Viewport Size Code:
Login | Create New Account


About | Classical Genetics | Timelines | What's New | What's Hot

About | Classical Genetics | Timelines | What's New | What's Hot


Bibliography Options Menu

Hide Abstracts   |   Hide Additional Links
Long bibliographies are displayed in blocks of 100 citations at a time. At the end of each block there is an option to load the next block.

Bibliography on: Squid-Vibrio Symbiosis

The Electronic Scholarly Publishing Project: Providing world-wide, free access to classic scientific papers and other scholarly materials, since 1993.


ESP: PubMed Auto Bibliography 20 Nov 2018 at 01:42 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®)

RevDate: 2018-11-14

Stabb EV (2018)

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

Molecular microbiology [Epub ahead of print].

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. This article is protected by copyright. All rights reserved.

RevDate: 2018-10-24

Bongrand C, EG Ruby (2018)

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

The ISME journal pii:10.1038/s41396-018-0305-8 [Epub ahead of print].

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: 2018-10-15

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): pii:mSystems00165-18.

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: 2018-10-09

Thompson CM, Tischler AH, Tarnowski DA, et al (2018)

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

Molecular microbiology [Epub ahead of print].

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, 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 biofilms and a mechanism for 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. This article is protected by copyright. All rights reserved.

RevDate: 2018-08-28

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 pii:ECE34266.

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: 2018-08-23

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 ~4Xs 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. This article is protected by copyright. All rights reserved.

RevDate: 2018-08-21

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 pii:1808302115 [Epub ahead of print].

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: 2018-08-01
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: 2018-07-26

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 pii:PONE-D-18-05396.

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: 2018-07-21

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

A genetic analysis reveals that a trio of regulators prevents biofilm formation by Vibrio fischeri.

Applied and environmental microbiology pii:AEM.01257-18 [Epub ahead of print].

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 only observed in vitro 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 syp, the genes 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: 2018-07-01

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): pii:mBio.01033-18.

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: 2018-07-08

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

An iterative synthetic approach to engineer a high-performing PhoB-specific reporter.

Applied and environmental microbiology pii:AEM.00603-18 [Epub ahead of print].

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 nineteen 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 "on" or low "off" expression. A second round of promoter design rationally constrained thirteen additional positions, leading to a reporter with high PhoB-dependent expression, essentially no background, and no other known regulatory elements. As expressed reporters, we used both stable and destabilized GFP, the latter with a half-life of eighty-one minutes in V. fischeri In culture, PhoB induced the reporter when phosphate was depleted 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 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 re-engineering.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 "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 thirty-four billion sequence variants, we identified bases correlated with favorable performance by screening only eighty-three 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: 2018-04-25

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

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

MicrobiologyOpen [Epub ahead of print].

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: 2018-04-15

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: 2018-06-28

Kerwin AH, SV Nyholm (2018)

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

mSphere, 3(2): pii:mSphere00531-17.

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: 2018-07-11

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): pii:JB.00016-18.

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: 2018-03-25

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: 2017-10-27
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: 2017-09-26

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: 2018-05-29
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: 2018-05-29
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: 2018-03-05
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: 2018-06-16
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: 2017-04-13

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 pii:srep46318.

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: 2018-02-22
CmpDate: 2018-01-08

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

Bactericidal Permeability-Increasing Proteins Shape Host-Microbe Interactions.

mBio, 8(2): pii:mBio.00040-17.

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: 2018-07-20
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: 2017-08-10
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 pii:PONE-D-16-34131.

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: 2017-08-16

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: 2018-02-08
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: 2018-07-11
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: 2017-02-20

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: 2017-11-28
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): pii:AEM.02470-16.

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: 2018-04-29
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.

RevDate: 2017-08-17
CmpDate: 2017-04-21

Kim HW, Hong YJ, Jo JI, et al (2017)

Raw ready-to-eat seafood safety: microbiological quality of the various seafood species available in fishery, hyper and online markets.

Letters in applied microbiology, 64(1):27-34.

Microbiological quality of 206 raw ready-to-eat seafood samples was investigated according to species (gizzard shad, halibut, rockfish, tuna, oyster and squid) and distribution channels (fishery, hyper and online market). Enumeration of aerobic plate count and total coliforms (TC) and pathogenic bacteria (Bacillus cereus, Staphylococcus aureus and Vibrio parahaemolyticus) was performed, and level of microbiological quality was classified into four groups: satisfactory, acceptable, unsatisfactory and unacceptable. Qualitative analysis was also performed for Escherichia coli and eight foodborne pathogens (B. cereus, E. coli O157:H7, Listeria monocytogenes, Salmonella spp., S. aureus, Vibrio cholerae, V. parahaemolyticus, and Vibrio vulnificus). Raw ready-to-eat seafood products revealed 0·5% at an unsatisfactory level and 4·9% at an unacceptable level due to ≥4 log CFU g-1 of TC in squid and ≥3 log CFU g-1 of V. parahaemolyticus in gizzard shad respectively. Gizzard shad was shown to be potentially hazardous, as its sashimi is eaten with its skin attached. Bacillus cereus, E. coli, S. aureus, V. parahaemolyticus and V. vulnificus were qualitatively detected. Samples from the fishery market showed higher detection rate especially in V. parahaemolyticus (21·6%) and V. vulnificus (1·7%) which indicates the need to improve microbiological safety of raw ready-to-eat seafood products in fishery market.

Raw ready-to-eat seafood products like sashimi can be easily contaminated with various bacteria from aquatic environments and human reservoirs, which subsequently bring about a risk in food poisoning due to no heating process before consumption. The results of this study provide comprehensive microbiological data on various species of raw ready-to-eat seafood from various distribution channels. It may contribute to establish reasonable standard and effective strategies to ensure a good microbiological quality of raw ready-to-eat seafood for the safety of meals, like sashimi and sushi.

RevDate: 2018-05-15
CmpDate: 2017-07-14

Dillon MM, Sung W, Sebra R, et al (2017)

Genome-Wide Biases in the Rate and Molecular Spectrum of Spontaneous Mutations in Vibrio cholerae and Vibrio fischeri.

Molecular biology and evolution, 34(1):93-109.

The vast diversity in nucleotide composition and architecture among bacterial genomes may be partly explained by inherent biases in the rates and spectra of spontaneous mutations. Bacterial genomes with multiple chromosomes are relatively unusual but some are relevant to human health, none more so than the causative agent of cholera, Vibrio cholerae Here, we present the genome-wide mutation spectra in wild-type and mismatch repair (MMR) defective backgrounds of two Vibrio species, the low-%GC squid symbiont V. fischeri and the pathogen V. cholerae, collected under conditions that greatly minimize the efficiency of natural selection. In apparent contrast to their high diversity in nature, both wild-type V. fischeri and V. cholerae have among the lowest rates for base-substitution mutations (bpsms) and insertion-deletion mutations (indels) that have been measured, below 10-3/genome/generation. Vibrio fischeri and V. cholerae have distinct mutation spectra, but both are AT-biased and produce a surprising number of multi-nucleotide indels. Furthermore, the loss of a functional MMR system caused the mutation spectra of these species to converge, implying that the MMR system itself contributes to species-specific mutation patterns. Bpsm and indel rates varied among genome regions, but do not explain the more rapid evolutionary rates of genes on chromosome 2, which likely result from weaker purifying selection. More generally, the very low mutation rates of Vibrio species correlate inversely with their immense population sizes and suggest that selection may not only have maximized replication fidelity but also optimized other polygenic traits relative to the constraints of genetic drift.

RevDate: 2017-02-20
CmpDate: 2016-09-23

Gromek SM, Suria AM, Fullmer MS, et al (2016)

Leisingera sp. JC1, a Bacterial Isolate from Hawaiian Bobtail Squid Eggs, Produces Indigoidine and Differentially Inhibits Vibrios.

Frontiers in microbiology, 7:1342.

Female members of many cephalopod species house a bacterial consortium in the accessory nidamental gland (ANG), part of the reproductive system. These bacteria are deposited into eggs that are then laid in the environment where they must develop unprotected from predation, pathogens, and fouling. In this study, we characterized the genome and secondary metabolite production of Leisingera sp. JC1, a member of the roseobacter clade (Rhodobacteraceae) of Alphaproteobacteria isolated from the jelly coat of eggs from the Hawaiian bobtail squid, Euprymna scolopes. Whole genome sequencing and MLSA analysis revealed that Leisingera sp. JC1 falls within a group of roseobacters associated with squid ANGs. Genome and biochemical analyses revealed the potential for and production of a number of secondary metabolites, including siderophores and acyl-homoserine lactones involved with quorum sensing. The complete biosynthetic gene cluster for the pigment indigoidine was detected in the genome and mass spectrometry confirmed the production of this compound. Furthermore, we investigated the production of indigoidine under co-culture conditions with Vibrio fischeri, the light organ symbiont of E. scolopes, and with other vibrios. Finally, both Leisingera sp. JC1 and secondary metabolite extracts of this strain had differential antimicrobial activity against a number of marine vibrios, suggesting that Leisingera sp. JC1 may play a role in host defense against other marine bacteria either in the eggs and/or ANG. These data also suggest that indigoidine may be partially, but not wholly, responsible for the antimicrobial activity of this squid-associated bacterium.

RevDate: 2018-04-09
CmpDate: 2017-11-06

Verma SC, T Miyashiro (2016)

Niche-Specific Impact of a Symbiotic Function on the Persistence of Microbial Symbionts within a Natural Host.

Applied and environmental microbiology, 82(19):5990-5996 pii:AEM.01770-16.

