scholarly journals FlrA, a σ54-Dependent Transcriptional Activator in Vibrio fischeri, Is Required for Motility and Symbiotic Light-Organ Colonization

2003 ◽  
Vol 185 (12) ◽  
pp. 3547-3557 ◽  
Author(s):  
Deborah S. Millikan ◽  
Edward G. Ruby
Keyword(s):  
Vibrio Fischeri ◽  
Transcriptional Activator ◽  
Pcr Analysis ◽  
Euprymna Scolopes ◽  
Wild Type ◽  
Flagellar Regulon ◽  
Colonization Factors ◽  
Recognition Motifs ◽  
Arbitrarily Primed Pcr ◽  
Organ Colonization

ABSTRACT Flagellum-mediated motility of Vibrio fischeri is an essential factor in the bacterium's ability to colonize its host, the Hawaiian squid Euprymna scolopes. To begin characterizing the nature of the flagellar regulon, we have cloned a gene, designated flrA, from V. fischeri that encodes a putative σ54-dependent transcriptional activator. Genetic arrangement of the flrA locus in V. fischeri is similar to motility master-regulator operons of Vibrio cholerae and Vibrio parahaemolyticus. In addition, examination of regulatory regions of a number of flagellar operons in V. fischeri revealed apparent σ54 recognition motifs, suggesting that the flagellar regulatory hierarchy is controlled by a similar mechanism to that described in V. cholerae. However, in contrast to its closest known relatives, flrA mutant strains of V. fischeri ES114 were completely abolished in swimming capability. Although flrA provided in trans restored motility to the flrA mutant, the complemented strain was unable to reach wild-type levels of symbiotic colonization in juvenile squid, suggesting a possible role for the proper expression of FlrA in regulating symbiotic colonization factors in addition to those required for motility. Comparative RNA arbitrarily primed PCR analysis of the flrA mutant and its wild-type parent revealed several differentially expressed transcripts. These results define a regulon that includes both flagellar structural genes and other genes apparently not involved in flagellum elaboration or function. Thus, the transcriptional activator FlrA plays an essential role in regulating motility, and apparently in modulating other symbiotic functions, in V. fischeri.

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.

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 Role for cheR of Vibrio fischeri in the Vibrio–squid symbiosis

2012 ◽  
Vol 58 (1) ◽  
pp. 29-38 ◽  
Author(s):  
Cindy R. DeLoney-Marino ◽  
Karen L. Visick
Keyword(s):  
Marine Bacterium ◽  
Wild Type Strain ◽  
Vibrio Fischeri ◽  
Bacterial Motility ◽  
Light Organ ◽  
Euprymna Scolopes ◽  
Wild Type ◽  
Acetylneuraminic Acid ◽  
Symbiotic Partner ◽  
Fourfold Excess

Upon hatching, the Hawaiian squid Euprymna scolopes is rapidly colonized by its symbiotic partner, the bioluminescent marine bacterium Vibrio fischeri . Vibrio fischeri cells present in the seawater enter the light organ of juvenile squid in a process that requires bacterial motility. In this study, we investigated the role chemotaxis may play in establishing this symbiotic colonization. Previously, we reported that V. fischeri migrates toward numerous attractants, including N-acetylneuraminic acid (NANA), a component of squid mucus. However, whether or not migration toward an attractant such as squid-derived NANA helps the bacterium to localize toward the light organ is unknown. When tested for the ability to colonize juvenile squid, a V. fischeri chemotaxis mutant defective for the methyltransferase CheR was outcompeted by the wild-type strain in co-inoculation experiments, even when the mutant was present in fourfold excess. Our results suggest that the ability to perform chemotaxis is an advantage during colonization, but not essential.

scholarly journals Population Dynamics of Vibrio fischeri during Infection of Euprymna scolopes

2003 ◽  
Vol 69 (10) ◽  
pp. 5928-5934 ◽  
Author(s):  
Jessica McCann ◽  
Eric V. Stabb ◽  
Deborah S. Millikan ◽  
Edward G. Ruby
Keyword(s):  
Population Dynamics ◽  
Vibrio Fischeri ◽  
Single Copy ◽  
Bacterial Cells ◽  
Euprymna Scolopes ◽  
Wild Type ◽  
Luminous Bacterium ◽  
Ambient Seawater ◽  
Light Emitting ◽  
Low Concentrations

ABSTRACT The luminous bacterium Vibrio fischeri colonizes a specialized light-emitting organ within its squid host, Euprymna scolopes. Newly hatched juvenile squid must acquire their symbiont from ambient seawater, where the bacteria are present at low concentrations. To understand the population dynamics of V. fischeri during colonization more fully, we used mini-Tn7 transposons to mark bacteria with antibiotic resistance so that the growth of their progeny could be monitored. When grown in culture, there was no detectable metabolic burden on V. fischeri cells carrying the transposon, which inserts in single copy in a specific intergenic region of the V. fischeri genome. Strains marked with mini-Tn7 also appeared to be equivalent to the wild type in their ability to infect and multiply within the host during coinoculation experiments. Studies of the early stages of colonization suggested that only a few bacteria became associated with symbiotic tissue when animals were exposed for a discrete period (3 h) to an inoculum of V. fischeri cells equivalent to natural population levels; nevertheless, all these hosts became infected. When three differentially marked strains of V. fischeri were coincubated with juvenile squid, the number of strains recovered from an individual symbiotic organ was directly dependent on the size of the inoculum. Further, these results indicated that, when exposed to low numbers of V. fischeri, the host may become colonized by only one or a few bacterial cells, suggesting that symbiotic infection is highly efficient.

