BACTERIA THAT GLOW IN THE DARK

Introduction
Perhaps the first studies of nematode-originated diseases were recorded by the physician Aldrovandi about 400 years ago (5). Aldrovandi also reported on luminescence in insects, but it wasn't until the mid-1970s that Poinar provided the first documentation of a parisitic nematode that employed a luminescent bacteria, Xenorhabdus luminescens, to invade insects as part of its life cycle (12). The Xenorhabdus genus is comprised of gram-negative, facultative anaerobes, and the only terrestrial members among three genera of luminescent bacteria (the others are marine).

Description of Life Cycle
Several species among two families of nematodes are each associated with a single bacterial species located in their intestines during their non-feeding infective stage. The nematodes enter the insect through the mouth or spiracles and trachea and penetrate the gut wall where they enter the hemocoel. In response to factors in the hemolymph, the nematode releases the bacterial symbionts, which rapidly grow in the hemolymph. After 20 hrs. of infection, light in the red spectrum is detectable and continues increasing in intensity for approximately 4 hrs. Without the bacteria, the nematode cannot complete its life cycle, because the bacteria provide nutrients through the use of extracellular proteases and lipases and inhibit the growth of other bacteria by producing antibiotics. Thus, the insect does not putrefy during the two-week stage when the nematodes feed and produce larvae. These bacteria do not live saprophytically in the soil. This is interesting since several clinical cases of luminescent wounds in humans have resulted in the isolation of bacteria identified as Xenorhabdus luminescens, although the temperature optimum of 37 C suggests an evolutionary association with warm-blooded hosts (2). Many early accounts suggest these wounds heal quickly, which is presumably due to the antibiotic production of the bacteria.

Purpose of Luminescence
As with many marine examples, the reasons for luminescence in Xenorhabdus are not clear. It apparently is important for the organism since the enzyme responsible, luciferase, accounts for as much as 5% of the cellular protein and 20% of the total oxygen consumption. In some strains the energy committed to bioluminescence is 10% of the total metabolism (9). Furthermore, nucleotide sequencing of the alpha (active) subunit of luciferases among the three genera have shown high conservation of this gene, as well as a close homology to Vibrio (8), (16).
It has been suggested that the light produced at night, as well as the red anthraquinone pigment by day, could attract other insects, or even animals, to feed on the insect cadavers, thus ensuring continuation of the nematode life cycle. However, research into the duration of the luminescent phase suggests the cadavers would be very dim by the time the infective-stage nematodes are ready to emerge (see Fig. 1) (13). Furthermore, such a function does not seem likely in the case of luminous wounds in humans.
Another suggestion is that the luciferase pathway can serve as a terminal oxidase, so that if the cytochrome pathway is impeded by such conditions as low oxygen or low iron, an electron sink would be available to continue aerobic metabolism (7). This idea is supported by evidence that luciferase in Photobacterium fischeri and Beneckea harveyi has an affinity for O2 far exceeding that of cytochrome oxidase (18). This would be a distinct advantage in situations where iron starvation, such as that caused by transferrin, is a threat: host organisms are known to produce transferrin as a defense mechanism. However, there is strong evidence that suggests luciferase itself is a heme-dependent enzyme (17), and therefore, in my view, would most likely be affected as well. However, it has also been postulated thermodynamically that the existence of the generation of ATP is possible at the level of NADH/Flavin oxidation-reduction (7).

Biochemistry The bioluminescent reaction among bacteria has been studied extensively. This has been facilitated by the slow turnover of the luciferase enzyme. The reaction can be expressed:
FMNH2 + RCHO + O2 ---------> FMN + H2O + RCOOH + hv (490 nm)
The substrates, a NADH-reduced riboflavin phosphate (FMNH2), and a long chain fatty aldehyde, are oxidized in the presence of oxygen and the enzyme and the resulting complex interacts with aldehyde as a monooxygenase to form an excited but highly stable intermediate, which decays slowly, resulting in the emission of light (8). It could be argued that there is some evolutionary connection between the cytochrome pathway and the luciferase system. Bacterial luciferase provides a path for electrons at the point where oxygen oxidizes flavin, and, in fact, cytochrome c1, cytochrome P-450, and a b-type cytochrome have been detected in luciferase of Photobacterium fischeri (17). Seen this way, luciferase may function as a terminal oxidase, as mentioned earlier, and accomplish metabolic redox balance via NADH without expending ATP (see Fig. 2) (6).
Nucleotide sequencing data indicates the luciferase of Xenorhabdus is closely related to those of other known luminous bacteria.(6) Bioluminescence (lux) genes are therefore similar for all luminescent bacteria. Five closely linked structural genes are involved: lux A and lux B are responsible for the alpha and beta subunits of luciferase, and lux C, D, and E encode the fatty acid reductase complex required for the generation and recycling of fatty acid to aldehyde. (9) A few other lux genes have been reported for the marine species, presumably coding for proteins required by the marine environment. In addition, a lux I gene is part of the operon and is believed to control autoinduction of lux gene expression. The existence of this gene has only been suggested for Xenorhabdus so far. This would ensure that luminescence would only develop when population density reaches a certain level, and therefore would suggest that the luminescent event itself is important to the organisms.