UNLABELLED: How the function of microbial symbionts is affected by their population/consortium structure within a host remains poorly understood. The symbiosis established between Euprymna scolopes and Vibrio fischeri is a well-characterized host-microbe association in which the function and structure of V. fischeri populations within the host are known: V. fischeri populations produce bioluminescence from distinct crypt spaces within a dedicated host structure called the light organ. Previous studies have revealed that luminescence is required for V. fischeri populations to persist within the light organ and that deletion of the lux gene locus, which is responsible for luminescence in V. fischeri, leads to a persistence defect. In this study, we investigated the impact of bioluminescence on V. fischeri population structure within the light organ. We report that the persistence defect is specific to crypt I, which is the most developmentally mature crypt space within the nascent light organ. This result provides insight into the structure/function relationship that will be useful for future mechanistic studies of squid-Vibrio symbiosis. In addition, our report highlights the potential impact of the host developmental program on the spatiotemporal dynamics of host-microbe interactions.

IMPORTANCE: Metazoan development and physiology depend on microbes. The relationship between the symbiotic function of microbes and their spatial structure within the host environment remains poorly understood. Here we demonstrate, using a binary symbiosis, that the host requirement for the symbiotic function of the microbial symbiont is restricted to a specific host environment. Our results also suggest a link between microbial function and host development that may be a fundamental aspect of the more complex host-microbe interactions.

RevDate: 2018-03-05
CmpDate: 2017-05-29

Aschtgen MS, Lynch JB, Koch E, et al (2016)

Rotation of Vibrio fischeri Flagella Produces Outer Membrane Vesicles That Induce Host Development.

Journal of bacteriology, 198(16):2156-2165 pii:JB.00101-16.

UNLABELLED: Using the squid-vibrio association, we aimed to characterize the mechanism through which Vibrio fischeri cells signal morphogenesis of the symbiotic light-emitting organ. The symbiont releases two cell envelope molecules, peptidoglycan (PG) and lipopolysaccharide (LPS) that, within 12 h of light organ colonization, act in synergy to trigger normal tissue development. Recent work has shown that outer membrane vesicles (OMVs) produced by V. fischeri are sufficient to induce PG-dependent morphogenesis; however, the mechanism(s) of OMV release by these bacteria has not been described. Like several genera of both beneficial and pathogenic bacteria, V. fischeri cells elaborate polar flagella that are enclosed by an extension of the outer membrane, whose function remains unclear. Here, we present evidence that along with the well-recognized phenomenon of blebbing from the cell's surface, rotation of this sheathed flagellum also results in the release of OMVs. In addition, we demonstrate that most of the development-inducing LPS is associated with these OMVs and that the presence of the outer membrane protein OmpU but not the LPS O antigen on these OMVs is important in triggering normal host development. These results also present insights into a possible new mechanism of LPS release by pathogens with sheathed flagella.

IMPORTANCE: Determining the function(s) of sheathed flagella in bacteria has been challenging, because no known mutation results only in the loss of this outer membrane-derived casing. Nevertheless, the presence of a sheathed flagellum in such host-associated genera as Vibrio, Helicobacter, and Brucella has led to several proposed functions, including physical protection of the flagella and masking of their immunogenic flagellins. Using the squid-vibrio light organ symbiosis, we demonstrate another role, that of V. fischeri cells require rotating flagella to induce apoptotic cell death within surface epithelium, which is a normal step in the organ's development. Further, we present evidence that this rotation releases apoptosis-triggering lipopolysaccharide in the form of outer membrane vesicles. Such release may also occur by pathogens but with different outcomes for the host.

RevDate: 2018-05-01
CmpDate: 2017-04-11

Salah Ud-Din AI, A Roujeinikova (2016)

The periplasmic sensing domain of Vibrio fischeri chemoreceptor protein A (VfcA): cloning, purification and crystallographic analysis.

Acta crystallographica. Section F, Structural biology communications, 72(Pt 5):382-385.

Flagella-mediated motility and chemotaxis towards nutrients are important characteristics of Vibrio fischeri that play a crucial role in the development of its symbiotic relationship with its Hawaiian squid host Euprymna scolopes. The V. fischeri chemoreceptor A (VfcA) mediates chemotaxis toward amino acids. The periplasmic sensory domain of VfcA has been crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 3350 as a precipitating agent. The crystals belonged to space group P1, with unit-cell parameters a = 39.9, b = 57.0, c = 117.0 Å, α = 88.9, β = 80.5, γ = 89.7°. A complete X-ray diffraction data set has been collected to 1.8 Å resolution using cryocooling conditions and synchrotron radiation.

RevDate: 2017-12-02
CmpDate: 2017-09-13

Bongrand C, Koch EJ, Moriano-Gutierrez S, et al (2016)

A genomic comparison of 13 symbiotic Vibrio fischeri isolates from the perspective of their host source and colonization behavior.

The ISME journal, 10(12):2907-2917.

Newly hatched Euprymna scolopes squid obtain their specific light-organ symbionts from an array of Vibrio (Allivibrio) fischeri strains present in their environment. Two genetically distinct populations of this squid species have been identified, one in Kaneohe Bay (KB), and another in Maunaloa Bay (MB), Oahu. We asked whether symbionts isolated from squid in each of these populations outcompete isolates from the other population in mixed-infection experiments. No relationship was found between a strain's host source (KB or MB) and its ability to competitively colonize KB or MB juveniles in a mixed inoculum. Instead, two colonization behaviors were identified among the 11 KB and MB strains tested: a 'dominant' outcome, in which one strain outcompetes the other for colonization, and a 'sharing' outcome, in which two strains co-colonize the squid. A genome-level comparison of these and other V. fischeri strains suggested that the core genomic structure of this species is both syntenous and highly conserved over time and geographical distance. We also identified ~250 Kb of sequence, encoding 194 dispersed orfs, that was specific to those strains that expressed the dominant colonization behavior. Taken together, the results indicate a link between the genome content of V. fischeri strains and their colonization behavior when initiating a light-organ symbiosis.

RevDate: 2018-04-06
CmpDate: 2017-09-20

Sun Y, LaSota ED, Cecere AG, et al (2016)

Intraspecific Competition Impacts Vibrio fischeri Strain Diversity during Initial Colonization of the Squid Light Organ.

Applied and environmental microbiology, 82(10):3082-3091 pii:AEM.04143-15.

UNLABELLED: Animal development and physiology depend on beneficial interactions with microbial symbionts. In many cases, the microbial symbionts are horizontally transmitted among hosts, thereby making the acquisition of these microbes from the environment an important event within the life history of each host. The light organ symbiosis established between the Hawaiian squid Euprymna scolopes and the bioluminescent bacterium Vibrio fischeri is a model system for examining how hosts acquire horizontally transmitted microbial symbionts. Recent studies have revealed that the light organ of wild-caught E. scolopes squid contains polyclonal populations of V. fischeri bacteria; however, the function and development of such strain diversity in the symbiosis are unknown. Here, we report our phenotypic and phylogenetic characterizations of FQ-A001, which is a V. fischeri strain isolated directly from the light organ of an E. scolopes individual. Relative to the type strain ES114, FQ-A001 exhibits similar growth in rich medium but displays increased bioluminescence and decreased motility in soft agar. FQ-A001 outcompetes ES114 in colonizing the crypt spaces of the light organs. Remarkably, we find that animals cocolonized with FQ-A001 and ES114 harbor singly colonized crypts, in contrast to the cocolonized crypts observed from competition experiments involving single genotypes. The results with our two-strain system suggest that strain diversity within the squid light organ is a consequence of diversity in the single-strain colonization of individual crypt spaces.

IMPORTANCE: The developmental programs and overall physiologies of most animals depend on diverse microbial symbionts that are acquired from the environment. However, the basic principles underlying how microbes colonize their hosts remain poorly understood. Here, we report our findings of bacterial strain competition within the coevolved animal-microbe symbiosis composed of the Hawaiian squid and bioluminescent bacterium Vibrio fischeri Using fluorescent proteins to differentially label two distinct V. fischeri strains, we find that the strains are unable to coexist in the same niche within the host. Our results suggest that strain competition for distinct colonization sites dictates the strain diversity associated with the host. Our study provides a platform for studying how strain diversity develops within a host.

RevDate: 2018-02-23
CmpDate: 2017-03-23

Brooks JF, MJ Mandel (2016)

The Histidine Kinase BinK Is a Negative Regulator of Biofilm Formation and Squid Colonization.

Journal of bacteriology, 198(19):2596-2607 pii:JB.00037-16.

UNLABELLED: Bacterial colonization of animal epithelial tissue is a dynamic process that relies on precise molecular communication. Colonization of Euprymna scolopes bobtail squid by Vibrio fischeri bacteria requires bacterial aggregation in host mucus as the symbiont transitions from a planktonic lifestyle in seawater to a biofilm-associated state in the host. We have identified a gene, binK (biofilm inhibitor kinase; VF_A0360), which encodes an orphan hybrid histidine kinase that negatively regulates the V. fischeri symbiotic biofilm (Syp) in vivo and in vitro We identified binK mutants as exhibiting a colonization advantage in a global genetic screen, a phenotype that we confirmed in controlled competition experiments. Bacterial biofilm aggregates in the host are larger in strains lacking BinK, whereas overexpression of BinK suppresses biofilm formation and squid colonization. Signaling through BinK is required for temperature modulation of biofilm formation at 28°C. Furthermore, we present evidence that BinK acts upstream of SypG, the σ(54)-dependent transcriptional regulator of the syp biofilm locus. The BinK effects are dependent on intact signaling in the RscS-Syp biofilm pathway. Therefore, we propose that BinK antagonizes the signal from RscS and serves as an integral component in V. fischeri biofilm regulation.