scholarly journals Role for Phosphoglucomutase in Vibrio fischeri-Euprymna scolopes Symbiosis

2002 ◽  
Vol 184 (18) ◽  
pp. 5121-5129 ◽  
Author(s):  
Cindy R. DeLoney ◽  
Therese M. Bartley ◽  
Karen L. Visick
Keyword(s):  
Vibrio Fischeri ◽  
Euprymna Scolopes ◽  
Wild Type ◽  
Galactose Metabolism ◽  
High Sequence Identity ◽  
E Coli ◽  
New Gene ◽  
Increased Sensitivity ◽  
Colonization Ability ◽  
Galactose Utilization

ABSTRACT Vibrio fischeri, a luminescent marine bacterium, specifically colonizes the light organ of its symbiotic partner, the Hawaiian squid Euprymna scolopes. In a screen for V. fischeri colonization mutants, we identified a strain that exhibited on average a 10-fold decrease in colonization levels relative to that achieved by wild-type V. fischeri. Further characterization revealed that this defect did not result from reduced luminescence or motility, two processes required for normal colonization. We determined that the transposon in this mutant disrupted a gene with high sequence identity to the pgm (phosphoglucomutase) gene of Escherichia coli, which encodes an enzyme that functions in both galactose metabolism and the synthesis of UDP-glucose. The V. fischeri mutant grew poorly with galactose as a sole carbon source and was defective for phosphoglucomutase activity, suggesting functional identity between E. coli Pgm and the product of the V. fischeri gene, which was therefore designated pgm. In addition, lipopolysaccharide profiles of the mutant were distinct from that of the parent strain and the mutant exhibited increased sensitivity to various cationic agents and detergents. Chromosomal complementation with the wild-type pgm allele restored the colonization ability to the mutant and also complemented the other noted defects. Unlike the pgm mutant, a galactose-utilization mutant (galK) of V. fischeri colonized juvenile squid to wild-type levels, indicating that the symbiotic defect of the pgm mutant is not due to an inability to catabolize galactose. Thus, pgm represents a new gene required for promoting colonization of E. scolopes by V. fischeri.

scholarly journals Roles of Vibrio fischeri and Nonsymbiotic Bacteria in the Dynamics of Mucus Secretion during Symbiont Colonization of the Euprymna scolopes Light Organ

2002 ◽  
Vol 68 (10) ◽  
pp. 5113-5122 ◽  
Author(s):  
Spencer V. Nyholm ◽  
Bart Deplancke ◽  
H. Rex Gaskins ◽  
Michael A. Apicella ◽  
Margaret J. McFall-Ngai
Keyword(s):  
Vibrio Fischeri ◽  
Mucus Secretion ◽  
Surface Epithelium ◽  
Light Organ ◽  
Euprymna Scolopes ◽  
Superficial Epithelium ◽  
Temporal And Spatial ◽  
Temporal And Spatial Characteristics ◽  
Bacterial Peptidoglycan ◽  
Organ Colonization

ABSTRACT During light organ colonization of the squid Euprymna scolopes by Vibrio fischeri, host-derived mucus provides a surface upon which environmental V. fischeri forms a biofilm and aggregates prior to colonization. In this study we defined the temporal and spatial characteristics of this process. Although permanent colonization is specific to certain strains of V. fischeri, confocal microscopy analyses revealed that light organ crypt spaces took up nonspecific bacteria and particles that were less than 2 μm in diameter during the first hour after hatching. However, within 2 h after inoculation, these cells or particles were not detectable, and further entry by nonspecific bacteria or particles appeared to be blocked. Exposure to environmental gram-negative or -positive bacteria or bacterial peptidoglycan caused the cells of the organ's superficial ciliated epithelium to release dense mucin stores at 1 to 2 h after hatching that were used to form the substrate upon which V. fischeri formed a biofilm and aggregated. Whereas the uncolonized organ surface continued to shed mucus, within 48 h of symbiont colonization mucus shedding ceased and the formation of bacterial aggregations was no longer observed. Eliminating the symbiont from the crypts with antibiotics restored the ability of the ciliated fields to secrete mucus and aggregate bacteria. While colonization by V. fischeri inhibited mucus secretion by the surface epithelium, secretion of host-derived mucus was induced in the crypt spaces. Together, these data indicate that although initiation of mucus secretion from the superficial epithelium is nonspecific, the inhibition of mucus secretion in these cells and the concomitant induction of secretion in the crypt cells are specific to natural colonization by V. fischeri.

