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Molecular biology of bacterial bioluminescence
Abstract
The cloning and expression of the lux genes from different luminescent bacteria including marine and terrestrial species have led to significant advances in our knowledge of the molecular biology of bacterial bioluminescence. All lux operons have a common gene organization of luxCDAB(F)E, with luxAB coding for luciferase and luxCDE coding for the fatty acid reductase complex responsible for synthesizing fatty aldehydes for the luminescence reaction, whereas significant differences exist in their sequences and properties as well as in the presence of other lux genes (I, R, F, G, and H). Recognition of the regulatory genes as well as diffusible metabolites that control the growth-dependent induction of luminescence (autoinducers) in some species has advanced our understanding of this unique regulatory mechanism in which the autoinducers appear to serve as sensors of the chemical or nutritional environment. The lux genes have now been transferred into a variety of different organisms to generate new luminescent species. Naturally dark bacteria containing the luxCDABE and luxAB genes, respectively, are luminescent or emit light on addition of aldehyde. Fusion of the luxAB genes has also allowed the expression of luciferase under a single promoter in eukaryotic systems. The ability to express the lux genes in a variety of prokaryotic and eukaryotic organisms and the ease and sensitivity of the luminescence assay demonstrate the considerable potential of the widespread application of the lux genes as reporters of gene expression and metabolic function.
... Autobioluminescence does not require the addition of a substrate by expressing the bacterial luciferase (lux) genes, including factors necessary for luminescence (11). The five genes that constitute the operon are involved in this bioluminescence and were harbored by genera, such as Vibrio, Photobacterium, and Xenorhabdus in nature (12). Namely, the bacterial luciferase subunits are encoded by the luxA and luxB. ...
... Namely, the bacterial luciferase subunits are encoded by the luxA and luxB. In contrast, the fatty acid reductase complex, which performs the biosynthesis of aldehyde substrates for the luminescence reaction, is encoded by the luxC, luxD, and luxE (9,12). In a series of luminescence reactions, reduced flavin mononucleotide (FMNH 2 ) is oxidized to flavin mononucleotide (FMN), and a long-chain aliphatic aldehyde is oxidized to the corresponding carboxylic acid. ...
... Both products are recycled under consumption of cellular energy and this is performed by a flavin reductase (FRP) which reduces the oxidized FMN to FMNH 2 . It has also been reported that the addition of the FRP gene to assays for bacterial luciferases will result in continuous light emission due to the regeneration of FMNH 2 and thus will eliminate the necessity for rapid mixing devices for injection of FMNH 2 combined with the light detection equipment (11,12). The luxABCDE-based bioluminescence system has been successfully employed to monitor the expression of certain genes and disease development in real-time in both Gram-negative and Gram-positive bacteria. ...
We established an autologous bioluminescent strain of R. equi and a method to evaluate its proliferation in vitro and in vivo quantitatively. This method overcomes the weakness of the fluorescence detection system that only measures the site of excitation light irradiation.
... Photorhabdus predominantly inhabits terrestrial ecosystems, whereas Vibrio, Aliivibrio, Photobacterium, and Shewanella are associated with marine and freshwater environments (Chatragadda 2020;Hastings and Nealson 1977;Meighen and Dunlap 1993;Nealson and Hastings 1979). Bacterial bioluminescence is driven by an enzymatic reaction catalyzed by luciferase, involving the oxidation of two substrates, reduced flavin mononucleotide (FMNH 2 ) and aldehyde (RCHO), that ultimately leads to light emission (Hastings and Nealson 1977;Meighen 1991;Meighen and Dunlap 1993). The components responsible for luminescence are encoded by the lux operon (Fig. 1). ...
... The light emission is generated through the reaction of molecular oxygen with FMNH 2 and a long-chain aldehyde, resulting in the production of flavin mononucleotide (FMN), water, and a corresponding carboxylic acid (RCOOH) as shown in Eq. 2 (Meighen and Dunlap 1993). In these luminous microbes, tetradecanal is proposed as the primary aldehyde substrate while heptanal also shows high-intensity bioluminescence (Meighen 1991;Meighen and Dunlap 1993). The oxidation of these aldehyde substrate produces a highenergy intermediate, which emits bioluminescence upon returning to its ground state (Da Silva Nunes-Halldorson and Duran 2003;Fleiss and Sarkisyan 2019). ...
Marine pollution threatens global ecosystems, underscoring the urgent need for robust and efficient monitoring systems. Microbial bioluminescence has emerged as a promising tool for pollution detection, offering unique advantages due to its simplicity, sensitivity, and ecological relevance. This review explores the fundamental principles of bacterial and dinoflagellate bioluminescence, ecological significance, and their applications in marine pollution monitoring. Bioluminescence-based detection systems are broadly categorized into whole-cell biosensors (WCBs) and enzyme-based biosensors. WCBs are further classified into recombinant organisms based WCBs (Class I and Class II WCBs) and wild-type organisms based WCBs (Class III WCBs), demonstrating distinct pollutant detection and stress-response monitoring capabilities. We highlight their potential to improve pollution monitoring strategies by critically evaluating these technologies. Integrating bioluminescence-based systems into current frameworks could significantly enhance the assessment of marine ecosystem health, facilitate timely pollution management, and support the conservation and sustainable use of marine resources.
... In addition, we observed a negative correlation between the TOP10 bioluminescence intensity and the amount of intratumoral thrombus as well as the degree of hypoxia within tumor, which is due to the fact that oxygen is an essential substrate for the bioluminescence reaction in the luxCDABE system and the oxygen content is positively correlated with the luminescence signal intensity. [28][29][30][31] Based on this, the process of TPZ treatment was effectively monitored by the TOP10 bioluminescence intensity assessment. Therefore, genetically engineered bacteria have great potential to boost tumor treatment and achieve visualization during the therapeutic course. ...
... To endow the TOP10 tumor therapeutic monitoring capability, the low pathogenicity Escherichia coli TOP10 was genetically modified with a recombinant plasmid containing the luxCDABE gene (pGEN-luxCDABE). The luxCDABE gene cluster encodes fatty acid reductase complex and luciferase, which can generate light by consuming the bioluminescent reaction substrates (reduced flavin, long-chain aldehyde, and oxygen) [28][29][30] (Figure 2a). Due to the advantages of non-destructiveness and easy detection, the lux-CDABE bioluminescent system has been used as a reporter gene in model studies of various microorganisms in environmental and biomedical contexts. ...
Tirapazamine (TPZ), an antitumor prodrug, can be activated in hypoxic environment. It specifically targets the hypoxic microenvironment of tumors and produces toxic free radicals. However, due to the tumor is not completely hypoxic, TPZ often fails to effectively treat the entire tumor tissue, resulting in suboptimal therapeutic outcomes. Herein, a low pathogenic Escherichia coli TOP10 is utilized to selectively colonize tumor tissues, disrupt blood vessels, and induce thrombus formation, leading to the expansion of hypoxic region and improving the therapeutic effect of TPZ. Additionally, a thermosensitive hydrogel is constructed by Pluronic F‐127 (F127), which undergoes gelation in situ at the tumor site, resulting in sustained release of TPZ. To monitor the therapeutic process, it is genetically modified TOP10 by integrating the bioluminescent system luxCDABE (TOP10‐Lux). The bioluminescent signal is associated with tumor hypoxia enhancement and thrombus formation, which is beneficial for therapeutic monitoring with bioluminescence imaging. In the murine colon cancer model, the TOP10‐Lux combined with TPZ‐loaded F127 hydrogel effectively suppressed tumor growth, and the treatment process is efficiently monitored. Together, this work employs genetically modified TOP10‐Lux to enhance the therapeutic efficacy of TPZ and monitor the treatment process, providing an effective strategy for bacteria‐based tumor‐targeted chemotherapy and treatment monitoring.
... The method has been commercialized, and it is rapid and automated. The limitations of this technique are the use of a bacterial strain with special growth requirements and test samples that may be turbid or stained or contain chlorine [4,5]. ...
... Unfortunately, these samples must contain microorganisms with a density of 5 × 10 4 to >10 8 CFU/mL, and there is no differentiation between dead and living cells. The apparatus is quite complicated and requires properly trained staff [4]. ...
tris-[(4,7-diphenyl-1,10-phenanthroline)ruthenium(II)] dichloride (Ru(DPP)3Cl2), a fluorescent sensor which is sensitive to the amount of oxygen in the sample, was applied using the fluorescent optical respirometry (FOR) technique. The oxygen in the samples quenches the fluorescence. The fluorescence intensity depends on the metabolic rate of the viable microorganisms. The effect of DMSO and plant extracts on bacteria was determined by FOR. It was shown that the MIC values obtained by FOR were consistent with the results of the MIC determinations using the method of serial dilutions; at the same time, the effects of concentrations lower than the growth-inhibitory concentrations on microbial cells were demonstrated. The FOR method enables the detection of multiplying bacteria in sterile and non-sterile pharmaceutical preparations in real time, which significantly shortens the time required to obtain results and allows the introduction of repair processes in the production. This method also allows for quick, unambiguous detection and the counting of the viable cells of aerobic microorganisms in non-sterile pharmaceuticals.
... To enhance the specificity of the biosensor, a group of authors led by G.B. Zavilgelsky developed biosensors based on an E. coli strain carrying a plasmid with the luxCDABE operon placed under the control of inducible promotors [9][10][11][12]. The genes of luxCDABE operon are the most universal part of the whole jointly transcribed lux operon of all known luminescent bacteria [3]. ...
... To detect the agents capable of inducing oxidative stress in a cell, biosensors with a multicopy recombinant plasmid were developed, fusing the lux operon from P. luminescens with the promoters of the catalase and superoxide dismutase genes [9,12]. The protein OxyR specifically reacts to an increase in the concentration of hydrogen peroxide and other peroxides in the bacterial cell and activates the promoter of the katG gene [6,13]. ...
The aim of this study was to assess the applicability of the bacterial lux biosensors for genotoxicological studies. Biosensors are the strains of E. coli MG1655 carrying a recombinant plasmid with the lux operon of the luminescent bacterium P. luminescens fused with the promoters of inducible genes: recA, colD, alkA, soxS, and katG. The genotoxicity of forty-seven chemical compounds was tested on a set of three biosensors pSoxS-lux, pKatG-lux and pColD-lux, which allowed us to estimate the oxidative and DNA-damaging activity of the analyzed drugs. The comparison of the results with the data on the mutagenic activity of these drugs from the Ames test showed a complete coincidence of the results for the 42 substances. First, using lux biosensors, we have described the enhancing effect of the heavy non-radioactive isotope of hydrogen deuterium (D2O) on the genotoxicity of chemical compounds as possible mechanisms of this effect. The study of the modifying effect of 29 antioxidants and radioprotectors on the genotoxic effects of chemical agents showed the applicability of a pair of biosensors pSoxS-lux and pKatG-lux for the primary assessment of the potential antioxidant and radioprotective activity of chemical compounds. Thus, the results obtained showed that lux biosensors can be successfully used to identify potential genotoxicants, radioprotectors, antioxidants, and comutagens among chemical compounds, as well as to study the probable mechanism of genotoxic action of test substance.
