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Bibliography on: Squid-Vibrio Symbiosis

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ESP: PubMed Auto Bibliography 17 Sep 2021 at 01:35 Created: 

Squid-Vibrio Symbiosis

The small bobtail squid (Euprymna scolopes) has a mutually beneficial relationship with bacteria called Vibrio fischeri that live on the squid's underside. The bacteria allow the squid to produce light, which then allows the squid to escape from things that might want to eat it. "The squid emit ventral luminescence that is often very, very close to the quality of light coming from the moon and stars at night," explains Margaret McFall-Ngai, Margaret McFall-Ngai, professor of medical microbiology and immunology at the University of Wisconsin-Madison. For fish looking up from below for something to eat, the squid are camouflaged against the moon or the starlight because they don't cast a shadow. "It's like a 'Klingon' cloaking device," she notes. But the Vibrio fischeri don't stay in the squid continuously. Every day, in response to the light cue of dawn, the squid vents 90 percent of the bacteria back into the seawater. "And then, while it's sitting quiescent in the sand, the bacteria grow up in the crypt so that when [the squid] comes out in the evening, it will have a full complement of luminous Vibrio fischeri," says McFall-Ngai.

Created with PubMed® Query: (squid OR euprymna) AND (vibrio OR symbiosis OR symbiotic OR endosymbiont) NOT pmcbook NOT ispreviousversion

Citations The Papers (from PubMed®)

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RevDate: 2021-08-09

Smith S, Salvato F, Garikipati A, et al (2021)

Activation of the type VI secretion system in the squid symbiont Vibrio fischeri requires the transcriptional regulator TasR and the structural proteins TssM and TssA.

Journal of bacteriology [Epub ahead of print].

Bacteria have evolved diverse strategies to compete for a niche, including the type VI secretion system (T6SS), a contact-dependent killing mechanism. T6SSs are common in bacterial pathogens, commensals, and beneficial symbionts, where they affect the diversity and spatial structure of host-associated microbial communities. Although T6SS gene clusters are often located on genomic islands (GIs), which may be transferred as a unit, the regulatory strategies that promote gene expression once the T6SS genes are transferred into a new cell are not known. We used the squid symbiont, Vibrio fischeri, to identify essential regulatory factors that control expression of a strain-specific T6SS encoded on a GI. We found that a transcriptional reporter for this T6SS is active only in strains that contain the T6SS-encoding GI, suggesting the GI encodes at least one essential regulator. A transposon screen identified seven mutants that could not activate the reporter. These mutations mapped exclusively to three genes on the T6SS-containing GI that encode two essential structural proteins (a TssA-like protein and TssM) and a transcriptional regulator (TasR). Using T6SS reporters, RT-PCR, competition assays, and differential proteomics, we found that all three genes are required for expression of many T6SS components, except for the TssA-like protein and TssM, which are constitutively expressed. Based on these findings, we propose a model whereby T6SS expression requires conserved structural proteins, in addition to the essential regulator TasR, and this ability to self-regulate may be a strategy to activate T6SS expression upon transfer of T6SS-encoding elements into a new bacterial host. Importance Interbacterial weapons like the T6SS are often located on mobile genetic elements and their expression is highly regulated. We found that two conserved structural proteins are required for T6SS expression in Vibrio fischeri. These structural proteins also contain predicted GTPase and GTP binding domains, suggesting their role in promoting T6SS expression may involve sensing the energetic state of the cell. Such a mechanism would provide a direct link between T6SS activation and cellular energy levels, providing a "checkpoint" to ensure the cell has sufficient energy to build such a costly weapon. Because these regulatory factors are encoded within the T6SS gene cluster, they are predicted to move with the genetic element to activate T6SS expression in a new host cell.

RevDate: 2021-07-21

Speare L, Woo M, Bultman KM, et al (2021)

Host-Like Conditions Are Required for T6SS-Mediated Competition among Vibrio fischeri Light Organ Symbionts.

mSphere [Epub ahead of print].

Bacteria employ diverse competitive strategies to enhance fitness and promote their own propagation. However, little is known about how symbiotic bacteria modulate competitive mechanisms as they compete for a host niche. The bacterium Vibrio fischeri forms a symbiotic relationship with marine animals and encodes a type VI secretion system (T6SS), which is a contact-dependent killing mechanism used to eliminate competitors during colonization of the Euprymna scolopes squid light organ. Like other horizontally acquired symbionts, V. fischeri experiences changes in its physical and chemical environment during symbiosis establishment. Therefore, we probed both environmental and host-like conditions to identify ecologically relevant cues that control T6SS-dependent competition during habitat transition. Although the T6SS did not confer a competitive advantage for V. fischeri strain ES401 under planktonic conditions, a combination of both host-like pH and viscosity was necessary for T6SS competition. For ES401, high viscosity activates T6SS expression and neutral/acidic pH promotes cell-cell contact for killing, and this pH-dependent phenotype was conserved in the majority of T6SS-encoding strains examined. We also identified a subset of V. fischeri isolates that engaged in T6SS-mediated competition at high viscosity under both planktonic and host-like pH conditions. T6SS phylogeny revealed that strains with pH-dependent phenotypes cluster together to form a subclade within the pH-independent strains, suggesting that V. fischeri may have recently evolved to limit competition to the host niche. IMPORTANCE Bacteria have evolved diverse strategies to compete for limited space and resources. Because these mechanisms can be costly to use, their expression and function are often restricted to specific environments where the benefits outweigh the costs. However, little is known about the specific cues that modulate competitive mechanisms as bacterial symbionts transition between free-living and host habitats. Here, we used the bioluminescent squid and fish symbiont Vibrio fischeri to probe for host and environmental conditions that control interbacterial competition via the type VI secretion system. Our findings identify a new host-specific cue that promotes competition among many but not all V. fischeri isolates, underscoring the utility of studying multiple strains to reveal how competitive mechanisms may be differentially regulated among closely related populations as they evolve to fill distinct niches.

RevDate: 2021-07-16

Dial CN, Eichinger SJ, Foxall R, et al (2021)

Quorum Sensing and Cyclic di-GMP Exert Control Over Motility of Vibrio fischeri KB2B1.

Frontiers in microbiology, 12:690459.

Bacterial motility is critical for symbiotic colonization by Vibrio fischeri of its host, the squid Euprymna scolopes, facilitating movement from surface biofilms to spaces deep inside the symbiotic organ. While colonization has been studied traditionally using strain ES114, others, including KB2B1, can outcompete ES114 for colonization for a variety of reasons, including superior biofilm formation. We report here that KB2B1 also exhibits an unusual pattern of migration through a soft agar medium: whereas ES114 migrates rapidly and steadily, KB2B1 migrates slowly and then ceases migration. To better understand this phenomenon, we isolated and sequenced five motile KB2B1 suppressor mutants. One harbored a mutation in the gene for the cAMP receptor protein (crp); because this strain also exhibited a growth defect, it was not characterized further. Two other suppressors contained mutations in the quorum sensing pathway that controls bacterial bioluminescence in response to cell density, and two had mutations in the diguanylate cyclase (DGC) gene VF_1200. Subsequent analysis indicated that (1) the quorum sensing mutations shifted KB2B1 to a perceived low cell density state and (2) the high cell density state inhibited migration via the downstream regulator LitR. Similar to the initial point mutations, deletion of the VF_1200 DGC gene increased migration. Consistent with the possibility that production of the second messenger c-di-GMP inhibited the motility of KB2B1, reporter-based measurements of c-di-GMP revealed that KB2B1 produced higher levels of c-di-GMP than ES114, and overproduction of a c-di-GMP phosphodiesterase promoted migration of KB2B1. Finally, we assessed the role of viscosity in controlling the quorum sensing pathway using polyvinylpyrrolidone and found that viscosity increased light production of KB2B1 but not ES114. Together, our data indicate that while the two strains share regulators in common, they differ in the specifics of the regulatory control over downstream phenotypes such as motility.

RevDate: 2021-07-17

Wasilko NP, Ceron JS, Baker ER, et al (2021)

Vibrio fischeri imports and assimilates sulfate during symbiosis with Euprymna scolopes.

Molecular microbiology [Epub ahead of print].

Sulfur is in cellular components of bacteria and is, therefore, an element necessary for growth. However, mechanisms by which bacteria satisfy their sulfur needs within a host are poorly understood. Vibrio fischeri is a bacterial symbiont that colonizes, grows, and produces bioluminescence within the light organ of the Hawaiian bobtail squid, which provides an experimental platform for investigating sulfur acquisition in vivo. Like other γ-proteobacteria, V. fischeri fuels sulfur-dependent anabolic processes with intracellular cysteine. Within the light organ, the abundance of a ΔcysK mutant, which cannot synthesize cysteine through sulfate assimilation, is attenuated, suggesting sulfate import is necessary for V. fischeri to establish symbiosis. Genes encoding sulfate-import systems of other bacteria that assimilate sulfate were not identified in the V. fischeri genome. A transposon mutagenesis screen implicated YfbS as a sulfate importer. YfbS is necessary for growth on sulfate and in the marine environment. During symbiosis, a ΔyfbS mutant is attenuated and strongly expresses sulfate-assimilation genes, which is a phenotype associated with sulfur-starved cells. Together, these results suggest V. fischeri imports sulfate via YfbS within the squid light organ, which provides insight into the molecular mechanisms by which bacteria harvest sulfur in vivo.

RevDate: 2021-06-30

Heath-Heckman E, M Nishiguchi (2021)

Leveraging Short-Read Sequencing to Explore the Genomics of Sepiolid Squid.

Integrative and comparative biology pii:6311815 [Epub ahead of print].

Due to their large size (∼3-5 Gb) and high repetitive content, the study of cephalopod genomes has historically been problematic. However, with the recent sequencing of several cephalopod genomes, including the Hawaiian bobtail squid (Euprymna scolopes), whole-genome studies of these molluscs are now possible. Of particular interest are the sepiolid or bobtail squids, many of which develop photophores in which bioluminescent bacterial symbionts reside. The variable presence of the symbiosis throughout the family allows us to determine regions of the genome that are under selection in symbiotic lineages, potentially providing a mechanism for identifying genes instrumental in the evolution of these mutualistic associations. To this end, we have used high-throughput sequencing to generate sequence from five bobtail squid genomes, four of which maintain symbioses with luminescent bacteria (E. hyllebergi, E. albatrossae, E. scolopes and Rondeletiola minor), and one of which does not (Sepietta neglecta). When we performed K-mer based heterozygosity and genome size estimations, we found that the Euprymna genus has a higher predicted genome size than other bobtail squid (∼ 5 Gb as compared to ∼ 4 Gb) and lower genomic heterozygosity. When we analyzed the repetitive content of the genomes, we found that genomes in the genus Euprymna appear to have recently acquired a significant quantity of LINE elements that are not found in its sister genus Rondeletiola or the closely related Sepietta. Using Abyss-2.0 and then Chromosomer with the published E. scolopes genome as a reference, we generated E. hyllebergi and E. albatrossae genomes of 1.54-1.57 Gb in size, but containing over 78-81% of eukaryotic single-copy othologs. The data we have generated will enable future whole-genome comparisons between these species to determine gene and regulatory content that differs between symbiotic and non-symbiotic lineages, as well as genes associated with symbiosis that are under selection.

RevDate: 2021-07-15
CmpDate: 2021-07-15

Kerwin AH, McAnulty SJ, SV Nyholm (2021)

Development of the Accessory Nidamental Gland and Associated Bacterial Community in the Hawaiian Bobtail Squid, Euprymna scolopes.

The Biological bulletin, 240(3):205-218.

AbstractThe Hawaiian bobtail squid, Euprymna scolopes, has a female reproductive organ called the accessory nidamental gland that contains a symbiotic bacterial consortium. These bacteria are deposited from the accessory nidamental gland into the squid's egg cases, where the consortium prevents microbial fouling. The symbiont community is environmentally transmitted and conserved across host populations, yet little is known about how the organ develops and is colonized by bacteria. In order to understand accessory nidamental gland development in E. scolopes, we characterized the gland during maturation by using histology and confocal and transmission electron microscopy. We found that an epithelial field formed first about four weeks after hatching, followed by the proliferation of numerous pores during what we hypothesize to be the initiation of bacterial recruitment (early development). Microscopy revealed that these pores were connected to ciliated invaginations that occasionally contained bacteria. During mid development, these epithelial fields expanded, and separate colonized tubules were observed below the epithelial layer that contained the pores and invaginations. During late development, the superficial epithelial fields appeared to regress as animals approached sexual maturity and were never observed in fully mature adults (about 2-3 months post-hatching), suggesting that they help facilitate bacterial colonization of the accessory nidamental gland. An analysis of 16S rRNA gene diversity in accessory nidamental glands from females of varying size showed that the bacterial community changed as the host approached sexual maturity, increasing in community evenness and shifting from a Verrucomicrobia-dominated to an Alphaproteobacteria-dominated consortium. Given the host's relationship with the well-characterized light organ symbiont Vibrio fischeri, our work suggests that the accessory nidamental gland of E. scolopes may have similar mechanisms to recruit bacteria from the environment. Understanding the developmental and colonization processes of the accessory nidamental gland will expand the use of E. scolopes as a model organism for studying bacterial consortia in marine symbioses.

RevDate: 2021-07-12

Nyholm SV, MJ McFall-Ngai (2021)

A lasting symbiosis: how the Hawaiian bobtail squid finds and keeps its bioluminescent bacterial partner.

Nature reviews. Microbiology [Epub ahead of print].

For more than 30 years, the association between the Hawaiian bobtail squid, Euprymna scolopes, and the bioluminescent bacterium Vibrio fischeri has been studied as a model system for understanding the colonization of animal epithelia by symbiotic bacteria. The squid-vibrio light-organ system provides the exquisite resolution only possible with the study of a binary partnership. The impact of this relationship on the partners' biology has been broadly characterized, including their ecology and evolutionary biology as well as the underlying molecular mechanisms of symbiotic dynamics. Much has been learned about the factors that foster initial light-organ colonization, and more recently about the maturation and long-term maintenance of the association. This Review synthesizes the results of recent research on the light-organ association and also describes the development of new horizons for E. scolopes as a model organism that promises to inform biology and biomedicine about the basic nature of host-microorganism interactions.

RevDate: 2021-06-25

Visick KL, Stabb EV, EG Ruby (2021)

A lasting symbiosis: how Vibrio fischeri finds a squid partner and persists within its natural host.

Nature reviews. Microbiology [Epub ahead of print].

As our understanding of the human microbiome progresses, so does the need for natural experimental animal models that promote a mechanistic understanding of beneficial microorganism-host interactions. Years of research into the exclusive symbiosis between the Hawaiian bobtail squid, Euprymna scolopes, and the bioluminescent bacterium Vibrio fischeri have permitted a detailed understanding of those bacterial genes underlying signal exchange and rhythmic activities that result in a persistent, beneficial association, as well as glimpses into the evolution of symbiotic competence. Migrating from the ambient seawater to regions deep inside the light-emitting organ of the squid, V. fischeri experiences, recognizes and adjusts to the changing environmental conditions. Here, we review key advances over the past 15 years that are deepening our understanding of these events.

RevDate: 2021-07-09

Ludvik DA, Bultman KM, MJ Mandel (2021)

Hybrid Histidine Kinase BinK Represses Vibrio fischeri Biofilm Signaling at Multiple Developmental Stages.

Journal of bacteriology, 203(15):e0015521.

The symbiosis between the Hawaiian bobtail squid, Euprymna scolopes, and its exclusive light organ symbiont, Vibrio fischeri, provides a natural system in which to study host-microbe specificity and gene regulation during the establishment of a mutually beneficial symbiosis. Colonization of the host relies on bacterial biofilm-like aggregation in the squid mucus field. Symbiotic biofilm formation is controlled by a two-component signaling (TCS) system consisting of regulators RscS-SypF-SypG, which together direct transcription of the symbiosis polysaccharide Syp. TCS systems are broadly important for bacteria to sense environmental cues and then direct changes in behavior. Previously, we identified the hybrid histidine kinase BinK as a strong negative regulator of V. fischeri biofilm regulation, and here we further explore the function of BinK. To inhibit biofilm formation, BinK requires the predicted phosphorylation sites in both the histidine kinase (H362) and receiver (D794) domains. Furthermore, we show that RscS is not essential for host colonization when binK is deleted from strain ES114, and imaging of aggregate size revealed no benefit to the presence of RscS in a background lacking BinK. Strains lacking RscS still suffered in competition. Finally, we show that BinK functions to inhibit biofilm gene expression in the light organ crypts, providing evidence for biofilm gene regulation at later stages of host colonization. Overall, this study provides direct evidence for opposing activities of RscS and BinK and yields novel insights into biofilm regulation during the maturation of a beneficial symbiosis. IMPORTANCE Bacteria are often in a biofilm state, and transitions between planktonic and biofilm lifestyles are important for pathogenic, beneficial, and environmental microbes. The critical nature of biofilm formation during Vibrio fischeri colonization of the Hawaiian bobtail squid light organ provides an opportunity to study development of this process in vivo using a combination of genetic and imaging approaches. The current work refines the signaling circuitry of the biofilm pathway in V. fischeri, provides evidence that biofilm regulatory changes occur in the host, and identifies BinK as one of the regulators of that process. This study provides information about how bacteria regulate biofilm gene expression in an intact animal host.

RevDate: 2021-06-11

Moriano-Gutierrez S, Ruby EG, MJ McFall-Ngai (2021)

MicroRNA-Mediated Regulation of Initial Host Responses in a Symbiotic Organ.

mSystems, 6(3):.

One of the most important events in an animal's life history is the initial colonization by its microbial symbionts, yet little is known about this event's immediate impacts on the extent of host gene expression or the molecular mechanisms controlling it. MicroRNAs (miRNAs) are short, noncoding RNAs that bind to target mRNAs, rapidly shaping gene expression by posttranscriptional control of mRNA translation and decay. Here, we show that, in the experimentally tractable binary squid-vibrio symbiosis, colonization of the light organ induces extensive changes in the miRNA transcriptome. Examination of the squid genome revealed the presence of evolutionarily conserved genes encoding elements essential for the production and processing of miRNAs. At 24 h postcolonization, 215 host miRNAs were detected in the light organ, 26 of which were differentially expressed in response to the symbionts. A functional enrichment analysis of genes potentially targeted by downregulation of certain miRNAs at the initiation of symbiosis revealed two major gene ontology (GO) term categories, neurodevelopment and tissue remodeling. This symbiont-induced downregulation is predicted to promote these activities in host tissues and is consistent with the well-described tissue remodeling that occurs at the onset of the association. Conversely, predicted targets of upregulated miRNAs, including the production of mucus, are consistent with attenuation of immune responses by symbiosis. Taken together, our data provide evidence that, at the onset of symbiosis, host miRNAs in the light organ drive alterations in gene expression that (i) orchestrate the symbiont-induced development of host tissues, and (ii) facilitate the partnership by dampening the immune response.IMPORTANCE Animals often acquire their microbiome from the environment at each generation, making the initial interaction of the partners a critical event in the establishment and development of a stable, healthy symbiosis. However, the molecular nature of these earliest interactions is generally difficult to study and poorly understood. We report that, during the initial 24 h of the squid-vibrio association, a differential expression of host miRNAs is triggered by the presence of the microbial partner. Predicted mRNA targets of these miRNAs were associated with regulatory networks that drive tissue remodeling and immune suppression, two major symbiosis-induced developmental outcomes in this and many other associations. These results implicate regulation by miRNAs as key to orchestrating the critical transcriptional responses that occur very early during the establishment of a symbiosis. Animals with more complex microbiota may have similar miRNA-driven responses as their association is initiated, supporting an evolutionary conservation of symbiosis-induced developmental mechanisms.