IMPORTANCE: Bacterial lifestyle transitions underlie the colonization of animal hosts from environmental reservoirs. Formation of matrix-enclosed, surface-associated aggregates (biofilms) is common in beneficial and pathogenic associations, but investigating the genetic basis of biofilm development in live animal hosts remains a significant challenge. Using the bobtail squid light organ as a model, we analyzed putative colonization factors and identified a histidine kinase that negatively regulates biofilm formation at the host interface. This work reveals a novel in vivo biofilm regulator that influences the transition of bacteria from their planktonic state in seawater to tight aggregates of cells in the host. The study enriches our understanding of biofilm regulation and beneficial colonization by an animal's microbiome.

RevDate: 2016-12-30
CmpDate: 2016-11-04

Seedevi P, Moovendhan M, Vairamani S, et al (2016)

Structural characterization and biomedical properties of sulfated polysaccharide from the gladius of Sepioteuthis lessoniana (Lesson, 1831).

International journal of biological macromolecules, 85:117-125.

Sulfated polysaccharide was extracted from the internal shell (gladius) of Sepioteuthis lessoniana. The sulfated polysaccharide contained 61.3% of carbohydrate, 0.8% of protein, 28.2% of ash and 1.33% of moisture respectively. The elemental composition was analyzed using CHNS/O analyzer. The molecular weight of sulfated polysaccharide determined through PAGE was found to be as 66 kDa. Monosaccharides analysis revealed that sulfated polysaccharide was composed of rhamnose, galactose, xylose and glucose. The structural features of sulfated polysaccharide were analyzed by FT-IR and NMR spectroscopy. Further the sulfated polysaccharide was evaluated for its antibacterial activity against selected human clinical pathogens, namely Staphylococcus aureus, Klebsiella pneumoniae, Salmonella typhi, Vibrio cholerae, Klebsiella oxytoca, Escherichia coli, Salmonella paratyphi, Proteus mirabilis, Vibrio parahaemolyticus and Streptococcus pyogenes using agar well diffusion method. The polysaccharide has showed good antibacterial activity and MIC and MBC have also been evaluated. The anticancer activity was tested against HeLa cell line by MTT assay. The Cytotoxic Concentration (CC50) was observed as 700 μg/ml and the maximum anticancer activity of 62.89% was recorded at 200 μg/ml; whereas, the lowest of 9.87% was observed at 25 μg/ml. In conclusion, the sulfated polysaccharide is an alternate, non-toxic and cheap source of substance that showed good antibacterial and anticancer acitivity.

RevDate: 2017-02-20
CmpDate: 2016-06-06

Kimbrough JH, EV Stabb (2015)

Antisocial luxO Mutants Provide a Stationary-Phase Survival Advantage in Vibrio fischeri ES114.

Journal of bacteriology, 198(4):673-687 pii:JB.00807-15.

UNLABELLED: The squid light organ symbiont Vibrio fischeri controls bioluminescence using two acyl-homoserine lactone pheromone-signaling (PS) systems. The first of these systems to be activated during host colonization, AinS/AinR, produces and responds to N-octanoyl homoserine lactone (C(8)-AHL). We screened activity of a P(ainS)-lacZ transcriptional reporter in a transposon mutant library and found three mutants with decreased reporter activity, low C(8)-AHL output, and other traits consistent with low ainS expression. However, the transposon insertions were unrelated to these phenotypes, and genome resequencing revealed that each mutant had a distinct point mutation in luxO. In the wild type, LuxO is phosphorylated by LuxU and then activates transcription of the small RNA (sRNA) Qrr, which represses ainS indirectly by repressing its activator LitR. The luxO mutants identified here encode LuxU-independent, constitutively active LuxO* proteins. The repeated appearance of these luxO mutants suggested that they had some fitness advantage during construction and/or storage of the transposon mutant library, and we found that luxO* mutants survived better and outcompeted the wild type in prolonged stationary-phase cultures. From such cultures we isolated additional luxO* mutants. In all, we isolated LuxO* allelic variants with the mutations P41L, A91D, F94C, P98L, P98Q, V106A, V106G, T107R, V108G, R114P, L205F, H319R, H324R, and T335I. Based on the current model of the V. fischeri PS circuit, litR knockout mutants should resemble luxO* mutants; however, luxO* mutants outcompeted litR mutants in prolonged culture and had much poorer host colonization competitiveness than is reported for litR mutants, illustrating additional complexities in this regulatory circuit.

IMPORTANCE: Our results provide novel insight into the function of LuxO, which is a key component of pheromone signaling (PS) cascades in several members of the Vibrionaceae. Our results also contribute to an increasingly appreciated aspect of bacterial behavior and evolution whereby mutants that do not respond to a signal from like cells have a selective advantage. In this case, although "antisocial" mutants locked in the PS signal-off mode can outcompete parents, their survival advantage does not require wild-type cells to exploit. Finally, this work strikes a note of caution for those conducting or interpreting experiments in V. fischeri, as it illustrates how pleiotropic mutants could easily and inadvertently be enriched in this bacterium during prolonged culturing.

RevDate: 2017-02-20
CmpDate: 2015-12-04

Augimeri RV, Varley AJ, JL Strap (2015)

Establishing a Role for Bacterial Cellulose in Environmental Interactions: Lessons Learned from Diverse Biofilm-Producing Proteobacteria.

Frontiers in microbiology, 6:1282.

Bacterial cellulose (BC) serves as a molecular glue to facilitate intra- and inter-domain interactions in nature. Biosynthesis of BC-containing biofilms occurs in a variety of Proteobacteria that inhabit diverse ecological niches. The enzymatic and regulatory systems responsible for the polymerization, exportation, and regulation of BC are equally as diverse. Though the magnitude and environmental consequences of BC production are species-specific, the common role of BC-containing biofilms is to establish close contact with a preferred host to facilitate efficient host-bacteria interactions. Universally, BC aids in attachment, adherence, and subsequent colonization of a substrate. Bi-directional interactions influence host physiology, bacterial physiology, and regulation of BC biosynthesis, primarily through modulation of intracellular bis-(3'→5')-cyclic diguanylate (c-di-GMP) levels. Depending on the circumstance, BC producers exhibit a pathogenic or symbiotic relationship with plant, animal, or fungal hosts. Rhizobiaceae species colonize plant roots, Pseudomonadaceae inhabit the phyllosphere, Acetobacteriaceae associate with sugar-loving insects and inhabit the carposphere, Enterobacteriaceae use fresh produce as vehicles to infect animal hosts, and Vibrionaceae, particularly Aliivibrio fischeri, colonize the light organ of squid. This review will highlight the diversity of the biosynthesis and regulation of BC in nature by discussing various examples of Proteobacteria that use BC-containing biofilms to facilitate host-bacteria interactions. Through discussion of current data we will establish new directions for the elucidation of BC biosynthesis, its regulation and its ecophysiological roles.

RevDate: 2018-03-12
CmpDate: 2016-10-12

Nikolakakis K, Monfils K, Moriano-Gutierrez S, et al (2016)

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

Applied and environmental microbiology, 82(2):696-704 pii:AEM.02856-15.

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

RevDate: 2016-10-19
CmpDate: 2016-07-26

Krasity BC, Troll JV, Lehnert EM, et al (2015)

Structural and functional features of a developmentally regulated lipopolysaccharide-binding protein.

mBio, 6(5):e01193-15 pii:mBio.01193-15.

UNLABELLED: Mammalian lipopolysaccharide (LPS) binding proteins (LBPs) occur mainly in extracellular fluids and promote LPS delivery to specific host cell receptors. The function of LBPs has been studied principally in the context of host defense; the possible role of LBPs in nonpathogenic host-microbe interactions has not been well characterized. Using the Euprymna scolopes-Vibrio fischeri model, we analyzed the structure and function of an LBP family protein, E. scolopes LBP1 (EsLBP1), and provide evidence for its role in triggering a symbiont-induced host developmental program. Previous studies showed that, during initial host colonization, the LPS of V. fischeri synergizes with peptidoglycan (PGN) monomer to induce morphogenesis of epithelial tissues of the host animal. Computationally modeled EsLBP1 shares some but not all structural features of mammalian LBPs that are thought important for LPS binding. Similar to human LBP, recombinant EsLBP1 expressed in insect cells bound V. fischeri LPS and Neisseria meningitidis lipooligosaccharide (LOS) with nanomolar or greater affinity but bound Francisella tularensis LPS only weakly and did not bind PGN monomer. Unlike human LBP, EsLBP1 did not bind N. meningitidis LOS:CD14 complexes. The eslbp1 transcript was upregulated ~22-fold by V. fischeri at 24 h postinoculation. Surprisingly, this upregulation was not induced by exposure to LPS but, rather, to the PGN monomer alone. Hybridization chain reaction-fluorescent in situ hybridization (HCR-FISH) and immunocytochemistry (ICC) localized eslbp1 transcript and protein in crypt epithelia, where V. fischeri induces morphogenesis. The data presented here provide a window into the evolution of LBPs and the scope of their roles in animal symbioses.

IMPORTANCE: Mammalian lipopolysaccharide (LPS)-binding protein (LBP) is implicated in conveying LPS to host cells and potentiating its signaling activity. In certain disease states, such as obesity, the overproduction of this protein has been a reliable biomarker of chronic inflammation. Here, we describe a symbiosis-induced invertebrate LBP whose tertiary structure and LPS-binding characteristics are similar to those of mammalian LBPs; however, the primary structure of this distantly related squid protein (EsLBP1) differs in key residues previously believed to be essential for LPS binding, suggesting that an alternative strategy exists. Surprisingly, symbiotic expression of eslbp1 is induced by peptidoglycan derivatives, not LPS, a pattern converse to that of RegIIIγ, an important mammalian immunity protein that binds peptidoglycan but whose gene expression is induced by LPS. Finally, EsLBP1 occurs along the apical surfaces of all the host's epithelia, suggesting that it was recruited from a general defensive role to one that mediates specific interactions with its symbiont.