... The oxidation reaction produced by this enzyme releases light at a wavelength of 490 nm. 35 This process is related to electron transport chain and therefore to respiration and gives an idea about the metabolic status as a chemical toxicity. The toxic compounds inhibit the bacterial metabolism, this is reflected in a decrease of light emission. ...
... The toxic compounds inhibit the bacterial metabolism, this is reflected in a decrease of light emission. 32,35,36 The employed methodology follows all the conditions and protocols established on the standardized tests for the determination of ecotoxicity in A. fischeri (UNE-EN ISO 2009). 37 The experiments have been carried out in triplicate for each tested solvent to ensure the reproducibility of the test. ...
The ecotoxicity of some deep eutectic solvents formed by glycerol derivatives have been studied in two aquatic biomodels: Aliivibrio fischeri (bacteria) and Raphidocelis subcapitata (algae). The results show that these chemicals are not toxic for aquatic media.
... To address this, bacterial biosensors engineered as "living diagnostics" have emerged. In engineered E. coli Nissle 1917, synthetic genetic circuits employ lactate-responsive promoters to drive tumor-specific expression of β-galactosidase (lacZ) and bioluminescent reporters (luxCDABE) (Meighen, 1991). The oral delivery strategy leverages the gut-liver axis, through which EcN crosses the intestinal barrier via bile acid transporters and colonizes hepatic metastases within 24 h, thereby avoiding systemic toxicity (Chien et al., 2021). ...
The demand for early disease detection, treatment monitoring, and personalized medicine is increasing, making it more imperative than ever to create effective, accurate, portable, intelligent, multifunctional diagnostic equipment. Bacteria possess a remarkable perception of their surroundings and have the capacity to adapt by altering the expression of specific genes. Bacteria interact with target substances and produce detectable signals in response to their presence or concentration. This unique property has been harnessed in the development of bacterial biosensors. Due to groundbreaking advancements in synthetic biology, genetic engineering now enables the creation of bacteria tailored with exceptional detecting traits. In addition to meeting a wide range of application needs, this allows quick and precise detection in intricate settings and offers a strong technological basis for early disease diagnosis and treatment monitoring. This article reviews the applications and recent advancements of bacterial biosensors in the medical field and discusses the challenges and obstacles that remain in their research and application.
... Bacterial luciferase genes (luxCDABE) are obtained from strains of luminescent bacteria, including species such as Vibrio fischeri, Vibrio harveyi, Photobacterium phosphoreum, and Photobacterium leiognathi. In all species mentioned, the luxA and lux B genes encode luciferase subunits, while luxCDE encodes a fatty acid reductase complex 33 . Bacterial luciferases are heterodimers that oxidize long-chain aldehydes with atmospheric oxygen using reduced flavin mononucleotide 32 . ...
The research used bacterial biosensors containing bacterial luciferase genes to monitor changes in the environment in real-time. In this work to express four different gene constructs: recA:luxCDABE, soxS:luxCDABE, micF:luxCDABE, and rpoB:luxCDABE in Escherichia coli SM lux biosensor after exposure to three different antibiotics (nalidixic acid, ampicillin, kanamycin) and diclofenac was determined. It was found that incubation of the E. coli SM strain in various concentrations of analytes results in differentiation in gene expression at each of the tested concentrations (from 0.625 to 10 µg/mL) and during all three measurements, in “time 0”, after 30 min. and after 1 h. The measurable signal is created as a result of the action of reporter genes (bacterial luciferase genes luxCDABE), present in genetically modified bacterial cells. E. coli luminescent bioreporters in the stationary phase were used. In the analysis of the induction of the promoter (regulatory proteins) to the control (0 µg/ml), the highest biosensor response was shown in the case of kanamycin concentration equal to 0.625 µg/mL after 1-h incubation. The highest increase express gene construct was found for micF:luxCDABE in E. coli SM343 lux biosensor, where the micF promoter induction relative to the control at a concentration of 0.625 µg/mL is 73.9%.
... Synthesis of the long-chain aldehyde is catalyzed by a fatty-acid reductase complex, which comprises three polypeptides: an NADPH-dependent acyl protein reductase (54 kDa), an acyl transferase (33 kDa), and an ATPdependent synthetase (42 kDa). This complex plays an essential role in the production of light in the absence of exogenously added aldehyde (Hastings et al., 1985;Meighen, 1991;Tinikul et al., 2013). Furthermore, the substantial amino acid residue and nucleotide sequence identity observed in luciferases from different species of luminous bacteria indicate a common evolutionary origin of luminescence in bacteria, consistent with the shared ancestry of these light-producing mechanisms. ...
Bioluminescence is a remarkable biological phenomenon where living organisms produce light through a chemical reaction, primarily involving the enzyme luciferase and the substrate luciferin. This process serves various ecological functions, including predation, communication and defense across different taxa such as bacteria, fungi and insects. Bioluminescent bacteria, predominantly found in marine environments, use this capability for symbiotic relationships, with species like Vibrio fischeri providing light to host organisms. Fungi display bioluminescence in a limited number of species, suggesting an evolutionary link across diverse lineages. In insects, bioluminescence plays roles in mating and predation, with fireflies showcasing intricate signalling systems that vary among species. Click beetles exhibit bioluminescence for communication and defense, while glowworms utilize light to attract prey. Recent advancements in genetic engineering, particularly using fluorescent proteins like Green Fluorescent Protein (GFP) in pest management, highlight the potential for bioluminescence in ecological monitoring and targeted pest control strategies. This review showcases bioluminescence in both microbes and insects that offers invaluable insights into the ecological roles and functions of these luminous organisms in their respective ecosystems.
... We describe herein the underlying principles of a quantitative chemical sensing (QCS) methodology for assessing the concentration of a wide range of target materials (TMs) in aqueous solutions. The methodology is based on bioluminescent sensor bacteria (bioreporters) [1][2][3][4], genetically engineered to bioluminesce upon exposure to a TM. Bioreporters have been employed as the core sensing elements in sensors that detect the presence of a TM in a sample [5]. ...
We present a generic quantitative chemical sensing methodology for assessing the concentration of a target material (TM) in an aqueous solution by using bioluminescent microbial bioreporters as the core sensing elements. Such bioreporters, genetically engineered to respond to the presence of a TM in their microenvironment by emitting bioluminescence, have previously been mostly designed to report the presence or absence of the TM in the sample. We extend this methodology to also assess the TM concentration, by exploiting the dose-dependency of the TM-induced luminescence. To overcome luminescence intensity variations due to bacterial batch differences and the ambient temperature, simultaneous measurements were carried out on sample solutions containing known concentrations of the TM. A “standard ratio” parameter, defined as the ratio between the two measurements, is shown to be independent of the bacterial batch and the temperature, and hence provides the conceptual basis for a generic quantitative chemical sensing methodology. Assessment of 2,4-dinitrotoluene (DNT) concentration in solutions is demonstrated with an accuracy of 2.5% over a wide dynamic range.
... A heterodimeric protein called bacterial luciferase contributes significantly to the bioluminescence process (Matheson and Lee 1981). Every luminous bacterium contains the lux gene, also known as the lux operon, which coordinates the expression of a group of genes to produce luciferase (Meighen 1991). Up to now, recombinant stains for bioassay have frequently been made using the lux operon of the luminous bacteria. ...
It is undeniable that removal efficiency is the main factor in coagulation-flocculation (C-F) process for wastewater treatment. However, as far as environmental safety is concerned, the ecotoxicological aspect of the C-F process needs to be examined further. In this study, a systematic review was performed based on publications related to the toxicity research in C-F technology for wastewater treatment. Through a series of screening steps, available toxicity studies were categorized into four themes, namely acute toxicity, phytotoxicity, cytotoxicity, and genotoxicity, which comprised 48 articles. A compilation of the methodologies executed for each theme was also outlined. The findings show that conventional metallic coagulants (e.g., alum, iron chloride, and iron sulfate) were less toxic when tested on test species such as Daphnia magna (water flea), Lattuca sativa (lettuce), and animal cells compared to synthetic polymers. Natural coagulants such as chitosan or Moringa oleifera were less toxic compared to metallic coagulants; however, inconsistent results were observed. Moreover, an advanced C-F (electrocoagulation) as well as integration between C-F and Fenton, adsorption, and photocatalytic does not significantly change the toxicological profile of the system. It was found that diverse coagulants and flocculants, species sensitivity, complexity in toxicity testing, and dynamic environmental conditions were some key challenges faced in this field. Finally, it was expected that advances in technology, interdisciplinary collaboration, and a growing awareness of environmental sustainability will drive efforts to develop more effective and eco-friendly coagulants and flocculants, improve toxicity testing methodologies, and enhance the overall efficiency and safety of water and wastewater treatment processes.
... A biosensor is an analytical instrument that combines a biological recognition component with a physical transducer to produce a quantifiable signal directly proportional to the concentration of the substances being analyzed [22,23]. Reporter proteins, such as those measured by GFP fluorescence (excitation/emission 488/533 nm) [24] or luciferase "hν~490 nm" [25], exemplify this progress. For instance, researchers have developed a quantitative autonomous bioluminescence reporter system with a wide dynamic range, utilizing the bioluminescence pathway from Neonothopanus Nambi, eliminating the need for external luciferase substrates and making it cost-effective and suitable for high-throughput applications [26]. ...
Plants have evolved intricate signaling pathways, which operate as networks governed by feedback to deal with stressors. Nevertheless, the sophisticated molecular mechanisms underlying these routes still need to be comprehended, and experimental validation poses significant challenges and expenses. Consequently, computational hypothesis evaluation gains prominence in understanding plant signaling dynamics. Biosensors are genetically modified to emit light when exposed to a particular hormone, such as abscisic acid (ABA), enabling quantification. We developed computational models to simulate the relationship between ABA concentrations and bioluminescent sensors utilizing the Hill equation and ordinary differential equations (ODEs), aiding better hypothesis development regarding plant signaling. Based on simulation results, the luminescence intensity was recorded for a concentration of 47.646 RLUs for 1.5 μmol, given the specified parameters and model assumptions. This method enhances our understanding of plant signaling pathways at the cellular level, offering significant benefits to the scientific community in a cost-effective manner. The alignment of these computational predictions with experimental results emphasizes the robustness of our approach, providing a cost-effective means to validate mathematical models empirically. The research intended to correlate the bioluminescence of biosensors with plant signaling and its mathematical models for quantified detection of specific plant hormone ABA.