RevDate: 2021-05-07

Zink KE, Ludvik DA, Lazzara PR, et al (2021)

A Small Molecule Coordinates Symbiotic Behaviors in a Host Organ.

mBio, 12(2):.

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.

RevDate: 2021-06-19

McFall-Ngai M, TCG Bosch (2021)

Animal development in the microbial world: The power of experimental model systems.

Current topics in developmental biology, 141:371-397.

The development of powerful model systems has been a critical strategy for understanding the mechanisms underlying the progression of an animal through its ontogeny. Here we provide two examples that allow deep and mechanistic insight into the development of specific animal systems. Species of the cnidarian genus Hydra have provided excellent models for studying host-microbe interactions and how metaorganisms function in vivo. Studies of the Hawaiian bobtail squid Euprymna scolopes and its luminous bacterial partner Vibrio fischeri have been used for over 30 years to understand the impact of a broad array of levels, from ecology to genomics, on the development and persistence of symbiosis. These examples provide an integrated perspective of how developmental processes work and evolve within the context of a microbial world, a new view that opens vast horizons for developmental biology research. The Hydra and the squid systems also lend an example of how profound insights can be discovered by taking advantage of the "experiments" that evolution had done in shaping conserved developmental processes.

RevDate: 2021-05-11
CmpDate: 2021-04-05

Cohen JJ, Eichinger SJ, Witte DA, et al (2021)

Control of Competence in Vibrio fischeri.

Applied and environmental microbiology, 87(6):.

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.

RevDate: 2021-02-26

Burgos HL, Burgos EF, Steinberger AJ, et al (2020)

Multiplexed Competition in a Synthetic Squid Light Organ Microbiome Using Barcode-Tagged Gene Deletions.

mSystems, 5(6):.

Beneficial symbioses between microbes and their eukaryotic hosts are ubiquitous and have widespread impacts on host health and development. The binary symbiosis between the bioluminescent bacterium Vibrio fischeri and its squid host Euprymna scolopes serves as a model system to study molecular mechanisms at the microbe-animal interface. To identify colonization factors in this system, our lab previously conducted a global transposon insertion sequencing (INSeq) screen and identified over 300 putative novel squid colonization factors in V. fischeri To pursue mechanistic studies on these candidate genes, we present an approach to quickly generate barcode-tagged gene deletions and perform high-throughput squid competition experiments with detection of the proportion of each strain in the mixture by barcode sequencing (BarSeq). Our deletion approach improves on previous techniques based on splicing by overlap extension PCR (SOE-PCR) and tfoX-based natural transformation by incorporating a randomized barcode that results in unique DNA sequences within each deletion scar. Amplicon sequencing of the pool of barcoded strains before and after colonization faithfully reports on known colonization factors and provides increased sensitivity over colony counting methods. BarSeq enables rapid and sensitive characterization of the molecular factors involved in establishing the Vibrio-squid symbiosis and provides a valuable tool to interrogate the molecular dialogue at microbe-animal host interfaces.IMPORTANCE Beneficial microbes play essential roles in the health and development of their hosts. However, the complexity of animal microbiomes and general genetic intractability of their symbionts have made it difficult to study the coevolved mechanisms for establishing and maintaining specificity at the microbe-animal host interface. Model symbioses are therefore invaluable for studying the mechanisms of beneficial microbe-host interactions. Here, we present a combined barcode-tagged deletion and BarSeq approach to interrogate the molecular dialogue that ensures specific and reproducible colonization of the Hawaiian bobtail squid by Vibrio fischeri The ability to precisely manipulate the bacterial genome, combined with multiplex colonization assays, will accelerate the use of this valuable model system for mechanistic studies of how environmental microbes-both beneficial and pathogenic-colonize specific animal hosts.

RevDate: 2021-06-14
CmpDate: 2021-06-14

Chavez-Dozal A, Soto W, MK Nishiguchi (2021)

Identification of a Transcriptomic Network Underlying the Wrinkly and Smooth Phenotypes of Vibrio fischeri.

Journal of bacteriology, 203(3):.

Vibrio fischeri is a cosmopolitan marine bacterium that oftentimes displays different colony morphologies, switching from a smooth to a wrinkly phenotype in order to adapt to changes in the environment. This wrinkly phenotype has also been associated with increased biofilm formation, an essential characteristic for V. fischeri to adhere to substrates, to suspended debris, and within the light organs of sepiolid squids. Elevated levels of biofilm formation are correlated with increased microbial survival of exposure to environmental stressors and the ability to expand niche breadth. Since V. fischeri has a biphasic life history strategy between its free-living and symbiotic states, we were interested in whether the wrinkly morphotype demonstrated differences in its expression profile in comparison to the naturally occurring and more common smooth variant. We show that genes involved in major biochemical cascades, including those involved in protein sorting, oxidative stress, and membrane transport, play a role in the wrinkly phenotype. Interestingly, only a few unique genes are specifically involved in macromolecule biosynthesis in the wrinkly phenotype, which underlies the importance of other pathways utilized for adaptation under the conditions in which Vibrio bacteria are producing this change in phenotype. These results provide the first comprehensive analysis of the complex form of genetic activation that underlies the diversity in morphologies of V. fischeri when switching between two different colony morphotypes, each representing a unique biofilm ecotype.IMPORTANCE The wrinkly bacterial colony phenotype has been associated with increased squid host colonization in V. fischeri The significance of our research is in identifying the genetic mechanisms that are responsible for heightened biofilm formation in V. fischeri This report also advances our understanding of gene regulation in V. fischeri and brings to the forefront a number of previously overlooked genetic networks. Several loci that were identified in this study were not previously known to be associated with biofilm formation in V. fischeri.

RevDate: 2021-07-16
CmpDate: 2021-03-26

Fidopiastis PM, Mariscal V, McPherson JM, et al (2021)

Vibrio fischeri Amidase Activity Is Required for Normal Cell Division, Motility, and Symbiotic Competence.

Applied and environmental microbiology, 87(3):.

N-Acetylmuramoyl-l-alanine amidases are periplasmic hydrolases that cleave the amide bond between N-acetylmuramic acid and alanine in peptidoglycan (PG). Unlike many Gram-negative bacteria that encode redundant periplasmic amidases, Vibrio fischeri appears to encode a single protein that is homologous to AmiB of Vibrio cholerae We screened a V. fischeri transposon mutant library for strains altered in biofilm production and discovered a biofilm-overproducing strain with an insertion in amiB (VF_2326). Further characterization of biofilm enhancement suggested that this phenotype was due to the overproduction of cellulose, and it was dependent on the bcsA cellulose synthase. Additionally, the amiB mutant was nonmotile, perhaps due to defects in its ability to septate during division. The amidase mutant was unable to compete with the wild type for the colonization of V. fischeri's symbiotic host, the squid Euprymna scolopes In single-strain inoculations, host squid inoculated with the mutant eventually became colonized but with a much lower efficiency than in squid inoculated with the wild type. This observation was consistent with the pleiotropic effects of the amiB mutation and led us to speculate that motile suppressors of the amiB mutant were responsible for the partially restored colonization. In culture, motile suppressor mutants carried point mutations in a single gene (VF_1477), resulting in a partial restoration of wild-type motility. In addition, these point mutations reversed the effect of the amiB mutation on cellulosic biofilm production. These data are consistent with V. fischeri AmiB possessing amidase activity; they also suggest that AmiB suppresses cellulosic biofilm formation but promotes successful host colonization.IMPORTANCE Peptidoglycan (PG) is a critical microbe-associated molecular pattern (MAMP) that is sloughed by cells of V. fischeri during symbiotic colonization of squid. Specifically, this process induces significant remodeling of a specialized symbiotic light organ within the squid mantle cavity. This phenomenon is reminiscent of the loss of ciliated epithelium in patients with whooping cough due to the production of PG monomers by Bordetella pertussis Furthermore, PG processing machinery can influence susceptibility to antimicrobials. In this study, we report roles for the V. fischeri PG amidase AmiB, including the beneficial colonization of squid, underscoring the urgency to more deeply understand PG processing machinery and the downstream consequences of their activities.

RevDate: 2021-05-21
CmpDate: 2021-01-04

Moriano-Gutierrez S, Bongrand C, Essock-Burns T, et al (2020)

The noncoding small RNA SsrA is released by Vibrio fischeri and modulates critical host responses.

PLoS biology, 18(11):e3000934.

The regulatory noncoding small RNAs (sRNAs) of bacteria are key elements influencing gene expression; however, there has been little evidence that beneficial bacteria use these molecules to communicate with their animal hosts. We report here that the bacterial sRNA SsrA plays an essential role in the light-organ symbiosis between Vibrio fischeri and the squid Euprymna scolopes. The symbionts load SsrA into outer membrane vesicles, which are transported specifically into the epithelial cells surrounding the symbiont population in the light organ. Although an SsrA-deletion mutant (ΔssrA) colonized the host to a normal level after 24 h, it produced only 2/10 the luminescence per bacterium, and its persistence began to decline by 48 h. The host's response to colonization by the ΔssrA strain was also abnormal: the epithelial cells underwent premature swelling, and host robustness was reduced. Most notably, when colonized by the ΔssrA strain, the light organ differentially up-regulated 10 genes, including several encoding heightened immune-function or antimicrobial activities. This study reveals the potential for a bacterial symbiont's sRNAs not only to control its own activities but also to trigger critical responses promoting homeostasis in its host. In the absence of this communication, there are dramatic fitness consequences for both partners.

RevDate: 2021-04-16
CmpDate: 2021-01-07

Koch EJ, Bongrand C, Bennett BD, et al (2020)

The cytokine MIF controls daily rhythms of symbiont nutrition in an animal-bacterial association.

Proceedings of the National Academy of Sciences of the United States of America, 117(44):27578-27586.

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 between Euprymna scolopes and Vibrio 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 of V. fischeri cells resides. Nocturnal migration of these macrophage-like cells, together with identification of an E. scolopes MIF (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.

RevDate: 2021-07-12
CmpDate: 2021-07-12

Christensen DG, Tepavčević J, KL Visick (2020)

Genetic Manipulation of Vibrio fischeri.

Current protocols in microbiology, 59(1):e115.

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.

RevDate: 2021-07-20
CmpDate: 2021-07-20

Bennett BD, Essock-Burns T, EG Ruby (2020)

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

mBio, 11(5):.

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.

RevDate: 2021-05-11
CmpDate: 2021-05-11

Benoist L, Houyvet B, Henry J, et al (2020)

In-Depth In Silico Search for Cuttlefish (Sepia officinalis) Antimicrobial Peptides Following Bacterial Challenge of Haemocytes.

Marine drugs, 18(9):.

Cuttlefish (Sepia officinalis) haemocytes are potential sources of antimicrobial peptides (AMPs). To study the immune response to Vibrio splendidus and identify new AMPs, an original approach was developed based on a differential transcriptomic study and an in-depth in silico analysis using multiple tools. Two de novo transcriptomes were retrieved from cuttlefish haemocytes following challenge by V. splendidus or not. A first analysis of the annotated transcripts revealed the presence of Toll/NF-κB pathway members, including newly identified factors such as So-TLR-h, So-IKK-h and So-Rel/NF-κB-h. Out of the eight Toll/NF-κB pathway members, seven were found up-regulated following V. splendidus challenge. Besides, immune factors involved in the immune response were also identified and up-regulated. However, no AMP was identified based on annotation or conserved pattern searches. We therefore performed an in-depth in silico analysis of unannotated transcripts based on differential expression and sequence characteristics, using several tools available like PepTraq, a homemade software program. Finally, five AMP candidates were synthesized. Among them, NF19, AV19 and GK28 displayed antibacterial activity against Gram-negative bacteria. Each peptide had a different spectrum of activity, notably against Vibrio species. GK28-the most active peptide-was not haemolytic, whereas NF19 and AV19 were haemolytic at concentrations between 50 and 100 µM, 5 to 10 times higher than their minimum inhibitory concentration.

RevDate: 2021-05-11
CmpDate: 2021-05-11

Chomicki G, Werner GDA, West SA, et al (2020)

Compartmentalization drives the evolution of symbiotic cooperation.

Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 375(1808):20190602.

Across the tree of life, hosts have evolved mechanisms to control and mediate interactions with symbiotic partners. We suggest that the evolution of physical structures that allow hosts to spatially separate symbionts, termed compartmentalization, is a common mechanism used by hosts. Such compartmentalization allows hosts to: (i) isolate symbionts and control their reproduction; (ii) reward cooperative symbionts and punish or stop interactions with non-cooperative symbionts; and (iii) reduce direct conflict among different symbionts strains in a single host. Compartmentalization has allowed hosts to increase the benefits that they obtain from symbiotic partners across a diversity of interactions, including legumes and rhizobia, plants and fungi, squid and Vibrio, insects and nutrient provisioning bacteria, plants and insects, and the human microbiome. In cases where compartmentalization has not evolved, we ask why not. We argue that when partners interact in a competitive hierarchy, or when hosts engage in partnerships which are less costly, compartmentalization is less likely to evolve. We conclude that compartmentalization is key to understanding the evolution of symbiotic cooperation. This article is part of the theme issue 'The role of the microbiome in host evolution'.

RevDate: 2021-05-27

Christensen DG, Marsden AE, Hodge-Hanson K, et al (2020)

LapG mediates biofilm dispersal in Vibrio fischeri by controlling maintenance of the VCBS-containing adhesin LapV.

Molecular microbiology, 114(5):742-761.

Efficient symbiotic colonization of the squid Euprymna scolopes by the bacterium Vibrio fischeri depends on bacterial biofilm formation on the surface of the squid's light organ. Subsequently, the bacteria disperse from the biofilm via an unknown mechanism and enter through pores to reach the interior colonization sites. Here, we identify a homolog of Pseudomonas fluorescens LapG as a dispersal factor that promotes cleavage of a biofilm-promoting adhesin, LapV. Overproduction of LapG inhibited biofilm formation and, unlike the wild-type parent, a ΔlapG mutant formed biofilms in vitro. Although V. fischeri encodes two putative large adhesins, LapI (near lapG on chromosome II) and LapV (on chromosome I), only the latter contributed to biofilm formation. Consistent with the Pseudomonas Lap system model, our data support a role for the predicted c-di-GMP-binding protein LapD in inhibiting LapG-dependent dispersal. Furthermore, we identified a phosphodiesterase, PdeV, whose loss promotes biofilm formation similar to that of the ΔlapG mutant and dependent on both LapD and LapV. Finally, we found a minor defect for a ΔlapD mutant in initiating squid colonization, indicating a role for the Lap system in a relevant environmental niche. Together, these data reveal new factors and provide important insights into biofilm dispersal by V. fischeri.

RevDate: 2021-07-06
CmpDate: 2021-06-18

Koch EJ, Moriano-Gutierrez S, Ruby EG, et al (2020)

The impact of persistent colonization by Vibrio fischeri on the metabolome of the host squid Euprymna scolopes.

The Journal of experimental biology, 223(Pt 16):.

Associations between animals and microbes affect not only the immediate tissues where they occur, but also the entire host. Metabolomics, the study of small biomolecules generated during metabolic processes, provides a window into how mutualistic interactions shape host biochemistry. The Hawaiian bobtail squid, Euprymna scolopes, is amenable to metabolomic studies of symbiosis because the host can be reared with or without its species-specific symbiont, Vibrio fischeri In addition, unlike many invertebrates, the host squid has a closed circulatory system. This feature allows a direct sampling of the refined collection of metabolites circulating through the body, a focused approach that has been highly successful with mammals. Here, we show that rearing E. scolopes without its natural symbiont significantly affected one-quarter of the more than 100 hemolymph metabolites defined by gas chromatography mass spectrometry analysis. Furthermore, as in mammals, which harbor complex consortia of bacterial symbionts, the metabolite signature oscillated on symbiont-driven daily rhythms and was dependent on the sex of the host. Thus, our results provide evidence that the population of even a single symbiont species can influence host hemolymph biochemistry as a function of symbiotic state, host sex and circadian rhythm.

RevDate: 2021-07-15
CmpDate: 2021-07-15

Suria AM, Tan KC, Kerwin AH, et al (2020)

Hawaiian Bobtail Squid Symbionts Inhibit Marine Bacteria via Production of Specialized Metabolites, Including New Bromoalterochromides BAC-D/D'.

mSphere, 5(4):.

The Hawaiian bobtail squid, Euprymna scolopes, has a symbiotic bacterial consortium in the accessory nidamental gland (ANG), a female reproductive organ that protects eggs against fouling microorganisms. To test the antibacterial activity of ANG community members, 19 bacterial isolates were screened for their ability to inhibit Gram-negative and Gram-positive bacteria, of which two strains were inhibitory. These two antibacterial isolates, Leisingera sp. ANG59 and Pseudoalteromonas sp. JC28, were subjected to further genomic characterization. Genomic analysis of Leisingera sp. ANG59 revealed a biosynthetic gene cluster encoding the antimicrobial compound indigoidine. The genome of Pseudoalteromonas sp. JC28 had a 14-gene cluster with >95% amino acid identity to a known bromoalterochromide (BAC) cluster. Chemical analysis confirmed production of known BACs, BAC-A/A' (compounds 1a/1b), as well as two new derivatives, BAC-D/D' (compounds 2a/2b). Extensive nuclear magnetic resonance (NMR) analyses allowed complete structural elucidation of compounds 2a/2b, and the absolute stereochemistry was unambiguously determined using an optimized Marfey's method. The BACs were then investigated for in vitro antibacterial, antifungal, and nitric oxide (NO) inhibitory activity. Compounds 1a/1b were active against the marine bacteria Bacillus algicola and Vibrio fischeri, while compounds 2a/2b were active only against B. algicola Compounds 1a/1b inhibited NO production via lipopolysaccharide (LPS)-induced inflammation in RAW264.7 macrophage cells and also inhibited the pathogenic fungus Fusarium keratoplasticum, which, coupled with their antibacterial activity, suggests that these polyketide-nonribosomal peptides may be used for squid egg defense against potential pathogens and/or fouling microorganisms. These results indicate that BACs may provide Pseudoalteromonas sp. JC28 an ecological niche, facilitating competition against nonsymbiotic microorganisms in the host's environment.IMPORTANCE Animals that deposit eggs must protect their embryos from fouling and disease by microorganisms to ensure successful development. Although beneficial bacteria are hypothesized to contribute to egg defense in many organisms, the mechanisms of this protection are only recently being elucidated. Our previous studies of the Hawaiian bobtail squid focused on fungal inhibition by beneficial bacterial symbionts of a female reproductive gland and eggs. Herein, using genomic and chemical analyses, we demonstrate that symbiotic bacteria from this gland can also inhibit other marine bacteria in vitro One bacterial strain in particular, Pseudoalteromonas sp. JC28, had broad-spectrum abilities to inhibit potential fouling bacteria, in part via production of novel bromoalterochromide metabolites, confirmed via genomic annotation of the associated biosynthetic gene cluster. Our results suggest that these bacterial metabolites may contribute to antimicrobial activity in this association and that such defensive symbioses are underutilized sources for discovering novel antimicrobial compounds.

