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Karen L. Visick - One of the best experts on this subject based on the ideXlab platform.
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Control of Competence in Vibrio fischeri.
Applied and environmental microbiology, 2021Co-Authors: Joshua J. Cohen, Jovanka Tepavčević, Steven J. Eichinger, Danae A. Witte, Connor J. Cook, Pat M. Fidopiastis, Karen L. VisickAbstract:ABSTRACT Vibrio species, including the squid symbiont Vibrio fischeri, become competent to take up DNA under specific conditions. For example, V. fischeri becomes competent when grown in the presence of chitin oligosaccharides or upon overproduction of the competence regulatory factor TfoX. While little is known about the regulatory pathway(s) that controls V. fischeri competence, this microbe encodes homologs of factors that control competence in the well-studied V. cholerae. To further develop V. fischeri as a genetically tractable organism, we evaluated the roles of some of these competence homologs. Using TfoX-overproducing cells, we found that competence depends upon LitR, the homolog of V. cholerae master quorum-sensing and competence regulator HapR, and upon homologs of putative pilus genes that in V. cholerae facilitate DNA uptake. Disruption of genes for negative regulators upstream of LitR, namely, the LuxO protein and the small RNA (sRNA) Qrr1, resulted in increased transformation frequencies. Unlike LitR-controlled light production, however, competence did not vary with cell density under tfoX overexpression conditions. Analogous to the case with V. cholerae, the requirement for LitR could be suppressed by loss of the Dns nuclease. We also found a role for the putative competence regulator CytR. Finally, we determined that transformation frequencies varied depending on the TfoX-encoding plasmid, and we developed a new dual tfoX and litR overexpression construct that substantially increased the transformation frequency of a less genetically tractable strain. By advancing the ease of genetic manipulation of V. fischeri, these findings will facilitate the rapid discovery of genes involved in physiologically relevant processes, such as biofilm formation and host colonization. IMPORTANCE The ability of bacteria to take up DNA (competence) and incorporate foreign DNA into their genomes (transformation) permits them to rapidly evolve and gain new traits and/or acquire antibiotic resistances. It also facilitates laboratory-based investigations into mechanisms of specific phenotypes, such as those involved in host colonization. Vibrio fischeri has long been a model for symbiotic bacterium-host interactions as well as for other aspects of its physiology, such as bioluminescence and biofilm formation. Competence of V. fischeri can be readily induced upon overexpression of the competence factor TfoX. Relatively little is known about the V. fischeri competence pathway, although homologs of factors known to be important in V. cholerae competence exist. By probing the importance of putative competence factors that control transformation of V. fischeri, this work deepens our understanding of the competence process and advances our ability to genetically manipulate this important model organism.
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Genetic Manipulation of Vibrio fischeri.
Current protocols in microbiology, 2020Co-Authors: David G. Christensen, Jovanka Tepavčević, Karen L. VisickAbstract:Vibrio fischeri is a nonpathogenic organism related to pathogenic Vibrio species. The bacterium has been used as a model organism to study symbiosis in the context of its association with its host, the Hawaiian bobtail squid Euprymna scolopes. The genetic tractability of this bacterium has facilitated the mapping of pathways that mediate interactions between these organisms. The protocols included here describe methods for genetic manipulation of V. fischeri. Following these protocols, the researcher will be able to introduce linear DNA via transformation to make chromosomal mutations, to introduce plasmid DNA via conjugation and subsequently eliminate unstable plasmids, to eliminate antibiotic resistance cassettes from the chromosome, and to randomly or specifically mutagenize V. fischeri with transposons. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Transformation of V. fischeri with linear DNA Basic Protocol 2: Plasmid transfer into V. fischeri via conjugation Support Protocol 1: Removing FRT-flanked antibiotic resistance cassettes from the V. fischeri genome Support Protocol 2: Eliminating unstable plasmids from V. fischeri Alternate Protocol 1: Introduction of exogenous DNA using a suicide plasmid Alternate Protocol 2: Site-specific transposon insertion using a suicide plasmid Alternate Protocol 3: Random transposon mutagenesis using a suicide plasmid.
