','.' Luminescent Bacteria: An Introduction

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1 ,_1' ','.' Luminescent Bacteria: An Introduction AuthorsKenneth Nealson Bioluminescence is widespread in living organisms, occurring in such diverse groups as jellyfish, earthworms, insects, squid, fish, algae, and bacteria (1). Bioluminescence is especially common in the unlit ocean depths. A variety of biochemical mechanisms have evolved to emit light at many different wavelengths. The reasons for the emission of light are not always clear, but it often appears to be a recognition device, a way of attracting mates or prey, confusing predators, or promoting schooling in the dark depths of the ocean. While many forms of life produce their own light, others have formed elaborate ectosymbiotic relationships with luminous bacteria that are sequestered within special light organs (Fig. 1 to 4). It is assumed that the hosts provide optimal conditions in these organs for the growth and luminance of the bacteria. These light organs contain a transparent lens and often have a layer of reflective tissue at the rear to maximize directional light emission (Fig. 1 and 2). Light output is often regulated by the host through systems that control the visibility of the light. Fish in the genera Photoblepharon and Anomalops harbor luminous bacteria in special pouches under the eyes (Fig. 1 to 4). The Anomalops control the visibility of light by rolling the light organ downward against a pocket of black tissue, while the Photobfepharon, or flashlight fish. have a fold of black tissue they can draw over the light organ when required (Fig. 3). The marine bacteria Vibrio fischeri has been isolated from the flashlight fish (Fig. 4 and 5). The majority of luminescent bacteria inhabit the ocean. Two genera of marine bacteria, Vibrio and Photobacterium, are among the most abundant luminous bacteria. They can be found in seawater and in the intestinal tract and on the body surfaces of marine animals. These bacteria are easily isolated by incubating a raw marine fish in a cold room for several days, after which the luminous patches that develop can be streaked for isolation on seawater-based agar medium. Their natural light emission is at a maximum near 490 nm. but mutants have been isolated or genetically produced which emit a variety of colors (Fig. 5b). Ught emission by these bacteria, as well as many other luminescent organisms, is mediated by the enzyme luciferase. In the presence of oxygen. FMNH" and a fatly acid aldehyde (R-CHO), luciferase catalyzes the oxidation of the FMNH2 to water, R-COOH, and excited FMN* which decays to ground state by emitting light. The genes for the luciferase system have been cloned from a number of luminescent species into bacteria (4, 5) (Fig. 5a). The only terrestrial luminescent bacterial genus known is Photorhabdus. Members of the Photorhabdus are mostly insect pathogens that exist in a complex symbiotic relationship with a family of insect pathogen nematodes (2, 3). Photorhabdus bacteria, carried by nematodes that invade insect larvae (Fig. 6), are released into the insect hemolymph, where they rapidly grow and kill the insect host. The dead insect subsequently serves as a source of nutrients for

2 nematode reproduction. The bacteria produce pigments that turn the insect carcass a red-orange color (Fig. 6a and b), antibiotics that inhibit the growth of other microbes, and light that causes the carcasses to become luminous (Fig. 6c). Luminescent systems are proving extremely valuable in a variety of molecular biological research areas as visible indicators (reporters) of gene regulation and as a way of following reactions that occur within a living cell (6, 7,8). These images can be used to illustrate to students the concept of luminescent systems. Luminescent Bacteria: IResource Type: Visual: ImagePublication Date: Prior 101/1/2002 Figure 1. A group of flashlight fish, Pholobfephron palpebralus, in the coral reefs of the Gulf of Eilat. The photograph was taken with a strobe light that was reflected off the reflective material at the back of the light organ and reflective areas of the lateral lines on the fish and the coral reef. Figure 2. The flashlight fish, P. pafpebratus. The comma-shaped, light organ is visible under the eye. The photograph was taken with a strobe light that was reflected off the reflective material at the back of the light organ. Figure 3. Light organ and covering lid of P. palpebralus. Close-up views of (a) the light organ and (b) the black membrane used to control light visibility from the light organ.