... Vibrio fischeri, also belonging to the Gammaproteobacteria, is a luminous marine bacterium that lives freely or in symbiosis with different species of fish and squid [89]. The most studied interaction in this microorganism is the symbiosis with the Hawaiian squid, Euprymna scolopes, inducing bioluminescence that the squid uses to avoid predation during nocturnal activity [90]. ...
Many bacteria have the ability to survive in challenging environments; however, they cannot all grow on standard culture media, a phenomenon known as the viable but non-culturable (VBNC) state. Bacteria commonly enter the VBNC state under nutrient-poor environments or under stressful conditions. This review explores the concept of the VBNC state, providing insights into the beneficial bacteria known to employ this strategy. The investigation covers different chemical and physical factors that can induce the latency state, cell features, and gene expression observed in cells in the VBNC state. The review also covers the significance and applications of beneficial bacteria, methods of evaluating bacterial viability, the ability of bacteria to persist in environments associated with higher organisms, and the factors that facilitate the return to the culturable state. Knowledge about beneficial bacteria capable of entering the VBNC state remains limited; however, beneficial bacteria in this state could face adverse environmental conditions and return to a culturable state when the conditions become suitable and continue to exert their beneficial effects. Likewise, this unique feature positions them as potential candidates for healthcare applications, such as the use of probiotic bacteria to enhance human health, applications in industrial microbiology for the production of prebiotics and functional foods, and in the beer and wine industry. Moreover, their use in formulations to increase crop yields and for bacterial bioremediation offers an alternative pathway to harness their beneficial attributes.
... The use of designer probiotics for cancer detection holds great potential for improving the accuracy and efficiency of cancer diagnosis, leading to better patient outcomes. (Danino et al., 2015;Heimann & Rosenberg, 2003;Meighen, 1991;Nemunaitis et al., 2003;Riedel et al., 2007;Toso et al., 2002). ...
Probiotic microorganisms have been used for therapeutic purposes for over a century, and recent advances in biotechnology and genetic engineering have opened up new possibilities for developing therapeutic approaches using indigenous probiotic microorganisms. Diseases are often related to metabolic and immunological factors, which play a critical role in their onset. With the help of advanced genetic tools, probiotics can be modified to produce or secrete important therapeutic peptides directly into mucosal sites, increasing their effectiveness. One potential approach to enhancing human health is through the use of designer probiotics, which possess immunogenic characteristics. These genetically engineered probiotics hold promise in providing novel therapeutic options. In addition to their immunogenic properties, designer probiotics can also be equipped with sensors and genetic circuits, enabling them to detect a range of diseases with remarkable precision. Such capabilities may significantly advance disease diagnosis and management. Furthermore, designer probiotics have the potential to be used in diagnostic applications, offering a less invasive and more cost‐effective alternative to conventional diagnostic techniques. This review offers an overview of the different functional aspects of the designer probiotics and their effectiveness on different diseases and also, we have emphasized their limitations and future implications. A comprehensive understanding of these functional attributes may pave the way for new avenues of prevention and the development of effective therapies for a range of diseases.
... The majority of known bioluminescent bacteria are gram-negative, facultatively anaerobic, and symbiotic [62]. All bioluminescent bacteria use the same fundamental process for light emission, in which photons are generated in an array of reactions requiring oxygen, nicotinamide adenine dinucleotide (NADH), FMN, and myristic aldehyde. ...
Photoproteins, luminescent proteins or optoproteins are a kind of light-response protein responsible for the conversion of light into biochemical energy that is used by some bacteria or fungi to regulate specific biological processes. Within these specific proteins, there are groups such as the photoreceptors that respond to a given light wavelength and generate reactions susceptible to being used for the development of high-novel applications, such as the optocontrol of metabolic pathways. Photoswitchable proteins play important roles during the development of new materials due to their capacity to change their conformational structure by providing/eliminating a specific light stimulus. Additionally, there are bioluminescent proteins that produce light during a heatless chemical reaction and are useful to be employed as biomarkers in several fields such as imaging, cell biology, disease tracking and pollutant detection. The classification of these optoproteins from bacteria and fungi as photoreceptors or photoresponse elements according to the excitation-emission spectrum (UV-Vis-IR), as well as their potential use in novel applications, is addressed in this article by providing a structured scheme for this broad area of knowledge.
... Because the lux operon (luxCDABE) encodes a luciferase enzyme and an aldehyde substrate, there is no need to add an exogenous substrate. [35]. Bioluminescence imaging (BLI) in live animal models facilitates tracking pathogens. ...
Virulent Aeromonas hydrophila (vAh) strains that cause motile Aeromonas septicemia (MAS) in farmed channel catfish (Ictalurus punctatus) have been an important problem for more than a decade. However, the routes of infection of vAh in catfish are not well understood. Therefore, it is critical to study the pathogenicity of vAh in catfish. To this goal, a new bioluminescence expression plasmid (pAKgfplux3) with the chloramphenicol acetyltransferase (cat) gene was constructed and mobilized into vAh strain ML09-119, yielding bioluminescent vAh (BvAh). After determining optimal chloramphenicol concentration, plasmid stability, bacteria number–bioluminescence relationship, and growth kinetics, the catfish were challenged with BvAh, and bioluminescent imaging (BLI) was conducted. Results showed that 5 to 10 µg/mL chloramphenicol was suitable for stable bioluminescence expression in vAh, with some growth reduction. In the absence of chloramphenicol, vAh could not maintain pAKgfplux3 stably, with the half-life being 16 h. Intraperitoneal injection, immersion, and modified immersion (adipose fin clipping) challenges of catfish with BvAh and BLI showed that MAS progressed faster in the injection group, followed by the modified immersion and immersion groups. BvAh was detected around the anterior mouth, barbels, fin bases, fin epithelia, injured skin areas, and gills after experimental challenges. BLI revealed that skin breaks and gills are potential attachment and entry portals for vAh. Once vAh breaches the skin or epithelial surfaces, it can cause a systemic infection rapidly, spreading to all internal organs. To our best knowledge, this is the first study that reports the development of a bioluminescent vAh and provides visual evidence for catfish–vAh interactions. Findings are expected to provide a better understanding of vAh pathogenicity in catfish.
... Many bioluminescent bacteria are characterized by induction of lux-operon expression depending on cell density (quorum sensing) and environmental conditions [10][11][12]. Also, commonly the glow of bacteria depends on various factors: cAMP concentration, iron level, oxygen concentration, osmolarity, etc [13]. However, for some representatives of the luminescent microflora, the mechanisms of luminosity regulation have not been identified. ...
For decades, transcription of Photorhabdus luminescens lux-operon was considered being constitutive. Therefore, this lux-operon has been used for measurements in non-specific bacterial luminescent biosensors. Here, the expression of Photorhabdus lux-operon under high temperature was studied. The expression was researched in the natural strain Photorhabdus temperata and in the heterologous system of Escherichia coli. P. temperata FV2201 bacterium was isolated from soil in the Moscow region (growth optimum 28 °C). We showed that its luminescence significantly increases when the temperature rises to 34 °C. The increase in luminescence is associated with an increase in the transcription of luxCDABE genes, which was confirmed by RT-PCR. The promoter of the lux-operon of the related bacterium P. luminescens ZM1 from the forests of Moldova, being cloned in the heterologous system of E. coli, is activated when the temperature rises from room temperature to 42 °C. When heat shock is caused by ethanol addition, transcription of lux-operon increases only in the natural strain of P. temperata, but not in the heterologous system of E. coli cells. In addition, the activation of the lux-operon of P. luminescens persists in E. coli strains deficient in both the rpoH and rpoE genes. These results indicate the presence of sigma 32 and sigma 24 independent heat-shock-like mechanism of regulation of the lux-operon of P. luminescens in the heterologous E. coli system.
... This compound oxidizes the fatty aldehyde forming its corresponding acid and a luciferasehydroxyflavin complex. This intermediate is decomposed slowly and emitting a blue-green light with its highest intensity at 490 nm (Meighen 1991). This process is completely linked to respiration, through the electron transport chain, and gives an idea about the metabolic status as a chemical toxicity. ...
The search of new solvents is currently focused on deep eutectic solvents (DES). However, there are not many ecotoxicological studies in different biomodels of DES that allow knowing how these chemicals affect to the environment along the trophic chain. In this manuscript, two DES at different proportion of water have been prepared and characterized from the ecotoxicological point of view. These solvents are glucose:choline chloride (2:5) and sorbitol:choline chloride (3:2) at different contents of water. To carry out the ecotoxicological study, three biomodels have been used: bacteria Aliivibrio fisheri (A. fisheri), crustacean Daphnia magna (D. magna) and algae Raphidocelis subcapitata (R. subcapitata). The obtained results show that the ecotoxicity of these chemicals depends on the biomodel used and the amount of water, being toxicity values lower for chemicals with higher water content. However, it is important to highlight that the ecotoxicity for all chemicals is quite low with effective concentrations, EC50 values above 1000 mg/L in all the studied cases.
... However, one of the substrates, aldehyde, is synthesized through an enzymatic reaction that is catalyzed by fatty acid reductase multienzyme complexes. In fact, the multienzyme complexes are expressed by genes luxC, luxD, and luxE (Meighen 1991;Sitnikov et al. 1995). Since the lux operons were identified, the heterologous expression of these lux operons has been extensively conducted in various host strains, such as Escherichia coli, Pseudomonas putida, and Saccharomyces cerevisiae (Thouand and Marks 2014). ...
