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

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 (right picture, panel A shows one lobe of the bilobed juvenile light organ). Powered by their flagella (left 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 (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 (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 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.

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 to decrease in number; (4) 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 (5) 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.

Selected References:

Reviews:

Visick, K.L. 2009. An intricate network of regulators controls biofilm formation and colonization by Vibrio fischeri. Mol Microbiol. (in press)

McFall-Ngai, M., 2008. Hawaiian bobtail squid. Curr Biol. 18:R1043-1044.

Visick, K.V. and M.J. McFall-Ngai. 2000. An exclusive contract: specificity in the Vibrio fischeri-Euprymna scolopes partnership. J. Bacteriol. 182:1779-1787.

McFall-Ngai, M.J. 1999. Consequences of evolution with bacterial symbionts: insights from the squid-Vibrio association. Annu. Rev. Ecol. Syst. 30:235-256.

Ruby, E. G. 1999. Ecology of a benign "infection": colonization of the squid luminous organ by Vibrio fischeri. p. 217-231. In: E. Rosenberg (ed.), Microbial ecology and infectious disease. ASM Press, Washington, D.C.

Ruby, E. G. and M. J. McFall-Ngai. 1999. Oxygen-utilizing reactions and symbiotic colonization of the squid light organ by Vibrio fischeri. Trends Microbiol. 7:414-20.

McFall-Ngai, M.J., and E.G. Ruby. 1998. Bobtail squid and their luminous bacteria: when first they meet. BioScience 48:257-265.

Ruby, E.G., and K.-H. Lee. 1998. The Vibrio fischeri-Euprymna scolopes light organ association: current ecological paradigms. Appl. Environ. Microbiol. 64:805-812.

Ruby, E.G. 1996. Development of a cooperative, bacterial-animal symbiosis: the Vibrio fischeri-Euprymna scolopes light organ symbiosis. Annu. Rev. Microbiol. 50:591-624.

Selected Research Publications:

Troll, J.V., D.M. Adin, A.M. Wier. N. Paquette, N. Silverman, W.E. Goldman, F.J. Stadermann, E.V. Stabb and M.J. McFall-Ngai. 2009. Peptidoglycan induces loss of a nuclear peptidoglycan recognition protein during host tissue development in a beneficial animal-bacterial symbiosis. Cell Microbiol.11:1114-1127.

Tong, D., N.S. Rozas, T.H. Oakley, J. Mitchell. N.J. Colley and M.J. McFall-Ngai. 2009. Evidence for light percepion in a bioluminescwnt organ. Proc Natl Acad Sci U S A.106:9836-9841

Nyholm, S.V., J.J. Stewart, E.G Ruby and M.J. McFall-Ngai. 2009. Recognition between symbiotic Vibrio fischeri and the haemocytes of Euprymna scolopes. Environ Microbiol. 11:483-493.

Mandel, M.J., M.S. Wollenberg, E.V. Stabb, K.L. Visick and E.G Ruby. 2009. A single regulatory gene is sufficient to alter bacterial host range. Nature. 458: 215-218

Wollenberg, M.S. and E.G. Ruby. 2009. Population structure of Vibrio fischeri within the light organs of Euprymna scolopes squid from Two Oahu (Hawaii) populations. Appl Environ Microbiol. 75:193-202.

Hussa, E.A., C.L. Darnell and K.L Visick. 2008. RscS functions upstream of SypG to control the syp locus biofilm formation in Vibrio fischeri. J Bacteriol. 190:4576-4583.

Jones. B.W., A. Maruyama, C.C Ouverney and M.K. Nishiguchi. 2006. Population structure between environmentally transmitted vibrios and bobtail squids using nested clade analysis. Mol Ecol. 15:4317-4329.

Koropatnick, T. A., J. T. Engle, M.A. Apicella, E. V. Stabb, W. E. Goldman, M.J. McFall-Ngai. 2004. Microbial factor-mediated development in a host-bacterial mutualism. Science. 306:1186-1188.

Kimbell, J., and M.J. McFall-Ngai. 2004. Symbiont-induced changes in host actin during the onset of a beneficial animal-bacterial association. Appl. Environ. Microbiol. 70:1434-1441.

DeLooney-Marino, C.R., A. J. Wolfe, K. L. Visick. 2003. Chemoattraction in Vibrio fischeri to serine, nucleosides, and N-acetylneuraminic acid, a component of squid light-organ mucous. Appl. Environ. Microbiol. 69:7527-7530.

Nishiguchi, M. K. and V. S. Nair. Evolution of symbiosis in the Vibrionaceae: a combined approach using molecules and physiology. Int. J. Syst. Evol. Microbiol. 54:2019-2026.

Fidopiastis PM, Miyamoto CM, Jobling MG, Meighen EA, Ruby EG. 2002. LitR, a new transcriptional activator in Vibrio fischeri, regulates luminescence and symbiotic light organ colonization. Mol. Microbiol. 45:131-43.

Millikan, D. S. and E. G. Ruby. 2002. Alterations in Vibrio fischeri motility correlate with a delay in symbiosis initiation and are associated with additional symbiotic colonization defects. Appl. Environ. Microbiol. 68:2519-2528.

Nishiguchi MK. 2002.Host-symbiont recognition in the environmentally transmitted sepiolid squid-Vibrio mutualism. Microb. Ecol. 44:10-8.

Ackersberg, F., C. Lupp, B. Feliciano and E. G. Ruby. 2001. Vibrio fischeri outer membrane protein OmpU plays a role in normal symbiotic colonization. J. Bacteriol. 183:6590-6597.