3 Luminescent Bacteria: II Resource Type: Visual: Image Publication Date: Prior to Fi ure 5 Figure t~""',.. Figure 5. Cloning of Vibn"o fischeri luciferase genes and color variants. (a) Cloned luciferase genes in Escherichia coli (4). Blue-green light emission at -490 nm. (b) V. fischer; isolates exhibiting different color emissions (4). Figure 6. Pigmentation variants of P. luminescens. Three (a, b, and c) color variants of natural isolates of P. luminescens. P. fuminescens isolates collected from infected larvae or Lheir nemalode hosts exhibit a wide range of colony colors. Figure 7. Normal and infected Galleria mellone/la larvae. (a) Normal tan-colored G. me/lonelfa larvae surrounding dead pigmented larvae infected with P. luminescens. (b) G. me/lonella larvae killed by a P. luminescens infection. The red-orange pigment is produced by the bacteria. (c) Dead luminescent G. mellonella larvae photographed by light emission of P. luminescens cells growing in the carcass. To see Figures 1 to 4, go to Luminescent Bacteria I. For more information, see Luminescent Bacteria: an introduction.

The Light-Organ Symbiosis of Vibrio fischeri and the Hawaiian squid, Euprymna sc%pes During Ihe day the bobtailed squid, Euprymna sc%pes, remains buried in the sand of shallow reef flats.

4 The Light-Organ Symbiosis of Vibrio fischeri and the Hawaiian squid, Euprymna sc%pes During Ihe day the bobtailed squid, Euprymna sc%pes, remains buried in the sand of shallow reef flats. As lhe sun sets, the nocturnal animal emerges from its safe hiding place and searches for food. In the moonlit night, the squid would appear as a dark silhouette when it swims through the water and would be easily detected by preditory fish from below. II is Ihoughllhallhe squid camouflages itself by projecting light downward from its light organ. Inside the light organ are luminescent bacteria, Vibrio fischeri, that produce the light. In September 2002, the genome sequence of V. fischeri became available to the public. The strain that was sequenced, ES114, was isolated from the light organ of E. sea/opes in For more information visit the home page of the Vibrio fischeri genome project. Symbionts and host influence each others development. When a juvenile squid hatches from the egg, it does not contain any symbionts (it is aposymbiotic). It needs to acquire the symbionls from the sea water before it can use its light organ. The light organ of such a hatchling has modifications that apparently aid the hatchling in obtaining the symbionts from the multitude of bacteria present in sea water. The most obvious modification are ciliated "arms" that circulate sea water over the pores of the empty light organ crypts (right picture, panel A shows one lobe of the bilobed juvenile light organ). Powered by their fiagelia (left picture, panel A), molile V. fischeri enter the pores of the light organ, move into the empty crypts and begin to grow rapidly. The presence of the symbionts influences the development of the host. The ciliated arms regress (righl picture, panel B) and the bacteria are packed tightly in the crypts (picture below on the right, the arrow points at the symbionts inside the crypt that is lined by epithelial cells). Several hours after the bacteria have entered the light organ, the symbionts change; they loose their nagella. decrease in size and begin to emit light (left picture, panel B). Within a few weeks after the bacteria colonize the squid, the fully developed light organ is present. The light organ possess a silvercolored reflector tissue, a shutter mechanism (the black ink sack), a transparent lens that covers the light organ and a yellow filter that changes the color of the emitted light (shown below left). This allows Ihe squid 10 control the amount of light that it emits.