RevDate: 2017-02-20
CmpDate: 2016-12-13

Aschtgen MS, Wetzel K, Goldman W, et al (2016)

Vibrio fischeri-derived outer membrane vesicles trigger host development.

Cellular microbiology, 18(4):488-499.

Outer membrane vesicles (OMV) are critical elements in many host-cell/microbe interactions. Previous studies of the symbiotic association between Euprymna scolopes and Vibrio fischeri had shown that within 12 h of colonizing crypts deep within the squid's light organ, the symbionts trigger an irreversible programme of tissue development in the host. Here, we report that OMV produced by V. fischeri are powerful contributors to this process. The first detectable host response to the OMV is an increased trafficking of macrophage-like cells called haemocytes into surface epithelial tissues. We showed that exposing the squid to other Vibrio species fails to induce this trafficking; however, addition of a high concentration of their OMV, which can diffuse into the crypts, does. We also provide evidence that tracheal cytotoxin released by the symbionts, which can induce haemocyte trafficking, is not part of the OMV cargo, suggesting two distinct mechanisms to induce the same morphogenesis event. By manipulating the timing and localization of OMV signal delivery, we showed that haemocyte trafficking is fully induced only when V. fischeri, the sole species able to reach and grow in the crypts, succeeds in establishing a sustained colonization. Further, our data suggest that the host's detection of OMV serves as a symbiotic checkpoint prior to inducing irreversible morphogenesis.

RevDate: 2018-03-19
CmpDate: 2016-10-17

Schwartzman JA, EG Ruby (2016)

A conserved chemical dialog of mutualism: lessons from squid and vibrio.

Microbes and infection, 18(1):1-10.

Microorganisms shape, and are shaped by, their environment. In host-microbe associations, this environment is defined by tissue chemistry, which reflects local and organism-wide physiology, as well as inflammatory status. We review how, in the squid-vibrio mutualism, both partners shape tissue chemistry, revealing common themes governing tissue homeostasis in animal-microbe associations.

RevDate: 2017-11-16
CmpDate: 2016-04-29

Carey J (2015)

News Feature: Intimate partnerships.

Proceedings of the National Academy of Sciences of the United States of America, 112(33):10071-10073.

RevDate: 2017-04-28
CmpDate: 2015-08-18

Thompson CM, KL Visick (2015)

Assessing the function of STAS domain protein SypA in Vibrio fischeri using a comparative analysis.

Frontiers in microbiology, 6:760.

Colonization of the squid Euprymna scolopes by Vibrio fischeri requires biofilm formation dependent on the 18-gene symbiosis polysaccharide locus, syp. One key regulator, SypA, controls biofilm formation by an as-yet unknown mechanism; however, it is known that SypA itself is regulated by SypE. Biofilm-proficient strains form wrinkled colonies on solid media, while sypA mutants form biofilm-defective smooth colonies. To begin to understand the function of SypA, we used comparative analyses and mutagenesis approaches. sypA (and the syp locus) is conserved in other Vibrios, including two food-borne human pathogens, Vibrio vulnificus (rbdA) and Vibrio parahaemolyticus (sypA VP). We found that both homologs could complement the biofilm defect of the V. fischeri sypA mutant, but their phenotypes varied depending on the biofilm-inducing conditions used. Furthermore, while SypAVP retained an ability to be regulated by SypE, RbdA was resistant to this control. To better understand SypA function, we examined the biofilm-promoting ability of a number of mutant SypA proteins with substitutions in conserved residues, and found many that were biofilm-defective. The most severe biofilm-defective phenotypes occurred when changes were made to a conserved stretch of amino acids within a predicted α-helix of SypA; we hypothesize that this region of SypA may interact with another protein to promote biofilm formation. Finally, we identified a residue required for negative control by SypE. Together, our data provide insights into the function of this key biofilm regulator and suggest that the SypA orthologs may play similar roles in their native Vibrio species.

RevDate: 2017-02-20
CmpDate: 2015-08-03

Sun Y, Verma SC, Bogale H, et al (2015)

NagC represses N-acetyl-glucosamine utilization genes in Vibrio fischeri within the light organ of Euprymna scolopes.

Frontiers in microbiology, 6:741.

Bacteria often use transcription factors to regulate the expression of metabolic genes in accordance to available nutrients. NagC is a repressor conserved among γ-proteobacteria that regulates expression of enzymes involved in the metabolism of N-acetyl-glucosamine (GlcNAc). The polymeric form of GlcNAc, known as chitin, has been shown to play roles in chemotactic signaling and nutrition within the light organ symbiosis established between the marine bacterium Vibrio fischeri and the Hawaiian squid Euprymna scolopes. Here, we investigate the impact of NagC regulation on the physiology of V. fischeri. We find that NagC repression contributes to the fitness of V. fischeri in the absence of GlcNAc. In addition, the inability to de-repress expression of NagC-regulated genes reduces the fitness of V. fischeri in the presence of GlcNAc. We find that chemotaxis toward GlcNAc or chitobiose, a dimeric form of GlcNAc, is independent of NagC regulation. Finally, we show that NagC represses gene expression during the early stages of symbiosis. Our data suggest that the ability to regulate gene expression with NagC contributes to the overall fitness of V. fischeri in environments that vary in levels of GlcNAc. Furthermore, our finding that NagC represses gene expression within the squid light organ during an early stage of symbiosis supports the notion that the ability of the squid to provide a source of GlcNAc emerges later in host development.

RevDate: 2017-07-23
CmpDate: 2015-12-04

Liu HH, He JY, Chi CF, et al (2015)

Identification and analysis of HSP70 from Sepiella maindroni under stress of Vibrio harveyi and Cd(2.).

Gene, 572(1):146-152.

The 70-kDa heat shock proteins (HSP70) play crucial roles in protecting cells against environmental stresses, such as heat shock, heavy metals and pathogenic bacteria. The full-length HSP70 cDNA of Sepiella maindroni (designated as SmHSP70, GenBank accession no. KJ739788) was 2109 bp, including an ORF of 1950 bp encoding a polypeptide of 649 amino acids with predicted pI/MW 5.24/71.30 kDa, a 62 bp-5'-UTR and a 97 bp-3'-UTR. BLASTp analysis and phylogenetic relationship strongly suggested that the amino acid sequence was a member of HSP70 family. Multiple sequence alignment revealed that SmHSP70 and other known HSP70 were highly conserved, especially in the regions of HSP70 family signatures, the bipartite nuclear targeting sequence, ATP/GTP-binding site motif and 'EEVD' motif. Time-dependent mRNA expression of SmHSP70 in the liver was recorded by quantitative real-time RT-PCR after Vibrio harveyi injection and Cd(2+) exposure. The results indicated that SmHSP70 played a significant role in mediating the environmental stress and immune response against pathogens.

RevDate: 2017-04-19
CmpDate: 2016-04-18

Pan M, Schwartzman JA, Dunn AK, et al (2015)

A Single Host-Derived Glycan Impacts Key Regulatory Nodes of Symbiont Metabolism in a Coevolved Mutualism.

mBio, 6(4):e00811 pii:mBio.00811-15.

UNLABELLED: Most animal-microbe mutualistic associations are characterized by nutrient exchange between the partners. When the host provides the nutrients, it can gain the capacity to shape its microbial community, control the stability of the interaction, and promote its health and fitness. Using the bioluminescent squid-vibrio model, we demonstrate how a single host-derived glycan, chitin, regulates the metabolism of Vibrio fischeri at key points in the development and maintenance of the symbiosis. We first characterized the pathways for catabolism of chitin sugars by V. fischeri, demonstrating that the Ccr-dependent phosphoenolpyruvate-pyruvate phosphotransferase system (PTS) prioritizes transport of these sugars in V. fischeri by blocking the uptake of non-PTS carbohydrates, such as glycerol. Next, we found that PTS transport of chitin sugars into the bacterium shifted acetate homeostasis toward a net excretion of acetate and was sufficient to override an activation of the acetate switch by AinS-dependent quorum sensing. Finally, we showed that catabolism of chitin sugars decreases the rate of cell-specific oxygen consumption. Collectively, these three metabolic functions define a physiological shift that favors fermentative growth on chitin sugars and may support optimal symbiont luminescence, the functional basis of the squid-vibrio mutualism.

IMPORTANCE: Host-derived glycans have recently emerged as a link between symbiont nutrition and innate immune function. Unfortunately, the locations at which microbes typically access host-derived glycans are inaccessible to experimentation and imaging, and they take place in the context of diverse microbe-microbe interactions, creating a complex symbiotic ecology. Here we describe the metabolic state of a single microbial symbiont in a natural association with its coevolved host and, by doing so, infer key points at which a host-controlled tissue environment might regulate the physiological state of its symbionts. We show that the presence of a regulatory glycan is sufficient to shift symbiont carbohydrate catabolism, acetate homeostasis, and oxygen consumption.

RevDate: 2015-07-19
CmpDate: 2016-04-25

Cornet V, Henry J, Goux D, et al (2015)

How Egg Case Proteins Can Protect Cuttlefish Offspring?.

PloS one, 10(7):e0132836 pii:PONE-D-15-10301.

