Bio-Luminescence

Bioluminescence in Bacteria

Ever wonder why some bacteria glow? Or what makes them luminesce? Bioluminescence has been of interest for many scientists. Bioluminescence refers to the visible light that is being emitted from an organic organism, created by a catalytic enzyme within the organism. This enzyme is called luciferase, and the substrates that are involved are called luciferins. The bioluminescent process is different from other organisms which is why it is of interest for scientists to study. The structures of luciferase differ between species of bacteria, and it’s difficult to study some structures, as I will talk about later on.

The consensus is that all luminous bacteria arose from one common ancestor, which is believed to have arose in Vibrionaceae lineage and gave rise to Aliivibrio, Photobacterium, and Vibrio. This idea came from the fact that there is a similarity in the lux gene sequence among them.

Luminous Bacteria Species

There are many species of luminescent bacteria, the majority of which inhabit marine environments while the others inhabit either terrestrial or freshwater environments. Most luminescent bacterial species have a symbiotic relationship with a host organism, such as squids, fish, nematodes, etc., but many species are capable of living without a host organism. An example of a symbiotic relationship made well-known by the movie Finding Nemo is with the Angler fish. This fish keeps the luminescent bacteria in a rod that extends out from its head, and the light emitted from the bacteria attracts prey. The bacteria, in return, gets nourishment from the Angler fish (Figure 1). Due to their symbiotic relationships, these bacterium cannot be separated from their host and cannot be cultured in a lab for extensive studies.

At least 30 species of bacteria carry the lux gene, which makes visible light (Table 1). All of these bacteria are Gram-negative and belong to the phylogenies of Gammaproteobacteria: Vibrionaceae, Enterobacteriaceae, and Shewanellacaea. Some members within those families do not illuminate, such as some members of the Vibrionaceae that lack lux genes (Dunlap, 2014). It should also be noted that only a few species of the Enterobacteriaceae and Shewanellacaea families have lux genes and are luminous.

Most luminous bacteria are further classified in three genera: Photobacterium, Vibrio, and Photorhabdus. Bacteria in marine environments tend to fall in the Photobacterium or Vibrio genera, while the terrestrial bacterium fall in the Photorhabdus genus. The marine bacteria differ in that Vibrio can live without a host if it wants to, whereas Photobacterium are almost always light organ symbionts (Lin & Meighen, 2009).

Luminous bacterium can be parasitic; Photobacterium and Vibrio infect crustacea, and Photorhabdus luminescens infect terrestrial insects (Lin & Meighen, 2009). Free-living bacteria can be found on the skin or in the gut of marine animals, and be considered non-specific parasites. Inside of the marine host’s gut, “…the extra-cellular chitinase produced on the cell wall of all luminous bacteria facilitate the decomposition of the ingested chitin (Lin & Meighen, 2009). The expression of luminescence is dependent on cell density; single bacterium that live free in the water are not going to emit as much light as bacteria that are in symbiotic relationships or that are grown in a culture (Meighen, 1993).

All luminous bacteria are the same in that they are all rod-shaped microorganisms that have flagellate, allowing them to move. These bacterium are also all gram-negative and are adapted to survive when molecular oxygen is limited. They all differ in that they have specific conditions in which they can thrive, such as nutritional requirements, temperature, pH, etc, which affects the reaction kinetics of light generation.

Biochemistry

In general, the bioluminescence reactions are either classified as direct or indirect. They are direct when, “…the chemical exothermicity is directly released into the product as it is formed,” and indirect when, “…a high-energy intermediate transferring its energy to a molecule not involved in chemistry” (Lee, 2016). Sometimes the indirect method is also called “sensitized” bioluminescence. Bacterial bioluminescence is an indirect mechanism.

The luminescence in bacteria is catalyzed with a unique luciferase that is approximately 80kD with homology to long-chain alkane monooxygenases (Lin & Meighen, 2009). To form a blue-green light, luciferase catalyzes the following reaction:

FMNH2 + O2 + RCHO FMN + H2O + RCOOH + light (90nm)

(Dunlap, 2014) (Figure 2).

