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 Symbiotic Characterization of Vibrio fischeri ES114 Mutants That Display Enhanced Luminescence in Culture

2013 ◽  
Vol 79 (7) ◽  
pp. 2480-2483 ◽  
Author(s):  
Noreen L. Lyell ◽  
Eric V. Stabb
Keyword(s):  
Vibrio Fischeri ◽  
Euprymna Scolopes ◽  
Content Type ◽  
Host Environment ◽  
Enhanced Luminescence

ABSTRACTVibrio fischeriES114 is a bioluminescent symbiont of the squidEuprymna scolopes. Like most isolates fromE. scolopes, ES114 produces only dim luminescence outside the host, even in dense cultures. We previously identified mutants with brighter luminescence, and here we report their symbiotic phenotypes, providing insights into the host environment.

scholarly journals Characterization of the Vibrio fischeri Fatty Acid Chemoreceptors, VfcB and VfcB2

2015 ◽  
Vol 82 (2) ◽  
pp. 696-704 ◽  
Author(s):  
K. Nikolakakis ◽  
K. Monfils ◽  
S. Moriano-Gutierrez ◽  
C. A. Brennan ◽  
E. G. Ruby
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Vibrio Fischeri ◽  
Euprymna Scolopes ◽  
Content Type ◽  
The Family ◽  
Ligand Interactions ◽  
Chemotaxis Proteins ◽  
Large Repertoire

ABSTRACTBacteria use a wide variety of methyl-accepting chemotaxis proteins (MCPs) to mediate their attraction to or repulsion from different chemical signals in their environment. The bioluminescent marine bacteriumVibrio fischeriis the monospecific symbiont of the Hawaiian bobtail squid,Euprymna scolopes, and encodes a large repertoire of MCPs that are hypothesized to be used during different parts of its complex, multistage lifestyle. Here, we report the initial characterization of two such MCPs fromV. fischerithat are responsible for mediating migration toward short- and medium-chain aliphatic (or fatty) acids. These receptors appear to be distributed among only members of the familyVibrionaceaeand are likely descended from a receptor that has been lost by the majority of the members of this family. While chemotaxis greatly enhances the efficiency of host colonization byV. fischeri, fatty acids do not appear to be used as a chemical cue during this stage of the symbiosis. This study presents an example of straight-chain fatty acid chemoattraction and contributes to the growing body of characterized MCP-ligand interactions.

scholarly journals HbtR, a Heterofunctional Homolog of the Virulence Regulator TcpP, Facilitates the Transition between Symbiotic and Planktonic Lifestyles in Vibrio fischeri

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Brittany D. Bennett ◽  
Tara Essock-Burns ◽  
Edward G. Ruby
Keyword(s):  
Vibrio Fischeri ◽  
Light Organ ◽  
Euprymna Scolopes ◽  
Regulatory Pathway ◽  
Content Type ◽  
Exopolysaccharide Production ◽  
Bioluminescent Bacterium ◽  
Expression Of Genes ◽  
Complex Process ◽  
Virulence Regulator

ABSTRACT The bioluminescent bacterium Vibrio fischeri forms a mutually beneficial symbiosis with the Hawaiian bobtail squid, Euprymna scolopes, in which the bacteria, housed inside a specialized light organ, produce light used by the squid in its nocturnal activities. Upon hatching, E. scolopes juveniles acquire V. fischeri from the seawater through a complex process that requires, among other factors, chemotaxis by the bacteria along a gradient of N-acetylated sugars into the crypts of the light organ, the niche in which the bacteria reside. Once inside the light organ, V. fischeri transitions into a symbiotic, sessile state in which the quorum-signaling regulator LitR induces luminescence. In this work we show that expression of litR and luminescence are repressed by a homolog of the Vibrio cholerae virulence factor TcpP, which we have named HbtR. Further, we demonstrate that LitR represses genes involved in motility and chemotaxis into the light organ and activates genes required for exopolysaccharide production. IMPORTANCE TcpP homologs are widespread throughout the Vibrio genus; however, the only protein in this family described thus far is a V. cholerae virulence regulator. Here, we show that HbtR, the TcpP homolog in V. fischeri, has both a biological role and regulatory pathway completely unlike those in V. cholerae. Through its repression of the quorum-signaling regulator LitR, HbtR affects the expression of genes important for colonization of the E. scolopes light organ. While LitR becomes activated within the crypts and upregulates luminescence and exopolysaccharide genes and downregulates chemotaxis and motility genes, it appears that HbtR, upon expulsion of V. fischeri cells into seawater, reverses this process to aid the switch from a symbiotic to a planktonic state. The possible importance of HbtR to the survival of V. fischeri outside its animal host may have broader implications for the ways in which bacteria transition between often vastly different environmental niches.