Sepia officinalis egg protection is ensured by a complex capsule produced by the female accessory genital glands and the ink bag. Our study is focused on the proteins constituting the main egg case. De novo transcriptomes from female genital glands provided essential databases for protein identification. A proteomic approach in SDS-PAGE coupled with MS unveiled a new egg case protein family: SepECPs, for Sepia officinalis Egg Case Proteins. N-glycosylation was demonstrated by PAS staining SDS-PAGE gels. These glycoproteins are mainly produced in the main nidamental glands. SepECPs share high sequence homology, especially in the signal peptide and the three cysteine-rich domains. SepECPs have a high number of cysteines, with conserved motifs involved in 3D-structure. SDS-PAGE showed that SepECPs could form dimers; this result was confirmed by TEM observations, which also revealed a protein network. This network is similar to the capsule network, and it associates these structural proteins with polysaccharides, melanin and bacteria to form a tight mesh. Its hardness and elasticity provide physical protection to the embryo. In addition, SepECPs also have bacteriostatic antimicrobial activity on GRAM- bacteria. By observing the SepECP / Vibrio aestuarianus complex in SEM, we demonstrated the ability of these proteins to agglomerate bacteria and thus inhibit their growth. These original proteins identified from the outer egg case ensure the survival of the species by providing physical and chemical protection to the embryos released in the environment without any maternal protection.

RevDate: 2015-08-13
CmpDate: 2016-05-24

Cornet V, Henry J, Corre E, et al (2015)

The Toll/NF-κB pathway in cuttlefish symbiotic accessory nidamental gland.

Developmental and comparative immunology, 53(1):42-46.

The female genital apparatus of decapod cephalopods contains a symbiotic accessory nidamental gland (ANG) that harbors bacterial symbionts. Although the ANG bacterial consortium is now well described, the impact of symbiosis on Sepia officinalis innate immunity pathways remains unknown. In silico analysis of the de novo transcriptome of ANG highlighted for the first time the existence of the NF-κB pathway in S. officinalis. Several signaling components were identified, i.e. five Toll-like receptors, eight signaling cascade features, and the immune response target gene iNOS, previously described as being involved in the initiation of bacterial symbiosis in a cephalopod gland. This work provides a first key for studying bacterial symbiosis and its impact on innate immunity in S. officinalis ANG.

RevDate: 2017-12-13
CmpDate: 2016-06-14

Colton DM, Stoudenmire JL, EV Stabb (2015)

Growth on glucose decreases cAMP-CRP activity while paradoxically increasing intracellular cAMP in the light-organ symbiont Vibrio fischeri.

Molecular microbiology, 97(6):1114-1127.

Proteobacteria often co-ordinate responses to carbon sources using CRP and the second messenger cyclic 3', 5'-AMP (cAMP), which combine to control transcription of genes during growth on non-glucose substrates as part of the catabolite-repression response. Here we show that cAMP-CRP is active and important in Vibrio fischeri during colonization of its host squid Euprymna scolopes. Moreover, consistent with a classical role in catabolite repression, a cAMP-CRP-dependent reporter showed lower activity in cells grown in media amended with glucose rather than glycerol. Surprisingly though, intracellular cAMP levels were higher in glucose-grown cells. Mutant analyses were consistent with predictions that CyaA was responsible for cAMP generation, that the EIIA(Glc) component of glucose transport could enhance cAMP production and that the phophodiesterases CpdA and CpdP consumed intracellular and extracellular cAMP respectively. However, the observation of lower cAMP levels in glycerol-grown cells seemed best explained by changes in cAMP export, via an unknown mechanism. Our data also indicated that cAMP-CRP activity decreased during growth on glucose independently of crp's native transcriptional regulation or cAMP levels. We speculate that some unknown mechanism, perhaps carbon-source-dependent post-translational modulation of CRP, may help control cAMP-CRP activity in V.fischeri.

RevDate: 2018-02-23
CmpDate: 2016-03-29

Singh P, Brooks JF, Ray VA, et al (2015)

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

Applied and environmental microbiology, 81(15):5223-5234.

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

RevDate: 2017-02-20
CmpDate: 2016-03-24

Nikolakakis K, Lehnert E, McFall-Ngai MJ, et al (2015)

Use of Hybridization Chain Reaction-Fluorescent In Situ Hybridization To Track Gene Expression by Both Partners during Initiation of Symbiosis.

Applied and environmental microbiology, 81(14):4728-4735.

The establishment of a productive symbiosis between Euprymna scolopes, the Hawaiian bobtail squid, and its luminous bacterial symbiont, Vibrio fischeri, is mediated by transcriptional changes in both partners. A key challenge to unraveling the steps required to successfully initiate this and many other symbiotic associations is characterization of the timing and location of these changes. We report on the adaptation of hybridization chain reaction-fluorescent in situ hybridization (HCR-FISH) to simultaneously probe the spatiotemporal regulation of targeted genes in both E. scolopes and V. fischeri. This method revealed localized, transcriptionally coregulated epithelial cells within the light organ that responded directly to the presence of bacterial cells while, at the same time, provided a sensitive means to directly show regulated gene expression within the symbiont population. Thus, HCR-FISH provides a new approach for characterizing habitat transition in bacteria and for discovering host tissue responses to colonization.

RevDate: 2018-04-23
CmpDate: 2016-02-04

Salazar KA, Joffe NR, Dinguirard N, et al (2015)

Transcriptome analysis of the white body of the squid Euprymna tasmanica with emphasis on immune and hematopoietic gene discovery.

PloS one, 10(3):e0119949 pii:PONE-D-14-31941.

In the mutualistic relationship between the squid Euprymna tasmanica and the bioluminescent bacterium Vibrio fischeri, several host factors, including immune-related proteins, are known to interact and respond specifically and exclusively to the presence of the symbiont. In squid and octopus, the white body is considered to be an immune organ mainly due to the fact that blood cells, or hemocytes, are known to be present in high numbers and in different developmental stages. Hence, the white body has been described as the site of hematopoiesis in cephalopods. However, to our knowledge, there are no studies showing any molecular evidence of such functions. In this study, we performed a transcriptomic analysis of white body tissue of the Southern dumpling squid, E. tasmanica. Our primary goal was to gain insights into the functions of this tissue and to test for the presence of gene transcripts associated with hematopoietic and immune processes. Several hematopoiesis genes including CPSF1, GATA 2, TFIID, and FGFR2 were found to be expressed in the white body. In addition, transcripts associated with immune-related signal transduction pathways, such as the toll-like receptor/NF-κβ, and MAPK pathways were also found, as well as other immune genes previously identified in E. tasmanica's sister species, E. scolopes. This study is the first to analyze an immune organ within cephalopods, and to provide gene expression data supporting the white body as a hematopoietic tissue.

RevDate: 2017-02-20
CmpDate: 2015-03-10

Collins AJ, Fullmer MS, Gogarten JP, et al (2015)

Comparative genomics of Roseobacter clade bacteria isolated from the accessory nidamental gland of Euprymna scolopes.

Frontiers in microbiology, 6:123.

The accessory nidamental gland (ANG) of the female Hawaiian bobtail squid, Euprymna scolopes, houses a consortium of bacteria including members of the Flavobacteriales, Rhizobiales, and Verrucomicrobia but is dominated by members of the Roseobacter clade (Rhodobacterales) within the Alphaproteobacteria. These bacteria are deposited into the jelly coat of the squid's eggs, however, the function of the ANG and its bacterial symbionts has yet to be elucidated. In order to gain insight into this consortium and its potential role in host reproduction, we cultured 12 Rhodobacterales isolates from ANGs of sexually mature female squid and sequenced their genomes with Illumina sequencing technology. For taxonomic analyses, the ribosomal proteins of 79 genomes representing both roseobacters and non-roseobacters along with a separate MLSA analysis of 33 housekeeping genes from Roseobacter organisms placed all 12 isolates from the ANG within two groups of a single Roseobacter clade. Average nucelotide identity analysis suggests the ANG isolates represent three genera (Leisingera, Ruegeria, and Tateyamaria) comprised of seven putative species groups. All but one of the isolates contains a predicted Type VI secretion system, which has been shown to be important in secreting signaling and/or effector molecules in host-microbe associations and in bacteria-bacteria interactions. All sequenced genomes also show potential for secondary metabolite production, and are predicted to be involved with the production of acyl homoserine lactones (AHLs) and/or siderophores. An AHL bioassay confirmed AHL production in three tested isolates and from whole ANG homogenates. The dominant symbiont, Leisingera sp. ANG1, showed greater viability in iron-limiting conditions compared to other roseobacters, possibly due to higher levels of siderophore production. Future comparisons will try to elucidate novel metabolic pathways of the ANG symbionts to understand their putative role in host development.

RevDate: 2015-04-04
CmpDate: 2016-01-29

Yazzie N, Salazar KA, MG Castillo (2015)

Identification, molecular characterization, and gene expression analysis of a CD109 molecule in the Hawaiian bobtail squid Euprymna scolopes.

Fish & shellfish immunology, 44(1):342-355.

All organisms have unique immune systems that help them identify and eliminate invading microorganisms. A group of evolutionary ancient molecules, the thioester-containing proteins (TEP) superfamily, are known to play an important immune role by aiding animal hosts in the recognition, destruction, and elimination of hazardous microorganisms and their products. Our laboratory focuses on studying the role of the immune system in the mutualistic relationship between the sepiolid squid, Euprymna scolopes and its bioluminescent symbiont Vibrio fischeri. In the present study, we report the identification of a novel TEP-like transcript expressed in the light organ of squid. Characterization of the full-length coding sequence showed a molecule of 4218 nucleotides, corresponding to 1406 amino acids. Further sequence analysis revealed it contained structural characteristics of A2M molecules, including the thioester and receptor-binding domains. Analysis using the predicted amino acid sequence suggested this transcript was a homologue of CD109 molecules, thus we named it E. scolopes-CD109 (Es-CD109). In addition to the light organ, we were able to detect and amplify Es-CD109 in 12 out of 14 adult squid tissues tested. Quantification experiments showed that Es-CD109 expression levels were significantly lower in the light organ of symbiotic compared to aposymbiotic juveniles, suggesting a possible down-regulation of the host immune response in the presence of the bacterial symbiont.