According to John Lee, in an aqueous solution FMNH2 oxidizes rapidly to FMN in the presence of dissolved oxygen, but when the bacterial luciferase catalyst was added, they don’t react as fast (2016). Hastings and Gibson proposed a series of intermediates in a linear mechanism with direct production of an excited state (Lee, 2016):

E-FMNH2 (I) + O2 E-FMN-H2O2 (II)

II + RCHO FMNH-OOCH(OH)R (III)

III FMNH-4a-OH (IV) hV + FMN + H2O

With the exception of intermediate II, the other intermediates are too short lived to identify the structures. The proposed structures are based on spectral properties. 25 years after it was proposed, intermediate II was proven using C-NMR to be 4a-hydroperoxy-FMNH: luciferase. Knowing that, it is reasonable to think that intermediate III is luciferase bound 4a-peroxyhemiacetal-FMNH. Observing that the final products are FMN and H2O, it is assumed that those products came from dehydration, so working backwards, that leaves 4a-hydroxy-FMNH: luciferase as intermediate IV, otherwise known as luciferase-hydroxyflavin (Lee, 2016).

Each bacteria has a different enzyme, for example: VH-luciferase in Vibrio harveyi, VF-luciferase in Vibrio fischeri, PL-luciferase in Photobacterium leiognathi, and PP-luciferase in Photobacterium phosphoreum (Lee, 2016). Their spectra broad bands tend to range between 472 to 505nm, which gives off a blue-green color. There is an exception to this with A. sifiae which maximum at 545nm and emits a yellow color. Color emission indicates, “…that the energy level of the photon that was produced when the excited electron on the flavin chromophore returns to the ground state” (Lin & Meighen, 2009).

Lux Genes

In order to emit light for long periods at a time, substrates constantly have to be supplied to the luciferase equation, which means substrates are a limiting factor. Because of this, the light emission must be maintained by various enzymes that can replenish the aldehyde substrate. Those enzymes are coded on the lux operon. Each lux gene codes for different aspects that come together in a specific order to create light emission. The luxA and luxB genes are the subunits of luciferase, luxC, luxD, and luxE are the subunits for fatty acid reductase (r, s, and t polypeptides) that synthesizes and recycles aldehyde substrates for luciferase, and the luxG is the subunit for flavin reductase (Dunlap, 2014).

Present in the lux operons of Photobacterium is the luxF gene, found between the luxB and luxE genes, which codes for nonfluorescent flavoprotein (Figure 3). This gene is undecided on whether it is really needed for the production of light, but it may serve another purpose for Photobacterium such as functioning as an inhibitory side product of the luciferase reaction (Dunlap, 2014). In the Photobacterium’s lux operon, genes can synthesis riboflavin (ribEBHA) by connecting ribEBHA to the end of luxCDADFEG, thus making a lux-rib¬ operon. Due to the fact that there is not a regulatory stopping site between the lux and rib genes means that they must be expressed from the same promoter starting at luxC (Dunlap, 2014). Many strains of P. leiognathi carry two lux-rib operons, both of which are functional. When comparing these operons in this species to other species’ lux and rib operons, it has been noticed that P. leiognathi’s lux-rib1 and lux-rib2 are more closely related. After analysis, it has been concluded that the lux-rib2 didn’t arise due to interspecies horizontal gene transfer or a gene duplication event, so it is believed that it was created from an intraspecies horizontal gene transfer, either from a lineage that had not been discovered or one that had gone extinct (Dunlap, 2014).

Aliivibrio species have two regulatory genes called luxI and luxR (Figure 3), which are in control of the lux operon transcription mechanism. The luxI gene codes for an acyl-homoserine lactone synthase and luxR codes for a receptor protein that interacts with luxI in order to start the transcription mechanism. LuxI and luxR do affect the light production of some bacteria, and each bacteria that has these genes requires different things in order to produce high luminescence.

Conclusion

Studying bioluminescence in bacteria is important for the understanding of how bioluminescence works in nature as well as potentially using it to further understand other complex biological phenomena. All bacteria have the same substrates for their bioluminescent reactions which include FMNH2,O2, and long fatty aldehydes. They are also catalyzed by luciferase (luxAB) with the fatty acid reductase complex (luxCDE). These together synthesize the long chain aldehyde substrate of tetradecanal (Lin & Meighen, 2009). Although much has been learned about bioluminescence in bacteria, such as the molecular mechanisms, structures and regulation of the lux enzymes, there is still much to learn.