RevDate: 2021-03-23
CmpDate: 2021-03-23

Parmar P, Shukla A, Goswami D, et al (2020)

Comprehensive depiction of novel heavy metal tolerant and EPS producing bioluminescent Vibrio alginolyticus PBR1 and V. rotiferianus PBL1 confined from marine organisms.

Microbiological research, 238:126526.

The current study depicts the isolation of luminescent bacteria from fish and squid samples that were collected from Veraval fish harbour. From Indian mackerel, total 14 and from squid, total 23 bioluminescent bacteria were isolated using luminescence agar medium. Two bioluminescent bacteria with highest relative luminescence intensity PBR1 and PBL1 were selected. These two isolates were subjected to detailed biochemical characterization and were tested positive for 5 out of 13 biochemical tests. Furthermore, both PBR1 and PBL1 were able to ferment cellobiose, dextrose, fructose, galactose, maltose, mannose, sucrose and trehalose with acid production. Based on 16S rRNA partial gene sequence analysis, PBR1 was identified as Vibrio alginolyticus and PBL1 as V. rotiferianus. Antibiotic susceptibility test using paper-disc method showed that PBR1 and PBL1 were sensitive to chloramphenicol, ciprofloxacin, co-trimoxazole, gatifloxacin, levofloxacin, linezolid ad roxithromycin out of 18 antibiotics tested. Moreover, both strains were evaluated for their exopolysachharide (EPS) producing ability where PBR1 and PBL1 were able to yield 1.34 g% (w/v) and 2.45 g% (w/v) EPS respectively from 5 g% (v/v) sucrose concentration. Heavy metal toxicity assessment was carried out using agar well diffusion method with eight heavy metals and both the strains were sensitive to As(III), Cd(II), Ce(II), Cr(III), Cu(II), Hg(II) and while they showed resistance to Pb(II) and Sr(II). Based on these results, a study was conducted to demonstrate bio-removal of Pb and Sr by EPS of PBR1 and PBL1. Fourier transform infrared (FTIR) spectra revealed the functional groups of EPS involved in interaction with the heavy metals. Owing to the sensitivity for the remaining heavy metals, these bioluminescent bacteria can be used further for the development of luminescence-based biosensor.

RevDate: 2021-05-25
CmpDate: 2021-04-09

Essock-Burns T, Bongrand C, Goldman WE, et al (2020)

Interactions of Symbiotic Partners Drive the Development of a Complex Biogeography in the Squid-Vibrio Symbiosis.

mBio, 11(3):.

Microbes live in complex microniches within host tissues, but how symbiotic partners communicate to create such niches during development remains largely unexplored. Using confocal microscopy and symbiont genetics, we characterized the shaping of host microenvironments during light organ colonization of the squid Euprymna scolopes by the bacterium Vibrio fischeri During embryogenesis, three pairs of invaginations form sequentially on the organ's surface, producing pores that lead to interior compressed tubules at different stages of development. After hatching, these areas expand, allowing V. fischeri cells to enter and migrate ∼120 μm through three anatomically distinct regions before reaching blind-ended crypt spaces. A dynamic gatekeeper, or bottleneck, connects these crypts with the migration path. Once V. fischeri cells have entered the crypts, the bottlenecks narrow, and colonization by the symbiont population becomes spatially restricted. The actual timing of constriction and restriction varies with crypt maturity and with different V. fischeri strains. Subsequently, starting with the first dawn following colonization, the bottleneck controls a lifelong cycle of dawn-triggered expulsions of most of the symbionts into the environment and a subsequent regrowth in the crypts. Unlike other developmental phenotypes, bottleneck constriction is not induced by known microbe-associated molecular patterns (MAMPs) or by V. fischeri -produced bioluminescence, but it does require metabolically active symbionts. Further, while symbionts in the most mature crypts have a higher proportion of live cells and a greater likelihood of expulsion at dawn, they have a lower resistance to antibiotics. The overall dynamics of these distinct microenvironments reflect the complexity of the host-symbiont dialogue.IMPORTANCE The complexity, inaccessibility, and time scales of initial colonization of most animal microbiomes present challenges for the characterization of how the bacterial symbionts influence the form and function of tissues in the minutes to hours following the initial interaction of the partners. Here, we use the naturally occurring binary squid-vibrio association to explore this phenomenon. Imaging of the spatiotemporal landscape of this symbiosis during its onset provides a window into the impact of differences in both host-tissue maturation and symbiont strain phenotypes on the establishment of a dynamically stable symbiotic system. These data provide evidence that the symbionts shape the host-tissue landscape and that tissue maturation impacts the influence of strain-level differences on the daily rhythms of the symbiosis, the competitiveness for colonization, and antibiotic sensitivity.

RevDate: 2021-04-16
CmpDate: 2021-04-16

Cohen SK, Aschtgen MS, Lynch JB, et al (2020)

Tracking the cargo of extracellular symbionts into host tissues with correlated electron microscopy and nanoscale secondary ion mass spectrometry imaging.

Cellular microbiology, 22(4):e13177.

Extracellular bacterial symbionts communicate biochemically with their hosts to establish niches that foster the partnership. Using quantitative ion microprobe isotopic imaging (nanoscale secondary ion mass spectrometry [NanoSIMS]), we surveyed localization of 15 N-labelled molecules produced by the bacterium Vibrio fischeri within the cells of the symbiotic organ of its host, the Hawaiian bobtail squid, and compared that with either labelled non-specific species or amino acids. In all cases, two areas of the organ's epithelia were significantly more 15 N enriched: (a) surface ciliated cells, where environmental symbionts are recruited, and (b) the organ's crypts, where the symbiont population resides in the host. Label enrichment in all cases was strongest inside host cell nuclei, preferentially in the euchromatin regions and the nucleoli. This permissiveness demonstrated that uptake of biomolecules is a general mechanism of the epithelia, but the specific responses to V. fischeri cells recruited to the organ's surface are due to some property exclusive to this species. Similarly, in the organ's deeper crypts, the host responds to common bacterial products that only the specific symbiont can present in that location. The application of NanoSIMS allows the discovery of such distinct modes of downstream signalling dependent on location within the host and provides a unique opportunity to study the microbiogeographical patterns of symbiotic dialogue.

RevDate: 2021-04-12

Dumen E, Ekici G, Ergin S, et al (2020)

Presence of Foodborne Pathogens in Seafood and Risk Ranking for Pathogens.

Foodborne pathogens and disease, 17(9):541-546.

This study aims at examining the contamination of coliform bacteria, Escherichia coli, Listeria monocytogenes, Vibrio vulnificus, and Vibrio cholerae, which carry extremely serious risks to the consumer health, in 700 seafood belonging to 4 different (raw sea fish, raw mussels, raw shrimp, and raw squid) categories. The total number of samples was determined as 700. When the obtained results were viewed in total, they were found to be 48.14%, 18.71%, 8.57%, and 3.42% for coliform bacteria, E. coli, L. monocytogenes, and V. vulnificus, respectively. V. cholerae, one of the factors studied, was not found. Conventional microbiological cultivation methods were used in the analysis stage as well as the real-time PCR method. This study aims at making a risk ranking modeling for consumer health based on product category and pathogens by interpreting the results of the analysis with statistical methods. According to the statistical analysis, significantly binary correlations were determined among some parameters that stimulate one another for reproducing. In the light of the obtained results of the study, it has been concluded that the studies of the most detailed examinations of the microbiological risks associated with seafood, forms of microbial pollution and microorganisms that cause deterioration in seafood and threaten consumer health and the path that their epidemiologies follow, are of primary importance to both protecting consumer health and obtaining safe and quality seafood.

RevDate: 2021-04-15
CmpDate: 2020-12-14

Bongrand C, Moriano-Gutierrez S, Arevalo P, et al (2020)

Using Colonization Assays and Comparative Genomics To Discover Symbiosis Behaviors and Factors in Vibrio fischeri.

mBio, 11(2):.

The luminous marine Gram-negative bacterium Vibrio (Aliivibrio) fischeri is the natural light organ symbiont of several squid species, including the Hawaiian bobtail squid, Euprymna scolopes, and the Japanese bobtail squid, Euprymna morsei Work with E. scolopes has shown how the bacteria establish their niche in the light organ of the newly hatched host. Two types of V. fischeri strains have been distinguished based upon their behavior in cocolonization competition assays in juvenile E. scolopes, i.e., (i) niche-sharing or (ii) niche-dominant behavior. This study aimed to determine whether these behaviors are observed with other V. fischeri strains or whether they are specific to those isolated from E. scolopes light organs. Cocolonization competition assays between V. fischeri strains isolated from the congeneric squid E. morsei or from other marine animals revealed the same sharing or dominant behaviors. In addition, whole-genome sequencing of these strains showed that the dominant behavior is polyphyletic and not associated with the presence or absence of a single gene or genes. Comparative genomics of 44 squid light organ isolates from around the globe led to the identification of symbiosis-specific candidates in the genomes of these strains. Colonization assays using genetic derivatives with deletions of these candidates established the importance of two such genes in colonization. This study has allowed us to expand the concept of distinct colonization behaviors to strains isolated from a number of squid and fish hosts.IMPORTANCE There is an increasing recognition of the importance of strain differences in the ecology of a symbiotic bacterial species and, in particular, how these differences underlie crucial interactions with their host. Nevertheless, little is known about the genetic bases for these differences, how they manifest themselves in specific behaviors, and their distribution among symbionts of different host species. In this study, we sequenced the genomes of Vibrio fischeri isolated from the tissues of squids and fishes and applied comparative genomics approaches to look for patterns between symbiont lineages and host colonization behavior. In addition, we identified the only two genes that were exclusively present in all V. fischeri strains isolated from the light organs of sepiolid squid species. Mutational studies of these genes indicated that they both played a role in colonization of the squid light organ, emphasizing the value of applying a comparative genomics approach in the study of symbioses.

RevDate: 2020-09-28

Septer AN, Speare L, Coleman CK, et al (2020)

Draft Genome Sequence of a Harveyi Clade Bacterium Isolated from Lolliguncula brevis Squid.

Microbiology resource announcements, 9(8):.

Vibrio species of the Harveyi clade are commonly found in free-living and host-associated marine habitats. Here, we report the draft genome sequence for a Harveyi clade bacterium, Vibrio sp. strain LB10LO1, which was isolated from the Atlantic brief squid Lolliguncula brevis.

RevDate: 2021-06-17
CmpDate: 2021-06-17

Truong TV, Holland DB, Madaan S, et al (2020)

High-contrast, synchronous volumetric imaging with selective volume illumination microscopy.

Communications biology, 3(1):74.

Light-field fluorescence microscopy uniquely provides fast, synchronous volumetric imaging by capturing an extended volume in one snapshot, but often suffers from low contrast due to the background signal generated by its wide-field illumination strategy. We implemented light-field-based selective volume illumination microscopy (SVIM), where illumination is confined to only the volume of interest, removing the background generated from the extraneous sample volume, and dramatically enhancing the image contrast. We demonstrate the capabilities of SVIM by capturing cellular-resolution 3D movies of flowing bacteria in seawater as they colonize their squid symbiotic partner, as well as of the beating heart and brain-wide neural activity in larval zebrafish. These applications demonstrate the breadth of imaging applications that we envision SVIM will enable, in capturing tissue-scale 3D dynamic biological systems at single-cell resolution, fast volumetric rates, and high contrast to reveal the underlying biology.

RevDate: 2020-10-16
CmpDate: 2020-10-16

Speare L, Smith S, Salvato F, et al (2020)

Environmental Viscosity Modulates Interbacterial Killing during Habitat Transition.

mBio, 11(1):.

Symbiotic bacteria use diverse strategies to compete for host colonization sites. However, little is known about the environmental cues that modulate interbacterial competition as they transition between free-living and host-associated lifestyles. We used the mutualistic relationship between Eupyrmna scolopes squid and Vibrio fischeri bacteria to investigate how intraspecific competition is regulated as symbionts move from the seawater to a host-like environment. We recently reported that V. fischeri uses a type VI secretion system (T6SS) for intraspecific competition during host colonization. Here, we investigated how environmental viscosity impacts T6SS-mediated competition by using a liquid hydrogel medium that mimics the viscous host environment. Our data demonstrate that although the T6SS is functionally inactive when cells are grown under low-viscosity liquid conditions similar to those found in seawater, exposure to a host-like high-viscosity hydrogel enhances T6SS expression and sheath formation, activates T6SS-mediated killing in as little as 30 min, and promotes the coaggregation of competing genotypes. Finally, the use of mass spectrometry-based proteomics revealed insights into how cells may prepare for T6SS competition during this habitat transition. These findings, which establish the use of a new hydrogel culture condition for studying T6SS interactions, indicate that V. fischeri rapidly responds to the physical environment to activate the competitive mechanisms used during host colonization.IMPORTANCE Bacteria often engage in interference competition to gain access to an ecological niche, such as a host. However, little is known about how the physical environment experienced by free-living or host-associated bacteria influences such competition. We used the bioluminescent squid symbiont Vibrio fischeri to study how environmental viscosity impacts bacterial competition. Our results suggest that upon transition from a planktonic environment to a host-like environment, V. fischeri cells activate their type VI secretion system, a contact-dependent interbacterial nanoweapon, to eliminate natural competitors. This work shows that competitor cells form aggregates under host-like conditions, thereby facilitating the contact required for killing, and reveals how V. fischeri regulates a key competitive mechanism in response to the physical environment.

RevDate: 2021-01-10
CmpDate: 2020-11-19

Cohen ML, Mashanova EV, Jagannathan SV, et al (2020)

Adaptation to pH stress by Vibrio fischeri can affect its symbiosis with the Hawaiian bobtail squid (Euprymna scolopes).

Microbiology (Reading, England), 166(3):262-277.

Many microorganisms engaged in host-microbe interactions pendulate between a free-living phase and a host-affiliated stage. How adaptation to stress during the free-living phase affects host-microbe associations is unclear and understudied. To explore this topic, the symbiosis between Hawaiian bobtail squid (Euprymna scolopes) and the luminous bacterium Vibrio fischeri was leveraged for a microbial experimental evolution study. V. fischeri experienced adaptation to extreme pH while apart from the squid host. V. fischeri was serially passaged for 2000 generations to the lower and upper pH growth limits for this microorganism, which were pH 6.0 and 10.0, respectively. V. fischeri was also serially passaged for 2000 generations to vacillating pH 6.0 and 10.0. Evolution to pH stress both facilitated and impaired symbiosis. Microbial evolution to acid stress promoted squid colonization and increased bioluminescence for V. fischeri, while symbiont adaptation to alkaline stress diminished these two traits. Oscillatory selection to acid and alkaline stress also improved symbiosis for V. fischeri, but the facilitating effects were less than that provided by microbial adaptation to acid stress. In summary, microbial adaptation to harsh environments amid the free-living phase may impact the evolution of host-microbe interactions in ways that were not formerly considered.

RevDate: 2020-10-20
CmpDate: 2020-10-20

Guckes KR, Cecere AG, Williams AL, et al (2020)

The Bacterial Enhancer Binding Protein VasH Promotes Expression of a Type VI Secretion System in Vibrio fischeri during Symbiosis.

Journal of bacteriology, 202(7):.

Vibrio fischeri is a bacterial symbiont that colonizes the light organ of the Hawaiian bobtail squid, Euprymna scolopes Certain strains of V. fischeri express a type VI secretion system (T6SS), which delivers effectors into neighboring cells that result in their death. Strains that are susceptible to the T6SS fail to establish symbiosis with a T6SS-positive strain within the same location of the squid light organ, which is a phenomenon termed strain incompatibility. This study investigates the regulation of the T6SS in V. fischeri strain FQ-A001. Here, we report that the expression of Hcp, a necessary structural component of the T6SS, depends on the alternative sigma factor σ54 and the bacterial enhancer binding protein VasH. VasH is necessary for FQ-A001 to kill other strains, suggesting that VasH-dependent regulation is essential for the T6SS of V. fischeri to affect intercellular interactions. In addition, this study demonstrates VasH-dependent transcription of hcp within host-associated populations of FQ-A001, suggesting that the T6SS is expressed within the host environment. Together, these findings establish a model for transcriptional control of hcp in V. fischeri within the squid light organ, thereby increasing understanding of how the T6SS is regulated during symbiosis.IMPORTANCE Animals harbor bacterial symbionts with specific traits that promote host fitness. Mechanisms that facilitate intercellular interactions among bacterial symbionts impact which bacterial lineages ultimately establish symbiosis with the host. How these mechanisms are regulated is poorly characterized in nonhuman bacterial symbionts. This study establishes a model for the transcriptional regulation of a contact-dependent killing machine, thereby increasing understanding of mechanisms by which different strains compete while establishing symbiosis.

RevDate: 2021-01-10
CmpDate: 2020-11-16

Li HW, Chen C, Kuo WL, et al (2019)

The Characteristics and Expression Profile of Transferrin in the Accessory Nidamental Gland of the Bigfin Reef Squid during Bacteria Transmission.

Scientific reports, 9(1):20163.