RevDate: 2016-11-25
CmpDate: 2015-12-21

Minamoto T, Takahashi N, Kitahara S, et al (2015)

Saccharification of β-chitin from squid pen by a fermentation method using recombinant chitinase-secreting Escherichia coli.

Applied biochemistry and biotechnology, 175(8):3788-3799.

Two strains [BL21(DE3) and HMS174(DE3)] of Escherichia coli harboring the recombinant chitinase expression plasmid pVP-Chi, which contains Vibrio parahaemolyticus chitinase gene with an attached signal sequence, were prepared. These E. coli transformants produced a large amount of recombinant chitinase, which hydrolyzes chitin to yield di-N-acetylchitobiose (GlcNAc)2, under the presence of isopropyl-1-thio-β-D-galactopyranoside (IPTG), and secreted the enzyme into their culture fluid with the aid of the signal peptide. Cultivation of these E. coli transformants in Luria-Bertani medium containing squid pen β-chitin and IPTG gave rise to the decomposition of this polysaccharide and the accumulation of (GlcNAc)2 in the culture fluid. Through these experiments, we confirmed that the use of strain HMS174(DE3) was preferable for the stable accumulation of (GlcNAc)2 in the culture fluid during cultivation owing to lower (GlcNAc)2 assimilation compared to BL21(DE3). Next, using E. coli HMS174(DE3) transformants, we conducted saccharification of different forms (fluffy fiber, flake, and powder) of β-chitin samples prepared from squid pens in Bacterion-N-KS(B)K medium containing 2 % of each sample under the presence of IPTG. In these experiments, (GlcNAc)2 was isolated with a more than 20 % stoichiometric yield from each culture supernatant through charcoal column chromatography followed by recrystallization.

RevDate: 2017-02-20
CmpDate: 2015-02-02

Yong E (2015)

Microbiology: Here's looking at you, squid.

Nature, 517(7534):262-264.

RevDate: 2017-02-20
CmpDate: 2016-01-19

Norsworthy AN, KL Visick (2015)

Signaling between two interacting sensor kinases promotes biofilms and colonization by a bacterial symbiont.

Molecular microbiology, 96(2):233-248.

Cells acclimate to fluctuating environments by utilizing sensory circuits. One common sensory pathway used by bacteria is two-component signaling (TCS), composed of an environmental sensor [the sensor kinase (SK)] and a cognate, intracellular effector [the response regulator (RR)]. The squid symbiont Vibrio fischeri uses an elaborate TCS phosphorelay containing a hybrid SK, RscS, and two RRs, SypE and SypG, to control biofilm formation and host colonization. Here, we found that another hybrid SK, SypF, was essential for biofilms by functioning downstream of RscS to directly control SypE and SypG. Surprisingly, although wild-type SypF functioned as an SK in vitro, this activity was dispensable for colonization. In fact, only a single non-enzymatic domain within SypF, the HPt domain, was critical in vivo. Remarkably, this domain within SypF interacted with RscS to permit a bypass of RscS's own HPt domain and SypF's enzymatic function. This represents the first in vivo example of a functional SK that exploits the enzymatic activity of another SK, an adaptation that demonstrates the elegant plasticity in the arrangement of TCS regulators.

RevDate: 2017-06-06
CmpDate: 2015-04-30

Kåhrström CT (2015)

Symbiosis: Sweet talking your partner.

Nature reviews. Microbiology, 13(2):66-67.

RevDate: 2018-02-23
CmpDate: 2015-04-30

Brooks JF, Gyllborg MC, Kocher AA, et al (2015)

TfoX-based genetic mapping identifies Vibrio fischeri strain-level differences and reveals a common lineage of laboratory strains.

Journal of bacteriology, 197(6):1065-1074.

Bacterial strain variation exists in natural populations of bacteria and can be generated experimentally through directed or random mutation. The advent of rapid and cost-efficient whole-genome sequencing has facilitated strain-level genotyping. Even with modern tools, however, it often remains a challenge to map specific traits to individual genetic loci, especially for traits that cannot be selected under culture conditions (e.g., colonization level or pathogenicity). Using a combination of classical and modern approaches, we analyzed strain-level variation in Vibrio fischeri and identified the basis by which some strains lack the ability to utilize glycerol as a carbon source. We proceeded to reconstruct the lineage of the commonly used V. fischeri laboratory strains. Compared to the wild-type ES114 strain, we identify in ES114-L a 9.9-kb deletion with endpoints in tadB2 and glpF; restoration of the missing portion of glpF restores the wild-type phenotype. The widely used strains ESR1, JRM100, and JRM200 contain the same deletion, and ES114-L is likely a previously unrecognized intermediate strain in the construction of many ES114 derivatives. ES114-L does not exhibit a defect in competitive squid colonization but ESR1 does, demonstrating that glycerol utilization is not required for early squid colonization. Our genetic mapping approach capitalizes on the recently discovered chitin-based transformation pathway, which is conserved in the Vibrionaceae; therefore, the specific approach used is likely to be useful for mapping genetic traits in other Vibrio species.

RevDate: 2017-07-14
CmpDate: 2015-04-30

Schwartzman JA, Koch E, Heath-Heckman EA, et al (2015)

The chemistry of negotiation: rhythmic, glycan-driven acidification in a symbiotic conversation.

Proceedings of the National Academy of Sciences of the United States of America, 112(2):566-571.

Glycans have emerged as critical determinants of immune maturation, microbial nutrition, and host health in diverse symbioses. In this study, we asked how cyclic delivery of a single host-derived glycan contributes to the dynamic stability of the mutualism between the squid Euprymna scolopes and its specific, bioluminescent symbiont, Vibrio fischeri. V. fischeri colonizes the crypts of a host organ that is used for behavioral light production. E. scolopes synthesizes the polymeric glycan chitin in macrophage-like immune cells called hemocytes. We show here that, just before dusk, hemocytes migrate from the vasculature into the symbiotic crypts, where they lyse and release particulate chitin, a behavior that is established only in the mature symbiosis. Diel transcriptional rhythms in both partners further indicate that the chitin is provided and metabolized only at night. A V. fischeri mutant defective in chitin catabolism was able to maintain a normal symbiont population level, but only until the symbiotic organ reached maturity (∼ 4 wk after colonization); this result provided a direct link between chitin utilization and symbiont persistence. Finally, catabolism of chitin by the symbionts was also specifically required for a periodic acidification of the adult crypts each night. This acidification, which increases the level of oxygen available to the symbionts, enhances their capacity to produce bioluminescence at night. We propose that other animal hosts may similarly regulate the activities of epithelium-associated microbial communities through the strategic provision of specific nutrients, whose catabolism modulates conditions like pH or anoxia in their symbionts' habitat.

RevDate: 2017-02-20
CmpDate: 2014-12-24

Soto W, MK Nishiguchi (2014)

Microbial experimental evolution as a novel research approach in the Vibrionaceae and squid-Vibrio symbiosis.

Frontiers in microbiology, 5:593.

The Vibrionaceae are a genetically and metabolically diverse family living in aquatic habitats with a great propensity toward developing interactions with eukaryotic microbial and multicellular hosts (as either commensals, pathogens, and mutualists). The Vibrionaceae frequently possess a life history cycle where bacteria are attached to a host in one phase and then another where they are free from their host as either part of the bacterioplankton or adhered to solid substrates such as marine sediment, riverbeds, lakebeds, or floating particulate debris. These two stages in their life history exert quite distinct and separate selection pressures. When bound to solid substrates or to host cells, the Vibrionaceae can also exist as complex biofilms. The association between bioluminescent Vibrio spp. and sepiolid squids (Cephalopoda: Sepiolidae) is an experimentally tractable model to study bacteria and animal host interactions, since the symbionts and squid hosts can be maintained in the laboratory independently of one another. The bacteria can be grown in pure culture and the squid hosts raised gnotobiotically with sterile light organs. The partnership between free-living Vibrio symbionts and axenic squid hatchlings emerging from eggs must be renewed every generation of the cephalopod host. Thus, symbiotic bacteria and animal host can each be studied alone and together in union. Despite virtues provided by the Vibrionaceae and sepiolid squid-Vibrio symbiosis, these assets to evolutionary biology have yet to be fully utilized for microbial experimental evolution. Experimental evolution studies already completed are reviewed, along with exploratory topics for future study.

RevDate: 2018-04-13
CmpDate: 2015-04-28

Brooks JF, Gyllborg MC, Cronin DC, et al (2014)

Global discovery of colonization determinants in the squid symbiont Vibrio fischeri.

Proceedings of the National Academy of Sciences of the United States of America, 111(48):17284-17289.

Animal epithelial tissue becomes reproducibly colonized by specific environmental bacteria. The bacteria (microbiota) perform critical functions for the host's tissue development, immune system development, and nutrition; yet the processes by which bacterial diversity in the environment is selected to assemble the correct communities in the host are unclear. To understand the molecular determinants of microbiota selection, we examined colonization of a simplified model in which the light organ of Euprymna scolopes squid is colonized exclusively by Vibrio fischeri bacteria. We applied high-throughput insertion sequencing to identify which bacterial genes are required during host colonization. A library of over 41,000 unique transposon insertions was analyzed before and after colonization of 1,500 squid hatchlings. Mutants that were reproducibly depleted following squid colonization represented 380 genes, including 37 that encode known colonization factors. Validation of select mutants in defined competitions against the wild-type strain identified nine mutants that exhibited a reproducible colonization defect. Some of the colonization factors identified included genes predicted to influence copper regulation and secretion. Other mutants exhibited defects in biofilm development, which is required for aggregation in host mucus and initiation of colonization. Biofilm formation in culture and in vivo was abolished in a strain lacking the cytoplasmic chaperone DnaJ, suggesting an important role for protein quality control during the elaboration of bacterial biofilm in the context of an intact host immune system. Overall these data suggest that cellular stress responses and biofilm regulation are critical processes underlying the reproducible colonization of animal hosts by specific microbial symbionts.