Stabb, E. V., K. A. Reich and E. G. Ruby. 2001. Vibrio fischeri genes hvnA and hvnB encode secreted NAD+-Glycohydrolyases. J. Bacteriol. 183:309-317.

Visick, K. L. and L. M. Skoufos. 2001. Two-component sensor required for normal symbiotic colonization of Euprymna scolopes by Vibrio fischeri. J. Bacteriol. 183:835-842.

Callahan, S. M. and P. V. Dunlap. 2000. LuxR- and acyl- homoserine- lactone- controlled non- lux genes define a quorum-sensing regulon in Vibrio fischeri. J. Bacteriol. 182:2811-2822.

Clays, M. F. and P. V. Dunlap. 2000. Aposymbiotic culture of the sepiolid squid Euprymna scolopes: role of symbiotic bacterium Vibrio fischeri in host growth, development, and light organ morphogenesis. J. Exp. Zool. 286:280-296.

Foster, J. S., M. A. Apicella, and M. J. McFall-Ngai. 2000. Vibrio fischeri lipopolysaccharide induces developmental apoptosis, but not complete morphogenesis, of the Euprymna scolopes symbiotic light organ. Dev. Biol. 226:242-254.

Graf, J. and E. G. Ruby. 2000. Novel effects of a transposon insertion in the Vibrio fischeri glnD gene: defects in iron uptake and symbiotic persistence in addition to nitrogen utilization. Mol. Microbiol. 37:168-179.

Lemus, J. D. and M. J. McFall-Ngai. 2000. Alterations in the proteome of the Euprymna scolopes light organ in response to symbiotic Vibrio fischeri. Appl. Environ. Microbiol. 66(9): 4091-4097.

Nishiguchi, M. K. 2000. Temperature affects species distribution in symbiotic populations of Vibrio spp. Appl. Environ. Microbiol. 66(8):3550-3555.

Nyholm, S.V., E. V. Stabb, E. G. Ruby and M. J. McFall-Ngai. 2000. Establishment of an animal-bacterial association: Recruiting symbiotic vibrios from the environment. PNAS 97(18):10231-10235.

Visick, K. L., J. Foster, J. Doino, M. McFall-Ngai and E. G. Ruby. 2000. Vibrio fischeri lux genes play an important role in colonization and development of the host light organ. J. Bacteriol. 182:4578-4586.

Small, A. L. and M. J. McFall-Ngai. 1999. Halide peroxidase in tissues that interact with bacteria in the host squid Euprymna scolopes. J. Cell. Biochem. 72:445-457.

Fidopiastis, P., S. von Boletzky, and E.G. Ruby. 1998. A new niche for Vibrio logei, the predominant light organ symbiont of squids of the genus Sepiola. J. Bacteriol. 180:59-64.

Graf, J. and E. G. Ruby. 1998. Host-derived amino acids support the proliferation of symbiotic bacteria. Proc. Natl. Acad. Sci. USA. 95:1818-1822.

LaMarcq, L.H., and M.J. McFall-Ngai. 1998. Induction of a gradual, reversible morphogenesis of its host's epithelial brush border by Vibrio fischeri. Infect. Immun. 66:777-785.

Nyholm, S. V. and M. J. McFall-Ngai. 1998. Sampling the light-organ microenvironment of Euprymna scolopes: description of a population of host cells in association with the bacterial symbiont Vibrio fischeri. Biol. Bull. 195:89-97.

Visick, K. L., and E. G. Ruby. 1998. The periplasmic, group III catalase of Vibrio fischeri is required for normal symbiotic competence, and is induced both by oxidative stress and by approach to stationary phase. J. Bacteriol. 180: 2087-2092.

Weis, W.M., A.L. Small, and M.J. McFall-Ngai. 1997. A peroxidase related to the mammalian antimicrobial protein myeloperoxidase in the Euprymna-Vibrio mutualism. Proc. Natl. Acad. Sci. USA. 93:13683-13688.

Boettcher, K.J., E.G. Ruby, and M.J. McFall-Ngai. 1996. Bioluminescence in the symbiotic squid Euprymna scolopes is controlled by a daily biological rhythm. J. Comp. Physiol. A. 179:65-73.

Visick, K. L. and E. G. Ruby. 1996. Construction and symbiotic competence of a luxA-deletion mutant of Vibrio fischeri. Gene 175:89-94.

Boettcher, K. J., and E. G. Ruby. 1995. Detection and quantification of Vibrio fischeri autoinducer from symbiotic squid light organs. J. Bacteriol. 177:1053-1058.

Doino, J.A., and M. J. McFall-Ngai. 1995. A transient exposure to symbiosis-competent bacteria induces light-organ morphogenesis in the host squid. Biol. Bull. 189:347-355.

Graf, J., P. V. Dunlap and E. G. Ruby. 1994. Effect of transposon-induced motility mutations on colonization of the host light organ by Vibrio fischeri. J. Bacteriol. 176:6986-6991.

Montgomery, M. K., and M. J. McFall-Ngai. 1994. Bacterial symbionts induce host organ morphogenesis during early postembryonic development of the squid Euprymna scolopes. Development 120:1719-1729.

Ruby, E. G. and L. M. Asato. 1993. Growth and flagellation of Vibrio fischeri during initiation of the sepiolid squid light organ symbiosis. Arch. Microbiol. 1159:160-167.

McFall-Ngai, M. J. and E. G. Ruby. 1991. Symbiont recognition and subsequent morphogenesis as early events in an animal-bacterial mutualism. Science 254:1491-1494.

Boettcher, K. J. and E. G. Ruby. 1990. Depressed light emission by symbiotic Vibrio fischeri of the sepiolid squid Euprymna scolopes. J. Bacteriol. 172:3701-3706.