scholarly journals Genetic Analysis of Trimethylamine N-Oxide Reductases in the Light Organ Symbiont Vibrio fischeri ES114

2008 ◽  
Vol 190 (17) ◽  
pp. 5814-5823 ◽  
Author(s):  
Anne K. Dunn ◽  
Eric V. Stabb
Keyword(s):  
Vibrio Fischeri ◽  
Reductase Activity ◽  
Anaerobic Respiration ◽  
Gene Arrangement ◽  
Light Organ ◽  
Euprymna Scolopes ◽  
Light Organs ◽  
Dependent Growth ◽  
Tmao Reductase ◽  
Active Promoter

ABSTRACT Trimethylamine N-oxide (TMAO) reductases are widespread in bacteria and often function in anaerobic respiration. The regulation and expression of TMAO reductase operons have been well studied in model genera such as Escherichia, Shewanella, and Rhodobacter, although TMAO reductases are present in many other bacteria, including the marine Vibrio species. The genome sequence of Vibrio fischeri revealed three putative TMAO reductase operons, and a previous report identified TMAO reductase activity in symbiotic V. fischeri isolates associated with the light organs of adult Hawaiian bobtail squid, Euprymna scolopes. We examined the roles and regulation of these three operons using mutational analyses and promoter-reporter fusions. We found that the torECA promoter, and to a lesser extent the torYZ and dmsABC promoters, were active during symbiotic colonization of juvenile E. scolopes; however, a V. fischeri strain lacking TMAO reductase activity displays no discernible colonization defect over the first 48 h. Our studies also revealed that torECA has the most active promoter of the putative TMAO reductase operons, and TorECA is the major contributor to TMAO-dependent growth in V. fischeri under the conditions tested. Interestingly, the transcriptional regulation of TMAO reductase operons in V. fischeri appears to differ from that in previously studied organisms, such as Escherichia coli, which may reflect differences in gene arrangement and bacterial habitat. This study lays the foundation for using V. fischeri as a model system for studying TMAO reductases in the Vibrionaceae.

scholarly journals A New Niche for Vibrio logei, the Predominant Light Organ Symbiont of Squids in the GenusSepiola

1998 ◽  
Vol 180 (1) ◽  
pp. 59-64 ◽  
Author(s):  
Pat M. Fidopiastis ◽  
Sigurd von Boletzky ◽  
Edward G. Ruby
Keyword(s):  
Coastal Waters ◽  
Vibrio Fischeri ◽  
Aliphatic Aldehyde ◽  
Mixed Population ◽  
Light Organ ◽  
Euprymna Scolopes ◽  
Light Organs ◽  
Luminous Bacteria ◽  
Animal Hosts ◽  
The Western Pacific

ABSTRACT Two genera of sepiolid squids—Euprymna, found primarily in shallow, coastal waters of Hawaii and the Western Pacific, and Sepiola, the deeper-, colder-water-dwelling Mediterranean and Atlantic squids—are known to recruit luminous bacteria into light organ symbioses. The light organ symbiont ofEuprymna spp. is Vibrio fischeri, but until now, the light organ symbionts of Sepiola spp. have remained inadequately identified. We used a combination of molecular and physiological characteristics to reveal that the light organs ofSepiola affinis and Sepiola robusta contain a mixed population of Vibrio logei and V. fischeri, with V. logei comprising between 63 and 100% of the bacteria in the light organs that we analyzed. V. logei had not previously been known to exist in such symbioses. In addition, this is the first report of two different species of luminous bacteria co-occurring within a single light organ. The luminescence of these symbiotic V. logei strains, as well as that of other isolates of V. logei tested, is reduced when they are grown at temperatures above 20°C, partly due to a limitation in the synthesis of aliphatic aldehyde, a substrate of the luminescence reaction. In contrast, the luminescence of the V. fischeri symbionts is optimal above 24°C and is not enhanced by aldehyde addition. Also, V. fischeri strains were markedly more successful than V. logei at colonizing the light organs of juvenile Euprymna scolopes, especially at 26°C. These findings have important implications for our understanding of the ecological dynamics and evolution of cooperative, and perhaps pathogenic, associations of Vibrio spp. with their animal hosts.

scholarly journals Population Structure of Vibrio fischeri within the Light Organs of Euprymna scolopes Squid from Two Oahu (Hawaii) Populations