The accessory nidamental gland (ANG) is a female reproductive organ found in most squid and cuttlefish that contains a consortium of bacteria. These symbiotic bacteria are transmitted from the marine environment and selected by the host through an unknown mechanism. In animals, a common antimicrobial mechanism of innate immunity is iron sequestration, which is based on the development of transferrin (TF)-like proteins. To understand this mechanism of host-microbe interaction, we attempted to characterize the role of transferrin in bigfin reef squid (Sepioteuthis lessoniana) during bacterial transmission. qPCR analysis showed that Tf was exclusively expressed in the outer layer of ANG,and this was confirmed by in situ hybridization, which showed that Tf was localized in the outer epithelial cell layer of the ANG. Western blot analysis indicated that TF is a soluble glycoprotein. Immunohistochemical staining also showed that TF is localized in the outer epithelial cell layer of the ANG and that it is mainly expressed in the outer layer during ANG growth. These results suggest that robust Tf mRNA and TF protein expression in the outer layer of the ANG plays an important role in microbe selection by the host during bacterial transmission.

RevDate: 2020-12-14
CmpDate: 2020-12-08

Ramos AF, Woods DF, Shanahan R, et al (2020)

A structure-function analysis of interspecies antagonism by the 2-heptyl-4-alkyl-quinolone signal molecule from Pseudomonas aeruginosa.

Microbiology (Reading, England), 166(2):169-179.

In recent years, the alkyl-quinolone molecular framework has already provided a rich source of bioactivity for the development of novel anti-infective compounds. Based on the quorum-sensing signalling molecules 4-hydroxy-2-heptylquinoline (HHQ) and 3,4-dihydroxy-2-heptylquinoline (PQS) from the nosocomial pathogen Pseudomonas aeruginosa, modifications have been developed with markedly enhanced anti-biofilm bioactivity towards important fungal and bacterial pathogens, including Candida albicans and Aspergillus fumigatus. Here we show that antibacterial activity of HHQ against Vibrionaceae is species-specific and it requires an exquisite level of structural fidelity within the alkyl-quinolone molecular framework. Antibacterial activity was demonstrated against the serious human pathogens Vibrio vulnificus and Vibrio cholerae as well as a panel of bioluminescent squid symbiont Allivibrio fischeri isolates. In contrast, Vibrio parahaemolyticus growth and biofilm formation was unaffected in the presence of HHQ and all the structural variants tested. In general, modification to almost all of the molecule except the alkyl-chain end, led to loss of activity. This suggests that the bacteriostatic activity of HHQ requires the concerted action of the entire framework components. The only exception to this pattern was deuteration of HHQ at the C3 position. HHQ modified with a terminal alkene at the quinolone alkyl chain retained bacteriostatic activity and was also found to activate PqsR signalling comparable to the native agonist. The data from this integrated analysis provides novel insights into the structural flexibility underpinning the signalling activity of the complex alkyl-quinolone molecular communication system.

RevDate: 2020-11-08
CmpDate: 2020-07-22

Stubbendieck RM, Li H, CR Currie (2019)

Convergent evolution of signal-structure interfaces for maintaining symbioses.

Current opinion in microbiology, 50:71-78.

Symbiotic microbes are essential to the ecological success and evolutionary diversification of multicellular organisms. The establishment and stability of bipartite symbioses are shaped by mechanisms ensuring partner fidelity between host and symbiont. In this minireview, we demonstrate how the interface of chemical signals and host structures influences fidelity between legume root nodules and rhizobia, Hawaiian bobtail squid light organs and Allivibrio fischeri, and fungus-growing ant crypts and Pseudonocardia. Subsequently, we illustrate the morphological diversity and widespread phylogenetic distribution of specialized structures used by hosts to house microbial symbionts, indicating the importance of signal-structure interfaces across the history of multicellular life. These observations, and the insights garnered from well-studied bipartite associations, demonstrate the need to concentrate on the signal-structure interface in complex and multipartite systems, including the human microbiome.

RevDate: 2021-04-02
CmpDate: 2021-01-14

Zink KE, Tarnowski DA, Mandel MJ, et al (2020)

Optimization of a minimal sample preparation protocol for imaging mass spectrometry of unsectioned juvenile invertebrates.

Journal of mass spectrometry : JMS, 55(4):e4458.

Tissue sections have long been the subject matter for the application of imaging mass spectrometry, but recently the technique has been adapted for many other purposes including bacterial colonies and 3D cell culture. Here, we present a simple preparation method for unsectioned invertebrate tissue without the need for fixing, embedding, or slicing. The protocol was used to successfully prepare a Hawaiian bobtail squid hatchling for analysis, and the resulting data detected ions that correspond to compounds present in the host only during its symbiotic colonization by Vibrio fischeri.

RevDate: 2020-10-30
CmpDate: 2020-06-24

Aschtgen MS, Brennan CA, Nikolakakis K, et al (2019)

Insights into flagellar function and mechanism from the squid-vibrio symbiosis.

NPJ biofilms and microbiomes, 5(1):32.

Flagella are essential and multifunctional nanomachines that not only move symbionts towards their tissue colonization site, but also play multiple roles in communicating with the host. Thus, untangling the activities of flagella in reaching, interacting, and signaling the host, as well as in biofilm formation and the establishment of a persistent colonization, is a complex problem. The squid-vibrio system offers a unique model to study the many ways that bacterial flagella can influence a beneficial association and, generally, other bacteria-host interactions. Vibrio fischeri is a bioluminescent bacterium that colonizes the Hawaiian bobtail squid, Euprymna scolopes. Over the last 15 years, the structure, assembly, and functions of V. fischeri flagella, including not only motility and chemotaxis, but also biofilm formation and symbiotic signaling, have been revealed. Here we discuss these discoveries in the perspective of other host-bacteria interactions.

RevDate: 2020-06-25
CmpDate: 2020-04-30

Kerwin AH, Gromek SM, Suria AM, et al (2019)

Shielding the Next Generation: Symbiotic Bacteria from a Reproductive Organ Protect Bobtail Squid Eggs from Fungal Fouling.

mBio, 10(5):.

The importance of defensive symbioses, whereby microbes protect hosts through the production of specific compounds, is becoming increasingly evident. Although defining the partners in these associations has become easier, assigning function to these relationships often presents a significant challenge. Here, we describe a functional role for a bacterial consortium in a female reproductive organ in the Hawaiian bobtail squid, Euprymna scolopes Bacteria from the accessory nidamental gland (ANG) are deposited into the egg jelly coat (JC), where they are hypothesized to play a defensive role during embryogenesis. Eggs treated with an antibiotic cocktail developed a microbial biomass primarily composed of the pathogenic fungus Fusarium keratoplasticum that infiltrated the JC, resulting in severely reduced hatch rates. Experimental manipulation of the eggs demonstrated that the JC was protective against this fungal fouling. A large proportion of the bacterial strains isolated from the ANG or JC inhibited F. keratoplasticum in culture (87.5%), while a similar proportion of extracts from these strains also exhibited antifungal activity against F. keratoplasticum and/or the human-pathogenic yeast Candida albicans (72.7%). Mass spectral network analyses of active extracts from bacterial isolates and egg clutches revealed compounds that may be involved in preventing microbial overgrowth. Several secondary metabolites were identified from ANG/JC bacteria and egg clutches, including the known antimicrobial lincomycin as well as a suite of glycerophosphocholines and mycinamicin-like compounds. These results shed light on a widely distributed but poorly understood symbiosis in cephalopods and offer a new source for exploring bacterial secondary metabolites with antimicrobial activity.IMPORTANCE Organisms must have strategies to ensure successful reproduction. Some animals that deposit eggs protect their embryos from fouling/disease with the help of microorganisms. Although beneficial bacteria are hypothesized to contribute to egg defense in some organisms, the mechanisms of this protection remain largely unknown, with the exception of a few recently described systems. Using both experimental and analytical approaches, we demonstrate that symbiotic bacteria associated with a cephalopod reproductive gland and eggs inhibit fungi. Chemical analyses suggest that these bacteria produce antimicrobial compounds that may prevent overgrowth from fungi and other microorganisms. Given the distribution of these symbiotic glands among many cephalopods, similar defensive relationships may be more common in aquatic environments than previously realized. Such defensive symbioses may also be a rich source for the discovery of new antimicrobial compounds.

RevDate: 2020-10-06
CmpDate: 2020-07-22

Bongrand C, EG Ruby (2019)

The impact of Vibrio fischeri strain variation on host colonization.

Current opinion in microbiology, 50:15-19.

Strain-level epidemiology is a key approach to understanding the mechanisms underlying establishment of any host-microbe association. The squid-vibrio light organ symbiosis has proven to be an informative and tractable experimental model in which to discover these mechanisms because it involves only one bacterial species, Vibrio fischeri. In this horizontally transmitted symbiosis, the squid presents nutrients to the bacteria located in a bilobed light-emitting organ, while the symbionts provide bioluminescence to their host. To initiate this association, V. fischeri cells go through several distinct stages: from free-living in the bacterioplankton, to forming a multicellular aggregation near pores on the light organ's surface, to migrating through the pores and into crypts deep in the light organ, where the symbiont population grows and luminesces. Because individual cells must successfully navigate these distinct regions, phenotypic differences between strains will have a strong impact on the composition of the population finally colonizing the squid. Here we review recent advances in our understanding of behavioral characteristics that differentially drive a strain's success, including its effectiveness of aggregation, the rapidity with which it reaches the deep crypts, and its deployment of type VI secretion.

RevDate: 2020-10-01
CmpDate: 2020-06-11

Schwartzman JA, Lynch JB, Flores Ramos S, et al (2019)

Acidic pH promotes lipopolysaccharide modification and alters colonization in a bacteria-animal mutualism.

Molecular microbiology, 112(4):1326-1338.

Environmental pH can be an important cue for symbiotic bacteria as they colonize their eukaryotic hosts. Using the model mutualism between the marine bacterium Vibrio fischeri and the Hawaiian bobtail squid, we characterized the bacterial transcriptional response to acidic pH experienced during the shift from planktonic to host-associated lifestyles. We found several genes involved in outer membrane structure were differentially expressed based on pH, indicating alterations in membrane physiology as V. fischeri initiates its symbiotic program. Exposure to host-like pH increased the resistance of V. fischeri to the cationic antimicrobial peptide polymixin B, which resembles antibacterial molecules that are produced by the squid to select V. fischeri from the ocean microbiota. Using a forward genetic screen, we identified a homolog of eptA, a predicted phosphoethanolamine transferase, as critical for antimicrobial defense. We used MALDI-MS to verify eptA as an ethanolamine transferase for the lipid-A portion of V. fischeri lipopolysaccharide. We then used a DNA pulldown approach to discover that eptA transcription is activated by the global regulator H-NS. Finally, we revealed that eptA promotes successful squid colonization by V. fischeri, supporting its potential role in initiation of this highly specific symbiosis.

RevDate: 2020-08-18
CmpDate: 2020-08-18

Cohen ML, Mashanova EV, Rosen NM, et al (2019)

Adaptation to temperature stress by Vibrio fischeri facilitates this microbe's symbiosis with the Hawaiian bobtail squid (Euprymna scolopes).

Evolution; international journal of organic evolution, 73(9):1885-1897.

For microorganisms cycling between free-living and host-associated stages, where reproduction occurs in both of these lifestyles, an interesting inquiry is whether adaptation to stress during the free-living stage can impact microbial fitness in the host. To address this topic, the mutualism between the Hawaiian bobtail squid (Euprymna scolopes) and the marine bioluminescent bacterium Vibrio fischeri was utilized. Using microbial experimental evolution, V. fischeri was selected to low (8°C), high (34°C), and fluctuating temperature stress (8°C/34°C) for 2000 generations. The temperatures 8°C and 34°C were the lower and upper growth limits, respectively. V. fischeri was also selected to benign temperatures (21°C and 28°C) for 2000 generations, which served as controls. V. fischeri demonstrated significant adaptation to low, high, and fluctuating temperature stress. V. fischeri did not display significant adaptation to the benign temperatures. Adaptation to stressful temperatures facilitated V. fischeri's ability to colonize the squid host relative to the ancestral lines. Bioluminescence levels also increased. Evolution to benign temperatures did not manifest these results. In summary, microbial adaptation to stress during the free-living stage can promote coevolution between hosts and microorganisms.

RevDate: 2020-09-03
CmpDate: 2020-06-04

Guckes KR, Cecere AG, Wasilko NP, et al (2019)

Incompatibility of Vibrio fischeri Strains during Symbiosis Establishment Depends on Two Functionally Redundant hcp Genes.

Journal of bacteriology, 201(19):.

Bacteria that have the capacity to fill the same niche will compete with one another for the space and resources available within an ecosystem. Such competition is heightened among different strains of the same bacterial species. Nevertheless, different strains often inhabit the same host. The molecular mechanisms that impact competition between different strains within the same host are poorly understood. To address this knowledge gap, the type VI secretion system (T6SS), which is a mechanism for bacteria to kill neighboring cells, was examined in the marine bacterium Vibrio fischeri Different strains of V. fischeri naturally colonize the light organ of the bobtail squid Euprymna scolopes The genome of FQ-A001, a T6SS-positive strain, features two hcp genes that are predicted to encode identical subunits of the T6SS. Coincubation assays showed that either hcp gene is sufficient for FQ-A001 to kill another strain via the T6SS in vitro Additionally, induction of hcp expression is sufficient to induce killing activity in an FQ-A001 mutant lacking both hcp genes. Squid colonization assays involving inocula of FQ-A001-derived strains mixed with ES114 revealed that both hcp genes must be deleted for FQ-A001 and ES114 to occupy the same space within the light organ. These experimental results provide insight into the genetic factors necessary for the T6SS of V. fischeri to function in vivo, thereby increasing understanding of the molecular mechanisms that impact strain diversity within a host.IMPORTANCE Different bacterial strains compete to occupy the same niche. The outcome of such competition can be affected by the type VI secretion system (T6SS), an intercellular killing mechanism of bacteria. Here an animal-bacterial symbiosis is used as a platform for study of the genetic factors that promote the T6SS-mediated killing of one strain by another. Identification of the molecular determinants of T6SS function in vivo contributes to the understanding of how different strains interact within a host.

RevDate: 2020-06-08
CmpDate: 2020-06-08

Lynch JB, Schwartzman JA, Bennett BD, et al (2019)

Ambient pH Alters the Protein Content of Outer Membrane Vesicles, Driving Host Development in a Beneficial Symbiosis.

Journal of bacteriology, 201(20):.

Outer membrane vesicles (OMVs) are continuously produced by Gram-negative bacteria and are increasingly recognized as ubiquitous mediators of bacterial physiology. In particular, OMVs are powerful effectors in interorganismal interactions, driven largely by their molecular contents. These impacts have been studied extensively in bacterial pathogenesis but have not been well documented within the context of mutualism. Here, we examined the proteomic composition of OMVs from the marine bacterium Vibrio fischeri, which forms a specific mutualism with the Hawaiian bobtail squid, Euprymna scolopes We found that V. fischeri upregulates transcription of its major outer membrane protein, OmpU, during growth at an acidic pH, which V. fischeri experiences when it transitions from its environmental reservoir to host tissues. We used comparative genomics and DNA pulldown analyses to search for regulators of ompU and found that differential expression of ompU is governed by the OmpR, H-NS, and ToxR proteins. This transcriptional control combines with nutritional conditions to govern OmpU levels in OMVs. Under a host-encountered acidic pH, V. fischeri OMVs become more potent stimulators of symbiotic host development in an OmpU-dependent manner. Finally, we found that symbiotic development could be stimulated by OMVs containing a homolog of OmpU from the pathogenic species Vibrio cholerae, connecting the role of a well-described virulence factor with a mutualistic element. This work explores the symbiotic effects of OMV variation, identifies regulatory machinery shared between pathogenic and mutualistic bacteria, and provides evidence of the role that OMVs play in animal-bacterium mutualism.IMPORTANCE Beneficial bacteria communicate with their hosts through a variety of means. These communications are often carried out by a combination of molecules that stimulate responses from the host and are necessary for development of the relationship between these organisms. Naturally produced bacterial outer membrane vesicles (OMVs) contain many of those molecules and can stimulate a wide range of responses from recipient organisms. Here, we describe how a marine bacterium, Vibrio fischeri, changes the makeup of its OMVs under conditions that it experiences as it goes from its free-living lifestyle to associating with its natural host, the Hawaiian bobtail squid. This work improves our understanding of how bacteria change their signaling profile as they begin to associate with their beneficial partner animals.

RevDate: 2021-01-09
CmpDate: 2020-06-22

Rader B, McAnulty SJ, SV Nyholm (2019)

Persistent symbiont colonization leads to a maturation of hemocyte response in the Euprymna scolopes/Vibrio fischeri symbiosis.

MicrobiologyOpen, 8(10):e858.

The binary association between the squid, Euprymna scolopes, and its symbiont, Vibrio fischeri, serves as a model system to study interactions between beneficial bacteria and the innate immune system. Previous research demonstrated that binding of the squid's immune cells, hemocytes, to V. fischeri is altered if the symbiont is removed from the light organ, suggesting that host colonization alters hemocyte recognition of V. fischeri. To investigate the influence of symbiosis on immune maturation during development, we characterized hemocyte binding and phagocytosis of V. fischeri and nonsymbiotic Vibrio harveyi from symbiotic (sym) and aposymbiotic (apo) juveniles, and wild-caught and laboratory-raised sym and apo adults. Our results demonstrate that while light organ colonization by V. fischeri did not alter juvenile hemocyte response, these cells bound a similar number of V. fischeri and V. harveyi yet phagocytosed only V. harveyi. Our results also indicate that long-term colonization altered the adult hemocyte response to V. fischeri but not V. harveyi. All hemocytes from adult squid, regardless of apo or sym state, both bound and phagocytosed a similar number of V. harveyi while hemocytes from both wild-caught and sym-raised adults bound significantly fewer V. fischeri, although more V. fischeri were phagocytosed by hemocytes from wild-caught animals. In contrast, hemocytes from apo-raised squid bound similar numbers of both V. fischeri and V. harveyi, although more V. harveyi cells were engulfed, suggesting that blood cells from apo-raised adults behaved similarly to juvenile hosts. Taken together, these data suggest that persistent colonization by the light organ symbiont is required for hemocytes to differentially bind and phagocytose V. fischeri. The cellular immune system of E. scolopes likely possesses multiple mechanisms at different developmental stages to promote a specific and life-long interaction with the symbiont.

RevDate: 2020-09-30

Septer AN (2019)

The Vibrio-Squid Symbiosis as a Model for Studying Interbacterial Competition.

mSystems, 4(3):.

The symbiosis between Euprymna scolopes squid and its bioluminescent bacterial symbiont, Vibrio fischeri, is a valuable model system to study a natural, coevolved host-microbe association. Over the past 30 years, researchers have developed and optimized many experimental methods to study both partners in isolation and during symbiosis. These powerful tools, along with a strong foundational knowledge about the system, position the Vibrio-squid symbiosis at the forefront of host-microbe interactions because this system is uniquely suited to investigation of symbiosis from both host and bacterial perspectives. Moreover, the ability to isolate and characterize different strains of V. fischeri has revealed exciting new insights about how different genotypes evolve to compete for a host niche, including deploying interbacterial weapons early during host colonization. This Perspective explores how interbacterial warfare influences the diversity and spatial structure of the symbiotic population, as well as the possible effects that intraspecific competition might have on the host.