scholarly journals CysK Plays a Role in Biofilm Formation and Colonization by Vibrio fischeri

2015 ◽  
Vol 81 (15) ◽  
pp. 5223-5234 ◽  
Author(s):  
Priyanka Singh ◽  
John F. Brooks ◽  
Valerie A. Ray ◽  
Mark J. Mandel ◽  
Karen L. Visick
Keyword(s):  
Colony Formation ◽  
Biofilm Formation ◽  
Parent Strain ◽  
Vibrio Fischeri ◽  
Euprymna Scolopes ◽  
Wild Type ◽  
Biosynthetic Genes ◽  
Content Type ◽  
Agar Surface ◽  
Control Activation

ABSTRACTA biofilm, or a matrix-embedded community of cells, promotes the ability of the bacteriumVibrio fischerito colonize its symbiotic host, the Hawaiian squidEuprymna scolopes. Biofilm formation and colonization depend onsyp, an 18-gene polysaccharide locus. To identify other genes necessary for biofilm formation, we screened for mutants that failed to form wrinkled colonies, a type of biofilm. We obtained several with defects in genes required for cysteine metabolism, includingcysH,cysJ,cysK, andcysN. ThecysKmutant exhibited the most severe wrinkling defect. It could be complemented with a wild-type copy of thecysKgene, which encodesO-acetylserine sulfhydrolase, or by supplementing the medium with additional cysteine. None of a number of other mutants defective for biosynthetic genes negatively impacted wrinkled colony formation, suggesting a specific role for CysK. CysK did not appear to control activation of Syp regulators or transcription of thesyplocus, but it did influence production of the Syp polysaccharide. Under biofilm-inducing conditions, thecysKmutant retained the same ability as that of the parent strain to adhere to the agar surface. ThecysKmutant also exhibited a defect in pellicle production that could be complemented by thecysKgene but not by cysteine, suggesting that, under these conditions, CysK is important for more than the production of cysteine. Finally, our data reveal a role forcysKin symbiotic colonization byV. fischeri. Although many questions remain, this work provides insights into additional factors required for biofilm formation and colonization byV. fischeri.

scholarly journals Contribution of pilA to Competitive Colonization of the Squid Euprymna scolopes by Vibrio fischeri

2003 ◽  
Vol 69 (2) ◽  
pp. 820-826 ◽  
Author(s):  
Eric V. Stabb ◽  
Edward G. Ruby
Keyword(s):  
Wild Type Strain ◽  
Vibrio Fischeri ◽  
Kanamycin Resistance ◽  
Euprymna Scolopes ◽  
Wild Type ◽  
Mutualistic Symbiosis ◽  
Surface Molecules ◽  
Type Iv ◽  
Mixed Inocula ◽  
Environmental Bacteria

ABSTRACT Vibrio fischeri colonizes the squid Euprymna scolopes in a mutualistic symbiosis. Hatchling squid lack these bacterial symbionts, and V. fischeri strains must compete to occupy this privileged niche. We cloned a V. fischeri gene, designated pilA, that contributes to colonization competitiveness and encodes a protein similar to type IV-A pilins. Unlike its closest known relatives, Vibrio cholerae mshA and vcfA, pilA is monocistronic and not clustered with genes associated with pilin export or assembly. Using wild-type strain ES114 as the parent, we generated an in-frame pilA deletion mutant, as well as pilA mutants marked with a kanamycin resistance gene. In mixed inocula, marked mutants were repeatedly outcompeted by ES114 (P < 0.05) but not by an unmarked pilA mutant, for squid colonization. In contrast, the ratio of mutant to ES114 CFUs did not change during 70 generations of coculturing. The competitive defect of pilA mutants ranged from 1.7- to 10-fold and was more pronounced when inocula were within the range estimated for V. fischeri populations in Hawaiian seawater (200 to 2,000 cells/ml) than when higher densities were used. ES114 also outcompeted a pilA mutant by an average of twofold at lower inoculum densities, when only a fraction of the squid became infected, most by only one strain. V. fischeri strain ET101, which was isolated from Euprymna tasmanica and is outcompeted by ES114, lacks pilA; however, 11 other diverse V. fischeri isolates apparently possess pilA. The competitive defect of pilA mutants suggests that cell surface molecules may play important roles in the initiation of beneficial symbioses in which animals must acquire symbionts from a mixed community of environmental bacteria.

scholarly journals LitR, a new transcriptional activator in Vibrio fischeri, regulates luminescence and symbiotic light organ colonization

2002 ◽  
Vol 45 (1) ◽  
pp. 131-143 ◽  
Author(s):  
Pat M. Fidopiastis ◽  
Carol M. Miyamoto ◽  
Michael G. Jobling ◽  
Edward A. Meighen ◽  
Edward G. Ruby
Keyword(s):  
Vibrio Fischeri ◽  
Transcriptional Activator ◽  
Light Organ ◽  
Organ Colonization
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