Recombinant luminescent Escherichia coli strains could be used to detect the toxicity of pure or mixed contaminants as a light-off sensor. In this work, the lux operon of Photobacterium phosphoreum T3 was identified for the first time. Recombinant luminescent E. coli strains were constructed via expressing the lux operons of P. phosphoreum T3 and Vibrio qinghaiensis Q67 in E. coli MG1655, and the optimal protectant containing 10% (w/v) trehalose and 4% sucrose was used to prepare the freeze-dried recombinant luminescent E. coli cells. Then, these freeze-dried E. coli cells were subjected to acute toxicity detection. The results showed that luminescent E. coli strains displayed sensitive toxic responses to BPA, nFe2O3, Cd, Pb, As, and Hg, for example, the EC50 values of BPA and nFe2O3 to luminescent E. coli strains ranged from 1.54 to 50.19 mg/l and 17.50 to 21.52 mg/l, respectively. Indeed, luminescent E. coli strains exhibited more sensitive responses to Cd, Pb, and Hg than the natural strain Q67. The results suggested that recombinant luminescent E. coli strains could be used for the detection of acute toxicity. Furthermore, the combined toxicities of BPA and nFe2O3, Hg, and Pb were measured, and the joint effects of these mixtures were evaluated with luminescent E. coli. The results indicated that the joint effects of BPA and nFe2O3 suggested to be synergistic or additive to luminescent E. coli, while the joint effects of heavy metals and nFe2O3 exhibited additivities. The cellular endocytosis for Fe2O3 nanoparticles was not observed, which could explain the additive instead of synergistic effects between heavy metals and nFe2O3.
Key points
• Sequence of the lux operon from P. phosphoreum T3 was reported for the first time.
• Recombinant luminescent E. coli was more sensitive to Cd, Pb, and Hg than Q67.
• Joint effects of BPA and nFe2O3 were synergistic or additive to luminescent E. coli.
Graphical abstract
... The lightemitting reaction is catalyzed by the enzyme luciferase encoded by luxAB. In the presence of molecular oxygen, luciferase catalyzes the oxidation of a long chain aldehyde (luciferin) into an aliphatic carboxylic acid using FMNH 2 (reduced flavin mononucleotide) as a cofactor (2). During the oxidation process, substrates form excited intermediate compounds and release photons in a broad spectrum (490 nm maximum) with a low quantum yield (<1; Figure 1) (3). ...
This work explores feasibility using a bioluminescent reporter to understand Salmonella-chicken interaction. The technology revealed that;
1. Salmonella Enteritidis (cause human salmonellosis) colonize rapidly in chicken (withing 1-1.5 days). This window is so narrow and challenging for the industry to control once the pathogen enters farm.
2. Technology also supported to show that genomic island SPI-1 and genes related iron homeostasis of Salmonella play an important role in colonization the yolk sac. Hence these gene products have future therapeutic potentials.
3. Bioluminescent reporter system has limitation using chickens as a model. Ex; unable to provide tracking of Salmonella in deeper tissues. However has the potential to be improved.
... The lightemitting reaction is catalyzed by the enzyme luciferase encoded by luxAB. In the presence of molecular oxygen, luciferase catalyzes the oxidation of a long chain aldehyde (luciferin) into an aliphatic carboxylic acid using FMNH 2 (reduced flavin mononucleotide) as a cofactor (2). During the oxidation process, substrates form excited intermediate compounds and release photons in a broad spectrum (490 nm maximum) with a low quantum yield (<1; Figure 1) (3). ...
The light emitting module lux operon (luxCDABE) of Photorhabdus luminescens can be integrated into a “dark” bacterium for expression under a suitable promoter. The technique has been used to monitor kinetics of infection, e.g., by studying gene expression in Salmonella using mouse models in vivo and ex vivo. Here, we applied the bioluminescence imaging (BLI) technique to track Salmonella Enteritidis (SEn) strains carrying the lux operon expressed under a constitutive promoter sequence (sigma 70) in chicken after oral challenge. Detectable photon signals were localized in the crop, small intestine, cecum, and yolk sac in orally gavaged birds. The level of colonization was determined by quantification of signal intensity and SEn prevalence in the cecum and yolk sac. Furthermore, an isogenic SEn mutant strain tagged with the lux operon allowed for us to assess virulence determinants regarding their role in colonization of the cecum and yolk sac. Interestingly, mutations of SPI-1(Salmonella Pathogenicity Island 1) and fur (ferric uptake regulator) showed significantly decreased colonization in yolk sac that was correlated with the BLI data. A similar trend was detected in a ΔtonB strain by analyzing enrichment culture data. The inherently low quantum yield, light scattering, and absorption by tissues did not facilitate detection of signals from live birds. However, the detection limit of lux operon has the potential to be improved by resonance energy transfer to a secondary molecule. As a proof-of-concept, we were able to show that sensitization of a fluorescent-bound molecule known as the lumazine protein (LumP) improved the limit of detection to a certain extent.
... Ion pumps coupled with active transport make up the remaining ATP-ases within the cell membrane. An enzyme can also perform additional remote activities, e.g., luciferase producing light during fireflies [84]. Viruses also accommodate enzymes for influencing cells, e.g., the reverse transcriptase and HIV-integrase, for viral flashes from the cell, like the influenza virus's neuraminidase [85]. ...
The book presents recent advances in the field of nanoenzymes and the immobilization of enzymes in nanomaterials. Applications include disease diagnosis, environmental clean-up, biosensor manufacturing, drug delivery and vaccine production.
... These fatty aldehydes drive the luxAB complexes, which use molecular oxygen and reduced flavin mononucleotide (FMNH 2 ) to produce fatty acid, water, oxidized flavin mononucleotide, and light. Therefore, an essential requirement of bioluminescence is the availability of critical reactants, such as non-anoic acid, FMNH 2 , and adenosine triphosphate (ATP) (Meighen, 1991). In this study, we showed a differential light production based on catabolite repression and gluconeogenesis for streptococcal and staphylococcal strains of similar lux-cassette design. ...
Streptococcus pyogenes ( S. pyogenes ) can thrive in its host during an infection, and, as a result, it must be able to respond to external stimuli and available carbon sources. The preclinical use of engineered pathogens capable of constitutive light production may provide real-time information on microbial-specific metabolic processes. In this study, we mapped the central metabolism of a luxABCDE -modified S. pyogenes Xen20 ( Strep . Xen20) to its de novo synthesis of luciferase substrates as assessed by the rate of light production in response to different environmental triggers. Previous characterization predicted that the lux operon was under the myo-inositol iolE promotor. In this study, we revealed that supplementation with myo-inositol generated increased Strep. Xen20 luminescence. Surprisingly, when supplemented with infection-relevant carbon sources, such as glucose or glycine, light production was diminished. This was presumably due to the scavenging of pyruvate by L -lactate dehydrogenase (LDH). Inhibition of LDH by its inhibitor, oxamate, partially restored luminescent signal in the presence of glucose, presumably by allowing the resulting pyruvate to proceed to acetyl-coenzyme A (CoA). This phenomenon appeared specific to the lactic acid bacterial metabolism as glucose or glycine did not reduce signal in an analogous luxABCDE -modified Gram-positive pathogen, Staph . Xen29. The Strep. Xen20 cells produced light in a concentration-dependent manner, inversely related to the amount of glucose present. Taken together, our measures of microbial response could provide new information regarding the responsiveness of S. pyogenes metabolism to acute changes in its local environments and cellular health.
Yeast cells can be utilized as whole-cell biosensors for the detection of an array of target analytes. This technology is derived from the latest molecular biology techniques enabling the modification of the yeast genome to enhance the cells’ metabolic properties. Current methods to produce correctly folded recombinant proteins, particularly receptors in yeast, are now highly successful, and the implementation of diverse reporting strategies is well documented. A myriad of yeast whole-cell biosensors are now utilized in various laboratories, and some are also commercially available. A major target of these biosensors is endocrine disruptors, a class of organic molecules that can disturb the vertebrate endocrine system and therefore pose a potential threat to the environment. Recent studies focused on building new biosensors using receptors such as G-protein-coupled receptors and the thyroid receptor. In this chapter, we will present a selection of newly developed biosensors and the diverse reporting strategies used to transduce the binding event into a measurable signal.
Bioluminescence imaging has become a valuable tool in biological research, offering several advantages over fluorescence-based techniques, including the absence of phototoxicity and photobleaching, along with a higher signal-to-noise ratio. Common bioluminescence imaging methods often require the addition of an external chemical substrate (luciferin), which can result in a decrease in luminescence intensity over time and limit prolonged observations. Since the bacterial bioluminescence system is genetically encoded for luciferase-luciferin production, it enables autonomous bioluminescence (auto-bioluminescence) imaging. However, its application to multiple reporters is restricted due to a limited range of color variants. Here, we report five-color auto-bioluminescence system named Nano-lanternX (NLX), which can be expressed in bacterial, mammalian, and plant hosts, thereby enabling auto-bioluminescence in various living organisms. Utilizing spectral unmixing, we achieved the successful observation of multicolor auto-bioluminescence, enabling detailed single-cell imaging across both bacterial and mammalian cells. We have also expanded the applications of the NLX system, such as multiplexed auto-bioluminescence imaging for gene expression, protein localization, and dynamics of biomolecules within living mammalian cells.
Quantum phenomena can be identified in different forms in organisms, ranging from the exploitation of light photons for information processes or energy conversion over bioluminescence to the ubiquitous energy conversions from state differences of protons and electrons. These overwhelming diversity and ubiquitous presence of quantum processes at all levels of biological life provides strong hints for quantum-based foundations of biological evolution. A quantum-based interpretation of biological processes facilitates—scale independent—comparisons of biological phenomena over all realms of organic life. However, the final textbook of quantum biological evolution is still not written, so it is only possible to formulate preliminary hypotheses.
Quorum sensing (QS) is a communication form between bacteria via small signal molecules that enables global gene regulation as a function of cell density. We applied a microfluidic mother machine to study the kinetics of the QS response of Pseudomonas aeruginosa bacteria to additions and withdrawals of signal molecules. We traced the fast buildup and the subsequent considerably slower decay of a population-level and single-cell-level QS response. We applied a mathematical model to explain the results quantitatively. We found significant heterogeneity in QS on the single-cell level, which may result from variations in quorum-controlled gene expression and protein degradation. Heterogeneity correlates with cell lineage history, too. We used single-cell data to define and quantitatively characterize the population-level quorum state. We found that the population-level QS response is well-defined. The buildup of the quorum is fast upon signal molecule addition. At the same time, its decay is much slower following signal withdrawal, and the quorum may be maintained for several hours in the absence of the signal. Furthermore, the quorum sensing response of the population was largely repeatable in subsequent pulses of signal molecules.