RevDate: 2020-10-01

Lutz HL, Ramírez-Puebla ST, Abbo L, et al (2019)

A Simple Microbiome in the European Common Cuttlefish, Sepia officinalis.

mSystems, 4(4):.

The European common cuttlefish, Sepia officinalis, is used extensively in biological and biomedical research, yet its microbiome remains poorly characterized. We analyzed the microbiota of the digestive tract, gills, and skin in mariculture-raised S. officinalis using a combination of 16S rRNA amplicon sequencing, quantitative PCR (qPCR), and fluorescence spectral imaging. Sequencing revealed a highly simplified microbiota consisting largely of two single bacterial amplicon sequence variants (ASVs) of Vibrionaceae and Piscirickettsiaceae. The esophagus was dominated by a single ASV of the genus Vibrio. Imaging revealed bacteria in the family Vibrionaceae distributed in a discrete layer that lines the esophagus. This Vibrio was also the primary ASV found in the microbiota of the stomach, cecum, and intestine, but occurred at lower abundance, as determined by qPCR, and was found only scattered in the lumen rather than in a discrete layer via imaging analysis. Treatment of animals with the commonly used antibiotic enrofloxacin led to a nearly 80% reduction of the dominant Vibrio ASV in the esophagus but did not significantly alter the relative abundance of bacteria overall between treated versus control animals. Data from the gills were dominated by a single ASV in the family Piscirickettsiaceae, which imaging visualized as small clusters of cells. We conclude that bacteria belonging to the Gammaproteobacteria are the major symbionts of the cuttlefish Sepia officinalis cultured from eggs in captivity and that the esophagus and gills are major colonization sites. IMPORTANCE Microbes can play critical roles in the physiology of their animal hosts, as evidenced in cephalopods by the role of Vibrio (Aliivibrio) fischeri in the light organ of the bobtail squid and the role of Alpha- and Gammaproteobacteria in the reproductive system and egg defense in a variety of cephalopods. We sampled the cuttlefish microbiome throughout the digestive tract, gills, and skin and found dense colonization of an unexpected site, the esophagus, by a microbe of the genus Vibrio, as well as colonization of gills by Piscirickettsiaceae. This finding expands the range of organisms and body sites known to be associated with Vibrio and is of potential significance for understanding host-symbiont associations, as well as for understanding and maintaining the health of cephalopods in mariculture.

RevDate: 2020-10-01

Bultman KM, Cecere AG, Miyashiro T, et al (2019)

Draft Genome Sequences of Type VI Secretion System-Encoding Vibrio fischeri Strains FQ-A001 and ES401.

Microbiology resource announcements, 8(20):.

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.

RevDate: 2019-08-20
CmpDate: 2019-08-20

Liu H, Huo L, Yu Q, et al (2019)

Molecular insights of a novel cephalopod toll-like receptor homologue in Sepiella japonica, revealing its function under the stress of aquatic pathogenic bacteria.

Fish & shellfish immunology, 90:297-307.

Toll-like receptors (TLRs) play an important role in defense response to pathogens in mollusk. In this study the first TLR from Sepiella japonica (named as SjTLR) was functionally characterized, and its full-length cDNA consisted of 3914bp (GenBank accession no. AQY56780.1) including an open reading frame of 3582bp, encoding a putative protein of 1193 amino acids. Its theoretical molecular weight was 137.87 KDa and the predicted isoelectric point was 3.69. The derived amino acids sequence comprised of an extracellular domain including 26 amino acids signal peptide and eleven leucine-rich repeats (LRR), capped with LRRCT and LRRNT followed by transmembrane domain and cytoplasmic Toll/IL-1R domain (TIR). In addition, 12 potential N-linked glycosylation sites were present in the ectodomain to influence protein trafficking, surface presentation and ligand recognition. Multiple sequence alignment and phylogenetic analysis revealed that SjTLR shared the highest similarity to that of Euprymna scolopes and they fell into the same clade. Real-time PCR showed SjTLR expressed constitutively in all tested tissues, including gill, liver, brain, muscle, intestine, heart, lobus opticus and stomach, but showed different expression levels with genders. The highest expression was in the liver, and the lowest was in stomach for both genders. The functional domain region sequences encoding LRRs domain protein and TIR domain containing protein (TcpB) were expressed in BL21(DE3) respectively and purified with Ni-NAT Superflow resin conforming to the expected molecular weight. The cellular localization of SjTLR in HEK293 cells was conducted and plasma membrane localization was detected. SjLRRs internalization upon the activation of LPS was also observed, and dramatic redistribution of SjLRRs in the cytoplasm with distinct perinuclear accumulation was found. After SjTLR transfection Toll/NF-κB signaling pathway was active in HEK293 treated with LPS and TNFɑ. The nuclear related genes may also be activated by NF-κB in the nucleus, and the corresponding mRNA was transferred through the intracellular signal transduction pathway, so that IL-6 cytokines could be synthesized and released. After infection by Vibrio parahemolyticus and Aeromonas hydrophila the expression of SjTLR were upregulated with time-dependent manner. These findings might be valuable for understanding the innate immune signaling pathways of S.japonica and enabling future studies on host-pathogen interactions.

RevDate: 2021-06-21

Patelunas AJ, MK Nishiguchi (2018)

Vascular architecture in the bacteriogenic light organ of Euprymna tasmanica (Cephalopoda: Sepiolidae).

Invertebrate biology : a quarterly journal of the American Microscopical Society and the Division of Invertebrate Zoology/ASZ, 137(3):240-249.

Symbiosis between southern dumpling squid, Euprymna tasmanica (Cephalopoda: Sepiolidae), and its luminescent symbiont, the bacterium Vibrio fischeri, provides an experimentally tractable system to examine interactions between the eukaryotic host and its bacterial partner. Luminescence emitted by the symbiotic bacteria provides light for the squid in a behavior termed "counter-illumination," which allows the squid to mask its shadow amidst downwelling moonlight. Although this association is beneficial, light generated from the bacteria requires large quantities of oxygen to maintain this energy-consuming reaction. Therefore, we examined the vascular network within the light organ of juveniles of E. tasmanica with and without V. fischeri. Vessel type, diameter, and location of vessels were measured. Although differences between symbiotic and aposymbiotic squid demonstrated that the presence of V. fischeri does not significantly influence the extent of vascular branching at early stages of symbiotic development, these finding do provide an atlas of blood vessel distribution in the organ. Thus, these results provide a framework to understand how beneficial bacteria influence the development of a eukaryotic closed vascular network and provide insight to the evolutionary developmental dynamics that form during mutualistic interactions.

RevDate: 2020-03-23
CmpDate: 2020-03-23

Moriano-Gutierrez S, Koch EJ, Bussan H, et al (2019)

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

Proceedings of the National Academy of Sciences of the United States of America, 116(16):7990-7999.

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 squid Euprymna scolopes and its luminous bacterial partner, Vibrio fischeri, to define the impact of colonization on transcriptomic networks in the host. A night-active predator, E. scolopes coordinates 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 in V. fischeri-specifically, deletion of the lux operon, 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.

RevDate: 2020-09-29

Xie T, Pang R, Wu Q, et al (2019)

Cold Tolerance Regulated by the Pyruvate Metabolism in Vibrio parahaemolyticus.

Frontiers in microbiology, 10:178.

Vibrio parahaemolyticus is a common foodborne pathogen found in seafood, and represents a major threat to human health worldwide. Low-temperature storage is an important seafood processing method, but is not sufficient to completely eliminate the bacteria and avoid foodborne illness. To determine the mechanisms behind such cold tolerance, RNA-seq and iTRAQ analyses were first performed to obtain the global transcriptomic and proteomic patterns of frozen squid and clinical V. parahaemolyticus isolates under cold conditions. The integrated analysis revealed the modulation of multiple pathways such as the co-occurrence of down-regulated pyruvate metabolism and up-regulated fatty acid biosynthesis, which likely contribute to V. parahaemolyticus cold tolerance. Furthermore, we found that increasing concentrations of pyruvate can reduce the fatty acid content to influence V. parahaemolyticus growth in cold conditions. Thus, regulation of pyruvate concentration may be an effective method to control this seafood-borne pathogen.

RevDate: 2020-03-09
CmpDate: 2020-02-12

Rotman ER, Bultman KM, Brooks JF, et al (2019)

Natural Strain Variation Reveals Diverse Biofilm Regulation in Squid-Colonizing Vibrio fischeri.

Journal of bacteriology, 201(9):.

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 by 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. Thus, this work 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.

RevDate: 2020-02-25
CmpDate: 2019-04-22

Girard L, Blanchet E, Stien D, et al (2019)

Evidence of a Large Diversity of N-acyl-Homoserine Lactones in Symbiotic Vibrio fischeri Strains Associated with the Squid Euprymna scolopes.

Microbes and environments, 34(1):99-103.

Vibrio fischeri possesses a complex AHL-mediated Quorum-sensing (QS) system including two pathways, LuxI/R (3-oxo-C6-HSL and C6-HSL) and AinS/R (C8-HSL), which are important for the regulation of physiological traits. Diverse QS-dependent functional phenotypes have been described in V. fischeri; however, AHL diversity is still underestimated. In the present study, we investigated AHL diversity in five symbiotic V. fischeri strains with distinct phenotypic properties using UHPLC-HRMS/MS. The results obtained (1) revealed an unexpectedly high diversity of signaling molecules, (2) emphasized the complexity of QS in V. fischeri, and (3) highlight the importance of understanding the specificity of AHL-mediated QS.

RevDate: 2020-02-25
CmpDate: 2019-02-08

Jung SW (2018)

A foodborne outbreak of gastroenteritis caused by Vibrio parahaemolyticus associated with cross-contamination from squid in Korea.

Epidemiology and health, 40:e2018056.

OBJECTIVES: Water-borne diseases caused by Vibrio parahemolyticus are often known to cause gastritis when raw or undercooked seafood is eaten. It is very rare that Vibrio gastritis caused by ingesting non-seafood products occurs on a large scale. On September 19, 2017, a large-scale Vibrio gastritis occurred after the city residents consumed food at a bazaar held in a welfare center in Jungnang-gu, Seoul.

METHODS: The total number of visitors was approximately 299, and 237 (79.3%) of them showed symptoms. Among those who showed symptoms, 116 (48.9%) consulted the hospital, and 53 (45.6%) were hospitalized. Among the 299 exposed individuals, 174 (58.1%) responded to this survey: 163 (93.6%) with and 11 (6.4%) without symptoms. This study was retrospectively conducted by investigating the exposed individuals. To investigate the spread of infection, medical staff of hospitals in the epidemic area were interviewed, exposed individuals surveyed, microbiological testing conducted, and ingredient handling and cooking processes investigated.

RESULTS: A total of 237 individuals, including 6 food handlers, were affected (prevalence, 79.2%). During the microbiological testing, V. parahemolyticus was found in 34 patients and 4 food handlers. In the consumption analysis, the relative risk of kimbap was 6.79 (confidence interval 1.10 to 41.69). In-depth investigation found that squid, an ingredient of Korean pancake, and egg sheets, an ingredient of kimbap, were prepared using the same cutting board and knife, which were thought to be the cause of cross-contamination that led to a large-scale outbreak of Vibrio gastritis.

CONCLUSIONS: A recent large-scale outbreak of Vibrio gastritis occurred due to the cross-contamination with kimbap during the preparation process of squid rather than the actual consumption of seafood. Thus, a more stringent hygiene management is necessary during the processing and management of food to prevent infections associated with V. parahemolyticus.

RevDate: 2020-03-09
CmpDate: 2019-04-05

Bosch TCG (2019)

Squid genomes in a bacterial world.

Proceedings of the National Academy of Sciences of the United States of America, 116(8):2799-2801.

RevDate: 2021-01-09
CmpDate: 2019-12-12

Soto W, Travisano M, Tolleson AR, et al (2019)

Symbiont evolution during the free-living phase can improve host colonization.

Microbiology (Reading, England), 165(2):174-187.

For micro-organisms cycling between free-living and host-associated stages, where reproduction occurs in both of these lifestyles, an interesting inquiry is whether evolution during the free-living stage can be positively pleiotropic to microbial fitness in a host environment. To address this topic, the squid host Euprymna tasmanica and the marine bioluminescent bacterium Vibrio fischeri were utilized. Microbial ecological diversification in static liquid microcosms was used to simulate symbiont evolution during the free-living stage. Thirteen genetically distinct V. fischeri strains from a broad diversity of ecological sources (e.g. squid light organs, fish light organs and seawater) were examined to see if the results were reproducible in many different genetic settings. Genetic backgrounds that are closely related can be predisposed to considerable differences in how they respond to similar selection pressures. For all strains examined, new mutations with striking and facilitating effects on host colonization arose quickly during microbial evolution in the free-living stage, regardless of the ecological context under consideration for a strain's genetic background. Microbial evolution outside a host environment promoted host range expansion, improved host colonization for a micro-organism, and diminished the negative correlation between biofilm formation and motility.

RevDate: 2020-03-09
CmpDate: 2019-05-03

Belcaid M, Casaburi G, McAnulty SJ, et al (2019)

Symbiotic organs shaped by distinct modes of genome evolution in cephalopods.

Proceedings of the National Academy of Sciences of the United States of America, 116(8):3030-3035.

Microbes have been critical drivers of evolutionary innovation in animals. To understand the processes that influence the origin of specialized symbiotic organs, we report the sequencing and analysis of the genome of Euprymna scolopes, a model cephalopod with richly characterized host-microbe interactions. We identified large-scale genomic reorganization shared between E. scolopes and Octopus bimaculoides and posit that this reorganization has contributed to the evolution of cephalopod complexity. To reveal genomic signatures of host-symbiont interactions, we focused on two specialized organs of E. scolopes: the light organ, which harbors a monoculture of Vibrio fischeri, and the accessory nidamental gland (ANG), a reproductive organ containing a bacterial consortium. Our findings suggest that the two symbiotic organs within E. scolopes originated by different evolutionary mechanisms. Transcripts expressed in these microbe-associated tissues displayed their own unique signatures in both coding sequences and the surrounding regulatory regions. Compared with other tissues, the light organ showed an abundance of genes associated with immunity and mediating light, whereas the ANG was enriched in orphan genes known only from E. scolopes Together, these analyses provide evidence for different patterns of genomic evolution of symbiotic organs within a single host.

RevDate: 2020-03-09
CmpDate: 2019-08-02

Wasilko NP, Larios-Valencia J, Steingard CH, et al (2019)

Sulfur availability for Vibrio fischeri growth during symbiosis establishment depends on biogeography within the squid light organ.

Molecular microbiology, 111(3):621-636.

The fitness of host-associated microbes depends on their ability to access nutrients in vivo. Identifying these mechanisms is significant for understanding how microbes have evolved to fill specific ecological niches within a host. Vibrio fischeri is a bioluminescent bacterium that colonizes and proliferates within the light organ of the Hawaiian bobtail squid, which provides an opportunity to study how bacteria grow in vivo. Here, the transcription factor CysB is shown to be necessary for V. fischeri both to grow on several sulfur sources in vitro and to establish symbiosis with juvenile squid. CysB is also found to regulate several genes involved in sulfate assimilation and to contribute to the growth of V. fischeri on cystine, which is the oxidized form of cysteine. A mutant that grows on cystine but not sulfate could establish symbiosis, suggesting that V. fischeri acquires nutrients related to this compound within the host. Finally, CysB-regulated genes are shown to be differentially expressed among the V. fischeri populations occupying the various colonization sites found within the light organ. Together, these results suggest the biogeography of V. fischeri populations within the squid light organ impacts the physiology of this symbiotic bacterium in vivo through CysB-dependent gene regulation.

RevDate: 2019-09-06
CmpDate: 2019-09-05

Stabb EV (2019)

Should they stay or should they go? Nitric oxide and the clash of regulators governing Vibrio fischeri biofilm formation.

Molecular microbiology, 111(1):1-5.

A key regulatory decision for many bacteria is the switch between biofilm formation and motile dispersal, and this dynamic is well illustrated in the light-organ symbiosis between the bioluminescent bacterium Vibrio fischeri and the Hawaiian bobtail squid. Biofilm formation mediated by the syp gene cluster helps V. fischeri transition from a dispersed planktonic lifestyle to a robust aggregate on the surface of the nascent symbiotic organ. However, the bacteria must then swim to pores and down into the deeper crypt tissues that they ultimately colonize. A number of positive and negative regulators control syp expression and biofilm formation, but until recently the environmental inputs controlling this clash between opposing regulatory mechanisms have been unclear. Thompson et al. have now shown that Syp-mediated biofilms can be repressed by a well-known host-derived molecule: nitric oxide. This regulation is accomplished by the NO sensor HnoX exerting control over the biofilm regulator HahK. The discoveries reported here by Thompson et al. cast new light on a critical early stage of symbiotic initiation in the V. fischeri-squid model symbiosis, and more broadly it adds to a growing understanding of the role(s) that NO and HnoX play in biofilm regulation by many bacteria.

RevDate: 2019-03-04
CmpDate: 2019-03-04

Lv T, Song T, Liu H, et al (2019)

Isolation and characterization of a virulence related Vibrio alginolyticus strain Wz11 pathogenic to cuttlefish, Sepia pharaonis.

Microbial pathogenesis, 126:165-171.

Vibrio alginolyticus is a ubiquitous marine opportunistic pathogen that can infect various hosts in marine environment. In the present study, V. alginolyticus strain Wz11 was isolated from diseased cuttlefish, Sepia pharaonis, with 20% of promoted death and high survival capacity in skin mucus and tissue liquid. Its growth, siderophore production, and expressions of haemolysin and swarming related genes were characterized under iron limited conditions. The minimal inhibitory concentration (MIC) of 2,2'-dipyridyl (DP) to V. alginolyticus strain Wz11 was 640 μM. While growth of V. alginolyticus strain Wz11 was inhibited by DP, production of iron-seizing substances, haemolytic activity and swarming motility were increased. Moreover, expressions of haemolysin related genes tlh, tdh and vah and flagellar related genes flgH, fliC, fliD and fliS were also characterized using real-time reverse transcriptase PCR. Expression of tdh was up-regulated to 7.7-fold, while expressions of tlh and vah were down-regulated to 0.016-fold and 0.03-fold, respectively. The expression of fliC, flgH, fliD and fliS was up-regulated to 4.9-, 3.8-, 8.6- and 4.5-fold, respectively. Concluded from our results suggested that V. alginolyticus strain Wz11 was considered as a potential pathogen of S. pharaonis, and iron level played an important role in the production of iron-seizing substances, and activities of haemolysin and bacterial swarming as well as their related gene expressions.

