Vibrio fischeri

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Vibrio fischeri
Scientific classification
Kingdom: Bacteria Phylum: Proteobacteria Class: Gammaproteobacteria Order: Vibrionales Family: Vibrionaceae Genus: Aliivibrio Species: V. fischeri
Binomial name
Vibrio fischeri

Description and significance

Vibrio fischeri is a gram-negative bioluminescent marine bacterium that forms mutually symbiotic relationships with various species of fish and squids. V. fischeri is a member of the Vibrionacea family of marine γ-proteobacteria which includes many species that have evolved both beneficial and harmful relationships with animals. The motile bacterium can be found living freely in the oceans or inside the microhabitats of its host's light organs. The proteins necessary for the production of bioluminescence are encoded in a set of genes called the lux operon. The bioluminescent bacteria produces light in a chemical reaction where luciferin, a substrate molecule, is oxidized by an enzyme, luciferase. As a result, energy in the form of blue-green light (480-490nm) is emitted. Bioluminescence and other metabolic functions in V. fischeri are regulated by a cell density dependent system called quorum sensing. Quorum sensing occurs through the production and accumulation of a signal molecule, N-acyl homoserine lactone (AHL); as a consequence individual bacteria do not luminesce. Since its discovery in V. fischeri, quorum sensing and it's respective signal molecules have been shown to regulate a variety of genes whose proteins are required for virulence factors, symbiosis, biofilm formation, plasmid transfer and morphogenesis in many gram-negative and gram-positive bacteria..
The symbiotic relationship between a strain of V. fischeri and its host, the Hawaiian bobtail squid Euprymna scolopes, has been studied extensively and represents a model system of bacteria/animal symbiosis. Comparative studies are examining this symbiosis model and pathogenic members within the genus vibrios, to detect patterns and similarities in bacteria/host relationships. The information may provide insights into the mechanisms behind pathogenesis and pathogenic/host relationships. The isolation and cloning of the lux genes from V. fischeri, and their use as a reporter, have provided scientists with another valuable visual tool to examine living organisms at the cellular level. Similarly, V. fischeri cells have been made commercially available and are used in the field of ecotoxicology to detect contaminants in the environment more quickly and cheaply than conventional methods.

Quorum sensing

Quorum sensing is a mechanism that allows bacteria to detect other bacteria using a signal molecule which then allows them to act collectively by altering gene expression. In V. fischeri different quorum sensing signal molecules function to regulate bioluminescence and other genes during different stages of cell growth.
The first AHL signal molecule to be recognized was N-(3-oxohexanoyl) homoserine lactone, produced by an enzyme encoded by the luxI gene. The product of the luxI gene induces bioluminescence genes luxICDABEG during late stage exponential growth, when cell densities are high. The luxI signal molecule is an autoinducer because it forms a positive feedback loop by transcribing its own genes. Bioluminescence occurs when the signal molecule diffuses freely across the plasma membrane into the extracellular environment. The autoinducer accumulates with increasing cell numbers. At a critical population number, the concentration of luxI signal molecule is greater outside than inside the cell. The difference in the concentration gradient of the luxI signal molecule causes it to diffuse back into the cell. When this happens, the signal molecule binds to a membrane bound transcriptional activator protein encoded by the luxR gene. The binding of the autoinducer results in a conformational change in the luxR protein, exposing the active site. The luxR and luxI products form a transcriptional complex that binds to the promoter region known as the lux box and transcription of the bioluminescence gene luxICDABEG, occurs.
A second signal molecule, also a AHL derivative N-(octanoyl) homoserine lactone produced by an enzyme encoded in the ainS gene, regulates bioluminescence genes during early growth periods consistent with low cell densities and regulatory genes critical for the proper functioning of the late stage quorum sensing process. The ainS signal molecule promotes late stage quorum sensing by deactivation a negative regulator of the litR gene. LitR is a positive regulator that promotes the transcription of luxR. Without luxR activity, the luxI signal molecule cannot bind to the promoter of the lux genes and bioluminescence would not occur. The ainS signal molecule can also directly bind to the transcriptional activator luxR to produce bioluminescence, although the emission is weaker. Mutational analysis of the ainS gene has shown that this signal molecule is crucial in the initial stages of colonization by V. fischeri into its host. Current research has shown that the ainS signal molecule also regulates motility genes and many other genes which have yet to be characterized. (ref,lupp)