2008 ◽  
Vol 75 (1) ◽  
pp. 193-202 ◽  
Author(s):  
M. S. Wollenberg ◽  
E. G. Ruby
Keyword(s):  
Population Structure ◽  
Vibrio Fischeri ◽  
Population Data ◽  
Intraspecific Diversity ◽  
Light Organ ◽  
Euprymna Scolopes ◽  
Individual Host ◽  
Light Organs ◽  
Sepiolid Squid ◽  
The Stability

ABSTRACT We resolved the intraspecific diversity of Vibrio fischeri, the bioluminescent symbiont of the Hawaiian sepiolid squid Euprymna scolopes, at two previously unexplored morphological and geographical scales. These scales ranged from submillimeter regions within the host light organ to the several kilometers encompassing two host populations around Oahu. To facilitate this effort, we employed both novel and standard genetic and phenotypic assays of light-organ symbiont populations. A V. fischeri-specific fingerprinting method and five phenotypic assays were used to gauge the genetic richness of V. fischeri populations; these methods confirmed that the symbiont population present in each adult host's light organ is polyclonal. Upon statistical analysis of these genetic and phenotypic population data, we concluded that the characteristics of symbiotic populations were more similar within individual host populations than between the two distinct Oahu populations of E. scolopes, providing evidence that local geographic symbiont population structure exists. Finally, to better understand the genesis of symbiont diversity within host light organs, the process of symbiosis initiation in newly hatched juvenile squid was examined both experimentally and by mathematical modeling. We concluded that, after the juvenile hatches, only one or two cells of V. fischeri enter each of six internal epithelium-lined crypts present in the developing light organ. We hypothesize that the expansion of different, crypt-segregated, clonal populations creates the polyclonal adult light-organ population structure observed in this study. The stability of the luminous-bacterium-sepiolid squid mutualism in the presence of a polyclonal symbiont population structure is discussed in the context of contemporary evolutionary theory.

scholarly journals An Expanded Transposon Mutant Library Reveals that Vibrio fischeri δ-Aminolevulinate Auxotrophs Can Colonize Euprymna scolopes

2016 ◽  
Vol 83 (5) ◽  
Author(s):  
Noreen L. Lyell ◽  
Alecia N. Septer ◽  
Anne K. Dunn ◽  
Drew Duckett ◽  
Julie L. Stoudenmire ◽  
...  
Keyword(s):  
Vibrio Fischeri ◽  
Light Organ ◽  
Mutant Library ◽  
Euprymna Scolopes ◽  
Simple Method ◽  
Content Type ◽  
Transposon Insertion ◽  
Rich Media ◽  
Show Evidence

ABSTRACT Libraries of defined mutants are valuable research tools but necessarily lack gene knockouts that are lethal under the conditions used in library construction. In this study, we augmented a Vibrio fischeri mutant library generated on a rich medium (LBS, which contains [per liter] 10 g of tryptone, 5 g of yeast extract, 20 g of NaCl, and 50 mM Tris [pH 7.5]) by selecting transposon insertion mutants on supplemented LBS and screening for those unable to grow on LBS. We isolated strains with insertions in alr, glr (murI), glmS, several heme biosynthesis genes, and ftsA, as well as a mutant disrupted 14 bp upstream of ftsQ. Mutants with insertions in ftsA or upstream of ftsQ were recovered by addition of Mg2+ to LBS, but their cell morphology and motility were affected. The ftsA mutant was more strongly affected and formed cells or chains of cells that appeared to wind back on themselves helically. Growth of mutants with insertions in glmS, alr, or glr was recovered with N-acetylglucosamine (NAG), d-alanine, or d-glutamate, respectively. We hypothesized that NAG, d-alanine, or d-glutamate might be available to V. fischeri in the Euprymna scolopes light organ; however, none of these mutants colonized the host effectively. In contrast, hemA and hemL mutants, which are auxotrophic for δ-aminolevulinate (ALA), colonized at wild-type levels, although mutants later in the heme biosynthetic pathway were severely impaired or unable to colonize. Our findings parallel observations that legume hosts provide Bradyrhizobium symbionts with ALA, but they contrast with virulence phenotypes of hemA mutants in some pathogens. The results further inform our understanding of the symbiotic light organ environment. IMPORTANCE By supplementing a rich yeast-based medium, we were able to recover V. fischeri mutants with insertions in conditionally essential genes, and further characterization of these mutants provided new insights into this bacterium's symbiotic environment. Most notably, we show evidence that the squid host can provide V. fischeri with enough ALA to support its growth in the light organ, paralleling the finding that legumes provide Bradyrhizobium ALA in symbiotic nodules. Taken together, our results show how a simple method of augmenting already rich media can expand the reach and utility of defined mutant libraries.