RevDate: 2021-02-25
CmpDate: 2019-08-06

Bongrand C, EG Ruby (2019)

Achieving a multi-strain symbiosis: strain behavior and infection dynamics.

The ISME journal, 13(3):698-706.

Strain diversity, while now recognized as a key driver underlying partner dynamics in symbioses, is usually difficult to experimentally manipulate and image in hosts with complex microbiota. To address this problem, we have used the luminous marine bacterium Vibrio fischeri, which establishes a symbiosis within the crypts of the nascent light organ of the squid Euprymna scolopes. Competition assays in newly hatched juvenile squid have shown that symbiotic V. fischeri are either niche-sharing "S strains", which share the light organ when co-inoculated with other S strains, or niche-dominant "D strains", which are typically found alone in the light organ after a co-colonization. To understand this D strain advantage, we determined the minimum time that different V. fischeri strains needed to initiate colonization and used confocal microscopy to localize the symbionts along their infection track. Further, we determined whether symbiont-induced host morphogenic events also occurred earlier during a D strain colonization. We conclude that D strains colonized more quickly than S strains. Nevertheless, light-organ populations in field-caught adult squid often contain both D and S strains. We determined experimentally that this symbiont population heterogeneity might be achieved in nature by a serial encounter of different strains in the environment.

RevDate: 2020-10-01

Kremer N, Koch EJ, El Filali A, et al (2018)

Persistent Interactions with Bacterial Symbionts Direct Mature-Host Cell Morphology and Gene Expression in the Squid-Vibrio Symbiosis.

mSystems, 3(5):.

In horizontally transmitted symbioses, structural, biochemical, and molecular features both facilitate host colonization by specific symbionts and mediate their persistent carriage. In the association between the squid Euprymna scolopes and its luminous bacterial partner Vibrio fischeri, the symbionts interact with two epithelial fields; they interact (i) transiently with the superficial ciliated field that potentiates colonization and regresses within days of colonization and (ii) persistently with the cells that line the internal crypts, whose ultrastructure changes in response to the symbionts. Development of the association creates conditions that promote the symbiotic partner over the lifetime of the host. To determine whether light organ maturation requires continuous interactions with V. fischeri or only the signaling that occurs during its initiation, we compared 4-week-old squid that were uncolonized with those colonized either persistently by wild-type V. fischeri or transiently by a V. fischeri mutant that triggers early events in morphogenesis but does not persist. Microscopic analysis of the light organs showed that, while morphogenesis of the superficial ciliated field is greatly accelerated by V. fischeri colonization, its eventual outcome is largely independent of colonization state. In contrast, the symbiont-induced changes in crypt cell shape require persistent host-symbiont interaction, reflected in the similarity between uncolonized and transiently colonized animals. Transcriptomic analyses reflected the microscopy results; host gene expression at 4 weeks was due primarily to the persistent interactions of host and symbiont cells. Further, the transcriptomic signature of specific pathways reflected the daily rhythm of symbiont release and regrowth and required the presence of the symbionts. IMPORTANCE A long-term relationship between symbiotic partners is often characterized by development and maturation of host structures that harbor the symbiont cells over the host's lifetime. To understand the mechanisms involved in symbiosis maintenance more fully, we studied the mature bobtail squid, whose light-emitting organ, under experimental conditions, can be transiently or persistently colonized by Vibrio fischeri or remain uncolonized. Superficial anatomical changes in the organ were largely independent of symbiosis. However, both the microanatomy of cells with which symbionts interact and the patterns of gene expression in the mature animal were due principally to the persistent interactions of host and symbiont cells rather than to a response to early colonization events. Further, the characteristic pronounced daily rhythm on the host transcriptome required persistent V. fischeri colonization of the organ. This experimental study provides a window into how persistent symbiotic colonization influences the form and function of host animal tissues.

RevDate: 2020-03-09
CmpDate: 2019-07-29

Thompson CM, Tischler AH, Tarnowski DA, et al (2019)

Nitric oxide inhibits biofilm formation by Vibrio fischeri via the nitric oxide sensor HnoX.

Molecular microbiology, 111(1):187-203.

Nitric oxide (NO) is an important defense molecule secreted by the squid Euprymna scolopes and sensed by the bacterial symbiont, Vibrio fischeri, via the NO sensor HnoX. HnoX inhibits colonization through an unknown mechanism. The genomic location of hnoX adjacent to hahK, a recently identified positive regulator of biofilm formation, suggested that HnoX may inhibit colonization by controlling biofilm formation, a key early step in colonization. Indeed, the deletion of hnoX resulted in early biofilm formation in vitro, an effect that was dependent on HahK and its putative phosphotransfer residues. An allele of hnoX that encodes a protein with increased activity severely delayed wrinkled colony formation. Control occurred at the level of transcription of the syp genes, which produce the polysaccharide matrix component. The addition of NO abrogated biofilm formation and diminished syp transcription, effects that required HnoX. Finally, an hnoX mutant formed larger symbiotic biofilms. This work has thus uncovered a host-relevant signal controlling biofilm and a mechanism for the inhibition of biofilm formation by V. fischeri. The study of V. fischeri HnoX permits us to understand not only host-associated biofilm mechanisms, but also the function of HnoX domain proteins as regulators of important bacterial processes.

RevDate: 2020-09-29

Coryell RL, Turnham KE, de Jesus Ayson EG, et al (2018)

Phylogeographic patterns in the Philippine archipelago influence symbiont diversity in the bobtail squid-Vibrio mutualism.

Ecology and evolution, 8(15):7421-7435.

Marine microbes encounter a myriad of biotic and abiotic factors that can impact fitness by limiting their range and capacity to move between habitats. This is especially true for environmentally transmitted bacteria that cycle between their hosts and the surrounding habitat. As geologic history, biogeography, and other factors such as water temperature, salinity, and physical barriers can inhibit bacterial movement to novel environments, we chose to examine the genetic architecture of Euprymna albatrossae (Mollusca: Cephalopoda) and their Vibrio fischeri symbionts in the Philippine archipelago using a combined phylogeographic approach. Eleven separate sites in the Philippine islands were examined using haplotype estimates that were examined via nested clade analysis to determine the relationship between E. albatrossae and V. fischeri populations and their geographic location. Identical analyses of molecular variance (AMOVA) were used to estimate variation within and between populations for host and symbiont genetic data. Host animals demonstrated a significant amount of variation within island groups, while symbiont variation was found within individual populations. Nested clade phylogenetic analysis revealed that hosts and symbionts may have colonized this area at different times, with a sudden change in habitat. Additionally, host data indicate restricted gene flow, whereas symbionts show range expansion, followed by periodic restriction to genetic flow. These differences between host and symbiont networks indicate that factors "outside the squid" influence distribution of Philippine V. fischeri. Our results shed light on how geography and changing environmental factors can impact marine symbiotic associations at both local and global scales.

RevDate: 2020-03-09

Koehler S, Gaedeke R, Thompson C, et al (2018)

The model squid-vibrio symbiosis provides a window into the impact of strain- and species-level differences during the initial stages of symbiont engagement.

Environmental microbiology [Epub ahead of print].

Among horizontally acquired symbioses, the mechanisms underlying microbial strain- and species-level specificity remain poorly understood. Here, confocal-microscopy analyses and genetic manipulation of the squid-vibrio association revealed quantitative differences in a symbiont's capacity to interact with the host during initial engagement. Specifically, dominant strains of Vibrio fischeri, 'D-type', previously named for their dominant, single-strain colonization of the squid's bioluminescent organ, were compared with 'S-type', or 'sharing', strains, which can co-colonize the organ. These D-type strains typically: (i) formed aggregations of 100s-1000s of cells on the light-organ surface, up to 3 orders of magnitude larger than those of S-type strains; (ii) showed dominance in co-aggregation experiments, independent of inoculum size or strain proportion; (iii) perturbed larger areas of the organ's ciliated surface; and, (iv) appeared at the pore of the organ approximately 4×s more quickly than S-type strains. At least in part, genes responsible for biofilm synthesis control the hyperaggregation phenotype of a D-type strain. Other marine vibrios produced relatively small aggregations, while an array of marine Gram-positive and -negative species outside of the Vibrionaceae did not attach to the organ's surface. These studies provide insight into the impact of strain variation on early events leading to establishment of an environmentally acquired symbiosis.

RevDate: 2019-06-10
CmpDate: 2018-10-05

Speare L, Cecere AG, Guckes KR, et al (2018)

Bacterial symbionts use a type VI secretion system to eliminate competitors in their natural host.

Proceedings of the National Academy of Sciences of the United States of America, 115(36):E8528-E8537.

Intraspecific competition describes the negative interaction that occurs when different populations of the same species attempt to fill the same niche. Such competition is predicted to occur among host-associated bacteria but has been challenging to study in natural biological systems. Although many bioluminescent Vibrio fischeri strains exist in seawater, only a few strains are found in the light-organ crypts of an individual wild-caught Euprymna scolopes squid, suggesting a possible role for intraspecific competition during early colonization. Using a culture-based assay to investigate the interactions of different V. fischeri strains, we found "lethal" and "nonlethal" isolates that could kill or not kill the well-studied light-organ isolate ES114, respectively. The killing phenotype of these lethal strains required a type VI secretion system (T6SS) encoded in a 50-kb genomic island. Multiple lethal and nonlethal strains could be cultured from the light organs of individual wild-caught adult squid. Although lethal strains eliminate nonlethal strains in vitro, two lethal strains could coexist in interspersed microcolonies that formed in a T6SS-dependent manner. This coexistence was destabilized upon physical mixing, resulting in one lethal strain consistently eliminating the other. When juvenile squid were coinoculated with lethal and nonlethal strains, they occupied different crypts, yet they were observed to coexist within crypts when T6SS function was disrupted. These findings, using a combination of natural isolates and experimental approaches in vitro and in the animal host, reveal the importance of T6SS in spatially separating strains during the establishment of host colonization in a natural symbiosis.

RevDate: 2018-11-27
CmpDate: 2018-11-27

Houyvet B, Zanuttini B, Corre E, et al (2018)

Design of antimicrobial peptides from a cuttlefish database.

Amino acids, 50(11):1573-1582.

No antimicrobial peptide has been identified in cephalopods to date. Annotation of transcriptomes or genomes using basic local alignment Search Tool failed to yield any from sequence identities. Therefore, we searched for antimicrobial sequences in the cuttlefish (Sepia officinalis) database by in silico analysis of a transcriptomic database. Using an original approach based on the analysis of cysteine-free antimicrobial peptides selected from our Antimicrobial Peptide Database (APD3), the online prediction tool of the Collection of Anti-Microbial Peptides (CAMPR3), and a homemade software program, we identified potential antibacterial sequences. Nine peptides less than 25 amino acids long were synthesized. The hydrophobic content of all nine of them ranged from 30 to 70%, and they could form alpha-helices. Three peptides possessed similarities with piscidins, one with BMAP-27, and five were totally new. Their antibacterial activity was evaluated on eight bacteria including the aquatic pathogens Vibrio alginolyticus, Aeromonas salmonicida, or human pathogens such as Salmonella typhimurium, Listeria monocytogenes, or Staphylococcus aureus. Despite the prediction of an antimicrobial potential for eight of the peptides, only two-GR21 and KT19-inhibited more than one bacterial strain with minimal inhibitory concentrations below 25 µM. Some sequences like VA20 and FK19 were hemolytic, while GR21 induced less than 10% of hemolysis on human blood cells at a concentration of 200 µM. GR21 was the only peptide derived from a precursor with a signal peptide, suggesting a real role in cuttlefish immune defense.

RevDate: 2019-06-10
CmpDate: 2019-01-17

Reddi G, Pruss K, Cottingham KL, et al (2018)

Catabolism of mucus components influences motility of Vibrio cholerae in the presence of environmental reservoirs.

PloS one, 13(7):e0201383.

Vibrio cholerae O1, the etiological agent of cholera, is a natural inhabitant of aquatic ecosystems. Motility is a critical element for the colonization of both the human host and its environmental reservoirs. In this study, we investigated the molecular mechanisms underlying the chemotactic response of V. cholerae in the presence of some of its environmental reservoirs. We found that, from the several oligosaccharides found in mucin, two specifically triggered motility of V. cholerae O1: N-acetylneuraminic acid (Neu5Ac) and N-acetylglucosamine (GlcNAc). We determined that the compounds need to be internally catabolized in order to trigger motility of V. cholerae. Interestingly, the catabolism of Neu5Ac and GlcNAc converges and the production of one molecule common to both pathways, glucosamine-6-phosphate (GlcN-6P), is essential to induce motility in the presence of both compounds. Mutants unable to produce GlcN-6P show greatly reduced motility towards mucin. Furthermore, we determined that the production of GlcN-6P is necessary to induce motility of V. cholerae in the presence of some of its environmental reservoirs such as crustaceans or cyanobacteria, revealing a molecular link between the two distinct modes of the complex life cycle of V. cholerae. Finally, cross-species comparisons revealed varied chemotactic responses towards mucin, GlcNAc, and Neu5Ac for environmental (non-pathogenic) strains of V. cholerae, clinical and environmental isolates of the human pathogens Vibrio vulnificus and Vibrio parahaemolyticus, and fish and squid isolates of the symbiotic bacterium Vibrio fischeri. The data presented here suggest nuance in convergent strategies across species of the same bacterial family for motility towards suitable substrates for colonization.

RevDate: 2019-10-10
CmpDate: 2019-10-09

Thompson CM, Marsden AE, Tischler AH, et al (2018)

Vibrio fischeri Biofilm Formation Prevented by a Trio of Regulators.

Applied and environmental microbiology, 84(19):.

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.

RevDate: 2019-12-27
CmpDate: 2019-01-24

Hendry TA, Freed LL, Fader D, et al (2018)

Ongoing Transposon-Mediated Genome Reduction in the Luminous Bacterial Symbionts of Deep-Sea Ceratioid Anglerfishes.

mBio, 9(3):.

Diverse marine fish and squid form symbiotic associations with extracellular bioluminescent bacteria. These symbionts are typically free-living bacteria with large genomes, but one known lineage of symbionts has undergone genomic reduction and evolution of host dependence. It is not known why distinct evolutionary trajectories have occurred among different luminous symbionts, and not all known lineages previously had genome sequences available. In order to better understand patterns of evolution across diverse bioluminescent symbionts, we de novo sequenced the genomes of bacteria from a poorly studied interaction, the extracellular symbionts from the "lures" of deep-sea ceratioid anglerfishes. Deep-sea anglerfish symbiont genomes are reduced in size by about 50% compared to free-living relatives. They show a striking convergence of genome reduction and loss of metabolic capabilities with a distinct lineage of obligately host-dependent luminous symbionts. These losses include reductions in amino acid synthesis pathways and abilities to utilize diverse sugars. However, the symbiont genomes have retained a number of categories of genes predicted to be useful only outside the host, such as those involved in chemotaxis and motility, suggesting that they may persist in the environment. These genomes contain very high numbers of pseudogenes and show massive expansions of transposable elements, with transposases accounting for 28 and 31% of coding sequences in the symbiont genomes. Transposon expansions appear to have occurred at different times in each symbiont lineage, indicating either independent evolutions of reduction or symbiont replacement. These results suggest ongoing genomic reduction in extracellular luminous symbionts that is facilitated by transposon proliferations.IMPORTANCE Many female deep-sea anglerfishes possess a "lure" containing luminous bacterial symbionts. Here we show that unlike most luminous symbionts, these bacteria are undergoing an evolutionary transition toward small genomes with limited metabolic capabilities. Comparative analyses of the symbiont genomes indicate that this transition is ongoing and facilitated by transposon expansions. This transition may have occurred independently in different symbiont lineages, although it is unclear why. Genomic reduction is common in bacteria that only live within host cells but less common in bacteria that, like anglerfish symbionts, live outside host cells. Since multiple evolutions of genomic reduction have occurred convergently in luminous bacteria, they make a useful system with which to understand patterns of genome evolution in extracellular symbionts. This work demonstrates that ecological factors other than an intracellular lifestyle can lead to dramatic gene loss and evolutionary changes and that transposon expansions may play important roles in this process.

RevDate: 2019-10-07
CmpDate: 2019-10-07

Stoudenmire JL, Essock-Burns T, Weathers EN, et al (2018)

An Iterative, Synthetic Approach To Engineer a High-Performance PhoB-Specific Reporter.

Applied and environmental microbiology, 84(14):.

Transcriptional reporters are common tools for analyzing either the transcription of a gene of interest or the activity of a specific transcriptional regulator. Unfortunately, the latter application has the shortcoming that native promoters did not evolve as optimal readouts for the activity of a particular regulator. We sought to synthesize an optimized transcriptional reporter for assessing PhoB activity, aiming for maximal "on" expression when PhoB is active, minimal background in the "off" state, and no control elements for other regulators. We designed specific sequences for promoter elements with appropriately spaced PhoB-binding sites, and at 19 additional intervening nucleotide positions for which we did not predict sequence-specific effects, the bases were randomized. Eighty-three such constructs were screened in Vibrio fischeri, enabling us to identify bases at particular randomized positions that significantly correlated with high-level "on" or low-level "off" expression. A second round of promoter design rationally constrained 13 additional positions, leading to a reporter with high-level PhoB-dependent expression, essentially no background, and no other known regulatory elements. As expressed reporters, we used both stable and destabilized variants of green fluorescent protein (GFP), the latter of which has a half-life of 81 min in V. fischeri In culture, PhoB induced the reporter when phosphate was depleted to a concentration below 10 μM. During symbiotic colonization of its host squid, Euprymna scolopes, the reporter indicated heterogeneous phosphate availability in different light-organ microenvironments. Finally, testing this construct in other members of the Proteobacteria demonstrated its broader utility. The results illustrate how a limited ability to predict synthetic promoter-reporter performance can be overcome through iterative screening and reengineering.IMPORTANCE Transcriptional reporters can be powerful tools for assessing when a particular regulator is active; however, native promoters may not be ideal for this purpose. Optimal reporters should be specific to the regulator being examined and should maximize the difference between the "on" and "off" states; however, these properties are distinct from the selective pressures driving the evolution of natural promoters. Synthetic promoters offer a promising alternative, but our understanding often does not enable fully predictive promoter design, and the large number of alternative sequence possibilities can be intractable. In a synthetic promoter region with over 34 billion sequence variants, we identified bases correlated with favorable performance by screening only 83 candidates, allowing us to rationally constrain our design. We thereby generated an optimized reporter that is induced by PhoB and used it to explore the low-phosphate response of V. fischeri This promoter design strategy will facilitate the engineering of other regulator-specific reporters.

