A highly efficient, thermostable and cadmium selective firefly luciferase suitable for ratiometric metal and pH biosensing and for sensitive ATP assays

Pelentir, Gabriel F; Bevilaqua, Vanessa R.; Viviani, Vadim R.

doi:10.1039/c9pp00174c

Cited by 18 publications

(16 citation statements)

References 45 publications

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“…The luciferin-luciferase assay is the most commonly used ATP-detection method in studies of antibiotic susceptibility [18-22, 28, 37] as well as the cleanliness of surfaces and medical equipment in healthcare facilities or food industries [32,[38][39][40]. Other methods involve ATP-sensing fluorescent proteins such as engineered luciferase probes expressed on the outer side of the plasma membrane [41,42] or use of fluorescence microscopy for real-time ATP measurement using a two-enzyme system [43].…”

Section: Existing Methods For Atp Detection In Antibiotic Susceptibility and Hygiene Testingmentioning

confidence: 99%

“…Online (real time) measurement of eATP in microbial cultures has not yet been described, presumably due to limited stability of the most widely available wild-type luciferases. The half-life of the WT firefly luciferase in assay buffer at room temperature is only 3 h which makes it difficult to use in growth experiments requiring longer time periods of monitoring [32,33]. In the last two decades several engineered firefly luciferases have been described with strongly improved thermostability in buffer [34][35][36].…”

Section: Atp-based Hygiene and Cell Viability Testingmentioning

confidence: 99%

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Real-time monitoring of extracellular ATP in bacterial cultures using thermostable luciferase

Ihssen¹,

Jovanović

Širec³

et al. 2021

PLoS ONE

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Adenosine triphosphate (ATP) is one of the most important indicators of cell viability. Extracellular ATP (eATP) is commonly detected in cultures of both eukaryotic and prokaryotic cells but is not the focus of current scientific research. Although ATP release has traditionally been considered to mainly occur as a consequence of cell destruction, current evidence indicates that ATP leakage also occurs during the growth phase of diverse bacterial species and may play an important role in bacterial physiology. ATP can be conveniently measured with high sensitivity in luciferase-based bioluminescence assays. However, wild-type luciferases suffer from low stability, which limit their use. Here we demonstrate that an engineered, thermostable luciferase is suitable for real-time monitoring of ATP release by bacteria, both in broth culture and on agar surfaces. Different bacterial species show distinct patterns of eATP accumulation and decline. Real-time monitoring of eATP allows for the estimation of viable cell number by relating luminescence onset time to initial cell concentration. Furthermore, the method is able to rapidly detect the effect of antibiotics on bacterial cultures as Ampicillin sensitive strains challenged with beta lactam antibiotics showed strongly increased accumulation of eATP even in the absence of growth, as determined by optical density. Patterns of eATP determined by real-time luminescence measurement could be used to infer the minimal inhibitory concentration of Ampicillin. Compared to conventional antibiotic susceptibility testing, the method presented here is faster and more sensitive, which is essential for better treatment outcomes and reducing the risk of inducing antibiotic resistance. Real-time eATP bioluminescence assays are suitable for different cell types, either prokaryotic or eukaryotic, thus, permitting their application in diverse fields of research. It can be used for example in the study of the role of eATP in physiology and pathophysiology, for monitoring microbial contamination or for antimicrobial susceptibility testing in clinical diagnostics.

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Section: Existing Methods For Atp Detection In Antibiotic Susceptibility and Hygiene Testingmentioning

confidence: 99%

Section: Atp-based Hygiene and Cell Viability Testingmentioning

confidence: 99%

Real-time monitoring of extracellular ATP in bacterial cultures using thermostable luciferase

Ihssen¹,

Jovanović

Širec³

et al. 2021

PLoS ONE

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“…Viviani and co-workers reported a technique to estimate the intracellular pH in E. coli using mutant firefly luciferases, 164 as well as a mutant firefly luciferase for ratiometric pH sensing and the selective detection of cadmium. 207 A genetically encoded pH sensitive reporter has also been recently reported that consists of a pH-sensitive GFP (superecliptic pHluorin) that emits at 510 nm, and a pH-stable luciferase Antares that emits at 580 nm. On the addition of furimazine, a ratiometric readout R 580/510 was indicative of pH and this system was shown to be functional both in vitro and in vivo using xenograft murine tumours to detect acidosis.…”

Section: Sensing Of Ph Metal Ions Reactive Oxygen Species (Ros) Enmentioning

confidence: 99%

Applications of bioluminescence in biotechnology and beyond

2021

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“…The brightness of BL reaction, which is a function of quantum yield, K m , V max , turnover rate, protein stability, and sensitivity to product inhibition [ 26 ], is another important parameter that has to be considered, for example, in microscopy. In order to overcome the drawbacks of natural enzymes, several brighter analogs of firefly luciferase and luciferases with increased thermostability have been generated with the help of site-directed mutagenesis methods [ 27 ].…”

Section: D-luciferin-dependent Luciferasesmentioning

confidence: 99%

Palette of Luciferases: Natural Biotools for New Applications in Biomedicine

2020

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Optoanalytical methods based on using genetically encoded bioluminescent enzymes,luciferases, allow one to obtain highly sensitive signals, are non-invasive, and require no external irradiation. Bioluminescence is based on the chemical reaction of oxidation of a low-molecular-weight substrate (luciferin) by atmospheric oxygen, which is catalyzed by an enzyme (luciferase). Relaxation of the luciferin oxidation product from its excited state is accompanied by a release of a quantum of light, which can be detected as an analytical signal.The ability to express luciferase genes in various heterological systems and high quantum yields of luminescence reactions have made these tools rather popular in biology and medicine. Amongseveral naturally available luciferases, a few have been found to be useful for practicalapplication. Luciferase size, the wavelength of its luminescence maximum, enzyme thermostability, optimal pH of the reaction, and the need for cofactors areparameters that may differ for luciferases from different groups of organisms, and this fact directly affects the choice of the application area for each enzyme. It is quite important to overview the whole range of currently available luciferases based ontheir biochemical properties before choosing one bioluminescent probe suitable for a specific application.

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A highly efficient, thermostable and cadmium selective firefly luciferase suitable for ratiometric metal and pH biosensing and for sensitive ATP assays

Abstract: The brighter and cadmium sensitive luciferase of Amydetes is suitable for sensitive ATP and biosensing assays.

Cited by 18 publications

References 45 publications

Real-time monitoring of extracellular ATP in bacterial cultures using thermostable luciferase

Real-time monitoring of extracellular ATP in bacterial cultures using thermostable luciferase

Applications of bioluminescence in biotechnology and beyond

Palette of Luciferases: Natural Biotools for New Applications in Biomedicine

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