Bioluminescence

Bioluminescence the ability of living organisms to produce light has evolved independently multiple times and occurs in a variety of marine species as well as some terrestrial forms. There are various mechanisms leading to light production and there are many different forms of luciferins and luciferases. However, all bioluminescence reactions require the use of molecular oxygen .
The bacterial luciferase is a multifunctional enzyme, required for both bioluminescence and aerobic respiration reactions. In the bioluminescence reaction, luciferase catalyses the dual oxidation of luciferin, a reduced riboflavin mononucleotide (FMNH₂) and an associated molecule, a long chain aldehyde. ATP is required to catalyze this reaction and the resulting products are light, water, oxyluciferin and carboxyl group. The luciferase enzyme is a heterodimer consisting of alpha and beta subunits, encoded by genes luxA and luxB. LuxCDE form the fatty acid reductase complex, the enzymes required for the production of the long chain fatty aldehyde. The enzyme required to reduce the flavin mononucleotide is encoded by the gene luxG.

Juvenile E. scolopes, symbiotic host of V. fischeri

Ecology

Outside from its primary hosts, V. fischeri can be found living freely as ‘marine snow’, in fecal pellets, as saprohytes , and amongst the microbial flora in the guts of marine animals. The heterotrophic bacteria is distributed in the pelagic zone of temperate and subtropical waters .

Symbiosis

The symbiosis benefits E.scolopes by using the bioluminescent bacteria in a camouflage strategy called ‘counter-illumination’. The light produced by the bacterium is projected ventrally by the squid, which mimics down dwelling moonlight when viewed from below. The squid effectively projects no shadow, and it can regulate the amount of bacterial illumination with the light organ . The squid provides housing and nutrients, especially carbon and nitrogen, in the form of proteins and peptides inside the crypts of the light organ . In providing a nutrient rich environment, the bacteria population increases to 1.0⁹ cells of which 90-95% are expelled every morning by the squid. The remaining bacteria in the light organ are replenished daily, and the squid becomes a vehicle to produce an abundance of V. fischeri . The symbiotic relationship between strains of V. fischeri and their particular hosts is highly specific . Newly hatched squids are born without the bacterium and must acquire them from the surrounding seawaters. In waters inhabited by E.scolopes, the abundance of V. fischeri constitutes only 0.1% of total microbes per mL of seawater, yet only the specific strain of V. fischeri can effectively colonize and remain in the light organ of the squid. The bacterium is housed inside the crypts of the light organ located in the squid’s mantle cavity. Once the bacterium is acquired and colonizes the crypts, morphogenesis occurs for both the bacterium and its host. The bacteria lose the flagella, and the cells decrease in size. As for the host, the ciliated fields used to obtain the bacterium undergo programmed cell death, and the light organ swells Colonization by the bacterium concurrently alters gene expression in the squid and the bacterium.

Inoculation pathway of V. fischeri into light organ of juvenile E. scolopes

Genome

To date, the complete genomes of two strains of V. fischeri, ES114 and MJII have been sequenced. MJ11 is isolated from the japanese pinecone fish, Monocentris japonica. Strain ES114 isolated from E.scolopes contains two circular chromosomes and a circular plasmid pES100. The genetic material is composed of DNA and the total genome is 4.284 Mbp in length. Chromosome 1, the larger of the two chromosomes contains 2586 genes, while Chromosome 2 and the plasmid contains 1175 and 57 genes respectively. The lux operon system, located on chromosome 2, contains luxI, luxR and luxCDABEG. .

Pathology

V. fischeri is non-pathogenic to humans but is pathogenic to some marine invertebrates. Within the Vibrios family there are three human pathogens V. cholerae, V.parahaemolytus, V. vulnificus.