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Vibrio fischeri Biofilm Formation Prevented by a Trio of Regulators.
Applied and Environmental Microbiology, 2018Co-Authors: Cecilia M. Thompson, Anne E. Marsden, Alice H. Tischler, Karen L. VisickAbstract:ABSTRACT Biofilms, complex communities of microorganisms surrounded by a self-produced matrix, facilitate attachment and provide protection to bacteria. A natural model used to study biofilm formation is the symbiosis between Vibrio fischeri and its host, the Hawaiian bobtail squid, Euprymna scolopes. Host-relevant biofilm formation is a tightly regulated process and is observed in vitro only with strains that have been genetically manipulated to overexpress or disrupt specific regulators, primarily two-component signaling (TCS) regulators. These regulators control biofilm formation by dictating the production of the symbiosis polysaccharide (Syp-PS), the major component of the biofilm matrix. Control occurs both at and below the level of transcription of the syp genes, which are responsible for Syp-PS production. Here, we probed the roles of the two known negative regulators of biofilm formation, BinK and SypE, by generating double mutants. We also mapped and evaluated a point mutation using natural transformation and linkage analysis. We examined traditional biofilm formation phenotypes and established a new assay for evaluating the start of biofilm formation in the form of microscopic aggregates in shaking liquid cultures, in the absence of the known biofilm-inducing signal calcium. We found that wrinkled colony formation is negatively controlled not only by BinK and SypE but also by SypF. SypF is both required for and inhibitory to biofilm formation. Together, these data reveal that these three regulators are sufficient to prevent wild-type V. fischeri from forming biofilms under these conditions. IMPORTANCE Bacterial biofilms promote attachment to a variety of surfaces and protect the constituent bacteria from environmental stresses, including antimicrobials. Understanding the mechanisms by which biofilms form will promote our ability to resolve them when they occur in the context of an infection. In this study, we found that Vibrio fischeri tightly controls biofilm formation using three negative regulators; the presence of a single one of these regulators was sufficient to prevent full biofilm development, while disruption of all three permitted robust biofilm formation. This work increases our understanding of the functions of specific regulators and demonstrates the substantial negative control that one benign microbe exerts over biofilm formation, potentially to ensure that it occurs only under the appropriate conditions.
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Tools for rapid genetic engineering of Vibrio fischeri
Applied and environmental microbiology, 2018Co-Authors: Karen L. Visick, Alice H. Tischler, Kelsey M. Hodge-hanson, Allison K. Bennett, Vincent MastrodomenicoAbstract:Vibrio fischeri is used as a model for number of processes, including symbiosis, quorum sensing, bioluminescence, and biofilm formation. Many of these studies depend on generating deletion mutants and complementing them. Engineering such strains, however, is a time-consuming, multi-step process that relies on cloning and subcloning. Here, we describe a set of tools that can be used to rapidly engineer deletions and insertions in the V. fischeri chromosome without cloning. We developed a uniform approach for generating deletions using PCR SOEing (Splicing by Overlap Extension) with antibiotic cassettes flanked by standardized linker sequences. PCR SOEing of the cassettes to sequences up- and downstream of the target gene generates a DNA product that can be directly introduced by natural transformation. Selection for the introduced antibiotic resistance marker yields the deletion of interest in a single step. Because these cassettes also contain FRT sequences flanking the resistance marker, Flp recombinase can be used to generate an unmarked, in-frame deletion. We developed a similar methodology and tools for the rapid insertion of specific genes at a benign site in the chromosome, for purposes such as complementation. Finally, we generated derivatives of these tools to facilitate different applications, such as inducible gene expression and assessing protein production. We demonstrated the utility of these tools by deleting and inserting genes known or predicted to be involved in motility. While developed for V. fischeri strain ES114, we anticipate that these tools can be adapted for use in other V. fischeri strains and, potentially, other microbes. Importance. Vibrio fischeri is a model organism for studying a variety of important processes, including symbiosis, biofilm formation, and quorum sensing. To facilitate investigation of these biological mechanisms, we developed approaches for rapidly generating deletions and insertions, and demonstrated their utility using two genes of interest. The ease, consistency, and speed of the engineering is facilitated by a set of antibiotic resistance cassettes with common linker sequences that can be amplified by PCR with universal primers and fused to adjacent sequences using splicing by overlap extension, then introduced directly into V. fischeri , eliminating the need for cloning and plasmid conjugation. The antibiotic cassettes are flanked by FRT sequences, permitting their removal using Flp recombinase. We augmented these basic tools with a family of constructs for different applications. We anticipate that these tools will greatly accelerate mechanistic studies of biological processes in V. fischeri , and potentially other Vibrio species.