Vibrio (Aliivibrio) fischeri’s initial rise to fame derived from its alluring production of blue-green light. Subsequent studies to probe the mechanisms underlying this bioluminescence helped the field discover the phenomenon now known as quorum sensing. Orthologs of quorum-sensing regulators (i.e., LuxR and LuxI) originally identified in V. fischeri were subsequently uncovered in a plethora of bacterial species, and analogous pathways were found in yet others. Over the past three decades, the study of this microbe has greatly expanded to probe the unique role of V. fischeri as the exclusive symbiont of the light organ of the Hawaiian bobtail squid, Euprymna scolopes. Buoyed by this optically amenable host and by persistent and insightful researchers who have applied novel and cross-disciplinary approaches, V. fischeri has developed into a robust model for microbe-host associations. It has contributed to our understanding of how bacteria experience and respond to specific, often fluxing environmental conditions and the mechanisms by which bacteria impact the development of their host. It has also deepened our understanding of numerous microbial processes such as motility and chemotaxis, biofilm formation and dispersal, and bacterial competition, and of the relevance of specific bacterial genes in the context of colonizing an animal host. Parallels in these processes between this symbiont and bacteria studied as pathogens are readily apparent, demonstrating functional conservation across diverse associations and permitting a reinterpretation of “pathogenesis.” Collectively, these advances built a foundation for microbiome studies and have positioned V. fischeri to continue to expand the frontiers of our understanding of the microbial world inside animals.
Luminescent bacteria–based biosensors are widely used for fast and sensitive monitoring of food safety, water quality, and other environmental pollutions. Recent advancements in biomedical engineering technology have led to improved portability, integration, and intelligence of these biotoxicity assays. Moreover, genetic engineering has played a significant role in the development of recombinant luminescent bacterial biosensors, enhancing both detection accuracy and sensitivity. This review provides an overview of recent advances in the development and applications of novel luminescent bacteria–based biosensors, and future perspectives and challenges in the cutting‐edge research, market translation, and practical applications of luminescent bacterial biosensing are discussed.
The discovery of the bioluminescence pathway in the fungus Neonothopanus nambi enabled engineering of eukaryotes with self-sustained luminescence. However, the brightness of luminescence in heterologous hosts was limited by performance of the native fungal enzymes. Here we report optimized versions of the pathway that enhance bioluminescence by one to two orders of magnitude in plant, fungal and mammalian hosts, and enable longitudinal video-rate imaging.
The microorganisms are provided by the Quorum sensing (QS) mechanism to communicate between inter-species. The signaling molecules known as autoinducers are essential to this QS communication mechanism. The most typical autoinducer is known as N-acyl-homoserine lactones (AHLs). QS enables bacteria to cooperate, live, compete, endure in the environment, or colonize the host. The proliferation of nearby bacterial cells is the most crucial factor to activate for QA. So, a wide of behaviors in bacteria are provided by QS including bioluminescence, swarming, biofilm, motility, stress survival, and virulence factors. However, some studies indicate that the QS-disputing mechanism reduces adequately population density and virulence accordingly. The QS manipulation methods such as AHL-degradation and mimic act give promising approaches to controlling plant pathogen bacteria. So, it can lead to an alternative way of supporting biological control. This review focuses on the QS mechanism in plant bacteria and the different disrupting mechanisms. It demonstrates a novel method in biocontrol and the major outcomes of plant protection.
There are a number of reporter systems that are useful for gene expression analysis in bacteria. However, at least in Salmonella, a versatile and simple luciferase reporter system that can be integrated precisely behind a promoter or gene of interest on a chromosome is not currently available. The luciferase operon luxCDABE from Photorhabdus luminescens has several advantages, including brightness, wide linear range, absence in most bacteria, stability at high temperature, and no substrate addition required for the assay. Here, a conjugation-mediated site-specific single-copy luciferase fusion system is developed. A reporter plasmid containing the conditional replication origin R6Kgγ, FRT-luxCDABE, and KmR marker was designed to be incorporated into the FRT site behind the promoter or gene of interest on the chromosome in cells expressing FLP. However, when this reporter plasmid was electroporated directly into such a S. enterica strain, no colonies appeared, likely due to the low transformation efficiency of this relatively large plasmid DNA. Meanwhile, the same reporter plasmid was successfully introduced and launched as an insert of an FRT-containing conjugative transfer plasmid from a mating E. coli strain to the same recipient S. enterica strain, as well as Citrobacter koseri. RcsB-dependent inducible luminescence from the constructed wzc-luxCDABE strains was confirmed. This system is feasible for detecting very low levels of transcription, even in Gram-negative bacterial species that are relatively difficult to genetically manipulate.
Luciferase-based gene reporters generating bioluminescence signals are important tools for biomedical research. Amongst the luciferases, flavin-dependent enzymes use the most economical chemicals. However, their applications in mammalian cells are limited due to their low signals compared to other systems. Here, we constructed Flavin Luciferase from Vibrio campbellii (Vc) for Mammalian Cell Expression (FLUXVc) by engineering luciferase from Vibrio campbellii (the most thermostable bacterial luciferase reported to date) and optimizing its expression and reporter assays in mammalian cells which can improve the bioluminescence light output by >400-fold as compared to the non-engineered version. We found that the FLUXVc reporter gene can be overexpressed in various cell lines and showed outstanding signal-to-background in HepG2 cells, significantly higher than that of firefly luciferase (Fluc). The combined use of FLUXVc/Fluc as target/control vectors gave the most stable signals, better than the standard set of Fluc(target)/Rluc(control). We also demonstrated that FLUXVc can be used for testing inhibitors of the NF-κB signaling pathway. Collectively, our results provide an optimized method for using the more economical flavin-dependent luciferase in mammalian cells.
This review aims to systematize data on the construction and applications of bacterial lux-biosensors in various fields ranging from investigation of gene regulation and regulatory networks to the new probiotics' search and ecotoxicological research. The typical technical solutions and devices required for diverse tasks applying lux-biosensors are reviewed. Aspects of the application of lux-biosensors in fundamental researches, such as the study of oxidative stress, heat shock, DNA-damaging, pro- and antioxidant activities, are also considered. This technology allows rapid screening of the biological activities of newly synthesized compounds, which could be applied as components for fuels, household chemicals, and drugs. Works related to the ecological state assessment on water resources are also described. The use of lux-biosensor complexes based on different organisms, including both gram-positive and gram-negative bacteria, makes toxicological investigations more comprehensive. Bacterial lux-biosensors based on Escherichia coli can be used as a model for evaluation of the effect of certain substances on the transmembrane potential in mitochondria, albeit with extrapolation to a certain extent. Another aspect that draws our interest is that biosensors are able to help predict some systemic properties of probiotics. In the future, it's quite promising to see more applications of lux-biosensors for environmental control, microbial-microbial interaction assessment, antioxidant action mechanism studies and toxicological studies in the development of new drugs.
The bacterial bioluminescence system enables the generation of light by living cells without the requirement of an external luciferin. Due to the relatively low light emission, many applications of bioluminescence imaging would benefit from an increase in brightness of this system. In this report, a new approach of mutagenesis and screening of the involved proteins is described that is based on the identification of mutants with improved properties under rate-limiting reaction conditions. Multiple rounds of screening in Escherichia coli resulted in the operon ilux2 that contains 26 new mutations in the fatty acid reductase complex which provides the aldehyde substrate for the bioluminescence reaction. Chromosomal integration of ilux2 yielded an autonomously bioluminescent E. coli strain with sixfold increased brightness compared to the previously described ilux operon. The ilux2 strain produces sufficient signal for the robust detection of individual cells and enables highly sensitive long-term imaging of bacterial propagation without a selection marker.
Consumption of live microorganisms “Probiotics” for health benefits and well-being is increasing worldwide. Their use as a therapeutic approach to confer health benefits has fascinated humans for centuries; however, its conceptuality gradually evolved with methodological advancement, thereby improving our understanding of probiotics-host interaction. However, the emerging concern regarding safety aspects of live microbial is enhancing the interest in non-viable or microbial cell extracts, as they could reduce the risks of microbial translocation and infection. Due to technical limitations in the production and formulation of traditionally used probiotics, the scientific community has been focusing on discovering new microbes to be used as probiotics. In many scientific studies, probiotics have been shown as potential tools to treat metabolic disorders such as obesity, type-2 diabetes, non-alcoholic fatty liver disease, digestive disorders (e.g., acute and antibiotic-associated diarrhea), and allergic disorders (e.g., eczema) in infants. However, the mechanistic insight of strain-specific probiotic action is still unknown. In the present review, we analyzed the scientific state-of-the-art regarding the mechanisms of probiotic action, its physiological and immuno-modulation on the host, and new direction regarding the development of next-generation probiotics. We discuss the use of recently discovered genetic tools and their applications for engineering the probiotic bacteria for various applications including food, biomedical applications, and other health benefits. Finally, the review addresses the future development of biological techniques in combination with clinical and preclinical studies to explain the molecular mechanism of action, and discover an ideal multifunctional probiotic bacterium.
Cell-based biosensors have powerful abilities to sense a variety of signal chemical molecules. However, compared with commercialized methods, whole-cell biosensors cannot meet the requirements for the lower sensitivity and faster response. Here, we reprogrammed a gene circuit by coupling split-lux cassette with a toggle switch for detecting heme ultra-sensitively. The resultant biosensor (named YES601) exhibited improved detection limit (0.12 ppm) and satisfied maximum induction ratio (more than 4000 folds) for lysed blood detection. Furthermore , we harnessed YES601 to detect the blood signal in the human urine and feces from the mice with DSS-induced colitis, and the results indicated that YES601 showed more satisfied sensitivity and maximum induction ratio compared with chemical method. This ultrasensitive blood biosensor will be applied to detect trace blood in vitro for early-stage diagnosis of serious diseases, and aiding the rapid development for application in diagnosis in vivo in the future.
Background:
The utilization of bioluminescent bacteria in environmental monitoring of water contaminates considers being a vital and powerful approach. This study aimed to isolate, optimize, and apply luminescent bacteria for toxicity monitoring of various toxicants in wastewater.
Results:
On the basis of light intensity, strain Vibrio sp. 6HFE was initially selected, physiologically/morphologically characterized, and identified using the 16SrDNA gene. The luminescence production was further optimized by employing statistical approaches (Plackett-Burman design and central composite design). The maximum bioluminescence intensity recorded 1.53 × 106 CPS using optimized medium containing (g/L), yeast extract (0.2g), CaCl2 (4.0), MgSO4 (0.1), and K2HPO4 (0.1) by 2.3-fold increase within 1h. The harnessing of Vibrio sp. 6HFE as a bioluminescent reporter for toxicity of organic solvents was examined using a bioluminescence inhibition assay. According to IC50 results, the toxicity order of such pollutants was chloroform > isoamyl > acetic acid > formamide > ethyl acetate > acetonitrile > DMSO > acetone > methanol. However, among eight heavy metals tested, the bioluminescence was most sensitive to Ag+ and Hg+ and least sensitive to Co2+ and Ni2+. Additionally, the bioluminescence was inhibited by benzene, catechol, phenol, and penta-chlorophenol at 443.1, 500, 535.1, and 537.4 ppm.