Significant Uses of Xenorhabdus
Biological Pest Control The use of nematodes for biological pest control began in the 1930s. Infective nematodes possess both chemoreceptors and mobility, allowing them to find pests effectively, and remain infective for months. The discovery of Xenorhabdus appeared promising because it kills the host so quickly that the nematodes have not had to adapt to a specific host life cycle, and thus can be very effective against a large number of insects, including Japanese beetles, root and vine weevils, fire ants, mole crickets, cutworms, and potato beetles (4).
Unfortunately, field evaluations have produced mixed results. This has been largely due to a lack of understanding of the types of conditions required to facilitate infestation, including abiotic factors such as soil moisture, temperature and solar radiation, as well as biotic factors such as nematode strain, and host stage and defenses. On this last point, it has been found that insects have devised many behavioral counter measures. For instance, nematodes seek out their hosts by responding to carbon dioxide released by the insect. Some pupae release carbon dioxide in bursts, with a 7 hr period between bursts. Other insects move around rapidly, have high defecation rates, wall off other infected insects, and push nematodes out of their mouths with their legs (4). Despite these problems, there remains much optimism as to the future use of these parasitic organisms to replace chemical insecticides, and their use is increasing rapidly.

Production of Antibiotics It was discovered very early that these bacteria produce a wide variety of antibiotics (10). This is not unusual. Many bacteria species have evolved this ability to give them an advantage over other organisms. Screening for new antibiotics is a large important and industry, but potential clinical antibiotics often fail to live up to their promise. It is still noteworthy to mention, however, that a wide range of both of gram-positive and gram-negative bacteria, as well as yeast, were found to be sensitive to all Xenorhabdus species studied, and that each of the Xenorhabdus species themselves were sensitive to each other (See Fig. 3) (1). The inhibitory compounds were purified and identified as either hydroxyl and acetoxyl-bearing indole derivatives, probably produced via a tryptophan pathway, or hydroxystilbene derivitives arising via polyketide pathways (3).

Light Emission Measurements Creative use of the luminescent qualities of this bacteria have been used both at the organismal and molecular levels. An example of the former involves research into aquatic food webs. It has not been until the last decade or so that bacteria have been recognized for their important contribution to the food chain. Bacterial production, in fact, accounts for at least 20% of the primary production in aquatic systems (12). Microflagellate phagotrophs are important consumer of aquatic bacteria, but information has been lacking concerning the relationship between these two organisms.
Electronic particle counter (EPC) techniques and/or microscopic methods have been used to measure such things as protozoan feeding rates to clearly define the food webs. These methods are associated with a host of problems. The ability of Xenorhabdus to survive in freshwater has allowed such activities as feeding rates to be determined with greater ease by means of photometer readings of bacterial light emission (14). The relatively large size of Xenorhabdus, however, has necessitated the use of smaller bacteria, since some microflagellates cannot feed on them. This has been facilitated by such experiments as the integration of the lux gene responsible for luminescence into the genome of E. Coli from Vibrio, and, recently, Xenorhabdus . (3) Light emission detection is relatively sensitive and easy to use and has been employed in studies of cellular metabolic functions. It has also led to the use of cDNA of luciferase genes as reporters of gene expression and regulation. For instance, the introduction of the lux phenotype into different bacterial species provides a convenient method for rapidly screening for the presence of specific bacteria in both lab and field experiments. This could be used as an early warning detection system for contaminating bacteria. Furthermore, agents that kill bacteria can be studied by monitoring light emission breakdown. In addition, enzyme assays utilizing light emission in both prokaryotes and eukaryotes will probably increase in the future. Although any of the three genera of luminescent bacteria could contribute lux genes for this purpose, the Xenorhabdus lux system may turn out to be the system of choice because it turns out that, among other favorable properties, its thermal stability is the highest of any of the lux systems studied. (t1/2 > 3 hr at 45 C) (8).