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Engineering Vibrio fischeri for Inducible Gene Expression.
The open microbiology journal, 2014Co-Authors: Jakob M. Ondrey, Karen L. VisickAbstract:The marine bacterium Vibrio fischeri serves as a model organism for a variety of natural phenomena, including symbiotic host colonization. The ease with which the V. fischeri genome can be manipulated contributes greatly to our ability to identify the factors involved in these phenomena. Here, we have adapted genetic tools for use in V. fischeri to promote our ability to conditionally control the expression of genes of interest. Specifically, we modified the commonly used mini-Tn5 transposon to contain an outward-facing, LacI-repressible/IPTG-inducible promoter, and inserted the lacI gene into the V. fischeri chromosome. Used together, these tools permit the identification and induction of genes that control specific phenotypes. To validate this approach, we identified IPTG-controllable motility mutants. We anticipate that the ability to randomly insert an inducible promoter into the genome of V. fischeri will advance our understanding of various aspects of the physiology of this microbe.
Edward G. Ruby - One of the best experts on this subject based on the ideXlab platform.
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Shedding light on bioluminescence regulation in Vibrio fischeri
Molecular microbiology, 2012Co-Authors: Tim Miyashiro, Edward G. RubyAbstract:The bioluminescence emitted by the marine bacterium Vibrio fischeri is a particularly striking result of individual microbial cells co-ordinating a group behaviour. The genes responsible for light production are principally regulated by the LuxR-LuxI quorum-sensing system. In addition to LuxR-LuxI, numerous other genetic elements and environmental conditions control bioluminescence production. Efforts to mathematically model the LuxR-LuxI system are providing insight into the dynamics of this autoinduction behaviour. The Hawaiian squid Euprymna scolopes forms a natural symbiosis with V. fischeri, and utilizes the symbiont-derived bioluminescence for certain nocturnal behaviours, such as counterillumination. Recent work suggests that the tissue with which V. fischeri associates not only can detect bioluminescence but may also use this light to monitor the V. fischeri population.
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Transcriptome Analysis of the Vibrio fischeri LuxR-LuxI Regulon
Journal of bacteriology, 2007Co-Authors: Luis Caetano M. Antunes, Amy L. Schaefer, Rosana B. R. Ferreira, Nan Qin, Ann M. Stevens, Edward G. Ruby, E. Peter GreenbergAbstract:The Vibrio fischeri quorum-sensing signal N-3-oxohexanoyl-l-homoserine lactone (3OC6-HSL) activates expression of the seven-gene luminescence operon. We used microarrays to unveil 18 additional 3OC6-HSL-controlled genes, 3 of which had been identified by other means previously. We show most of these genes are regulated by the 3OC6-HSL-responsive transcriptional regulator LuxR directly. This demonstrates that V. fischeri quorum sensing regulates a substantial number of genes other than those involved in light production.
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Vibrio fischeri and its host: it takes two to tango.
Current opinion in microbiology, 2006Co-Authors: Karen L. Visick, Edward G. RubyAbstract:The association of Vibrio fischeri and Euprymna scolopes provides insights into traits essential for symbiosis, and the signals and pathways of bacteria-induced host development. Recent studies have identified important bacterial colonization factors, including those involved in motility, bioluminescence and biofilm formation. Surprising links between symbiosis and pathogenesis have been revealed through discoveries that nitric oxide is a component of the host defense, and that V. fischeri uses a cytotoxin-like molecule to induce host development. Technological advances in this system include the genome sequence of V. fischeri, an expressed sequence tagged library for E. scolopes and the availability of dual-fluorescence markers and confocal microscopy to probe symbiotic structures and the dynamics of colonization.