scholarly journals Critical symbiont signals drive both local and systemic changes in diel and developmental host gene expression

2019 ◽  
Vol 116 (16) ◽  
pp. 7990-7999 ◽  
Author(s):  
Silvia Moriano-Gutierrez ◽  
Eric J. Koch ◽  
Hailey Bussan ◽  
Kymberleigh Romano ◽  
Mahdi Belcaid ◽  
...  
Keyword(s):  
Gene Expression ◽  
Visual Cues ◽  
Vibrio Fischeri ◽  
Single Mutation ◽  
Light Organ ◽  
Euprymna Scolopes ◽  
Transcriptional Responses ◽  
Transcriptional Signature ◽  
The Impact ◽  
Organ Colonization

The colonization of an animal’s tissues by its microbial partners creates networks of communication across the host’s body. We used the natural binary light-organ symbiosis between the squidEuprymna scolopesand its luminous bacterial partner,Vibrio fischeri, to define the impact of colonization on transcriptomic networks in the host. A night-active predator,E. scolopescoordinates the bioluminescence of its symbiont with visual cues from the environment to camouflage against moon and starlight. Like mammals, this symbiosis has a complex developmental program and a strong day/night rhythm. We determined how symbiont colonization impacted gene expression in the light organ itself, as well as in two anatomically remote organs: the eye and gill. While the overall transcriptional signature of light organ and gill were more alike, the impact of symbiosis was most pronounced and similar in light organ and eye, both in juvenile and adult animals. Furthermore, the presence of a symbiosis drove daily rhythms of transcription within all three organs. Finally, a single mutation inV. fischeri—specifically, deletion of theluxoperon, which abrogates symbiont luminescence—reduced the symbiosis-dependent transcriptome of the light organ by two-thirds. In addition, while the gills responded similarly to light-organ colonization by either the wild-type or mutant, luminescence was required for all of the colonization-associated transcriptional responses in the juvenile eye. This study defines not only the impact of symbiont colonization on the coordination of animal transcriptomes, but also provides insight into how such changes might impact the behavior and ecology of the host.

scholarly journals The cytokine MIF controls daily rhythms of symbiont nutrition in an animal–bacterial association

2020 ◽  
Vol 117 (44) ◽  
pp. 27578-27586 ◽  
Author(s):  
Eric J. Koch ◽  
Clotilde Bongrand ◽  
Brittany D. Bennett ◽  
Susannah Lawhorn ◽  
Silvia Moriano-Gutierrez ◽  
...  
Keyword(s):  
Macrophage Migration Inhibitory Factor ◽  
Vibrio Fischeri ◽  
Diurnal Rhythms ◽  
Light Organ ◽  
Euprymna Scolopes ◽  
Daily Rhythms ◽  
Western Blots ◽  
Periodic Movement ◽  
Nocturnal Migration

The recent recognition that many symbioses exhibit daily rhythms has encouraged research into the partner dialogue that drives these biological oscillations. Here we characterized the pivotal role of the versatile cytokine macrophage migration inhibitory factor (MIF) in regulating a metabolic rhythm in the model light-organ symbiosis betweenEuprymna scolopesandVibrio fischeri. As the juvenile host matures, it develops complex daily rhythms characterized by profound changes in the association, from gene expression to behavior. One such rhythm is a diurnal shift in symbiont metabolism triggered by the periodic provision of a specific nutrient by the mature host: each night the symbionts catabolize chitin released from hemocytes (phagocytic immune cells) that traffic into the light-organ crypts, where the population ofV. fischericells resides. Nocturnal migration of these macrophage-like cells, together with identification of anE. scolopesMIF (EsMIF) in the light-organ transcriptome, led us to ask whether EsMIF might be the gatekeeper controlling the periodic movement of the hemocytes. Western blots, ELISAs, and confocal immunocytochemistry showed EsMIF was at highest abundance in the light organ. Its concentration there was lowest at night, when hemocytes entered the crypts. EsMIF inhibited migration of isolated hemocytes, whereas exported bacterial products, including peptidoglycan derivatives and secreted chitin catabolites, induced migration. These results provide evidence that the nocturnal decrease in EsMIF concentration permits the hemocytes to be drawn into the crypts, delivering chitin. This nutritional function for a cytokine offers the basis for the diurnal rhythms underlying a dynamic symbiotic conversation.