Future Work Although field research experiments utilizing the Xenorhabdus system as an agricultural pest control have had mixed results, the future looks very promising. This is because many successes have been recorded, while the sources of error in the less successful attempts do not appear to be insurmountable. As a greater understanding grows of the particular conditions for optimum infection of pests, the reasons cited that render this a good system should create less dependency on chemical methods of control.
Photometric measurement methods have several advantages over other tedious and error-prone techniques. Utilization of the light emission characteristics of luminescent bacteria in general, and, perhaps, Xenorhabdus in particular (due to its unique thermal stability), will certainly take a useful role in research. This includes studies conducted at the organismal level, but certainly at the molecular level as well. Results should improve since it has been discovered that certain combinations of alpha and beta subunits among genera yield very high luciferase activity (16). In addition, continuous light emission can be accomplished in some assays by adding flavin reductases and NAD(P)H (8). Coupling enzyme assays, metabolic functions, or gene expression to the bacterial luminescence reaction is certainly just the beginning of the future exploitation of the resources this fascinating organism provides.

References l. Akhurst, R. J. 1982. Antibiotic activity of Xenorhabdus spp., bacteria symbiotically associated with insect pathogenic nematodes of the families Heterorhabditidae and Steinernematidae. J. Gen. Micro. 128: 3061 -3065.

2. Colepicolo, Pio, Ki-Woong Cho, George Poinar, and J. Hastings. 1989. Growth and Luminescence of the bacterium Xenorhabdus luminescens from a human wound. Applied and Environ. Microbio., 55: 2601-2606.

3. Frackman, Susan, Michael Anhalt, and K. Nealson. 1990. Cloning, organization, and expression of the bioluminescence genes os Xenorhabdux luminescens. 1990. J. of Bacteriology, 172:5767-5773.

4. Gaugler, Randy. 1988. Ecological considerations in the biological control of soil-inhabiting insects with entomopathogenic nematodes. Agric., Ecosyst. and Environ., 24: 351-360.

5. Harvey, E. N., A History of Luminescence, Vol. 4, 1957. The American Philosophical Society.

6. Hastings, J. W. 1983. Bilogical diversity, chemical mechanisms, and the evolutionary origins of bioluminescent systems. J. Mol. Evol. 19:309-321.

7. Makemson, John C. and J. Hastings. 1986. Luciferase-dependent growth of cytochrome-deficient Vibrio harveyi. 1986. FEMS Microbio. Ecology 38:79-85.

8. Meighen, Edward A. 1991. Molecular biology of bacterial bioluminescence. Microbio. Rev. 55:123-142.

9. Nealson, K. H., and J. Hastings. 1979. Bacterial bioluminescence: its control and ecological significance. Microbio. Rev. 43:496-518.

10. Paul, Valerie J., Sally Frautschy, William Fenical and Kenneth Nealson. 1981. Antibiotics in microbial ecology. Isolation and structure assignment of several new antibacterial compounds from the insect-sybiotic bacteria Xenerhabdus spp. 1981. J. Chem. Ecol. 7:589-597.

11. Poinar, George, and Gerard Thomas. 1980. Growth and luminescence of the symbiotic bacteria associated with the terrestrial nematode, Heterorhabditis Bacteriophora. Soil Biol. Biochem. 12:5-10.

12. Porter, Karen, Evelyn Sherr, Barry Sherr, Michael Pace, and Robert Sanders. 1985. Protozoa in planktonic food webs. J. Protozool. 32:409-415.

13. Richardson, William, Thomas Schmidt, and Kenneth Nealson. 1988. Identification of an anthraquinone pigment and a hydroxystilbene antibiotic from Xenorhabdus luminescens. Appl. and Environ. Microbiol. 54:1602-1605.

14. Seale, Dianne B., Martin Boraas, Dale Holen, and Kenneth Nealson. 1990. Use of bioluminescent bacteria, Xenorhabdus luminiscens, to measure predation on bacteria by freshwater microflagellates. FEMS Microbio. Ecol. 73:31-40.

15. Akhurst, R. J. 1980. Morphological and Functional Dimorphism in Xenorhabdus spp., bacteria symbiotically associated with the insect pathogenic nematodes Neoaplectana and Heterorhabditis. J. Gen. Microbio. 121:303-309.

16. Schmidt, Thomas, Karl Kopecky, and Kenneth Nealson. 1989. Bioluminescence of the insect pathogen Xenorhabdus luminescens. Appl. and Environ. Microbio. 55:2607-2612.

17. Danilov, V. S., and Yu. A Malkov. 1986. Eff of heme ligands of CO and cyanide on the reaction catalyzed by bacterial luciferase. Biokhimiya, 51:782-787.

18. Shumikhin, Danilov, Malkov, and N. S. Egorov. 1980. Biokhimiya, 45:1576-1581.

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