The impact of LuxF on light intensity in bacterial bioluminescence.

The enzymes involved in bacterial bioluminescence are encoded in the lux operon with a conserved gene order of luxCDABEG. Some photobacterial strains carry an additional gene, termed luxF, which produces the LuxF protein, whose function and influence on bacterial bioluminescence is still uncertain. The LuxF protein binds the flavin derivative 6-(3'-(R)-myristyl)-flavin mononucleotide (myrFMN), which is generated as a side product in the luciferase-catalyzed reaction. This study utilized an Escherichia coli (E. coli) based lux operon expression system where the lux operons of Photobacterium leiognathi subsp. mandapamensis 27561 or of Photobacterium leiognathi subsp. leiognathi 25521, namely luxCDAB(F)EG, were cloned into a single expression vector. Exclusion of luxF gene from the lux operon enabled novel insights into the role of LuxF protein in light emission. E. coli cultures harboring and expressing the genes of the lux operon including luxF gene emit more light than without luxF gene. Furthermore, isolation of the tightly bound flavin derivative revealed the presence of at least three different flavin derivatives. Analysis by UV/Vis absorption and NMR spectroscopy as well as mass spectrometry showed that the flavin derivatives bear fatty acids of various chain lengths. This distribution of FMN derivatives is vastly different to what was found in bioluminescent bacteria and indicates that the luciferase is supplied with a range of aldehyde substrates in E. coli.

Molecular Mechanisms of Bacterial Bioluminescence.

Bioluminescence refers to the production of light by living organisms. Bioluminescent bacteria with a variety of bioluminescence emission characteristics have been identified in Vibrionaceae, Shewanellaceae and Enterobacteriaceae. Bioluminescent bacteria are mainly found in marine habitats and they are either free-floating, sessile or have specialized to live in symbiosis with other marine organisms. On the molecular level, bioluminescence is enabled by a cascade of chemical reactions catalyzed by enzymes encoded by the lux operon with the gene order luxCDABEG. The luxA and luxB genes encode the α- and ß- subunits, respectively, of the enzyme luciferase producing the light emitting species. LuxC, luxD and luxE constitute the fatty acid reductase complex, responsible for the synthesis of the long-chain aldehyde substrate and luxG encodes a flavin reductase. In bacteria, the heterodimeric luciferase catalyzes the monooxygenation of long-chain aliphatic aldehydes to the corresponding acids utilizing reduced FMN and molecular oxygen. The energy released as a photon results from an excited state flavin-4a-hydroxide, emitting light centered around 490 nm. Advances in the mechanistic understanding of bacterial bioluminescence have been spurred by the structural characterization of protein encoded by the lux operon. However, the number of available crystal structures is limited to LuxAB (Vibrio harveyi), LuxD (Vibrio harveyi) and LuxF (Photobacterium leiognathi). Based on the crystal structure of LuxD and homology models of LuxC and LuxE, we provide a hypothetical model of the overall structure of the LuxCDE fatty acid reductase complex that is in line with biochemical observations.

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria.

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite this similarity, these bacteria show extreme variations in characteristics like growth behavior, intensity of light emission or regulation of bioluminescence. The method presented here is a newly developed assay that combines recording of cell growth and bioluminescent light emission intensity over time utilizing a plate reader. The resulting growth and light emission characteristics can be linked to important features of the respective bacterial strain, such as quorum sensing regulation. The cultivation of a range of bioluminescent bacteria requires a specific medium (e.g., artificial sea water medium) and defined temperatures. The easy to handle, non-bioluminescent standard-research bacterium Escherichia coli (E. coli), on the other hand, can be cultivated inexpensively in high quantities in laboratory scale. Exploiting E. coli by introducing a plasmid containing the whole lux operon can simplify experimental conditions and additionally opens up many possibilities for future applications. The expression of all lux genes utilizing an E. coli expression strain was achieved by construction of an expression plasmid via Gibson cloning and insertion of four fragments containing seven lux genes and three rib genes of the lux-rib operon into a pET28a vector. E. coli based lux gene expression can be induced and controlled via Isopropyl-ß-D-thiogalactopyranosid (IPTG) addition resulting in bioluminescent E. coli cells. The advantages of this system are to avoid quorum sensing regulation restrictions and complex medium compositions along with non-standard growth conditions, such as defined temperatures. This system enables analysis of lux genes and their interplay, by the exclusion of the respective gene from the lux operon, or even addition of novel genes, exchanging the luxAB genes from one bacterial strain by another, or analyzing protein complexes, such as luxCDE.

Evidence for the generation of myristylated FMN by bacterial luciferase.

The genes responsible for the light production in bioluminescent bacteria are present as an operon, luxCDABEG. Many strains of Photobacteria carry an additional gene, termed luxF. X-ray crystallographic analysis of LuxF revealed the presence of four molecules of a flavin derivative, i.e. 6-(3'-(R)-myristyl) flavin adenine mononucleotide (myrFMN) non-covalently bound to the homodimer. In the present study, we exploited the binding of myrFMN to recombinant apo-LuxF to explore the occurrence of myrFMN in various bioluminescent bacteria. MyrFMN was detected in all bacterial strains tested including Vibrio and Aliivibrio indicating that it is more widely occurring in bioluminescent bacteria than previously assumed. We also show that apo-LuxF captures myrFMN and thereby relieves the inhibitory effect on luciferase activity. Thus our results provide support for the hypothesis that LuxF acts as a scavenger of myrFMN in bioluminescent bacteria. However, the source of myrFMN remained obscure. To address this issue, we established a cofactor regeneration enzyme-catalyzed cascade reaction that supports luciferase activity in vitro for up to 3 days. This approach enabled us to unambiguously demonstrate that myrFMN is generated in the bacterial bioluminescent reaction. Based on this finding we postulate a reaction mechanism for myrFMN generation that is based on the luciferase reaction.

Synthesis of α,ß-unsaturated aldehydes as potential substrates for bacterial luciferases.

Bacterial luciferase catalyzes the monooxygenation of long-chain aldehydes such as tetradecanal to the corresponding acid accompanied by light emission with a maximum at 490nm. In this study even numbered aldehydes with eight, ten, twelve and fourteen carbon atoms were compared with analogs having a double bond at the α,ß-position. These α,ß-unsaturated aldehydes were synthesized in three steps and were examined as potential substrates in vitro. The luciferase of Photobacterium leiognathi was found to convert these analogs and showed a reduced but significant bioluminescence activity compared to tetradecanal. This study showed the trend that aldehydes, both saturated and unsaturated, with longer chain lengths had higher activity in terms of bioluminescence than shorter chain lengths. The maximal light intensity of (E)-tetradec-2-enal was approximately half with luciferase of P. leiognathi, compared to tetradecanal. Luciferases of Vibrio harveyi and Aliivibrio fisheri accepted these newly synthesized substrates but light emission dropped drastically compared to saturated aldehydes. The onset and the decay rate of bioluminescence were much slower, when using unsaturated substrates, indicating a kinetic effect. As a result the duration of the light emission is doubled. These results suggest that the substrate scope of bacterial luciferases is broader than previously reported.