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complete genome sequence of Vibrio fischeri a symbiotic bacterium with pathogenic congeners
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Edward G. Ruby, Anne K. Dunn, M Urbanowski, J Campbell, M Faini, Robert P Gunsalus, P Lostroh, Claudia Lupp, Jessica R Mccann, Deborah S. MillikanAbstract:Vibrio fischeri belongs to the Vibrionaceae, a large family of marine γ-proteobacteria that includes several dozen species known to engage in a diversity of beneficial or pathogenic interactions with animal tissue. Among the small number of pathogenic Vibrio species that cause human diseases are Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus, the only members of the Vibrionaceae that have had their genome sequences reported. Nonpathogenic members of the genus Vibrio, including a number of beneficial symbionts, make up the majority of the Vibrionaceae, but none of these species has been similarly examined. Here we report the genome sequence of V. fischeri ES114, which enters into a mutualistic symbiosis in the light organ of the bobtail squid, Euprymna scolopes. Analysis of this sequence has revealed surprising parallels with V. cholerae and other pathogens.
Eric V. Stabb - One of the best experts on this subject based on the ideXlab platform.
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Symbiotic Characterization of Vibrio fischeri ES114 Mutants That Display Enhanced Luminescence in Culture
Applied and environmental microbiology, 2013Co-Authors: Noreen L. Lyell, Eric V. StabbAbstract:Vibrio fischeri ES114 is a bioluminescent symbiont of the squid Euprymna scolopes. Like most isolates from E. 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.
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Effective Mutagenesis of Vibrio fischeri by Using Hyperactive Mini-Tn5 Derivatives
Applied and environmental microbiology, 2008Co-Authors: Noreen L. Lyell, Anne K. Dunn, Jeffrey L. Bose, Susan L. Vescovi, Eric V. StabbAbstract:We have developed a transposon mutagenesis system for Vibrio fischeri ES114 that utilizes a hyperactive mutant Tn5 transposase (E54K and M56A) and optimized transposon ends. Using a conjugation-based procedure, we obtained independent single-insertion mini-Tn5 mutants at a rate of approximately 10(-6). This simple and inexpensive technique represents a significant improvement over previous methods for transposon mutagenesis of V. fischeri and should be applicable to many other bacteria.
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The twin arginine translocation system contributes to symbiotic colonization of Euprymna scolopes by Vibrio fischeri.
FEMS microbiology letters, 2008Co-Authors: Anne K. Dunn, Eric V. StabbAbstract:In many bacteria, the twin arginine translocation (Tat) system transports folded proteins across the cytoplasmic membrane, and these proteins can play a role in symbiotic or pathogenic infections. A role for the Vibrio fischeri Tat system was identified during symbiotic colonization of its host Euprymna scolopes, demonstrating a function for the Tat system in host colonization by a member of the Vibrionaceae. Using bioinformatics, mutant analyses, and green fluorescent protein fusions, a set of Tat-targeted proteins in V. fischeri was identified.
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Correlation between Osmolarity and Luminescence of Symbiotic Vibrio fischeri Strain ES114
Journal of bacteriology, 2004Co-Authors: Eric V. Stabb, Melissa S. Butler, Dawn M. AdinAbstract:Vibrio fischeri isolates from Euprymna scolopes are dim in culture but bright in the host. We found the luminescence of V. fischeri to be correlated with external osmolarity both in culture and in this symbiosis. Luminescence enhancement by osmolarity was independent of the lux promoter and unaffected by autoinducers or the level of lux expression, but the addition of an aldehyde substrate for luciferase raised the luminescence of cells grown at high and low osmolarities to the same high level. V. fischeri culture media have lower osmolarities than are typical in seawater or in cephalopods, partially accounting for the bacterium's low light output in culture.
Mark J. Mandel - One of the best experts on this subject based on the ideXlab platform.
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Draft Genome Sequences of Type VI Secretion System-Encoding Vibrio fischeri Strains FQ-A001 and ES401.