5 Colonization mutants. One important question is: What genes do the bacteria require to successfully colonize the squid? This can be addressed in Lhis symbiosis because the bacteria are culturable, the symbiosis can be initiated experimentally (a colonization assay was developed), and genetics are available for the bacteria (transposon mutagenesis, allelic exchange, and stable plasmid vectors). Several genes that are important in the symbiotic interaction have already been discovered and, as more knowledge is obtained, this will allow a comparison of these "symbiotic" factors to the "virulence" factors of Vibrio cho/erae, the causative agent of cholera. The mutants that have been tested so far can be grouped into five different classes: (1) initiation mutants, these are mutants that cannot colonize the light organ to a detectable level; (2) accommodation mutants, these are mutants that can grow inside the light organ, but do not reach the same level of colonization as the wild type strain; (3) persistence mutants colonize as well as the wild type strain until after a certain time point they begin 10 decrease in number; (4) competition mutants, these mutants can colonize and persist just as well as Ihe wild type strain when the mutant strain enters the light organ alone, but if the wild type strain is also present, the mutant is outcompeted by the wild type strain and does not reach its normal level of colonization; and (5) none, no defect in symbiotic association was detected using the standard conditions. The microenvironment; Another interesting feature of this symbiotic interaction is that every morning the squid expels 90% of the symbiont population from the light organ. The released symbionts probably serve as the inoculum for the newly hatched squid that need to obtain the symbiont from the sea water. The "venting" is done by releasing a thick paste consisting of cells and a surrounding matrix. This venting behavior can be induced artificially and thus provided an opportunity to investigate the environment inside the crypt. The first surprising discovery was this "environment" was very rich in amino acids and did not just contain simple carbon compounds that would be energetically cheaper for the host to synthesize. This explained why mutants of a symbiotic strain that were auxotrophic for amino acids could proliferate so well. The next study revealed another surprise. In addition to the bacteria, between 1000 and host cells were released from the crypts. These cells resemble macrophage like hemocytes of mollusks. These findings provide a glimpse at the complex nature of bacteria animal symbiosis and also at the novel discoveries laying ahead.

6 Evidence for oxidative stress occuring inside the light organ A common defense mechanism that animals use to protect themselves against pathogenic bacteria is to synthesize and release toxic oxygen radicals. A well-studied example is the oxidative burst and the release of hydrogen peroxide and oxygen radicals inside the phagosomes of neutrophiles. One of the enzymes involved is a myeloperoxidase that catalyses the synthesis of hypohalous acid from halide ions and hydrogen peroxide. Interestingly, one of the most abundant mrna's in the light organ encodes a halide peroxidase that catalyses a similar reaction. So are the symbionts exposed to oxidative stress and if so how can they overcome these adverse conditions? One way for the bacteria to protect themselves against the halide peroxidase would be to remove the substrate, hydrogen peroxide. A catalase mutant that was unable to degrade hydrogen peroxide Lo water was shown to be unable to compete with the wildtype bacterium suggesting a that the expression of the catalase provided the bacteria with a competitive advantage. Experiments analysing the host tissue indicated that when the light organ was colonized with the symbionts less halide peroxidase was synthesized, thus in some way the symbionts appear to influence the gene expression of the host. One interpretation of these experiments is that an animal may respond to pathogenic and cooperative bacteria in a very similar manner and that the magnitude of the response is regulated. Perhaps the lines between being a cooperative symbiont and a pathogen are not as clear cut. Initiation of the symbiosis One fundamental question in symbioses is how the symbionts are transmitted from one generation to the next. In the V. fischeri-e. sc%pes symbiosis it is known that juvenile squids need to aquire the symbiotic bacteria from the seawater. This type of transmission is called horizontal transmission. In the seawater where the squid live, only 500 V. fischer; are found in one milliliter of water and only 2/1 OOOth of a milliliter is pumped by the light organ every second. So 1 V. fischeri bacterium is washed by the opening of the light organ every second, but how do these bacteria find the pores of the light organ? How do can they stop as they swirl by in a rapid water current? Answers to these questions were published in study by Nyholm et al. The squid secretes a mucoid substance that is twirljed between the "arms" of the juvenile light organ and is held just above the pores. V. fischeri becomes entrapped inside the mucus, proliferates there and then moves to pores of the light organ and enters the organ.

NAD + + H 2 O C (+1) 2 e -

NAD + + H 2 O C (+1) 2 e - Bioluminescence Bioluminescence is the biochemical emission of visible light by living organisms. As such, it is a special category of chemiluminescence. (Do not confuse bioluminescence with phosphorescence,