scholarly journals Bacterial Quorum-Sensing Regulation Induces Morphological Change in a Key Host Tissue during the Euprymna scolopes-Vibrio fischeri Symbiosis

mBio ◽  
2021 ◽  
Author(s):  
T. Essock-Burns ◽  
B. D. Bennett ◽  
D. Arencibia ◽  
S. Moriano-Gutierrez ◽  
M. Medeiros ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Morphological Change ◽  
Vibrio Fischeri ◽  
Host Tissue ◽  
Light Organ ◽  
Euprymna Scolopes ◽  
Content Type ◽  
Bacterial Quorum Sensing ◽  
Colonization Factors ◽  
Bacterial Quorum

Interbacterial signaling within a host-associated population can have profound effects on the behavior of the bacteria, for instance, in their production of virulence/colonization factors; in addition, such signaling can dictate the nature of the outcome for the host, in both pathogenic and beneficial associations. Using the monospecific squid-vibrio model of symbiosis, we examined how quorum-sensing regulation by the Vibrio fischeri population induces a biogeographic tissue phenotype that promotes the retention of this extracellular symbiont within the light organ of its host, Euprymna scolopes .

scholarly journals A Small Molecule Coordinates Symbiotic Behaviors in a Host Organ

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Katherine E. Zink ◽  
Denise A. Ludvik ◽  
Phillip R. Lazzara ◽  
Terry W. Moore ◽  
Mark J. Mandel ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Small Molecules ◽  
Vibrio Fischeri ◽  
Imaging Mass Spectrometry ◽  
Light Organ ◽  
Euprymna Scolopes ◽  
Content Type ◽  
Bacterial Luminescence ◽  
Microbe Interactions ◽  
Host Microbe Interactions

ABSTRACT The lifelong relationship between the Hawaiian bobtail squid Euprymna scolopes and its microbial symbiont Vibrio fischeri represents a simplified model system for studying microbiome establishment and maintenance. The bacteria colonize a dedicated symbiotic light organ in the squid, from which bacterial luminescence camouflages the host in a process termed counterillumination. The squid host hatches without its symbionts, which must be acquired from the ocean amidst a diversity of nonbeneficial bacteria, such that precise molecular communication is required for initiation of the specific relationship. Therefore it is likely there are specialized metabolites used in the light organ microenvironment to modulate these processes. To identify small molecules that may influence the establishment of this symbiosis, we used imaging mass spectrometry to analyze metabolite production in V. fischeri with altered biofilm production, which correlates directly to colonization capability in its host. “Biofilm-up” and “biofilm-down” mutants were compared to a wild-type strain, and ions that were more abundantly produced by the biofilm-up mutant were detected. Using a combination of structural elucidation and synthetic chemistry, one such signal was determined to be a diketopiperazine, cyclo(d-histidyl-l-proline). This diketopiperazine modulated luminescence in V. fischeri and, using imaging mass spectrometry, was directly detected in the light organ of the colonized host. This work highlights the continued need for untargeted discovery efforts in host-microbe interactions and showcases the benefits of the squid-Vibrio system for identification and characterization of small molecules that modulate microbiome behaviors. IMPORTANCE The complexity of animal microbiomes presents challenges to defining signaling molecules within the microbial consortium and between the microbes and the host. By focusing on the binary symbiosis between Vibrio fischeri and Euprymna scolopes, we have combined genetic analysis with direct imaging to define and study small molecules in the intact symbiosis. We have detected and characterized a diketopiperazine produced by strong biofilm-forming V. fischeri strains that was detectable in the host symbiotic organ, and which influences bacterial luminescence. Biofilm formation and luminescence are critical for initiation and maintenance of the association, respectively, suggesting that the compound may link early and later development stages, providing further evidence that multiple small molecules are important in establishing these beneficial relationships.