scholarly journals Dominance of Vibrio fischeri in Secreted Mucus outside the Light Organ of Euprymna scolopes: the First Site of Symbiont Specificity

2003 ◽  
Vol 69 (7) ◽  
pp. 3932-3937 ◽  
Author(s):  
Spencer V. Nyholm ◽  
Margaret J. McFall-Ngai
Keyword(s):  
Fluorescent Protein ◽  
Vibrio Fischeri ◽  
Signal Molecules ◽  
Natural Seawater ◽  
Gram Negative Bacteria ◽  
Light Organ ◽  
Bacterial Cells ◽  
Euprymna Scolopes ◽  
Gram Negative ◽  
Green Fluorescent

ABSTRACT Previous studies of the Euprymna scolopes-Vibrio fischeri symbiosis have demonstrated that, during colonization, the hatchling host secretes mucus in which gram-negative environmental bacteria amass in dense aggregations outside the sites of infection. In this study, experiments with green fluorescent protein-labeled symbiotic and nonsymbiotic species of gram-negative bacteria were used to characterize the behavior of cells in the aggregates. When hatchling animals were exposed to 103 to 106 V. fischeri cells/ml added to natural seawater, which contains a mix of approximately 106 nonspecific bacterial cells/ml, V. fischeri cells were the principal bacterial cells present in the aggregations. Furthermore, when animals were exposed to equal cell numbers of V. fischeri (either a motile or a nonmotile strain) and either Vibrio parahaemolyticus or Photobacterium leiognathi, phylogenetically related gram-negative bacteria that also occur in the host's habitat, the symbiont cells were dominant in the aggregations. The presence of V. fischeri did not compromise the viability of these other species in the aggregations, and no significant growth of V. fischeri cells was detected. These findings suggested that dominance results from the ability of V. fischeri either to accumulate or to be retained more effectively within the mucus. Viability of the V. fischeri cells was required for both the formation of tight aggregates and their dominance in the mucus. Neither of the V. fischeri quorum-sensing compounds accumulated in the aggregations, which suggested that the effects of these small signal molecules are not critical to V. fischeri dominance. Taken together, these data provide evidence that the specificity of the squid-vibrio symbiosis begins early in the interaction, in the mucus where the symbionts aggregate outside of the light organ.

scholarly journals Vibrio fischeri Flagellin A Is Essential for Normal Motility and for Symbiotic Competence during Initial Squid Light Organ Colonization

2004 ◽  
Vol 186 (13) ◽  
pp. 4315-4325 ◽  
Author(s):  
Deborah S. Millikan ◽  
Edward G. Ruby
Keyword(s):  
Vibrio Fischeri ◽  
Complex Structure ◽  
Immunoblot Analysis ◽  
Soft Agar ◽  
Light Organ ◽  
Euprymna Scolopes ◽  
Wild Type ◽  
Mixed Inoculum ◽  
Mutant Cells ◽  
Organ Colonization

ABSTRACT The motile bacterium Vibrio fischeri is the specific bacterial symbiont of the Hawaiian squid Euprymna scolopes. Because motility is essential for initiating colonization, we have begun to identify stage-specific motility requirements by creating flagellar mutants that have symbiotic defects. V. fischeri has six flagellin genes that are uniquely arranged in two chromosomal loci, flaABCDE and flaF. With the exception of the flaA product, the predicted gene products are more similar to each other than to flagellins of other Vibrio species. Immunoblot analysis indicated that only five of the six predicted proteins were present in purified flagella, suggesting that one protein, FlaF, is unique with respect to either its regulation or its function. We created mutations in two genes, flaA and flaC. Compared to a flaC mutant, which has wild-type flagellation, a strain having a mutation in the flaA gene has fewer flagella per cell and exhibits a 60% decrease in its rate of migration in soft agar. During induction of light organ symbiosis, colonization by the flaA mutant is impaired, and this mutant is severely outcompeted when it is presented to the animal as a mixed inoculum with the wild-type strain. Furthermore, flaA mutant cells are preferentially expelled from the animal, suggesting either that FlaA plays a role in adhesion or that normal motility is an advantage for retention within the host. Taken together, these results show that the flagellum of V. fischeri is a complex structure consisting of multiple flagellin subunits, including FlaA, which is essential both for normal flagellation and for motility, as well as for effective symbiotic colonization.

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