RevDate: 2017-02-20
CmpDate: 2015-04-14

Pankey MS, Minin VN, Imholte GC, et al (2014)

Predictable transcriptome evolution in the convergent and complex bioluminescent organs of squid.

Proceedings of the National Academy of Sciences of the United States of America, 111(44):E4736-42.

Despite contingency in life's history, the similarity of evolutionarily convergent traits may represent predictable solutions to common conditions. However, the extent to which overall gene expression levels (transcriptomes) underlying convergent traits are themselves convergent remains largely unexplored. Here, we show strong statistical support for convergent evolutionary origins and massively parallel evolution of the entire transcriptomes in symbiotic bioluminescent organs (bacterial photophores) from two divergent squid species. The gene expression similarities are so strong that regression models of one species' photophore can predict organ identity of a distantly related photophore from gene expression levels alone. Our results point to widespread parallel changes in gene expression evolution associated with convergent origins of complex organs. Therefore, predictable solutions may drive not only the evolution of novel, complex organs but also the evolution of overall gene expression levels that underlie them.

RevDate: 2017-02-20
CmpDate: 2015-06-18

Schleicher TR, VerBerkmoes NC, Shah M, et al (2014)

Colonization state influences the hemocyte proteome in a beneficial squid-Vibrio symbiosis.

Molecular & cellular proteomics : MCP, 13(10):2673-2686.

The squid Euprymna scolopes and the luminescent bacterium Vibrio fischeri form a highly specific beneficial light organ symbiosis. Not only does the host have to select V. fischeri from the environment, but it must also prevent subsequent colonization by non-symbiotic microorganisms. Host macrophage-like hemocytes are believed to play a role in mediating the symbiosis with V. fischeri. Previous studies have shown that the colonization state of the light organ influences the host's hemocyte response to the symbiont. To further understand the molecular mechanisms behind this process, we used two quantitative mass-spectrometry-based proteomic techniques, isobaric tags for relative and absolute quantification (iTRAQ) and label-free spectral counting, to compare and quantify the adult hemocyte proteomes from colonized (sym) and uncolonized (antibiotic-treated/cured) squid. Overall, iTRAQ allowed for the quantification of 1,024 proteins with two or more peptides. Thirty-seven unique proteins were determined to be significantly different between sym and cured hemocytes (p value < 0.05), with 20 more abundant proteins and 17 less abundant in sym hemocytes. The label-free approach resulted in 1,241 proteins that were identified in all replicates. Of 185 unique proteins present at significantly different amounts in sym hemocytes (as determined by spectral counting), 92 were more abundant and 93 were less abundant. Comparisons between iTRAQ and spectral counting revealed that 30 of the 37 proteins quantified via iTRAQ exhibited trends similar to those identified by the label-free method. Both proteomic techniques mutually identified 16 proteins that were significantly different between the two groups of hemocytes (p value < 0.05). The presence of V. fischeri in the host light organ influenced the abundance of proteins associated with the cytoskeleton, adhesion, lysosomes, proteolysis, and the innate immune response. These data provide evidence that colonization by V. fischeri alters the hemocyte proteome and reveals proteins that may be important for maintaining host-symbiont specificity.

RevDate: 2017-02-20
CmpDate: 2015-03-04

Chavez-Dozal AA, Gorman C, Lostroh CP, et al (2014)

Gene-swapping mediates host specificity among symbiotic bacteria in a beneficial symbiosis.

PloS one, 9(7):e101691 pii:PONE-D-13-47418.

Environmentally acquired beneficial associations are comprised of a wide variety of symbiotic species that vary both genetically and phenotypically, and therefore have differential colonization abilities, even when symbionts are of the same species. Strain variation is common among conspecific hosts, where subtle differences can lead to competitive exclusion between closely related strains. One example where symbiont specificity is observed is in the sepiolid squid-Vibrio mutualism, where competitive dominance exists among V. fischeri isolates due to subtle genetic differences between strains. Although key symbiotic loci are responsible for the establishment of this association, the genetic mechanisms that dictate strain specificity are not fully understood. We examined several symbiotic loci (lux-bioluminescence, pil = pili, and msh-mannose sensitive hemagglutinin) from mutualistic V. fischeri strains isolated from two geographically distinct squid host species (Euprymna tasmanica-Australia and E. scolopes-Hawaii) to determine whether slight genetic differences regulated host specificity. Through colonization studies performed in naïve squid hatchlings from both hosts, we found that all loci examined are important for specificity and host recognition. Complementation of null mutations in non-native V. fischeri with loci from the native V. fischeri caused a gain in fitness, resulting in competitive dominance in the non-native host. The competitive ability of these symbiotic loci depended upon the locus tested and the specific squid species in which colonization was measured. Our results demonstrate that multiple bacterial genetic elements can determine V. fischeri strain specificity between two closely related squid hosts, indicating how important genetic variation is for regulating conspecific beneficial interactions that are acquired from the environment.

RevDate: 2014-09-11
CmpDate: 2015-05-15

McFall-Ngai MJ (2014)

The importance of microbes in animal development: lessons from the squid-vibrio symbiosis.

Annual review of microbiology, 68:177-194.

Developmental biology is among the many subdisciplines of the life sciences being transformed by our increasing awareness of the role of coevolved microbial symbionts in health and disease. Most symbioses are horizontally acquired, i.e., they begin anew each generation. In such associations, the embryonic period prepares the animal to engage with the coevolved partner(s) with fidelity following birth or hatching. Once interactions are underway, the microbial partners drive maturation of tissues that are either directly associated with or distant from the symbiont populations. Animal alliances often involve complex microbial communities, such as those in the vertebrate gastrointestinal tract. A series of simpler-model systems is providing insight into the basic rules and principles that govern the establishment and maintenance of stable animal-microbe partnerships. This review focuses on what biologists have learned about the developmental trajectory of horizontally acquired symbioses through the study of the binary squid-vibrio model.

RevDate: 2014-08-26
CmpDate: 2015-01-23

He JY, Chi CF, HH Liu (2014)

Identification and analysis of an intracellular Cu/Zn superoxide dismutase from Sepiella maindroni under stress of Vibrio harveyi and Cd2+.

Developmental and comparative immunology, 47(1):1-5.

Superoxide dismutases (SODs) are ubiquitous family of metalloenzymes involved in protecting organisms from excess reactive oxygen species damage. In this paper, a novel intracellular Cu/ZnSOD from Sepiella maindroni (designated as SmSOD) was identified and characterized. The full-length cDNA sequence of SmSOD (GenBank accession No. KF908850) was 709 bp containing an open reading frame (ORF) of 459 bp, encoding 153 amino acid residues peptide with predicted pI/MW (6.02/15.75 kDa), a 131 bp-5'- and 116 bp-3'- untranslated region (UTR). BLASTn analysis and phylogenetic relationship strongly suggested that the sequence shared high similarity with known Cu/Zn SODs. Several highly conserved motifs, including two typical Cu/Zn SOD family domains, two conserved Cu-/Zn-binding sites (H-47, H-49, H-64, H-120 for Cu binding, and H-64, H-72, H-81, D-84 for Zn binding) and intracellular disulfide bond (C-58 and C-146), were also identified in SmSOD. Time-dependent mRNA expression of SmSOD in hepatopancreas was recorded by quantitative real-time RT-PCR after Vibrio harveyi injection and Cd(2+) exposure. The results indicated that SmSOD was an acute-phase protein involved in the immune responses against pathogens and biological indicator for metal contaminants in aquatic environment.

RevDate: 2017-11-16
CmpDate: 2015-01-06

Kremer N, Schwartzman J, Augustin R, et al (2014)

The dual nature of haemocyanin in the establishment and persistence of the squid-vibrio symbiosis.

Proceedings. Biological sciences, 281(1785):20140504 pii:rspb.2014.0504.

We identified and sequenced from the squid Euprymna scolopes two isoforms of haemocyanin that share the common structural/physiological characteristics of haemocyanin from a closely related cephalopod, Sepia officinalis, including a pronounced Bohr effect. We examined the potential roles for haemocyanin in the animal's symbiosis with the luminous bacterium Vibrio fischeri. Our data demonstrate that, as in other cephalopods, the haemocyanin is primarily synthesized in the gills. It transits through the general circulation into other tissues and is exported into crypt spaces that support the bacterial partner, which requires oxygen for its bioluminescence. We showed that the gradient of pH between the circulating haemolymph and the matrix of the crypt spaces in adult squid favours offloading of oxygen from the haemocyanin to the symbionts. Haemocyanin is also localized to the apical surfaces and associated mucus of a juvenile-specific epithelium on which the symbionts gather, and where their specificity is determined during the recruitment into the association. The haemocyanin has an antimicrobial activity, which may be involved in this enrichment of V. fischeri during symbiont initiation. Taken together, these data provide evidence that the haemocyanin plays a role in shaping two stages of the squid-vibrio partnership.

RevDate: 2016-10-19
CmpDate: 2015-03-30

Heath-Heckman EA, Gillette AA, Augustin R, et al (2014)

Shaping the microenvironment: evidence for the influence of a host galaxin on symbiont acquisition and maintenance in the squid-Vibrio symbiosis.

Environmental microbiology, 16(12):3669-3682.