scholarly journals Squid-Derived Chitin Oligosaccharides Are a Chemotactic Signal during Colonization by Vibrio fischeri

2012 ◽  
Vol 78 (13) ◽  
pp. 4620-4626 ◽  
Author(s):  
Mark J. Mandel ◽  
Amy L. Schaefer ◽  
Caitlin A. Brennan ◽  
Elizabeth A. C. Heath-Heckman ◽  
Cindy R. DeLoney-Marino ◽  
...  
Keyword(s):  
Vibrio Fischeri ◽  
Attachment Site ◽  
Light Organ ◽  
Euprymna Scolopes ◽  
Luminous Bacterium ◽  
Content Type ◽  
Light Emitting ◽  
Marine Luminous Bacterium ◽  
The Family ◽  
Host Generation

ABSTRACTChitin, a polymer ofN-acetylglucosamine (GlcNAc), is noted as the second most abundant biopolymer in nature. Chitin serves many functions for marine bacteria in the familyVibrionaceae(“vibrios”), in some instances providing a physical attachment site, inducing natural genetic competence, and serving as an attractant for chemotaxis. The marine luminous bacteriumVibrio fischeriis the specific symbiont in the light-emitting organ of the Hawaiian bobtail squid,Euprymna scolopes. The bacterium provides the squid with luminescence that the animal uses in an antipredatory defense, while the squid supports the symbiont's nutritional requirements.V. fischericells are harvested from seawater during each host generation, andV. fischeriis the only species that can complete this process in nature. Furthermore, chitin is located in squid hemocytes and plays a nutritional role in the symbiosis. We demonstrate here that chitin oligosaccharides produced by the squid host serve as a chemotactic signal for colonizing bacteria.V. fischeriuses the gradient of host chitin to enter the squid light organ duct and colonize the animal. We provide evidence that chitin serves a novel function in an animal-bacterial mutualism, as an animal-produced bacterium-attracting synomone.

scholarly journals Characterization of RelA in Acinetobacter baumannii

2020 ◽  
Vol 202 (12) ◽  
Author(s):  
María Pérez-Varela ◽  
Aimee R. P. Tierney ◽  
Ju-Sim Kim ◽  
Andrés Vázquez-Torres ◽  
Philip Rather
Keyword(s):  
Acinetobacter Baumannii ◽  
Galleria Mellonella ◽  
Multidrug Resistant ◽  
Cell Physiology ◽  
Content Type ◽  
Content Model ◽  
Transposon Insertion ◽  
Downstream Effectors

ABSTRACT In response to nutrient depletion, the RelA and SpoT proteins generate the signaling molecule (p)ppGpp, which then controls a number of downstream effectors to modulate cell physiology. In Acinetobacter baumannii strain AB5075, a relA ortholog (ABUW_3302) was identified by a transposon insertion that conferred an unusual colony phenotype. An in-frame deletion in relA (ΔrelA) failed to produce detectable levels of ppGpp when amino acid starvation was induced with serine hydroxamate. The ΔrelA mutant was blocked from switching from the virulent opaque colony variant (VIR-O) to the avirulent translucent colony variant (AV-T), but the rate of AV-T to VIR-O switching was unchanged. In addition, the ΔrelA mutation resulted in a pronounced hypermotile phenotype on 0.35% agar plates. This hypermotility was dependent on the activation of a LysR regulator ABUW_1132, which was required for expression of AbaR, a LuxR family quorum-sensing regulator. In the ΔrelA mutant, ABUW_1132 was also required for the increased expression of an operon composed of the ABUW_3766-ABUW_3773 genes required for production of the surfactant-like lipopeptide acinetin 505. Additional phenotypes identified in the ΔrelA mutant included (i) cell elongation at high density, (ii) reduced formation of persister cells tolerant to colistin and rifampin, and (iii) decreased virulence in a Galleria mellonella model. IMPORTANCE Acinetobacter baumannii is a pathogen of worldwide importance. Due to the increasing prevalence of antibiotic resistance, these infections are becoming increasingly difficult to treat. New therapies are required to combat multidrug-resistant isolates. The role of RelA in A. baumannii is largely unknown. This study demonstrates that like in other bacteria, RelA controls a variety of functions, including virulence. Strategies to inhibit the activity of RelA and the resulting production of ppGpp could inhibit virulence and may represent a new therapeutic approach.

