotic cells and then implanted into small animal models for direct internal imaging of reporter signals without the need to anesthetize or remove the animal from its natural habitat, offering unparalleled opportunities for studying changes in physiology and compound bioavailability under a wide range of conditions. Similarly, these cells could be complexed with microcircuitry capable of initiating hormone or therapeutic compound dosage. In this fashion, the lux-expressing cells could be programed to continuously monitor the body for specific target compounds, acting as real-time biosentinels that detect changes in physiology and whose resultant changes in bioluminescent output could trigger the release of counteractive compounds. This would allow the development of fully autonomous, implantable dose/response therapeutic devices.

Conclusions
While the use of bacterial bioluminescence as a reporter system has been employed for quite a long time, it is still a continually developing reporter system. There are many examples from the recent literature that demonstrate new and creative lux-based bacterial biosensors that are employed for myriad sensing applications, and there are also recent examples showing how modification of these genes has expanded their usage to new applications that were previously thought impossible. The unique ability of the lux system to produce a bioluminescent signal without exogenous substrate input has ensured that it will continue to find use in basic and applied scientific research for years to come. Whether or not it continues to be improved for function in eukaryotic cells may well decide the true extent of lux usage in the future, however, for the time being it remains both an interesting and practical example of the benefits available from an optical reporter system.