Conclusion:
Vibrio sp. 6HFE succeeded in pollution detection at four different environmental and wastewater samples revealing its efficiency in ecotoxicity monitoring.
Superparamagnetic iron oxide nanoparticles (SPIONs) can be used as imaging agents to differentiate between normal and diseased tissue or track cell movement. Magnetic particle imaging (MPI) detects the magnetic properties of SPIONs, providing quantitative and sensitive image data. MPI performance depends on the size, structure, and composition of nanoparticles. Magnetotactic bacteria produce magnetosomes with properties similar to those of synthetic nanoparticles, and these can be modified by mutating biosynthetic genes. The use of Magnetospirillum gryphiswaldense, MSR-1 with a mamJ deletion, containing clustered magnetosomes instead of typical linear chains, resulted in improved MPI signal and resolution. Bioluminescent MSR-1 with the mamJ deletion were administered into tumor-bearing and healthy mice. In vivo bioluminescence imaging revealed the viability of MSR-1, and MPI detected signals in livers and tumors. The development of living contrast agents offers opportunities for imaging and therapy with multimodality imaging guiding development of these agents by tracking the location, viability, and resulting biological effects.
Immobilized enzymes are now a significant and appropriate area of modern technologies.
Immobilization of enzymes on nanoparticles (NPs) especially magnetic nanoparticles
(MNPs) not only increase the stability of the enzymes by protecting the active site but also facilitates the separation mode. Immobilized technology is considered effective in context of running cost to exercise immobilized enzymes technique. Nowadays, variety of
magnetic nanoparticles are available such as chitin-chitosan magnetic nanoparticles, Fe3O4 magnetic nanoparticles, bacteriophages T4 capsid novozym-435 etc. which are quite fit for loading enzymes and to use fruitfully. The main focus in this piece of work is that how immobilized enzymes are helpful in different biomedical uses and what kind of enzymes and nanoparticles could be hyphenated to take advantage in health care sectors. Different method of enzymes immobilization will also be discussed in details including both physical methods and chemical methods of loading enzymes on nanoparticles.
Bioluminescence (BL) is an amazing natural phenomenon whose visible light is produced by living organisms. BL phenomenon is quite pervasive and has been observed in 17 phyla of 4 kingdoms. This fascinating natural phenomenon has unceasingly attracted people’s curiosity from ancient era to today. For a very long time, we can only receive some sporadic and static information from experimental observations, the mechanism of most BL remains is unclear. How the chemical reaction of BL process is initiated? Where the energy for light emission comes from? How does the light emitter produce? What is the light emitter for a wild bioluminescent organism? How to regain luciferin for next bioluminescence when it is used up? The luciferin is utilized forthwith or stored and release for subsequent light emission? What factors affect the color and strength of a bioluminescence? How to artificially tune the bioluminescence for special application? Computational BL plays unreplaceable role in answering these mechanistic questions. In contrast with experimental BL, computational BL came very late. In the past two decades, computational BL has touched nearly all the bioluminescent systems with chemical bases via the method of multiscale simulation. In this review, the author firstly introduced the history, types and general chemical process of BL. Then, the computational scheme on BL was briefly epitomized. Using firefly BL as a paradigmatic case, the author summarized theoretical investigation on the six stages of general chemical process in a BL cycle: luciferin oxidation, peroxide thermolysis, light emission, luciferin regeneration, luciferin storage and luciferin release. At each stage, the available theoretical studies of other bioluminescent organisms are briefly introduced and compared with the firefly system. Basing on the mechanistic understanding, the author reviewed the up-to-date theoretical design on bioluminescent systems. Again, the firefly was mainly focused on, and the other possible systems were just briefly introduced. This review summarized the theoretical studies to date on BL and addressed the status, critical challenges and future prospects of computational BL.
The nucleotide sequence of the 1.85-kilobase EcoRI fragment from Vibrio harveyi that was cloned using a mixed-sequence synthetic oligonucleotide probe (Cohn, D. H., Ogden, R. C., Abelson, J. N., Baldwin, T. O., Nealson, K. H., Simon, M. I., and Mileham, A. J. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 120-123) has been determined. The alpha subunit-coding region (luxA) was found to begin at base number 707 and end at base number 1771. The alpha subunit has a calculated molecular weight of 40,108 and comprises a total of 355 amino acid residues. There are 34 base pairs separating the start of the alpha subunit structural gene and a 669-base open reading frame extending from the proximal EcoRI site. At the 3' end of the luxA coding region there are 26 bases between the end of the structural gene and the start of the luxB structural gene. Approximately two-thirds of the alpha subunit was sequenced by protein chemical techniques. The amino acid sequence implied by the DNA sequence, with few exceptions, confirmed the chemically determined sequence. Regions of the alpha subunit thought to comprise the active center were found to reside in two discrete and relatively basic regions, one from around residues 100-115 and the second from around residues 280-295.
A new genus, Xenorha bdus, is created to accommodate large, gram-negative, rod-shaped, facultatively anaerobic, entomopathogenic bacteria which are intimately associated with entomogenous nematodes. The normal habitat of these bacteria is the intestinal lumen of nematodes or the body cavity of host insects into which they have been introduced by the nematodes. The genus is placed in the family Enterobacteriaceae since the bacteria possess most of the important characteristics of this family. Xenorhabdus differs from other genera of Enterobacteriaceae in large cell size, failure to reduce nitrates to nitrites, intimate association with entomogenous nematodes, entomopathogenesis, and immunological characteristics. The type species is Xenorhabdus nematophilus (Poinar and Thomas) comb. nov. (synonym: Achromobacter nematophilus Poinar and Thomas). Xenorhabdus luminescens sp. nov., a bioluminescent, entomopathogenic bacterium isolated from the intestinal lumen of an entomogenous nematode, Heterorhabditis bacteriophora, is also described. In addition to their immunological differences, the two species are dissimilar in that X. luminescens is positive for bioluminescence and catalase activity, whereas X. nematophilus is not. The type strain of X. nematophilus is ATCC 19061, and that of X. luminescens is strain Hb (= ATCC 29999). In 1965, we (9) published a description of the bacterium Achromo bacter nematophilus Poinar and Thomas, which was found in association with strain DD-136 of the nematode Neoaplectuna carpocapsae Weiser (8). Further studies
The synthesis of the luminous system of the marine luminous bacterium Photobacterium fischeri is subject to a complex, self-regulated control system called autoinduction. The bacteria produce an autoinducer which accumulates in the medium at a constant rate (as a function of cell growth). When autoinducer reaches a critical concentration it stimulates, at the level of transcription, the synthesis of the luminous system. Autoinduction is thus viewed as an environmental sensing mechanism, which curtails the synthesis of the luminous system under dilute conditions. For several isolates of P. fischeri it was found that variations in luminescence intensity could be accounted for by correlated variations in autoinducer production.
Eleven marine luminous isolates, which could not be identified with previously studied species of luminous marine bacteria,
were subjected to an extensive characterization. The results indicated that these strains were phenotypically similar, had
a G+C content in their DNA of 45 mol%, and differed from all previously characterized luminous species by their inability
to ferment sugars. On the basis of these and other properties, the 11 luminous strains were assigned to the genusAlteromonas and given the species designationA hanedai. Strain 281 (ATCC 33224) has been designated as the type strain of this new species.
For most people, bioluminescence is represented by the flash of the firefly or the ‘‘phosphorescence” that frequently occurs on agitating the surface of ocean water. Because of the ease of obtaining material, the firefly’s bioluminescence reaction was one of the first to receive detailed biochemical study, with the result that this system is the archetype of a variety of enzymatic processes that produce light in the many bioluminescent organisms, ranging from the marine bacteria to the large luminous beetles from South America. What is usually understood by the term bioluminescence is a cold-light emission of high efficiency that has a biological function for the organism concerned, although what this function is is still conjectural in many cases. In contrast to bioluminescence, a great number of biological processes have been observed to be accompanied by the emission of a very low level of light, so-called biological chemiluminescence. This observation and the fact that bioluminescence is so widespread among many phyla (although rarely are many members of a phylum bioluminescent) has led to the suggestion that low-level light emission arose very early in evolution, and that the efficient bioluminescence was a secondary adaptation that enabled some species to compete more effectively in their biological niches.
The nucleotide sequence of the luciferase gene from the firefly Photinus pyralis was determined from the analysis of cDNA and genomic clones. The gene contains six introns, all less than 60 bases in length. The 5' end of the luciferase mRNA was determined by both S1 nuclease analysis and primer extension. Although the luciferase cDNA clone lacked the six N-terminal codons of the open reading frame, we were able to reconstruct the equivalent of a full-length cDNA using the genomic clone as a source of the missing 5' sequence. The full-length, intronless luciferase gene was inserted into mammalian expression vectors and introduced into monkey (CV-1) cells in which enzymatically active firefly luciferase was transiently expressed. In addition, cell lines stably expressing firefly luciferase were isolated. Deleting a portion of the 5'-untranslated region of the luciferase gene removed an upstream initiation (AUG) codon and resulted in a twofold increase in the level of luciferase expression. The ability of the full-length luciferase gene to activate cryptic or enhancerless promoters was also greatly reduced or eliminated by this 5' deletion. Assaying the expression of luciferase provides a rapid and inexpensive method for monitoring promoter activity. Depending on the instrumentation employed to detect luciferase activity, we estimate this assay to be from 30- to 1,000-fold more sensitive than assaying chloramphenicol acetyltransferase expression.
A rapid, quantitative assay for long-chain aldehydes based on bacterial luminescence was developed. Significant luminescent responses were obtained with both saturated and unsaturated aldehydes of 12-18 carbons. Maximum responses were obtained with the 14- and 16-carbon compounds, including those that are known insect sex pheromones. The bioluminescent response was linearly related to the amount of aldehyde over a 10(4)-10(5) concentration range with as little as 0.1 pmol (∼20 pg) of aldehyde being detected. The bioluminescent assay represents a new quantitative tool for rapidly measuring aldehyde pheromones of insects.
The linked structural genes lux A and lux B, encoding bacterial luciferase of a marine bacterium Vibrio harveyi, were fused with the nitrogenase nifD promoter from Bradyrhizobium japonicum and with the P1 promoter of pBR322. Both fusions were integrated into the B. japonicum chromosome by site-specific recombination. Soybean roots infected with the two types of rhizobium transconjugants formed nitrogen-fixing nodules that produced bright blue-green light. Cells containing the P1 promoter/lux AB fusion resulted in continuously expressed bioluminescence in both free-living rhizobium and in nodule bacteriods. However, when under control of the nifD promoter, luciferase activity was found only in introgen-fixing nodules. Light emission from bacteroids allowed us to visualize and to photograph nodules expressing this marker gene fusion in vivo at various levels of resolution, including within single, living plant cells. Localization of host cells containing nitrogen-fixing bacteroids within nodule tissue was accomplished using low-light video microscopy aided by realtime image processing techniques developed specifically to enhance extreme low-level luminescent images.