Microbiology resource announcements, 2019Co-Authors: Katherine M. Bultman, Tim Miyashiro, Andrew G. Cecere, Alecia N. Septer, Mark J. MandelAbstract:ABSTRACT The type VI secretion system (T6SS) facilitates lethal competition between bacteria through direct contact. Comparative genomics has facilitated the study of these systems in Vibrio fischeri, which colonizes the squid host Euprymna scolopes. Here, we report the draft genome sequences of two lethal V. fischeri strains that encode the T6SS, FQ-A001 and ES401.
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natural strain variation reveals diverse biofilm regulation in squid colonizing Vibrio fischeri
bioRxiv, 2019Co-Authors: Ella Rotman, Mark J. Mandel, Katherine M. Bultman, Mattias C. Gyllborg, John F Brooks, Hector L Burgos, Michael S WollenbergAbstract:ABSTRACT The mutualistic symbiont Vibrio fischeri builds a symbiotic biofilm during colonization of squid hosts. Regulation of the exopolysaccharide component, termed Syp, has been examined in strain ES114, where production is controlled by a phosphorelay that includes the inner membrane hybrid histidine kinase RscS. Most strains that lack RscS or encode divergent RscS proteins cannot colonize a squid host unless RscS from a squid symbiont is heterologously expressed. In this study, we examine V. fischeri isolates worldwide to understand the landscape of biofilm regulation during beneficial colonization. We provide a detailed study of three distinct evolutionary groups of V. fischeri and find that while the RscS-Syp biofilm pathway is required in one of the groups, two other groups of squid symbionts require Syp independent of RscS. Mediterranean squid symbionts, including V. fischeri SR5, colonize without an RscS homolog encoded in their genome. Additionally, Group A V. fischeri strains, which form a tightly-related clade of Hawaii isolates, have a frameshift in rscS and do not require the gene for squid colonization or competitive fitness. These same strains have a frameshift in sypE, and we provide evidence that this Group A sypE allele leads to an upregulation in biofilm activity. This work thus describes the central importance of Syp biofilm in colonization of diverse isolates, and demonstrates that significant evolutionary transitions correspond to regulatory changes in the syp pathway. IMPORTANCE Biofilms are surface-associated, matrix-encased bacterial aggregates that exhibit enhanced protection to antimicrobial agents. Previous work has established the importance of biofilm formation by a strain of luminous Vibrio fischeri bacteria as the bacteria colonize their host, the Hawaiian bobtail squid. In this study, expansion of this work to many natural isolates revealed that biofilm genes are universally required, yet there has been a shuffling of the regulators of those genes. This work provides evidence that even when bacterial behaviors are conserved, dynamic regulation of those behaviors can underlie evolution of the host colonization phenotype. Furthermore, this work emphasizes the importance of investigating natural diversity as we seek to understand molecular mechanisms in bacteria.
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Chemoreceptor VfcA Mediates Amino Acid Chemotaxis in Vibrio fischeri
Applied and environmental microbiology, 2013Co-Authors: Caitlin A. Brennan, Cindy R. Deloney-marino, Mark J. MandelAbstract:Flagellar motility and chemotaxis by Vibrio fischeri are important behaviors mediating the colonization of its mutualistic host, the Hawaiian bobtail squid. However, none of the 43 putative methyl-accepting chemotaxis proteins (MCPs) encoded in the V. fischeri genome has been previously characterized. Using both an available transposon mutant collection and directed mutagenesis, we isolated mutants for 19 of these genes, and screened them for altered chemotaxis to six previously identified chemoattractants. Only one mutant was defective in responding to any of the tested compounds; the disrupted gene was thus named vfcA (Vibrio fischeri chemoreceptor A; locus tag VF_0777). In soft-agar plates, mutants disrupted in vfcA did not exhibit the serine-sensing chemotactic ring, and the pattern of migration in the mutant was not affected by the addition of exogenous serine. Using a capillary chemotaxis assay, we showed that, unlike wild-type V. fischeri, the vfcA mutant did not undergo chemotaxis toward serine and that expression of vfcA on a plasmid in the mutant was sufficient to restore the behavior. In addition to serine, we demonstrated that alanine, cysteine, and threonine are strong attractants for wild-type V. fischeri and that the attraction is also mediated by VfcA. This study thus provides the first insights into how V. fischeri integrates information from one of its 43 MCPs to respond to environmental stimuli.
