Title: The impact of LuxF on light intensity in bacterial bioluminescence
Journal: Journal of Photochemistry and Photobiology B: Biology
Published: 2020
Background
Bacterial bioluminescence is driven by the lux operon, which encodes the luciferase enzyme system responsible for light emission. While the core gene cluster follows a conserved order (luxCDABEG), some strains, such as Photobacterium leiognathi subsp. mandapamensis, carry an additional gene—luxF. The LuxF protein binds a specific flavin derivative, 6-(3'-(R)-myristyl)-FMN (myrFMN), a side product of the bioluminescent reaction.
Previously, the function of LuxF was unclear. This study used a recombinant E. coli system expressing lux operons with and without luxF to investigate its biological role. The presence of luxF was shown to significantly enhance light emission by preventing the inhibitory effects of myrFMN on luciferase activity. Additionally, the study confirmed that LuxF acts as a scavenger for myrFMN and identified several flavin derivatives with varying fatty acid side chains. This highlights LuxF's regulatory role in optimizing bioluminescence through flavin derivative sequestration.
Materials & Methods
Reagents
Commercial reagents including flavin mononucleotide (FMN) and isopropyl-β-D-thiogalactopyranosid (IPTG) were purchased from Sigma-Aldrich, Thermo Fisher Scientific, or VWR, and used without further purification.
Instrumentation
- In vivo activity assays: Performed using a CLARIOstar plate reader on 24-well black-walled plates with glass bottoms. Measurements (OD650 and bioluminescence) were taken every 10 minutes for 10 hours at 28 °C.
- HPLC: Conducted using a Dionex UltiMate 3000 system and Atlantis dC18 column, with detection at multiple wavelengths (280, 370, 450 nm). Four flavin references (FAD, FMN, riboflavin, myrFMN) were characterized by retention time and absorbance.
- HPLC-MS: Performed with an Agilent 1200 Series LC-MS using ESI in negative scan mode (m/z 100–800).
- NMR: Conducted with a Varian INOVA 500 MHz spectrometer. ^1H and ^13C spectra were obtained in CD3OD with solvent suppression and standard acquisition settings. HSQC spectra were also recorded.
Design & Expression of lux Operon
Multiple expression vectors containing different variants of the lux operon (with or without luxF) were constructed using Gibson Assembly. Vectors were based on sequences from Photobacterium leiognathi subspecies. Constructs were confirmed by sequencing. E. coli Top10 cells were transformed, cultured in LB/kanamycin, and induced with IPTG for light production at 28 °C for ~44 hours.
Extraction of myrFMN and Flavin Derivatives
Cell pellets were lysed using lysozyme and sonication, followed by acidified extraction using ethyl acetate:butanol. The extract was incubated with His-tagged apo-LuxF for flavin binding, then purified via affinity chromatography using a HisTrap column. Final purification included additional solvent extraction and HPLC analysis.
In vivo Bioluminescence Assay
The bioluminescence and OD650 of modified E. coli and bacterial strains were measured simultaneously using a customized script. Cultures were standardized to OD650 = 0.1 in 24-well plates, and bioluminescence was measured over time. Data were processed using MARS software, Excel, and Origin, with blank corrections and statistical analysis (mean, SD) applied.
Results
LuxF Enhances Bioluminescence Intensity
Experiments using E. coli expression systems with and without the luxF gene confirmed that LuxF increases bioluminescence. Cells expressing the lux operon containing luxF showed higher light intensity compared to those without it. This enhancement was observed both in engineered E. coli and in natural strains of Photobacterium—whether or not they naturally contained luxF.
Bioluminescence Measurement System
A plate reader assay measured both light emission and cell density over time. Non-induced controls and background levels confirmed the observed light was due to induced gene expression. Light emission began after approximately 6 hours of incubation.
Formation of myrFMN and Other Flavin Derivatives
The study confirmed that myrFMN (6-(3'-(R)-myristyl)-FMN), an inhibitory side product of the luciferase reaction, is generated in E. coli expressing the lux operon. LuxF likely functions by binding myrFMN, reducing its inhibitory effect on luciferase.
In addition to myrFMN, two new flavin derivatives were discovered using HPLC, mass spectrometry, and NMR. These compounds had similar UV/Vis spectra and appeared to be FMN molecules modified with longer or branched fatty acid chains.
Structural Analysis of Flavin Derivatives
One derivative was further analyzed using NMR, but due to low yields and impurities, complete structural elucidation was not possible. Mass differences suggested the presence of pentadecanoylFMN and stearylFMN, although their exact identities remain unclear.
LuxF Timing and Inhibitor Accumulation
The beneficial effect of LuxF became noticeable 1–2 hours after induction, suggesting that myrFMN accumulation and subsequent inhibition of luciferase is a time-dependent process. LuxF mitigates this inhibition as myrFMN builds up.
Fatty Acid Source Uncertainty
The origin of aldehyde substrates used by luciferase in E. coli is unclear. Unlike in natural hosts, E. coli may not efficiently supply myristoyl-CoA, possibly leading to the formation of alternate FMN derivatives. This raises the possibility of limited interaction between LuxCDE and E. coli's fatty acid synthase (FAS).
HPLC-UV/Vis and HPLC-MS analysis of isolated flavin derivatives as side products of the bioluminescent reaction.
Comparison of the 1H NMR-spectra of alkylated FMN derivative (compound 2) and FMN.