RevDate: 2021-01-09
CmpDate: 2019-01-21

Peyer SM, Kremer N, MJ McFall-Ngai (2018)

Involvement of a host Cathepsin L in symbiont-induced cell death.

MicrobiologyOpen, 7(5):e00632.

The cathepsin L gene of the host squid, Euprymna scolopes, is upregulated during the first hours of colonization by the symbiont Vibrio fischeri. At this time, the symbiotic organ begins cell death-mediated morphogenesis in tissues functional only at the onset of symbiosis. The goal of this study was to determine whether Cathepsin L, a cysteine protease associated with apoptosis in other animals, plays a critical role in symbiont-induced cell death in the host squid. Sequence analysis and biochemical characterization demonstrated that the protein has key residues and domains essential for Cathepsin L function and that it is active within the pH range typical of these proteases. With in situ hybridization and immunocytochemistry, we localized the transcript and protein, respectively, to cells interacting with V. fischeri. Activity of the protein occurred along the path of symbiont colonization. A specific Cathepsin L, nonspecific cysteine protease, and caspase inhibitor each independently attenuated activity and cell death to varying degrees. In addition, a specific antibody decreased cell death by ~50%. Together these data provide evidence that Cathepsin L is a critical component in the symbiont-induced cell death that transforms the host tissues from a colonization morphology to one that promotes the mature association.

RevDate: 2020-09-29

Seehafer K, Brophy S, Tom SR, et al (2018)

Ontogenetic and Experience-Dependent Changes in Defensive Behavior in Captive-Bred Hawaiian Bobtail Squid, Euprymna scolopes.

Frontiers in physiology, 9:299.

Cephalopod molluscs are known for their extensive behavioral repertoire and their impressive learning abilities. Their primary defensive behaviors, such as camouflage, have received detailed study, but knowledge is limited to intensive study of relatively few species. A considerable challenge facing cephalopod research is the need to establish new models that can be captive bred, are tractable for range of different experimental procedures, and that will address broad questions in biological research. The Hawaiian Bobtail Squid (Euprymna scolopes) is a small, tropical cephalopod that has a long history of research in the field of microbial symbiosis, but offers great promise as a novel behavioral and neurobiological model. It can be bred in the laboratory through multiple generations, one of the few species of cephalopod that can meet this requirement (which is incorporated in regulations such as EU directive 2010/63/EU). Additionally, laboratory culture makes E. scolopes an ideal model for studying ontogeny- and experience-dependent behaviors. In this study, we show that captive bred juvenile and adult E. scolopes produce robust, repeatable defensive behaviors when placed in an exposed environment and presented with a visual threat. Further, adult and juvenile squid employ different innate defensive behaviors when presented with a size-matched model predator. When a 10-min training procedure was repeated over three consecutive days, defensive behaviors habituated in juvenile squid for at least 5 days after training, but memory did not appear to persist for 14 days. In contrast, adult squid did not show any evidence of long-term habituation memory. Thus we conclude that this species produces a range of quantifiable, modifiable behaviors even in a laboratory environment where ecologically-relevant, complex behavioral sequences may not reliably occur. We suggest that the lack of long-term memory in adult squid may be related to their less escalated initial response to the mimic, and thus indicates less motivation to retain memory and not necessary inability to form memory. This is the first demonstration of age-related differences in defensive behaviors in Euprymna, and the first record of habituation in this experimentally tractable genus of squid.

RevDate: 2019-03-12
CmpDate: 2019-03-12

Kerwin AH, SV Nyholm (2018)

Reproductive System Symbiotic Bacteria Are Conserved between Two Distinct Populations of Euprymna scolopes from Oahu, Hawaii.

mSphere, 3(2):.

Female Hawaiian bobtail squid, Euprymna scolopes, harbor a symbiotic bacterial community in a reproductive organ, the accessory nidamental gland (ANG). This community is known to be stable over several generations of wild-caught bobtail squid but has, to date, been examined for only one population in Maunalua Bay, Oahu, HI. This study assessed the ANG and corresponding egg jelly coat (JC) bacterial communities for another genetically isolated host population from Kaneohe Bay, Oahu, HI, using 16S amplicon sequencing. The bacterial communities from the ANGs and JCs of the two populations were found to be similar in richness, evenness, phylogenetic diversity, and overall community composition. However, the Kaneohe Bay samples formed their own subset within the Maunalua Bay ANG/JC community. An Alteromonadaceae genus, BD2-13, was significantly higher in relative abundance in the Kaneohe Bay population, and several Alphaproteobacteria taxa also shifted in relative abundance between the two groups. This variation could be due to local adaptation to differing environmental challenges, to localized variability, or to functional redundancy among the ANG taxa. The overall stability of the community between the populations further supports a crucial functional role that has been hypothesized for this symbiosis. IMPORTANCE In this study, we examined the reproductive ANG symbiosis found in two genetically isolated populations of the Hawaiian bobtail squid, Euprymna scolopes. The stability of the community reported here provides support for the hypothesis that this symbiosis is under strong selective pressure, while the observed differences suggest that some level of local adaptation may have occurred. These two host populations are frequently used interchangeably as source populations for research. Euprymna scolopes is an important model organism and offers the opportunity to examine the interplay between a binary and a consortial symbiosis in a single model host. Understanding the inherent natural variability of this association will aid in our understanding of the conservation, function, transmission, and development of the ANG symbiosis.

RevDate: 2019-08-29
CmpDate: 2019-08-29

Tischler AH, Lie L, Thompson CM, et al (2018)

Discovery of Calcium as a Biofilm-Promoting Signal for Vibrio fischeri Reveals New Phenotypes and Underlying Regulatory Complexity.

Journal of bacteriology, 200(15):.

Vibrio fischeri uses biofilm formation to promote symbiotic colonization of its squid host, Euprymna scolopes Control over biofilm formation is exerted at the level of transcription of the symbiosis polysaccharide (syp) locus by a complex set of two-component regulators. Biofilm formation can be induced by overproduction of the sensor kinase RscS, which requires the activities of the hybrid sensor kinase SypF and the response regulator SypG and is negatively regulated by the sensor kinase BinK. Here, we identify calcium as a signal that promotes biofilm formation by biofilm-competent strains under conditions in which biofilms are not typically observed (growth with shaking). This was true for RscS-overproducing cells as well as for strains in which only the negative regulator binK was deleted. The latter results provided, for the first time, an opportunity to induce and evaluate biofilm formation without regulator overexpression. Using these conditions, we determined that calcium induces both syp-dependent and bacterial cellulose synthesis (bcs)-dependent biofilms at the level of transcription of these loci. The calcium-induced biofilms were dependent on SypF, but SypF's Hpt domain was sufficient for biofilm formation. These data suggested the involvement of another sensor kinase(s) and led to the discovery that both RscS and a previously uncharacterized sensor kinase, HahK, functioned in this pathway. Together, the data presented here reveal both a new signal and biofilm phenotype produced by V. fischeri cells, the coordinate production of two polysaccharides involved in distinct biofilm behaviors, and a new regulator that contributes to control over these processes.IMPORTANCE Biofilms, or communities of surface-attached microorganisms adherent via a matrix that typically includes polysaccharides, are highly resistant to environmental stresses and are thus problematic in the clinic and important to study. Vibrio fischeri forms biofilms to colonize its symbiotic host, making this organism useful for studying biofilms. Biofilm formation depends on the syp polysaccharide locus and its regulators. Here, we identify a signal, calcium, that induces both SYP-PS and cellulose-dependent biofilms. We also identify a new syp regulator, the sensor kinase HahK, and discover a mutant phenotype for the sensor kinase RscS. This work thus reveals a specific biofilm-inducing signal that coordinately controls two polysaccharides, identifies a new regulator, and clarifies the regulatory control over biofilm formation by V. fischeri.

RevDate: 2020-09-30

Sepehri S, Eriksson E, Kalaboukhov A, et al (2018)

Volume-amplified magnetic bioassay integrated with microfluidic sample handling and high-Tc SQUID magnetic readout.

APL bioengineering, 2(1):016102.

A bioassay based on a high-Tc superconducting quantum interference device (SQUID) reading out functionalized magnetic nanoparticles (fMNPs) in a prototype microfluidic platform is presented. The target molecule recognition is based on volume amplification using padlock-probe-ligation followed by rolling circle amplification (RCA). The MNPs are functionalized with single-stranded oligonucleotides, which give a specific binding of the MNPs to the large RCA coil product, resulting in a large change in the amplitude of the imaginary part of the ac magnetic susceptibility. The RCA products from amplification of synthetic Vibrio cholera target DNA were investigated using our SQUID ac susceptibility system in microfluidic channel with an equivalent sample volume of 3 μl. From extrapolation of the linear dependence of the SQUID signal versus concentration of the RCA coils, it is found that the projected limit of detection for our system is about 1.0 × 105 RCA coils (0.2 × 10-18 mol), which is equivalent to 66 fM in the 3 μl sample volume. This ultra-high magnetic sensitivity and integration with microfluidic sample handling are critical steps towards magnetic bioassays for rapid detection of DNA and RNA targets at the point of care.

RevDate: 2019-03-01
CmpDate: 2018-12-19

Kinosita Y, Kikuchi Y, Mikami N, et al (2018)

Unforeseen swimming and gliding mode of an insect gut symbiont, Burkholderia sp. RPE64, with wrapping of the flagella around its cell body.

The ISME journal, 12(3):838-848.

A bean bug symbiont, Burkholderia sp. RPE64, selectively colonizes the gut crypts by flagella-mediated motility: however, the mechanism for this colonization remains unclear. Here, to obtain clues to this mechanism, we characterized the swimming motility of the Burkholderia symbiont under an advanced optical microscope. High-speed imaging of cells enabled the detection of turn events with up to 5-ms temporal resolution, indicating that cells showed reversal motions (θ ~ 180°) with rapid changes in speed by a factor of 3.6. Remarkably, staining of the flagellar filaments with a fluorescent dye Cy3 revealed that the flagellar filaments wrap around the cell body with a motion like that of a ribbon streamer in rhythmic gymnastics. A motility assay with total internal reflection fluorescence microscopy revealed that the left-handed flagellum wound around the cell body and propelled it forward by its clockwise rotation. We also detected periodic-fluorescent signals of flagella on the glass surface, suggesting that flagella possibly contacted the solid surface directly and produced a gliding-like motion driven by flagellar rotation. Finally, the wrapping motion was also observed in a symbiotic bacterium of the bobtail squid, Aliivibrio fischeri, suggesting that this motility mode may contribute to migration on the mucus-filled narrow passage connecting to the symbiotic organ.

RevDate: 2019-02-15
CmpDate: 2019-02-07

Schwartz WJ (2017)

Embodied Clocks.

Journal of biological rhythms, 32(6):503-504.

RevDate: 2018-12-02
CmpDate: 2017-10-27

Zepeda EA, Veline RJ, RJ Crook (2017)

Rapid Associative Learning and Stable Long-Term Memory in the Squid Euprymna scolopes.

The Biological bulletin, 232(3):212-218.

Learning and memory in cephalopod molluscs have received intensive study because of cephalopods' complex behavioral repertoire and relatively accessible nervous systems. While most of this research has been conducted using octopus and cuttlefish species, there has been relatively little work on squid. Euprymna scolopes Berry, 1913, a sepiolid squid, is a promising model for further exploration of cephalopod cognition. These small squid have been studied in detail for their symbiotic relationship with bioluminescent bacteria, and their short generation time and successful captive breeding through multiple generations make them appealing models for neurobiological research. However, little is known about their behavior or cognitive ability. Using the well-established "prawn-in-the-tube" assay of learning and memory, we show that within a single 10-min trial E. scolopes learns to inhibit its predatory behavior, and after three trials it can retain this memory for at least 12 d. Rapid learning and very long-term retention were apparent under two different training schedules. To our knowledge, this study is the first demonstration of learning and memory in this species as well as the first demonstration of associative learning in any squid.

RevDate: 2018-07-24
CmpDate: 2018-07-24

Guan Z, Cai T, Liu Z, et al (2017)

Origin of the Reflectin Gene and Hierarchical Assembly of Its Protein.

Current biology : CB, 27(18):2833-2842.e6.

Cephalopods, the group of animals including octopus, squid, and cuttlefish, have remarkable ability to instantly modulate body coloration and patterns so as to blend into surrounding environments [1, 2] or send warning signals to other animals [3]. Reflectin is expressed exclusively in cephalopods, filling the lamellae of intracellular Bragg reflectors that exhibit dynamic iridescence and structural color change [4]. Here, we trace the possible origin of the reflectin gene back to a transposon from the symbiotic bioluminescent bacterium Vibrio fischeri and report the hierarchical structural architecture of reflectin protein. Intrinsic self-assembly, and higher-order assembly tightly modulated by aromatic compounds, provide insights into the formation of multilayer reflectors in iridophores and spherical microparticles in leucophores and may form the basis of structural color change in cephalopods. Self-assembly and higher-order assembly in reflectin originated from a core repeating octapeptide (here named protopeptide), which may be from the same symbiotic bacteria. The origin of the reflectin gene and assembly features of reflectin protein are of considerable biological interest. The hierarchical structural architecture of reflectin and its domain and protopeptide not only provide insights for bioinspired photonic materials but also serve as unique "assembly tags" and feasible molecular platforms in biotechnology.

RevDate: 2019-01-18
CmpDate: 2018-05-29

Nawroth JC, Guo H, Koch E, et al (2017)

Motile cilia create fluid-mechanical microhabitats for the active recruitment of the host microbiome.

Proceedings of the National Academy of Sciences of the United States of America, 114(36):9510-9516.

We show that mucociliary membranes of animal epithelia can create fluid-mechanical microenvironments for the active recruitment of the specific microbiome of the host. In terrestrial vertebrates, these tissues are typically colonized by complex consortia and are inaccessible to observation. Such tissues can be directly examined in aquatic animals, providing valuable opportunities for the analysis of mucociliary activity in relation to bacteria recruitment. Using the squid-vibrio model system, we provide a characterization of the initial engagement of microbial symbionts along ciliated tissues. Specifically, we developed an empirical and theoretical framework to conduct a census of ciliated cell types, create structural maps, and resolve the spatiotemporal flow dynamics. Our multiscale analyses revealed two distinct, highly organized populations of cilia on the host tissues. An array of long cilia ([Formula: see text]25 [Formula: see text]m) with metachronal beat creates a flow that focuses bacteria-sized particles, at the exclusion of larger particles, into sheltered zones; there, a field of randomly beating short cilia ([Formula: see text]10 [Formula: see text]m) mixes the local fluid environment, which contains host biochemical signals known to prime symbionts for colonization. This cilia-mediated process represents a previously unrecognized mechanism for symbiont recruitment. Each mucociliary surface that recruits a microbiome such as the case described here is likely to have system-specific features. However, all mucociliary surfaces are subject to the same physical and biological constraints that are imposed by the fluid environment and the evolutionary conserved structure of cilia. As such, our study promises to provide insight into universal mechanisms that drive the recruitment of symbiotic partners.

RevDate: 2018-12-02
CmpDate: 2018-05-29

Viegas J (2017)

Profile of Margaret J. McFall-Ngai.

Proceedings of the National Academy of Sciences of the United States of America, 114(36):9494-9496.

RevDate: 2018-01-17
CmpDate: 2018-01-17

Song W, Mu C, Li R, et al (2017)

Peroxiredoxin 1 from cuttlefish (Sepiella maindroni): Molecular characterization of development and its immune response against Vibrio alginolyticus.

Fish & shellfish immunology, 67:596-603.

The aim of this work was constructive to understand the function of peroxiredoxin (PRDX) family member Peroxiredoxin 1 in Sepiella maindroni (SmPrx1) through molecular mechanisms of reproduction, embryonic development and immune responses to Vibrio alginolyticus. The full-length cDNA of SmPrx1 was of 1062 bp, contains a 5' untranslated region (UTR) of 79bp, a 3' UTR of 359 bp, an open reading frame of 624 bp encoding 207 amino acids. The conserved peroxidase catalytic center "FYPLDFTFVCPTEI" and "GEVCPA" were observed in the sequence of SmPrx1; this indicated that it was a member of 2-Cys Prx. Quantitative real-time (qRT)-PCR assays revealed that SmPrx1 was ubiquitously expressed in all examined tissues, muscle, ink sac, liver, ovary, testis, intestine, gill and totally blood cells, and showed high levels in testis. SmPrx1 mRNA was ubiquitously detected in all tested tissues, and the expression was comparatively high in testis, hemocyte, liver and ovary. Moreover, the SmPrx1 gene transcript was detected at all five stages of embryonic development phases that were respectively the zygote stage, the pre-embryonic stage, the organogenesis stage, the morphological integrity stage, the pre-hatching stage. The general tendency of expression was gradually increased and rapidly decreased. High expressed in progenitive tissues and embryonic development exhibit the proliferation-associated protein characterization like in mammal. The expression levels of SmPrx1 in liver and hemocytes grew swiftly and quickly reached peak value after Vibrio alginolyticus challenge. As hours passed by, the expression level began to reduce and resumed to normal levels after 48 h. The antioxidant activity and peroxidase activity of SmPrx1 were 6.17 U/mg. The results showed that the recombined protein of SmPrx1 had antioxidant activity and was the importance part of the antioxidant system in Sepiella maindroni. This study provides useful information to help further understand the functional mechanism of Prx 1 in marine cephalopod immunity.

RevDate: 2017-12-18
CmpDate: 2017-12-18

Li R, Xu Z, Mu C, et al (2017)

Molecular cloning and characterization of a hemocyanin from Sepiella maindroni.

Fish & shellfish immunology, 67:228-243.

Hemocyanins are respiratory proteins occurring freely dissolved in the hemolymph of many arthropods and molluscs. Hemocyanin and hemocyanin-derived peptides have been linked to key aspects of innate immunity. In the present study, the full-length cDNA encoding hemocyanin in Sepiella maindroni (SmHc) was cloned and characterized. Bioinformatic analysis predicted that SmHc contains one open reading frame of 10,032 bp and encodes a polypeptide of 3343 amino acids. Sequence analysis showed that the predicted protein sequence of SmHc contained eight functional units (FUs). Phylogenic analysis revealed that SmHc clustered with the mollusc Hcs. Quantitative real-time PCR assay detected SmHc transcripts were in a wide range of tissues, but mainly distributed in gills. After hypoxia or bacterial challenge, the expression level of SmHc in the gills was significantly higher than that of the control group. These results suggested that SmHc might play important roles in oxygen transport and the modulation of immune response in S. maindroni.