Eight luminescent strains of Vibrio, isolated from diverse geographic locations, were classified as serovar non-O1 Vibrio cholerae by morphological, physiological, and numerical taxonomy analysis. All isolates possessed overall DNA base composition of 47–48 mol % guanine plus cytosine. Based on DNA-DNA hybridization, luminescent strains, as a group, exhibited ≥ 70 % base sequence complementarity with serovar O1 V. cholerae and < 40 % with other Vibrio spp. examined. Results of the hybridization studies confirm that Vibrio albensis is synonymous with V. cholerae and distinct from other luminescent vibrios. In addition, the luminescence system in V. cholerae was investigated as a characteristic of V. cholerae, to provide new information for the description of the species. In most cases, autoinducer from V. harveyi strain 392S3 induced synthesis of the luminescent system in strains of V. cholerae and vice versa. Unlike the case of V. harveyi 392S3, in V. cholerae strain P287 the synthesis of the luminescent system is impaired at low oxygen and, also in contrast to V. harveyi strain 392S3, dark mutants (K variants) of the three V. cholerae examined occur at extremely low frequency.
Taking advantage of a specially constructed vector, luciferase LuxA and LuxB subunits were connected in frame to different amino acid linkers to reproduce a series of monomeric luciferase enzymes. A comparison of their activities in E. coli cells demonstrated that the length of the linkers positively affected activity. One luciferase fusion gene was expressed in plant cells, and we showed that this gene activity could be monitored directly without destructive sampling.
The gene of bioluminescent bacteria encodes the acyl-protein synthetase component of the fatty acid reductase complex. The complex is responsible for converting tetradecanoic acid to the aldehyde which serves as a substrate in the luciferase-catalyzed reaction. The nucleotide sequence of the gene of was determined and the amino acid sequence of the acyl-protein synthetase deduced. The protein consists of 378 amino acid residues and has a molecular weight of 42,965 daltons. Alignment of the enzyme with the acyl-protein synthetase showed 62% identity.
The ability to measure gene expression with light is revolutionizing biotechnology. It is now possible to visualize transcription or translation and localize it to particular domains, cells or organelles of any organism. Moreover, this can be done repeatedly, noninvasively, and with unsurpassed sensitivity.
Bioluminescence and the synthesis of luciferase inVibrio harveyi growing in a minimal medium are repressible by iron; this is not significantly reversed by cyclic adenosine 3',5'-monophosphate (cAMP). Cultures grown with added iron emit less light and possess less luciferase per cell than those grown under conditions of limiting iron; this may have significance in relation to the function of luciferase as an electron carrier. With iron, and with glycerol as the sole carbon and energy source, the addition of glucose causes further repression, both transient and permanent, and this is only partially reversible by cAMP. Without iron, glucose addition results in only a small and transient repression, but this is fully reversible by cAMP. The inability of cAMP to reverse iron-influenced repression may be explained by both a low rate of transport of cAMP into the bacteria and increased intracellular levels of cyclic nucleotide phosphodiesterase.
A sensitive reverse-phase high pressure liquid chromatographic assay for formation of AMP coupled with analysis of aldehyde production has been used to characterize the properties of the fatty acid reductase complex of Photobacterium phosphoreum. The enzyme complex, which consists of three different polypeptides (34 000, 50 000, and 58 000), has a high affinity for ATP (Km = 20 nM) and shows highest specificity with C14 fatty acids. Activation of the fatty acid is efficiently coupled to the reduction step showing a stoichiometry of one molecule of fatty acid reduced to aldehyde and one molecule of NADPH oxidized for every molecule of ATP converted to AMP. Reconstituted fatty acid reductase (50 000 and 58 000) shows an ATP hydrolase activity that is independent of NADPH with the maximum amount of AMP formed limited by the amount of fatty acid in the assay, consistent with acyl-protein turnover experiments and the channeling of fatty acids to form acyl thioesters (−NADPH) or aldehyde (+NADPH). Addition of the 34 000 polypeptide to the reconstituted enzyme results in stimulation of AMP formation (−NADPH) to a level far exceeding the amount of fatty acid, showing that the fatty acid can be recycled by the 34 000 protein through its thioesterase activity. Also the 34 000 protein is responsible for a two- to three-fold stimulation in the rate of ATP hydrolysis, suggesting that it can be involved in the stabilization of the enzyme complex.
We have engineered a two subunit luciferase enzyme into a single functional polypeptide chain using site specific mutagenesis. We have determined, using low light video imaging, that the activity of this novel enzyme is similar to wild type luciferase when synthesized at low temperatures (15-20°C), but that it is sensitive in vivo to higher temperatures. We have used the gene encoding the monomeric bacterial luciferase as a gene marker in prokaryotic and eukaryotic organisms. Combined with low light video image analysis, it is a practical and powerful tool for quantitatively monitoring gene expression in vivo.
Bioluminescence (lux) genes from Vibrio fischeri were cloned as a promoter–less gene set into the broad–host–range vector, pUCD4, resulting in the recombinant plasmid pUCD607 that was mobilized into a variety of plant pathogenic and symbiotic bacteria. All bacteria harboring the lux genes under the control of a constitutive promoter in pUCD607 constitutively bioluminesced. Virulence remained unaffected in pathogens carrying pUCD607, and bacterial invasion in host plant tissues was visually followed. The light generated is quantifiable and directly reflects the growth of the pathogen population in host tissue even before the onset of visual symptoms. pUCD607 is relatively stable in bacteria in planta in the absence of selective pressure. Bioluminescence can thus be successfully used to tag genetically engineered bacteria for subsequent monitoring during interactions with plants and in determining their fate in the environment.
This chapter discusses the techniques for cloning and analyzing bioluminescence genes from marine bacteria. The isolation by recombinant DNA techniques of genes for bioluminescence (lux) from marine bacteria has resulted in a rapid expansion of knowledge of the biochemical activities necessary for light production and of the regulatory mechanisms, which govern the expression of these functions. The chapter describes a variety of genetic methodologies for cloning DNA fragments encoding luminescence functions, eliciting expression of luminescence genes, defining individual lux genes and transcriptional units containing lux genes, identifying the products of cloned lux genes, exploring the regulatory control of lux genes, and using lux gene fusions to measure transcriptional control of other gene systems. Most genetic analysis has been performed with luminescent Vibrio, and the attention is confined to bacteria of this genus.
Genes encoding at least five polypeptides are required to produce the enzymes necessary for bioluminescence in marine bacteria. These include genes coding for the two subunits of luciferase (α and β) and the three polypeptides of a fatty acid reductase complex, which supplies aldehyde for the bioluminescent reaction. Two regulatory polypeptides have been identified in the Vibriofischeri luminescence system. Transfer of the genes from luminescent bacteria into Escherichia coli and the study of their regulation can now readily be accomplished as very rapid and highly sensitive techniques are available for detection of the cloned genes and the analyses of the mRNAs and gene products. Selection of clones containing genes from the bioluminescence operons is generally based on the detection of light emission. Only the luciferase genes need be present as the aldehyde substrate can be supplied exogenously to E. coli and sufficient FMNH2 and O2 are present intracellularly to allow generation of light. Screening may be accomplished by the following approaches: visual scanning, exposure of colonies to film, scintillation counter readings, and hybridization probes.
The regulation of bioluminescence in Vibrio fischeri involves both an autoregulatory mechanism and the adenosine cyclic 3′,5′-phosphate/crp system. The lux regulon from V. fischeri strain MJ-1, consisting of two operons, L (left) and R (right), and at least seven genes, luxR (L operon) and luxICDABE (R operon) and the intervening region, functions in laboratory strains of Escherichia coli [Engebrecht, J., Nealson, K., & Silverman, M. (1983) Cell (Cambridge, Mass.) 32, 773-781], The regulatory region, consisting of luxR, encoding the regulatory protein, and luxI, encoding a function required for synthesis of the autoinducer, and the intervening region of V. fischeri strain ATCC 7744 has been cloned and the nucleotide sequence determined. The regulatory protein is an Mr 28 518 polypeptide consisting of 250 amino acid residues; the I protein is an Mr 21937 polypeptide consisting of 193 amino acid residues. The luxR gene, the only known gene of the L operon, is transcribed in the opposite direction to the direction of transcription of the other genes of the lux regulon. There are 218 base pairs that separate the 5′ end of the open reading frame of the luxR gene from the 5′ end of the open reading frame of the luxI gene, the first gene in the rightward operon. In this region, there are both an apparent catabolite repressor protein binding site and an inverted repeat structure that may serve as protein binding sites for the regulation of bioluminescence.
The subunit structures of the fatty acid reductase complex from Photobacterium phosphoreum and its three component enzymes (designated acyl-CoA reductase, acyl-protein synthetase, and acyltransferase) have been investigated by cross-linking with bifunctional reagents, gel filtration, complementation experiments, and analysis of the polypeptide composition by quantitative protein staining. The acyl-CoA component eluted on gel filtration at a position [Mr 223 (±13) × 103] corresponding to a tetrameric structure. Protein bands corresponding to the dimer, trimer, and tetramer could be seen on sodium dodecyl sulfatepolyacrylamide gel electrophoresis after treatment of the enzyme with bifunctional cross-linking reagents. The dependence of the distribution of cross-linked species on the concentration and type of reagent indicated that acyl-CoA reductase has D2 symmetry with the subunits arranged as a set of dimers. In contrast, both the acyl-protein synthetase and acyl-transferase enzymes behaved as monomeric species. The acyl-protein synthetase interacted with both the acyl-transferase and the acyl-CoA reductase whereas no interaction was found between the latter two enzymes. Four molecules of the acyl-protein synthetase monomer were required to completely complement the tetrameric acyl-CoA reductase, while a simple 1:1 heterodimer complex was formed between the acyl-transferase and acyl-protein synthetase subunits. Analysis of the polypeptide composition of the fatty acid reductase complex indicated that the complex is composed of four polypeptides each of the acyl-CoA reductase and acyl-protein synthetase enzymes and variable amounts (two to four polypeptides) of the acyl-transferase enzyme with the acyl-protein synthetase playing a central metabolic and structural role between the transferase and reductase subunits.
