Symbionts

Symbiosis

The Light Organ Symbiosis of Vibrio Fischeri and the Hawaiian Aquid, Euprymna Scolopes

squid During the day the bobtailed squid, Euprymna scolopes, remains buried in the sand of shallow reef flats. As the 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. It is thought that the 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. scolopes in 1988. 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 symbionts 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 (top right picture, panel A shows one lobe of the bilobed juvenile light organ). Powered by their flagella (lower right picture, panel A), motile 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 by apoptosis (top right 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 flagella, decrease in size and begin to emit light (lower right 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 silver-colored 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 the squid to control the amount of light that it emits.

Colonization Mutants

One important question is: What genes do the bacteria require to successfully colonize the squid? This can be addressed in this 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 cholerae, the causative agent of cholera.

mutants analysis The mutants that have been tested so far can be grouped into five different classes:

initiation mutants, these are mutants that cannot colonize the light organ to a detectable level;
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;
persistence mutants colonize as well as the wild type strain until after a certain time point they begin to decrease in number;
competition mutants, these mutants can colonize and persist just as well as the 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
none, no defect in symbiotic association was detected using the standard conditions.

The following mutants have been tested for symbiotic competence (modified from Ruby, 1996).

Phenotypic Defect	Gene	Colonization Phenotype	Reference
Motility	mot (?)	Initiation	Graf et al.
Flagellum	fla (?)	Initiation	Graf et al.
None Detected	rscS	Initiation	Visick & Skoufos
Amino Acid Synthesis	various	Accommodation	Graf & Ruby
Luminescence	luxA	Accommodation/Persistence	Visick et al.
Autoinducer Synthesis	luxl	Accommodation/Persistence	Visick et al.
Autoinduction	luxR	Accommodation/Persistence	Visick et al.
Siderophore Production	glnD	Persistence	Graf & Ruby
Catalase Synthesis	katA	Competition	Visick & Ruby
None Detected	qsrP	Competition	Callahan & Dunlap
Outer Membrane Protein	ompU	Slow Colonization	Ackersberg et al.
ToxR Synthesis	toxR	None	Reich & Schoolnik
NAD+ -Glycohydrolase	hvnAB	None	Stabb,Reich, & Ruby
Sensitivity to Antimicrobial Peptides	sapABCDF	Accommodation/Competition	Lupp et al.
Regulatory Proteins	litR	Dominates Wild Type	Fidopiastis et al.
Transcriptional Activator	FlrA	Initiation	Millikan & Ruby
Sigma 54	rpoN	Initiation	Wolfe et al.

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 10000 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.

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 to water was shown to be unable to compete with the wild-type 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. scolopes 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. fischeri are found in one milliliter of water and only 2/1000th 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 twirlled 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.

Investigators who study the V. fischeri-E. scolopes symbiosis

The research on the symbiosis of V. fischeri and E. scolopes was pioneered by Margaret J. McFall-Ngai, who works on the animal side, and Edward G. Ruby, who is a microbiologist. Both hold positions at the University of Wisconsin at Madison.
Paul V. Dunlap works on bacterial luminescence and the identification of genes that are co-regulated with light production and therefore might play a role in symbiosis.

Michelle K. Nishigushi works on the evolutionary biology of this symbiosis at the New Mexico State University.

Spencer Nyholm at the University of Connecticut.

Eric Stabb is broadly interested in the genetics, physiology, and signaling pathways of isymbionts. In particularly, we are exploring the regulation of bioluminescence and addressing why bioluminescence enables V. fischeri symbionts to fully colonize the host light organ.

Karen L. Visick is a molecular geneticist at Loyola University in Chicago and was instrumental in developing many of the molecular tools that are presently used to identify genes required by the symbionts to properly function in this symbiosis. Her current research includes a two-component regulator that is involved in the symbiosis.

Cheryl Whistler at the University of New Hampshire.