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 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 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 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 Vibrio fischeri Genes hvnA andhvnB Encode Secreted NAD+-Glycohydrolases

2001 ◽  
Vol 183 (1) ◽  
pp. 309-317 ◽  
Author(s):  
Eric V. Stabb ◽  
Karl A. Reich ◽  
Edward G. Ruby
Keyword(s):  
Vibrio Fischeri ◽  
Amino Acid Sequences ◽  
Open Reading Frame ◽  
Light Organ ◽  
Euprymna Scolopes ◽  
Wild Type ◽  
Reading Frame ◽  
Host Signaling ◽  
Culture Supernatants ◽  
Lines Of Evidence

ABSTRACT HvnA and HvnB are proteins secreted by Vibrio fischeriES114, an extracellular light organ symbiont of the squidEuprymna scolopes, that catalyze the transfer of ADP-ribose from NAD+ to polyarginine. Based on this activity, HvnA and HvnB were presumptively designated mono-ADP-ribosyltransferases (ARTases), and it was hypothesized that they mediate bacterium-host signaling. We have clonedhvnA and hvnB from strain ES114.hvnA appears to be expressed as part of a four-gene operon, whereas hvnB is monocistronic. The predicted HvnA and HvnB amino acid sequences are 46% identical to one another and share 44% and 34% identity, respectively, with an open reading frame present in the Pseudomonas aeruginosa genome. Four lines of evidence indicate that HvnA and HvnB mediate polyarginine ADP-ribosylation not by ARTase activity, but indirectly through an NAD+-glycohydrolase (NADase) activity that releases free, reactive, ADP-ribose: (i) like other NADases, and in contrast to the ARTase cholera toxin, HvnA and HvnB catalyzed ribosylation of not only polyarginine but also polylysine and polyhistidine, and ribosylation was inhibited by hydroxylamine; (ii) HvnA and HvnB cleaved 1,N 6-etheno-NAD+ and NAD+; (iii) incubation of HvnA and HvnB with [32P]NAD+ resulted in the production of ADP-ribose; and (iv) purified HvnA displayed an NADase V max of 400 mol min−1 mol−1, which is within the range reported for other NADases and 102- to 104-fold higher than the minor NADase activity reported in bacterial ARTase toxins. Construction and analysis of an hvnA hvnB mutant revealed no other NADase activity in culture supernatants of V. fischeri, and this mutant initiated the light organ symbiosis and triggered regression of the light organ ciliated epithelium in a manner similar to that for the wild type.

scholarly journals Colony spreading of the gliding bacterium Flavobacterium johnsoniae in the absence of the motility adhesin SprB

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Keiko Sato ◽  
Masami Naya ◽  
Yuri Hatano ◽  
Yoshio Kondo ◽  
Mari Sato ◽  
...  
Keyword(s):  
Soft Agar ◽  
Gliding Motility ◽  
Wild Type ◽  
Initial Cell ◽  
Flavobacterium Johnsoniae ◽  
Seeking Behavior ◽  
Colony Spreading ◽  
Gliding Bacterium ◽  
Mutant Cells ◽  
Scanning Electron

AbstractColony spreading of Flavobacterium johnsoniae is shown to include gliding motility using the cell surface adhesin SprB, and is drastically affected by agar and glucose concentrations. Wild-type (WT) and ΔsprB mutant cells formed nonspreading colonies on soft agar, but spreading dendritic colonies on soft agar containing glucose. In the presence of glucose, an initial cell growth-dependent phase was followed by a secondary SprB-independent, gliding motility-dependent phase. The branching pattern of a ΔsprB colony was less complex than the pattern formed by the WT. Mesoscopic and microstructural information was obtained by atmospheric scanning electron microscopy (ASEM) and transmission EM, respectively. In the growth-dependent phase of WT colonies, dendritic tips spread rapidly by the movement of individual cells. In the following SprB-independent phase, leading tips were extended outwards by the movement of dynamic windmill-like rolling centers, and the lipoproteins were expressed more abundantly. Dark spots in WT cells during the growth-dependent spreading phase were not observed in the SprB-independent phase. Various mutations showed that the lipoproteins and the motility machinery were necessary for SprB-independent spreading. Overall, SprB-independent colony spreading is influenced by the lipoproteins, some of which are involved in the gliding machinery, and medium conditions, which together determine the nutrient-seeking behavior.

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 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.

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