scholarly journals Characterization of htrB and msbB Mutants of the Light Organ Symbiont Vibrio fischeri

2007 ◽  
Vol 74 (3) ◽  
pp. 633-644 ◽  
Author(s):  
Dawn M. Adin ◽  
Nancy J. Phillips ◽  
Bradford W. Gibson ◽  
Michael A. Apicella ◽  
Edward G. Ruby ◽  
...  
Keyword(s):  
Lipid A ◽  
Vibrio Fischeri ◽  
Sodium Dodecyl ◽  
Chromosome 1 ◽  
Chromosome 2 ◽  
Euprymna Scolopes ◽  
Host Interactions ◽  
Lipid A Structure ◽  
Bacterial Lipid

ABSTRACT Bacterial lipid A is an important mediator of bacterium-host interactions, and secondary acylations added by HtrB and MsbB can be critical for colonization and virulence in pathogenic infections. In contrast, Vibrio fischeri lipid A stimulates normal developmental processes in this bacterium's mutualistic host, Euprymna scolopes, although the importance of lipid A structure in this symbiosis is unknown. To further examine V. fischeri lipid A and its symbiotic function, we identified two paralogs of htrB (designated htrB1 and htrB2) and an msbB gene in V. fischeri ES114 and demonstrated that these genes encode lipid A secondary acyltransferases. htrB2 and msbB are found on the Vibrio “housekeeping” chromosome 1 and are conserved in other Vibrio species. Mutations in htrB2 and msbB did not impair symbiotic colonization but resulted in phenotypic alterations in culture, including reduced motility and increased luminescence. These mutations also affected sensitivity to sodium dodecyl sulfate, kanamycin, and polymyxin, consistent with changes in membrane permeability. Conversely, htrB1 is located on the smaller, more variable vibrio chromosome 2, and an htrB1 mutant was wild-type-like in culture but appeared attenuated in initiating the symbiosis and was outcompeted 2.7-fold during colonization when mixed with the parent. These data suggest that htrB2 and msbB play conserved general roles in vibrio biology, whereas htrB1 plays a more symbiosis-specific role in V. fischeri.

scholarly journals Genes Contributing to Staphylococcus aureus Fitness in Abscess- and Infection-Related Ecologies

mBio ◽  
2014 ◽  
Vol 5 (5) ◽  
Author(s):  
Michael D. Valentino ◽  
Lucy Foulston ◽  
Ama Sadaka ◽  
Veronica N. Kos ◽  
Regis A. Villet ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
New Drugs ◽  
Insertion Mutant ◽  
Mutant Library ◽  
Entire Genome ◽  
Antibiotic Resistant ◽  
Content Type ◽  
Transposon Insertion ◽  
Genes Encoding ◽  
Novel Targets

ABSTRACTStaphylococcus aureusis a leading cause of both community- and hospital-acquired infections that are increasingly antibiotic resistant. The emergence ofS. aureusresistance to even last-line antibiotics heightens the need for the development of new drugs with novel targets. We generated a highly saturated transposon insertion mutant library in the genome ofS. aureusand used Tn-seq analysis to probe the entire genome, with unprecedented resolution and sensitivity, for genes of importance in infection. We further identified genes contributing to fitness in various infected compartments (blood and ocular fluids) and compared them to genes required for growth in rich medium. This resulted in the identification of 426 genes that were important forS. aureusfitness during growth in infection models, including 71 genes that could be considered essential for survival specifically during infection. These findings highlight novel as well as previously known genes encoding virulence traits and metabolic pathways important forS. aureusproliferation at sites of infection, which may represent new therapeutic targets.IMPORTANCEStaphylococcus aureuscontinues to be a leading cause of antibiotic-resistant community and nosocomial infection. With the bacterium’s acquisition of resistance to methicillin and, more recently, vancomycin, the need for the development of new drugs with novel targets is urgent. Applying a highly saturated Tn-seq mutant library to analyze fitness and growth requirements in a murine abscess and in various infection-relevant fluids, we identifiedS. aureustraits that enable it to survive and proliferate during infection. This identifies potential new targeting opportunities for the development of novel therapeutics.

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.

Sign in / Sign up

Export Citation Format

Share Document