Autoluminescent Plants

Prospects of obtaining plants glowing in the dark have captivated the imagination of scientists and layman alike. While light emission has been developed into a useful marker of gene expression, bioluminescence in plants remained dependent on externally supplied substrate. Evolutionary conservation of the prokaryotic gene expression machinery enabled expression of the six genes of the lux operon in chloroplasts yielding plants that are capable of autonomous light emission. This work demonstrates that complex metabolic pathways of prokaryotes can be reconstructed and function in plant chloroplasts and that transplastomic plants can emit light that is visible by naked eye

LuxG Is a Functioning Flavin Reductase for Bacterial Luminescence

The 'luxG' gene is part of the 'lux' operon of marine luminous bacteria. 'luxG' has been proposed to be a flavin reductase that supplies reduced flavin mononucleotide (FMN) for bacterial luminescence. However, this role has never been established because the gene product has not been successfully expressed and characterized. In this study, 'luxG' from 'Photobacterium leiognathi' TH1 was cloned and expressed in 'Escherichia coli' in both native and C-terminal His6-tagged forms. Sequence analysis indicates that the protein consists of 237 amino acids, corresponding to a subunit molecular mass of 26.3 kDa. Both expressed forms of 'LuxG' were purified to homogeneity, and their biochemical properties were characterized. Purified 'LuxG' is homodimeric and has no bound prosthetic group. The enzyme can catalyze oxidation of NADH in the presence of free flavin, indicating that it can function as a flavin reductase in luminous bacteria. NADPH can also be used as a reducing substrate for the 'LuxG' reaction, but with much less efficiency than NADH. With NADH and FMN as substrates, a Lineweaver-Burk plot revealed a series of convergent lines characteristic of a ternary-complex kinetic model. From steady-state kinetics data at 4°C pH 8.0, Km for NADH, Km for FMN, and kcat were calculated to be 15.1 μM, 2.7 μM, and 1.7 s‾¹, respectively. Coupled assays between LuxG and luciferases from 'P. leiognathi' TH1 and Vibrio campbellii also showed that LuxG could supply FMNH‾ for light emission in vitro. A 'luxG' gene knockout mutant of 'P. leiognathi' TH1 exhibited a much dimmer luminescent phenotype compared to the native 'P. leiognathi' TH1, implying that 'LuxG' is the most significant source of FMNH‾ for the luminescence reaction in vivo

Biochemistry and Genetics of Bacterial Bioluminescence

Bacterial light production involves enzymes-luciferase, fatty acid reductase, and flavin reductase-and substrates-reduced flavin mononucleotide and long-chain fatty aldehyde-that are specific to bioluminescence in bacteria. The bacterial genes coding for these enzymes, luxA and luxB for the subunits of luciferase; luxC, luxD, and luxE for the components of the fatty acid reductase; and luxG for flavin reductase, are found as an operon in light-emitting bacteria, with the gene order, luxCDABEG. Over 30 species of marine and terrestrial bacteria, which cluster phylogenetically in Aliivibrio, Photobacterium, and Vibrio (Vibrionaceae), Shewanella (Shewanellaceae), and Photorhabdus (Enterobacteriaceae), carry lux operon genes. The luminescence operons of some of these bacteria also contain genes involved in the synthesis of riboflavin, ribEBHA, and in some species, regulatory genes luxI and luxR are associated with the lux operon. In well-studied cases, lux genes are coordinately expressed in a population density-responsive, self-inducing manner called quorum sensing. The evolutionary origins and physiological function of bioluminescence in bacteria are not well understood but are thought to relate to utilization of oxygen as a substrate in the luminescence reaction