Acylation of proteins with [3H]tetradecanoic acid (+ATP) has been demonstrated in extracts of different strains of luminescent bacteria. The labeled polypeptides from Photobacterium phosphoreum (34K and 50K) have been identified as being involved in the acyl-protein synthetase activity that is part of a purified fatty acid reductase complex responsible for synthesis of long-chain aldehydes for the bioluminescent reaction. The two polypeptides (34K and 50K) have been separated from the acyl-CoA reductase enzyme (58K) of the complex and resolved from each other, and the 50K polypeptide was further purified to >95% homogeneity. Acylation of the 50K polypeptide, alone, occurred at a low rate; however, the rate and level of acylation were greatly stimulated by the addition of either the 34K or the 58K polypeptide. Cold chase experiments demonstrated that the acylated 50K polypeptide turned over in the presence of the 58K polypeptide but not in a mixture containing only the 34K and 50K polypeptides. Furthermore, the acylated 50K polypeptide could function as the immediate substrate for the fatty acyl-CoA reductase enzyme (58K), being reduced with NADPH to aldehyde. The 34K polypeptide was acylated only when all three polypeptides (34K, 50K, and 58K) were present. Fatty acid reductase activity could be restored by mixing of only the 58K (acyl-CoA reductase) and 50K polypeptides, showing that the 50K polypeptide is responsible for fatty acid activation in the fatty acid reductase complex and raising the question of what role the 34K polypeptide plays in fatty acid utilization in the luminescent system.
Comparison of the nucleotide sequences and gene organization of the lux systems from Vibrio harveyi and Vibrio fischeri has demonstrated that the location and order of the lux structural genes are highly conserved whereas considerable divergence has arisen in the location and/or presence of lux regulatory genes in the two marine bacteria. The order of the lux structural genes (luxCDABE) are identical in the two bacteria with the three fatty acid reductase genes (luxCDE) required for aldehyde biosynthesis flanking the luciferase genes (luxAB). Complementation in trans of the upstream V. fischeri DNA containing the luxC and luxD structural genes with the downstream luxA, B and E genes of V. harveyi resulted in luminescent Escherichia coli that did not require any exogenous aldehyde. These results indicate that the light-emitting systems are very similar in the two bacteria.
However, the lux regulatory systems in these two bacteria appear to have clearly diverged. In V. harveyi, an open reading frame of >40 codons does not exist within 600 bp of the start of luxC in contrast to the luxl regulatory gene present in the V. fischeri lux system. An open reading frame of 615 bp is present farther upstream of luxC with the same direction of transcription and approximate location as the luxR regulatory gene of V. fischeri; however, apparent homologies do not exist between the two genes. The similarities in organization of the lux structural genes and the differences in the existence and/or location of lux regulatory genes in the two Vibrio species raises the question of how these marine bacteria can have a similar growth-dependent regulation of luminescence expression.
Recombinant bacteria which express only the luxA and luxB genes have a bioluminescent phenotype providing exogenous long chain aldehyde is supplied in the medium or as a vapour. As an integral part of the biochemistry of photon emission, sub-saturating concentrations of aldehyde are reflected as sub-maximal cellular bio-luminescence. This may be correlated in a kinetic analysis to provide apparent Km values. An analysis of such data across a spectrum of Gram-positive and Gramnegative bacteria was used to provide a limit for the sensitivity of aldehyde detection. The future development of a rapid bioluminescence-based rancidity test in foods and oils is discussed.
Fatty acid reductase from the luminescent bacterium Photobacterium phosphoreum catalyzes the NADPH- and ATP-dependent conversion of fatty acid into the corresponding aldehyde required for light emission. The mechanism involves acyl-AMP formation, followed by acylation of the synthetase subunit, acyl transfer to the reductase subunit and reduction with NADPH. Modification with N-ethylmaleimide of a highly reactive thiol in the acylprotein synthetase subunit prevents acylation but not acyl-AMP formation, indicating that these two steps take place at different sites in the synthetase subunit. Prior acylation of the enzyme, but not binding of fatty acid and ATP, protects against inactivation by N-ethylmaleimide. Treatment of the acylated enzyme with neutral hydroxylamine results in deacylation. Moreover, the proteolytic peptide maps of the synthetase labelled with and with [3H]tetradecanoic acid appear to be identical. These results indicate that acylation takes place at a specific cysteine residue in the synthetase subunit. Interaction with the reductase subunit, which stimulates the acylation of the synthetase subunit, partially protects the acylation site against modification with N-ethylmaleimide. Aminoacid analysis gave molecular weights of 34 000, 52 000 and 57 000 for the transferase, synthetase and reductase polypeptides, respectively, based on best fit integral analyses, in good agreement with molecular weights determined by SDS gel electrophoresis. The average hydrophobicity and the content of polar residues indicate that the synthetase subunit as well as the transferase subunit is less hydrophobic than the reductase subunit, in agreement with the isolation of the two former subunits as monomers and the latter subunit as a tetramer on resolution from the fatty acid reductase complex.
Regulatory mutants of the luminescent bacterium, Vibrio harveyi, have been isolated whose light emission can be stimulated by extracts of the growth media. Chloroform extracts of conditioned media in which V. harveyi has been grown can increase light emission in one of the dark mutants, D34, over 103-fold. An increase in the level of the mRNA and the enzymes associated with the lux system can also be demonstrated.
Analysis of the expression of the lux system in Escherichia coli transformed with DNA from the D34 regulatory mutant demonstrates that the mutation resides outside the luciferase structural genes. The results suggest that the decrease in light emission in the regulatory mutants may be due to a mutation in synthesis of an autoinducer analogous to that found for the Vibrio fischeri lux system.
In some luminous bacterial species, it is postulated that luciferase is “autoinduced” by a substance produced by the bacteria
themselves. This hypothesis was confirmed. In experiments with growing cultures that were subjected to repeated subculturing
into or dialysis against fresh medium, which should prevent the autoinducer from accumulating, the normal synthesis of luciferase
and the development of luminescence did not occur.
The autoinduction and glucose repression of luciferase synthesis in batch cultures and continuous cultures of Vibrio fischeri were investigate. As previously reported, a lag in luciferase synthesis occurred in glycerol-grown batch cultures and addition of d-glucose to the medium extended the lag period. A phosphate-limited chemostat culture with d-glucose as energy source (specific growth rate, =0.45 h-1) contained uninduced levels of luciferase. Luciferase activity increased to an induced level upon addition of c-AMP or autoinducer to such a chemostat culture while cell mass remained constant. Furthermore, when of a phosphate-limited chemostat culture containing d-glucose as energy source was decreased from 0.45 to 0.30 h-1, luciferase activity increased from an uniduced to induced level. After exogenously added c-AMP or autoinducer was diluted out of a phosphate-limited continous culture or after was increased to 0.45 h-1, luciferase activity remained at an induced level. Apparently, luciferase in V. fischeri was subject to a catabolite repression by d-glucose that could be overridden by autoinduction or by an autogenous control element.
Several strains of four species of luminous marine bacteria were maintained in a chemostat at a constant dilution rate and a variety of steady state densities by carbon (glycerol) limitation in order to study the relationship between culture density and bioluminescence activity. In general, luminescence per cell was constant at high culture density, and decreased dramatically at low culture density. For Vibrio fischeri, luminescence decreased to nondectable levels when the culture was maintained at low density; such dark cells were stimulated to synthesize luciferase and became luminous within minutes when purified autoinducer was added to the chemostat. Two strains, Photobacterium phosphoreum NZ11D and Photobacterium leiognathi S1, did not show the decrease in light intensity at low culture density that was characteristic of all other strains tested; they appeared to be constitutive for bioluminescence.
It has been previously demonstrated that luciferase synthesis in the luminous marine bacteria, Beneckea harveyi and Photobacterium fischeri is induced only when sufficient concentrations of metabolic products (autoinducers) of these bacteria accumulate in growth media. Thus, when cells are cultured in liquid medium there is a lag in luciferase synthesis. A quantitative bioassay for B. harveyi autoinducer was developed and it was shown that many marine bacteria produce a substance that mimics its action, but in different amounts, (20–130% of the activity produced by B. harveyi) depending on the species and strain. This is referred to as alloinduction. None of the bacteria tested produced detectable quantities of inducer for P. fischeri luciferase synthesis. These findings may have significance with respect to the ecology of B. harveyi and P. fischeri.
Luciferase (Lux)-encoding sequences are very useful as reporter genes. However, a drawback when applying Vibrio harveyi Lux as a reporter enzyme in eukaryotic cells, is that it is a heterodimeric enzyme, thus requiring simultaneous synthesis of both Lux subunits to be active. To overcome this disadvantage, luxA and luxB genes encoding the A and B subunits of this light-emitting heterodimeric Lux, were fused and expressed in Escherichia coli. Comparative analysis of four fused monomeric Lux enzymes by in vivo enzyme assay, immunoblotting and partial enzyme purification, showed that the fused Lux were active both as AB or as BA monomers, albeit at different levels (up to 80% activity for AB and up to 2% for BA, as compared with the wild type binary A + B construct). One of the LuxAB fusion proteins was stably expressed in calli of Nicotiana tabacum, and displayed very high Lux activity, thus demonstrating its potential as a reporter enzyme in eukaryotic systems.
The luxA and luxB genes of bioluminescent bacteria encode the α and β subunits of luciferase, respectively. Sequences of the luxA and luxB genes of Xenorhabdus luminescens, the only terrestrial bioluminescent bacterium known, were determined and the amino acid sequence of luciferase deduced. The α subunit was found to contain 360 amino acids and has a calculated molecular weight of 41,005 Da, while the β subunit contains 327 amino acids and has a calculated molecular weight of 37,684 Da. Alignment of this luciferase with the luciferases of three marine bacteria showed 196 (or 55%) conserved residues in the α subunit and 114 (or 35%) conserved residues in the β subunit. The highest degree of homology between any two species was between the luciferases of X. luminescens and Vibrio harveyi with 84% identity in the α subunits and 59% identity in the β subunits.
Genes encoding luminescence of Photobacterium leiognathi have been cloned in Escherichia coli. The luminescent clones were readily apparent. Among them, a clone containing a recombinant plasmid with a 13.5-kb insertion was identified. This DNA fragment contained all of the luminescence-encoding genes. The luciferase-encoding genes (lux) in this DNA fragment were localized. We have sequenced a part of the cloned lux region and identified the luxA, luxB and luxG genes encoding the α and β subunits of luciferase and a γ protein with an Mr of 26 180, respectively. The analysis of deduced amino acid sequences and comparison with known luciferase sequences from Vibrio harveyi, indicate the common origin of these proteins.