Bacterial Luminescence
Bacterial Bioluminescence is most commonly found in aquatic bacteria, such as Vibrio Fischeri. Through quorum sensing, Vibrio Fischeri's bioluminescence genes remain inactive at low cell densities. However, as the bacteria forms symbiotic relationships with host organisms high cell densities activate these genes. This leads to light emission which helps camouflage the host. Bioluminescence is achieved through an enzyme-substrate reaction, involving the oxidation of FMNH₂ and RCHO catalysed by luciferase.
The chemical reaction for bacterial luminescence using luciferin can be summarized as follows:
- FMNH₂ (Reduced Flavin Mononucleotide): Form of luciferin used in bacterial luminescence, and the substrate which is oxidized.
- RCHO (Long-Chain Fatty Aldehyde): This is a substrate provided by the bacteria, which varies depending on the species. It is oxidized to the corresponding fatty acid (RCOOH) during the reaction.
- O₂ (Oxygen): Crucial for the oxidation process.
- Luciferase: Enzyme that catalyzes the conversion of FMNH₂ and RCHO into FMN and RCOOH while producing light.
- FMN (Flavin Mononucleotide): Oxidized form of luciferin as a product of the reaction.
- RCOOH (Fatty Acid): The product created from the oxidation of the long-chain fatty aldehyde.
The luxA and luxB genes encode for the a and b subunits of a heterodimeric protein of bacterial luciferase, respectively (Kaku, 2021). Fusion mutations of LuxAB are done to imitate the bacterial luciferase systam in eukaryotes but at most conserve 80% of the Lux cassette’s brightness.
The luxC, luxD and luxE genes code for the reactants that will help form the fatty aldehyde substrate (luciferin) that reacts with luciferase. However, by prucing an external substrate, the bacteria can save the time and energy spent on this biosynthesis. There also isnt a need for this cycle to be renewable as new soil samples added to the bacteria filter would need a new bacteria sample as well.
Challenges in Fluorescence and Luminescence Detection
Bioluminescence emits energy through an enzyme-substrate reaction while fluorescence absorbs a high-energy wavelength and then reflects a lower-energy one. Both of these involve emission spectra which are generated when excited electrons return to lower energy states, releasing energy in the form of light (photons). This process occurs after the electrons have been elevated from their ground state to higher energy levels by absorbing energy which is characterized by the absorption spectra.
Compared to fluorescence imaging, bioluminescence imaging does not need external excitation illumination that can cause problems such as phototoxicity, photobleaching, and autofluorescence from the specimen. A wider variety of spectral information can be obtained from bioluminescence compared to fluorescence because the excitation illumination does not interfere with the spectral analysis. Bacterial luciferase-based reporter is a valuable tool because of its high signal-to-noise ratio and ease of operation. However, there are some challenges to this approach too.
The application of bacterial bioluminescence imaging has been limited because of the low brightness. That seven-times increment of bacterial bioluminescence allows imaging of single E. coli cells with improved spatiotemporal resolution However, image acquisition using this enhanced luminescence still requires about 10 min of exposure time, which would be difficult to capture biological phenomena that change rapidly. Therefore, a higher luminescence intensity is expected to allow observation of biological phenomena that change within minutes or even less.
Our Solution: The LuxB-Venus Fusion
In the study “ Bioluminescence Resonance Energy Transfer (BRET) Imaging in Plant Seedlings and Mammalian Cells”, Venus was fused with LuxA or LuxB at their N- or C-terminus in order to improve brightness and the time taken to see results. This chimeric protein allowed for significant improvement in brightness. LuxB: Venus’ enhanced bioluminescence is owed to BRET, as the fusion of N- and C-terminal Venus to LuxB did not improve the expression levels of their fused proteins. However, the Luminescence spectrum exhibited a high BRET efficiency when Venus was fused to the C-terminus of LuxB.
Without altering the Venus protein, a derivative of YFP, bioluminescence was 5 times higher for LuxB: Venus + LuxA than LuxA + LuxB
BRET Imaging
Bioluminescence Resonance Energy Transfer involves a resonance energy transfer between a bioluminescent donor and a fluorescent acceptor. The donor emits photons intrinsically as it undergoes bioluminescence, additional energy to induce fluorescence excitation is unnecessary. BRET relies on non-radiative energy transfer from the donor to the acceptor when they are in close proximity (typically within 10 nm). For this transfer to be effective, the emission spectrum of the donor must overlap with the absorption spectrum of the acceptor. In the case of the Venus-Lux B fusion mutation, the emission spectrum peak in the wavelength of the luciferase is from 490nm while the fluorescence emission maximum is about 528nm. This overlap allows BRET to happen.
Slight changes in spatial arrangements and orientation can influence BRET greatly. As there is a broader amount of emission wavelengths, the camera is more likely to pick up a clear signal (See hardware page). Influencing protein folding and rigidity through mutations can significantly improve the effectiveness of BRET.
References
Bioluminescence Imaging Vs. Fluorescence Imaging Modalities | GoldBio
Difference Between Emission and Absorption Spectra - Comparison Chart