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Draft Genome Sequence of Vibrio fischeri SR5, a Strain Isolated from the Light Organ of the Mediterranean Squid Sepiola robusta
Journal of bacteriology, 2012Co-Authors: Mattias C. Gyllborg, Jason W. Sahl, David C. Cronin, David A. Rasko, Mark J. MandelAbstract:Here, we describe the draft genome sequence of Vibrio fischeri SR5, a squid symbiotic isolate from Sepiola robusta in the Mediterranean Sea. This 4.3-Mbp genome sequence represents the first V. fischeri genome from an S. robusta symbiont and the first from outside the Pacific Ocean.
Anne K. Dunn - One of the best experts on this subject based on the ideXlab platform.
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Vibrio fischeri metabolism: symbiosis and beyond.
Advances in microbial physiology, 2012Co-Authors: Anne K. DunnAbstract:Vibrio fischeri is a bioluminescent, Gram-negative marine bacterium that can be found free living and in a mutualistic association with certain squids and fishes. Over the past decades, the study of V. fischeri has led to important discoveries about bioluminescence, quorum sensing, and the mechanisms that underlie beneficial host-microbe interactions. This chapter highlights what has been learned about metabolic pathways in V. fischeri, and how this information contributes to a broader understanding of the role of bacterial metabolism in host colonization by both beneficial and pathogenic bacteria, as well as in the growth and survival of free-living bacteria.
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Effective Mutagenesis of Vibrio fischeri by Using Hyperactive Mini-Tn5 Derivatives
Applied and environmental microbiology, 2008Co-Authors: Noreen L. Lyell, Anne K. Dunn, Jeffrey L. Bose, Susan L. Vescovi, Eric V. StabbAbstract:We have developed a transposon mutagenesis system for Vibrio fischeri ES114 that utilizes a hyperactive mutant Tn5 transposase (E54K and M56A) and optimized transposon ends. Using a conjugation-based procedure, we obtained independent single-insertion mini-Tn5 mutants at a rate of approximately 10(-6). This simple and inexpensive technique represents a significant improvement over previous methods for transposon mutagenesis of V. fischeri and should be applicable to many other bacteria.
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The twin arginine translocation system contributes to symbiotic colonization of Euprymna scolopes by Vibrio fischeri.
FEMS microbiology letters, 2008Co-Authors: Anne K. Dunn, Eric V. StabbAbstract:In many bacteria, the twin arginine translocation (Tat) system transports folded proteins across the cytoplasmic membrane, and these proteins can play a role in symbiotic or pathogenic infections. A role for the Vibrio fischeri Tat system was identified during symbiotic colonization of its host Euprymna scolopes, demonstrating a function for the Tat system in host colonization by a member of the Vibrionaceae. Using bioinformatics, mutant analyses, and green fluorescent protein fusions, a set of Tat-targeted proteins in V. fischeri was identified.
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complete genome sequence of Vibrio fischeri a symbiotic bacterium with pathogenic congeners
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Edward G. Ruby, Anne K. Dunn, M Urbanowski, J Campbell, M Faini, Robert P Gunsalus, P Lostroh, Claudia Lupp, Jessica R Mccann, Deborah S. MillikanAbstract:Vibrio fischeri belongs to the Vibrionaceae, a large family of marine γ-proteobacteria that includes several dozen species known to engage in a diversity of beneficial or pathogenic interactions with animal tissue. Among the small number of pathogenic Vibrio species that cause human diseases are Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus, the only members of the Vibrionaceae that have had their genome sequences reported. Nonpathogenic members of the genus Vibrio, including a number of beneficial symbionts, make up the majority of the Vibrionaceae, but none of these species has been similarly examined. Here we report the genome sequence of V. fischeri ES114, which enters into a mutualistic symbiosis in the light organ of the bobtail squid, Euprymna scolopes. Analysis of this sequence has revealed surprising parallels with V. cholerae and other pathogens.