Application to Biotechnology

The lux genes isolated from various bioluminescent organisms, in combination with the use of recombinant DNA technology, have had wide applications in the field of science. Applications for bioluminescent genes have reinforced the many innovative applications for green fluorescent protein (GFP), which is isolated from jellyfish. Differences in specific applications have to do with the different chemistries of light production. One of the primary uses of bioluminescent genes is as a reporter gene. A reporter gene allows scientists to visually track proteins and molecular processes occurring inside of living organisms. Scientists use reporter genes to uncover various characteristics of proteins, determine the functions of genes, and to describe regulatory regions of the genome (methods Gheysens). In clinical research, bioluminescence and fluorescence imaging is used to observe physiological changes during disease progression in patients and to observe how disease alters molecular processes in patients. The V. fischeri bacteria and its isolated lux operon are being used to detect pollution in the environment. In using the whole-cell approach, non-specific contamination is detected by the decrease in cell luminescence brought upon by cell death or metabolic failure. Methods to detect for specific contaminants utilize the lux operon and a regulatory region that is specific to the target compound.

Current Research

Dr. E.G. Ruby has been studying V. fischeri in the symbiosis model to provide insight into the molecular and biochemical pathways of pathogenic bacterial colonization of animal tissue. In his recent paper, he and his colleagues found that a regulatory gene in V. fischeri, the rscS, was necessary for establishment for symbiosis by V. fischeri and its host E.scolopes. Comparative genomic studies of the rscS gene in different strains of V. fischeri isolated from squid and fish hosts revealed that the squid isolates all had a conserved rscS gene. From the fish isolates, five out of the ten fish also had a conserved rscS gene, but highly divergent from the squid isolates. In the experiment, the rscs gene was inserted into the MJ11 strains isolated from fish that cannot effectively colonize squid. With the transformation, the MJ11 strains were able to colonize the light organ as effectively as the natural symbionts.

References

Fuqua,C., Winans,S., Greenberg, P. Census and consensus in bacterial ecosystems: the luxR-luxI family of quorum-sensing transciptional regulators. Annual Review of Microbiology Vol. 50: 727-751 (Volume publication date October 1996) (doi:10.1146/annurev.micro.50.1.727)

Herring, P.J. and Widder, E.A.2001. Bioluminescence in Plankton and Nekton. In: Steele, J.H., Thorpe, S.A. and Turekian, K.K. editors, Encyclopedia of Ocean Science, Vol. 1, 308-317. Academic Press, San Diego. Accessed from "International society of bioluminescence and chemiluminescence", <http://www.isbc.unibo.it/Files/BC_PlanktonNekton.htm> (updated 10/2009; accessed 03/20/09)

McFall-Ngai, M. J. 2000. Negotiations between animals and bacteria: the 'diplomacy' of the squid-vibrio symbiosis. Comp. Biochem. Physiol. A. Mol. Integr. Physiol., 126(4), 471-480.

NCBI. Entrez genome project. <http://www.ncbi.nlm.nih.gov/sites/entrez?Db=genomeprj&Cmd=Retrieve&list_uids=12986>(updated 2/09;accessed 03/29/09)

Ruby, E. G., M. Urbanowski, J. Campbell, A. Dunn, M. Faini, R. Gunsalus, P. Lostroh, C. Lupp, J. McCann, D. Millikan, A. Schaefer, E. Stabb, A. Stevens, K. Visick, C. Whistler, and E. P. Greenberg. 2005. Complete genome sequence of Vibrio fischeri: a symbiotic bacterium with pathogenic congeners. Proc. Natl. Acad. Sci. USA 102:3004-3009

Ruby, E. G., McFall-Ngai, M. J. Oxygen-utilizing reactions and symbiotic colonization of the squid light organ by Vibrio fischeri. Trends in microbiology. 415 Volume 7, issue , 10October 1999, Pages 414-420. Accessed from ScienceDirect

Thompson, F.L., Austin, B., Swings, J. The biology of Vibrios. 2006. ASM press. Virginia.

Visick, K., Ruby, E. G. Vibrio fischeri and its host: it takes two to tango. Current Opinion in Microbiology. Volume 9, Issue 6, December 2006, Pages 632-638. Accessed from ScienceDirect.

Willey, J.M., Sherwood, L.M., Woolverton, C. J. Prescott, Harley, and Klein's Microbiology. 7th Ed. 2008. McGraw-Hill. New York.