RevDate: 2020-12-09
CmpDate: 2018-02-19

Sabrina Pankey M, Foxall RL, Ster IM, et al (2017)

Host-selected mutations converging on a global regulator drive an adaptive leap towards symbiosis in bacteria.

eLife, 6:.

Host immune and physical barriers protect against pathogens but also impede the establishment of essential symbiotic partnerships. To reveal mechanisms by which beneficial organisms adapt to circumvent host defenses, we experimentally evolved ecologically distinct bioluminescent Vibrio fischeri by colonization and growth within the light organs of the squid Euprymna scolopes. Serial squid passaging of bacteria produced eight distinct mutations in the binK sensor kinase gene, which conferred an exceptional selective advantage that could be demonstrated through both empirical and theoretical analysis. Squid-adaptive binK alleles promoted colonization and immune evasion that were mediated by cell-associated matrices including symbiotic polysaccharide (Syp) and cellulose. binK variation also altered quorum sensing, raising the threshold for luminescence induction. Preexisting coordinated regulation of symbiosis traits by BinK presented an efficient solution where altered BinK function was the key to unlock multiple colonization barriers. These results identify a genetic basis for microbial adaptability and underscore the importance of hosts as selective agents that shape emergent symbiont populations.

RevDate: 2021-02-05
CmpDate: 2017-07-06

Pan S, Nikolakakis K, Adamczyk PA, et al (2017)

Model-enabled gene search (MEGS) allows fast and direct discovery of enzymatic and transport gene functions in the marine bacterium Vibrio fischeri.

The Journal of biological chemistry, 292(24):10250-10261.

Whereas genomes can be rapidly sequenced, the functions of many genes are incompletely or erroneously annotated because of a lack of experimental evidence or prior functional knowledge in sequence databases. To address this weakness, we describe here a model-enabled gene search (MEGS) approach that (i) identifies metabolic functions either missing from an organism's genome annotation or incorrectly assigned to an ORF by using discrepancies between metabolic model predictions and experimental culturing data; (ii) designs functional selection experiments for these specific metabolic functions; and (iii) selects a candidate gene(s) responsible for these functions from a genomic library and directly interrogates this gene's function experimentally. To discover gene functions, MEGS uses genomic functional selections instead of relying on correlations across large experimental datasets or sequence similarity as do other approaches. When applied to the bioluminescent marine bacterium Vibrio fischeri, MEGS successfully identified five genes that are responsible for four metabolic and transport reactions whose absence from a draft metabolic model of V. fischeri caused inaccurate modeling of high-throughput experimental data. This work demonstrates that MEGS provides a rapid and efficient integrated computational and experimental approach for annotating metabolic genes, including those that have previously been uncharacterized or misannotated.

RevDate: 2018-12-02
CmpDate: 2018-08-01

Davidson SK (2017)

The squid insurance plan: female Euprymna scolopes add potentially protective bacteria to the egg coats of their clutches.

Environmental microbiology, 19(6):2112-2114.

RevDate: 2019-01-11
CmpDate: 2018-11-27

Casaburi G, Goncharenko-Foster I, Duscher AA, et al (2017)

Transcriptomic changes in an animal-bacterial symbiosis under modeled microgravity conditions.

Scientific reports, 7:46318.

Spaceflight imposes numerous adaptive challenges for terrestrial life. The reduction in gravity, or microgravity, represents a novel environment that can disrupt homeostasis of many physiological processes. Additionally, it is becoming increasingly clear that an organism's microbiome is critical for host health and examining its resiliency in microgravity represents a new frontier for space biology research. In this study, we examine the impact of microgravity on the interactions between the squid Euprymna scolopes and its beneficial symbiont Vibrio fischeri, which form a highly specific binary mutualism. First, animals inoculated with V. fischeri aboard the space shuttle showed effective colonization of the host light organ, the site of the symbiosis, during space flight. Second, RNA-Seq analysis of squid exposed to modeled microgravity conditions exhibited extensive differential gene expression in the presence and absence of the symbiotic partner. Transcriptomic analyses revealed in the absence of the symbiont during modeled microgravity there was an enrichment of genes and pathways associated with the innate immune and oxidative stress response. The results suggest that V. fischeri may help modulate the host stress responses under modeled microgravity. This study provides a window into the adaptive responses that the host animal and its symbiont use during modeled microgravity.

RevDate: 2020-12-09
CmpDate: 2018-01-08

Chen F, Krasity BC, Peyer SM, et al (2017)

Bactericidal Permeability-Increasing Proteins Shape Host-Microbe Interactions.

mBio, 8(2):.

We characterized bactericidal permeability-increasing proteins (BPIs) of the squid Euprymna scolopes, EsBPI2 and EsBPI4. They have molecular characteristics typical of other animal BPIs, are closely related to one another, and nest phylogenetically among invertebrate BPIs. Purified EsBPIs had antimicrobial activity against the squid's symbiont, Vibrio fischeri, which colonizes light organ crypt epithelia. Activity of both proteins was abrogated by heat treatment and coincubation with specific antibodies. Pretreatment under acidic conditions similar to those during symbiosis initiation rendered V. fischeri more resistant to the antimicrobial activity of the proteins. Immunocytochemistry localized EsBPIs to the symbiotic organ and other epithelial surfaces interacting with ambient seawater. The proteins differed in intracellular distribution. Further, whereas EsBPI4 was restricted to epithelia, EsBPI2 also occurred in blood and in a transient juvenile organ that mediates hatching. The data provide evidence that these BPIs play different defensive roles early in the life of E. scolopes, modulating interactions with the symbiont.IMPORTANCE This study describes new functions for bactericidal permeability-increasing proteins (BPIs), members of the lipopolysaccharide-binding protein (LBP)/BPI protein family. The data provide evidence that these proteins play a dual role in the modulation of symbiotic bacteria. In the squid-vibrio model, these proteins both control the symbiont populations in the light organ tissues where symbiont cells occur in dense monoculture and, concomitantly, inhibit the symbiont from colonizing other epithelial surfaces of the animal.

RevDate: 2019-01-12
CmpDate: 2017-09-22

Thompson LR, Nikolakakis K, Pan S, et al (2017)

Transcriptional characterization of Vibrio fischeri during colonization of juvenile Euprymna scolopes.

Environmental microbiology, 19(5):1845-1856.

The marine bacterium Vibrio fischeri is the monospecific symbiont of the Hawaiian bobtail squid, Euprymna scolopes, and the establishment of this association involves a number of signaling pathways and transcriptional responses between both partners. We report here the first full RNA-Seq dataset representing host-associated V. fischeri cells from colonized juvenile E. scolopes, as well as comparative transcriptomes under both laboratory and simulated marine planktonic conditions. These data elucidate the broad transcriptional changes that these bacteria undergo during the early stages of symbiotic colonization. We report several previously undescribed and unexpected transcriptional responses within the early stages of this symbiosis, including gene expression patterns consistent with biochemical stresses inside the host, and metabolic patterns distinct from those reported in associations with adult animals. Integration of these transcriptional data with a recently developed metabolic model of V. fischeri provides us with a clearer picture of the metabolic state of symbionts within the juvenile host, including their possible carbon sources. Taken together, these results expand our understanding of the early stages of the squid-vibrio symbiosis, and more generally inform the transcriptional responses underlying the activities of marine microbes during host colonization.

RevDate: 2018-11-13
CmpDate: 2017-08-10

Marsden AE, Grudzinski K, Ondrey JM, et al (2017)

Impact of Salt and Nutrient Content on Biofilm Formation by Vibrio fischeri.

PloS one, 12(1):e0169521.

Vibrio fischeri, a marine bacterium and symbiont of the Hawaiian bobtail squid Euprymna scolopes, depends on biofilm formation for successful colonization of the squid's symbiotic light organ. Here, we investigated if culture conditions, such as nutrient and salt availability, affect biofilm formation by V. fischeri by testing the formation of wrinkled colonies on solid media. We found that V. fischeri forms colonies with more substantial wrinkling when grown on the nutrient-dense LBS medium containing NaCl relative to those formed on the more nutrient-poor, seawater-salt containing SWT medium. The presence of both tryptone and yeast extract was necessary for the production of "normal" wrinkled colonies; when grown on tryptone alone, the colonies displayed a divoting phenotype and were attached to the agar surface. We also found that the type and concentration of specific seawater salts influenced the timing of biofilm formation. Of the conditions assayed, wrinkled colony formation occurred earliest in LBS(-Tris) media containing 425 mM NaCl, 35 mM MgSO4, and 5 mM CaCl2. Pellicle formation, another measure of biofilm development, was also enhanced in these growth conditions. Therefore, both nutrient and salt availability contribute to V. fischeri biofilm formation. While growth was unaffected, these optimized conditions resulted in increased syp locus expression as measured by a PsypA-lacZ transcriptional reporter. We anticipate these studies will help us understand how the natural environment of V. fischeri affects its ability to form biofilms and, ultimately, colonize E. scolopes.

RevDate: 2020-10-01

McAnulty SJ, SV Nyholm (2016)

The Role of Hemocytes in the Hawaiian Bobtail Squid, Euprymna scolopes: A Model Organism for Studying Beneficial Host-Microbe Interactions.

Frontiers in microbiology, 7:2013.

Most, if not all, animals engage in associations with bacterial symbionts. Understanding the mechanisms by which host immune systems and beneficial bacteria communicate is a fundamental question in the fields of immunology and symbiosis. The Hawaiian bobtail squid (Euprymna scolopes) engages in two known symbioses; a binary relationship with the light organ symbiont Vibrio fischeri, and a bacterial consortium within a specialized organ of the female reproductive system, the accessory nidamental gland (ANG). E. scolopes has a well-developed circulatory system that allows immune cells (hemocytes) to migrate into tissues, including the light organ and ANG. In the association with V. fischeri, hemocytes are thought to have a number of roles in the management of symbiosis, including the recognition of non-symbiotic bacteria and the contribution of chitin as a nutrient source for V. fischeri. Hemocytes are hypothesized to recognize bacteria through interactions between pattern recognition receptors and microbe-associated molecular patterns. Colonization by V. fischeri has been shown to affect the bacteria-binding behavior, gene expression, and proteome of hemocytes, indicating that the symbiont can modulate host immune function. In the ANG, hemocytes have also been observed interacting with the residing bacterial community. As a model host, E. scolopes offers a unique opportunity to study how the innate immune system interacts with both a binary and consortial symbiosis. This mini review will recapitulate what is known about the role of hemocytes in the light organ association and offer future directions for understanding how these immune cells interact with multiple types of symbioses.

RevDate: 2020-12-09
CmpDate: 2018-01-02

Peyer SM, Heath-Heckman EAC, MJ McFall-Ngai (2017)

Characterization of the cell polarity gene crumbs during the early development and maintenance of the squid-vibrio light organ symbiosis.

Development genes and evolution, 227(6):375-387.

The protein Crumbs is a determinant of apical-basal cell polarity and plays a role in apoptosis of epithelial cells and their protection against photodamage. Using the squid-vibrio system, a model for development of symbiotic partnerships, we examined the modulation of the crumbs gene in host epithelial tissues during initiation and maintenance of the association. The extracellular luminous symbiont Vibrio fischeri colonizes the apical surfaces of polarized epithelia in deep crypts of the Euprymna scolopes light organ. During initial colonization each generation, symbiont harvesting is potentiated by the biochemical and biophysical activity of superficial ciliated epithelia, which are several cell layers from the crypt epithelia where the symbionts reside. Within hours of crypt colonization, the symbionts induce the cell death mediated regression of the remote superficial ciliated fields. However, the crypt cells directly interacting with the symbiont are protected from death. In the squid host, we characterized the gene and encoded protein during light organ morphogenesis and in response to symbiosis. Features of the protein sequence and structure, phylogenetic relationships, and localization patterns in the eye supported assignment of the squid protein to the Crumbs family. In situ hybridization revealed that the crumbs transcript shows opposite expression at the onset of symbiosis in the two different regions of the light organ: elevated levels in the superficial epithelia were attenuated whereas low levels in the crypt epithelia were turned up. Although a rhythmic association in which the host controls the symbiont population over the day-night cycle begins in the juvenile upon colonization, cycling of crumbs was evident only in the adult organ with peak expression coincident with maximum symbiont population and luminescence. Our results provide evidence that crumbs responds to symbiont cues that induce developmental apoptosis and to symbiont population dynamics correlating with luminescence-based stress throughout the duration of the host-microbe association.

RevDate: 2018-12-02
CmpDate: 2017-08-22

Kerwin AH, SV Nyholm (2017)

Symbiotic bacteria associated with a bobtail squid reproductive system are detectable in the environment, and stable in the host and developing eggs.

Environmental microbiology, 19(4):1463-1475.

Female Hawaiian bobtail squid, Euprymna scolopes, have an accessory nidamental gland (ANG) housing a bacterial consortium that is hypothesized to be environmentally transmitted and to function in the protection of eggs from fouling and infection. The composition, stability, and variability of the ANG and egg jelly coat (JC) communities were characterized and compared to the bacterial community composition of the surrounding environment using Illumina sequencing and transmission electron microscopy. The ANG bacterial community was conserved throughout hosts collected from the wild and was not affected by maintaining animals in the laboratory. The core symbiotic community was composed of Alphaproteobacteria and Opitutae (a class of Verrucomicrobia). Operational taxonomic units representing 94.5% of the average ANG abundance were found in either the seawater or sediment, which is consistent with the hypothesis of environmental transmission between generations. The bacterial composition of the JC was stable during development and mirrored that of the ANG. Bacterial communities from individual egg clutches also grouped with the ANG of the female that produced them. Collectively, these data suggest a conserved role of the ANG/JC community in host reproduction. Future directions will focus on determining the function of this symbiotic community, and how it may change during ANG development.

RevDate: 2020-10-01

Mandel MJ, AK Dunn (2016)

Impact and Influence of the Natural Vibrio-Squid Symbiosis in Understanding Bacterial-Animal Interactions.

Frontiers in microbiology, 7:1982.

Animals are colonized by bacteria, and in many cases partners have co-evolved to perform mutually beneficial functions. An exciting and ongoing legacy of the past decade has been an expansion of technology to enable study of natural associations in situ/in vivo. As a result, more symbioses are being examined, and additional details are being revealed for well-studied systems with a focus on the interactions between partners in the native context. With this framing, we review recent literature from the Vibrio fischeri-Euprymna scolopes symbiosis and focus on key studies that have had an impact on understanding bacteria-animal interactions broadly. This is not intended to be a comprehensive review of the system, but rather to focus on particular studies that have excelled at moving from pattern to process in facilitating an understanding of the molecular basis to intriguing observations in the field of host-microbe interactions. In this review we discuss the following topics: processes regulating strain and species specificity; bacterial signaling to host morphogenesis; multiple roles for nitric oxide; flagellar motility and chemotaxis; and efforts to understand unannotated and poorly annotated genes. Overall these studies demonstrate how functional approaches in vivo in a tractable system have provided valuable insight into general principles of microbe-host interactions.

RevDate: 2020-12-09
CmpDate: 2017-11-27

Lyell NL, Septer AN, Dunn AK, et al (2017)

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

Applied and environmental microbiology, 83(5):.

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.

RevDate: 2019-05-08
CmpDate: 2017-06-05

Heath-Heckman EA, Foster J, Apicella MA, et al (2016)

Environmental cues and symbiont microbe-associated molecular patterns function in concert to drive the daily remodelling of the crypt-cell brush border of the Euprymna scolopes light organ.

Cellular microbiology, 18(11):1642-1652.

Recent research has shown that the microbiota affects the biology of associated host epithelial tissues, including their circadian rhythms, although few data are available on how such influences shape the microarchitecture of the brush border. The squid-vibrio system exhibits two modifications of the brush border that supports the symbionts: effacement and repolarization. Together these occur on a daily rhythm in adult animals, at the dawn expulsion of symbionts into the environment, and symbiont colonization of the juvenile host induces an increase in microvillar density. Here we sought to define how these processes are related and the roles of both symbiont colonization and environmental cues. Ultrastructural analyses showed that the juvenile-organ brush borders also efface concomitantly with daily dawn-cued expulsion of symbionts. Manipulation of the environmental light cue and juvenile symbiotic state demonstrated that this behaviour requires the light cue, but not colonization. In contrast, symbionts were required for the observed increase in microvillar density that accompanies post dawn brush-border repolarization; this increase was induced solely by host exposure to phosphorylated lipid A of symbiont cells. These data demonstrate that a partnering of environmental and symbiont cues shapes the brush border and that microbe-associated molecular patterns play a role in the regulation of brush-border microarchitecture.

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In the early 1990's, Robert Robbins was a faculty member at Johns Hopkins, where he directed the informatics core of GDB — the human gene-mapping database of the international human genome project. To share papers with colleagues around the world, he set up a small paper-sharing section on his personal web page. This small project evolved into The Electronic Scholarly Publishing Project.

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In 1995, Robbins became the VP/IT of the Fred Hutchinson Cancer Research Center in Seattle, WA. Soon after arriving in Seattle, Robbins secured funding, through the ELSI component of the US Human Genome Project, to create the original ESP.ORG web site, with the formal goal of providing free, world-wide access to the literature of classical genetics.

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Although the methods of molecular biology can seem almost magical to the uninitiated, the original techniques of classical genetics are readily appreciated by one and all: cross individuals that differ in some inherited trait, collect all of the progeny, score their attributes, and propose mechanisms to explain the patterns of inheritance observed.

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In reading the early works of classical genetics, one is drawn, almost inexorably, into ever more complex models, until molecular explanations begin to seem both necessary and natural. At that point, the tools for understanding genome research are at hand. Assisting readers reach this point was the original goal of The Electronic Scholarly Publishing Project.

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Usage of the site grew rapidly and has remained high. Faculty began to use the site for their assigned readings. Other on-line publishers, ranging from The New York Times to Nature referenced ESP materials in their own publications. Nobel laureates (e.g., Joshua Lederberg) regularly used the site and even wrote to suggest changes and improvements.

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When the site began, no journals were making their early content available in digital format. As a result, ESP was obliged to digitize classic literature before it could be made available. For many important papers — such as Mendel's original paper or the first genetic map — ESP had to produce entirely new typeset versions of the works, if they were to be available in a high-quality format.

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Early support from the DOE component of the Human Genome Project was critically important for getting the ESP project on a firm foundation. Since that funding ended (nearly 20 years ago), the project has been operated as a purely volunteer effort. Anyone wishing to assist in these efforts should send an email to Robbins.

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With the development of methods for adding typeset side notes to PDF files, the ESP project now plans to add annotated versions of some classical papers to its holdings. We also plan to add new reference and pedagogical material. We have already started providing regularly updated, comprehensive bibliographies to the ESP.ORG site.

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Papers in Classical Genetics

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Selected Bibliographies

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