Most bacterial species make transitions between habitats, such as switching from free living to symbiotic niches. We provide evidence that a galaxin protein, EsGal1, of the squid Euprymna scolopes participates in both: (i) selection of the specific partner Vibrio fischeri from the bacterioplankton during symbiosis onset and, (ii) modulation of V. fischeri growth in symbiotic maintenance. We identified two galaxins in transcriptomic databases and showed by quantitative reverse-transcriptase polymerase chain reaction that one (esgal1) was dominant in the light organ. Further, esgal1 expression was upregulated by symbiosis, a response that was partially achieved with exposure to symbiont cell-envelope molecules. Confocal immunocytochemistry of juvenile animals localized EsGal1 to the apical surfaces of light-organ epithelia and surrounding mucus, the environment in which V. fischeri cells aggregate before migration into the organ. Growth assays revealed that one repeat of EsGal1 arrested growth of Gram-positive bacterial cells, which represent the cell type first 'winnowed' during initial selection of the symbiont. The EsGal1-derived peptide also significantly decreased the growth rate of V. fischeri in culture. Further, when animals were exposed to an anti-EsGal1 antibody, symbiont population growth was significantly increased. These data provide a window into how hosts select symbionts from a rich environment and govern their growth in symbiosis.

RevDate: 2017-02-20
CmpDate: 2014-10-08

Koropatnick T, Goodson MS, Heath-Heckman EA, et al (2014)

Identifying the cellular mechanisms of symbiont-induced epithelial morphogenesis in the squid-Vibrio association.

The Biological bulletin, 226(1):56-68.

The symbiotic association between the Hawaiian bobtail squid Euprymna scolopes and the luminous marine bacterium Vibrio fischeri provides a unique opportunity to study epithelial morphogenesis. Shortly after hatching, the squid host harvests bacteria from the seawater using currents created by two elaborate fields of ciliated epithelia on the surface of the juvenile light organ. After light organ colonization, the symbiont population signals the gradual loss of the ciliated epithelia through apoptosis of the cells, which culminates in the complete regression of these tissues. Whereas aspects of this process have been studied at the morphological, biochemical, and molecular levels, no in-depth analysis of the cellular events has been reported. Here we describe the cellular structure of the epithelial field and present evidence that the symbiosis-induced regression occurs in two steps. Using confocal microscopic analyses, we observed an initial epithelial remodeling, which serves to disable the function of the harvesting apparatus, followed by a protracted regression involving actin rearrangements and epithelial cell extrusion. We identified a metal-dependent gelatinolytic activity in the symbiont-induced morphogenic epithelial fields, suggesting the involvement of Zn-dependent matrix metalloproteinase(s) (MMP) in light organ morphogenesis. These data show that the bacterial symbionts not only induce apoptosis of the field, but also change the form, function, and biochemistry of the cells as part of the morphogenic program.

RevDate: 2014-03-05
CmpDate: 2016-03-29

Guillemin K, AS Rolig (2014)

A twist in the tail.

eLife, 3:e02386.

Lipopolysaccharide molecules released by the bacteria Vibrio fischeri when it rotates its flagella prompts its host, the Hawaiian bobtail squid, to prepare for its arrival.

RevDate: 2017-11-09
CmpDate: 2015-11-24

Brennan CA, Hunt JR, Kremer N, et al (2014)

A model symbiosis reveals a role for sheathed-flagellum rotation in the release of immunogenic lipopolysaccharide.

eLife, 3:e01579.

Bacterial flagella mediate host-microbe interactions through tissue tropism during colonization, as well as by activating immune responses. The flagellar shaft of some bacteria, including several human pathogens, is encased in a membranous sheath of unknown function. While it has been hypothesized that the sheath may allow these bacteria to evade host responses to the immunogenic flagellin subunit, this unusual structural feature has remained an enigma. Here we demonstrate that the rotation of the sheathed flagellum in both the mutualist Vibrio fischeri and the pathogen Vibrio cholerae promotes release of a potent bacteria-derived immunogen, lipopolysaccharide, found in the flagellar sheath. We further present a new role for the flagellar sheath in triggering, rather than circumventing, host immune responses in the model squid-vibrio symbiosis. Such an observation not only has implications for the study of bacterial pathogens with sheathed flagella, but also raises important biophysical questions of sheathed-flagellum function. DOI:

RevDate: 2017-02-24
CmpDate: 2014-10-13

McFall-Ngai M (2014)

Divining the essence of symbiosis: insights from the squid-vibrio model.

PLoS biology, 12(2):e1001783 pii:PBIOLOGY-D-13-04098.

Biology has a big elephant in the room. Researchers are learning that microorganisms are critical for every aspect of the biosphere's health. Even at the scale of our own bodies, we are discovering the unexpected necessity and daunting complexity of our microbial partners. How can we gain an understanding of the form and function of these "ecosystems" that are an individual animal? This essay explores how development of experimental model systems reveals basic principles that underpin the essence of symbiosis and, more specifically, how one symbiosis, the squid-vibrio association, provides insight into the persistent microbial colonization of animal epithelial surfaces.

RevDate: 2017-02-20
CmpDate: 2014-11-15

Soto W, Rivera FM, MK Nishiguchi (2014)

Ecological diversification of Vibrio fischeri serially passaged for 500 generations in novel squid host Euprymna tasmanica.

Microbial ecology, 67(3):700-721.

Vibrio fischeri isolated from Euprymna scolopes (Cephalopoda: Sepiolidae) was used to create 24 lines that were serially passaged through the non-native host Euprymna tasmanica for 500 generations. These derived lines were characterized for biofilm formation, swarming motility, carbon source utilization, and in vitro bioluminescence. Phenotypic assays were compared between "ES" (E. scolopes) and "ET" (E. tasmanica) V. fischeri wild isolates to determine if convergent evolution was apparent between E. tasmanica evolved lines and ET V. fischeri. Ecological diversification was observed in utilization of most carbon sources examined. Convergent evolution was evident in motility, biofilm formation, and select carbon sources displaying hyperpolymorphic usage in V. fischeri. Convergence in bioluminescence (a 2.5-fold increase in brightness) was collectively evident in the derived lines relative to the ancestor. However, dramatic changes in other properties--time points and cell densities of first light emission and maximal light output and emergence of a lag phase in growth curves of derived lines--suggest that increased light intensity per se was not the only important factor. Convergent evolution implies that gnotobiotic squid light organs subject colonizing V. fischeri to similar selection pressures. Adaptation to novel hosts appears to involve flexible microbial metabolism, establishment of biofilm and swarmer V. fischeri ecotypes, and complex changes in bioluminescence. Our data demonstrate that numerous alternate fitness optima or peaks are available to V. fischeri in host adaptive landscapes, where novel host squids serve as habitat islands. Thus, V. fischeri founder flushes occur during the initiation of light organ colonization that ultimately trigger founder effect diversification.

RevDate: 2017-02-20
CmpDate: 2013-12-20

Norsworthy AN, KL Visick (2013)

Gimme shelter: how Vibrio fischeri successfully navigates an animal's multiple environments.

Frontiers in microbiology, 4:356.

Bacteria successfully colonize distinct niches because they can sense and appropriately respond to a variety of environmental signals. Of particular interest is how a bacterium negotiates the multiple, complex environments posed during successful infection of an animal host. One tractable model system to study how a bacterium manages a host's multiple environments is the symbiotic relationship between the marine bacterium, Vibrio fischeri, and its squid host, Euprymna scolopes. V. fischeri encounters many different host surroundings ranging from initial contact with the squid to ultimate colonization of a specialized organ known as the light organ. For example, upon recognition of the squid, V. fischeri forms a biofilm aggregate outside the light organ that is required for efficient colonization. The bacteria then disperse from this biofilm to enter the organ, where they are exposed to nitric oxide, a molecule that can act as both a signal and an antimicrobial. After successfully managing this potentially hostile environment, V. fischeri cells finally establish their niche in the deep crypts of the light organ where the bacteria bioluminesce in a pheromone-dependent fashion, a phenotype that E. scolopes utilizes for anti-predation purposes. The mechanism by which V. fischeri manages these environments to outcompete all other bacterial species for colonization of E. scolopes is an important and intriguing question that will permit valuable insights into how a bacterium successfully associates with a host. This review focuses on specific molecular pathways that allow V. fischeri to establish this exquisite bacteria-host interaction.

RevDate: 2017-07-18
CmpDate: 2014-09-30

Studer SV, Schwartzman JA, Ho JS, et al (2014)

Non-native acylated homoserine lactones reveal that LuxIR quorum sensing promotes symbiont stability.

Environmental microbiology, 16(8):2623-2634.

Quorum sensing, a group behaviour coordinated by a diffusible pheromone signal and a cognate receptor, is typical of bacteria that form symbioses with plants and animals. LuxIR-type N-acyl L-homoserine (AHL) quorum sensing is common in Gram-negative Proteobacteria, and many members of this group have additional quorum-sensing networks. The bioluminescent symbiont Vibrio fischeri encodes two AHL signal synthases: AinS and LuxI. AinS-dependent quorum sensing converges with LuxI-dependent quorum sensing at the LuxR regulatory element. Both AinS- and LuxI-mediated signalling are required for efficient and persistent colonization of the squid host, Euprymna scolopes. The basis of the mutualism is symbiont bioluminescence, which is regulated by both LuxI- and AinS-dependent quorum sensing, and is essential for maintaining a colonization of the host. Here, we used chemical and genetic approaches to probe the dynamics of LuxI- and AinS-mediated regulation of bioluminescence during symbiosis. We demonstrate that both native AHLs and non-native AHL analogues can be used to non-invasively and specifically modulate induction of symbiotic bioluminescence via LuxI-dependent quorum sensing. Our data suggest that the first day of colonization, during which symbiont bioluminescence is induced by LuxIR, is a critical period that determines the stability of the V. fischeri population once symbiosis is established.

RevDate: 2017-02-20
CmpDate: 2014-09-04

Peyer SM, Pankey MS, Oakley TH, et al (2014)

Eye-specification genes in the bacterial light organ of the bobtail squid Euprymna scolopes, and their expression in response to symbiont cues.

Mechanisms of development, 131:111-126.

The squid Euprymna scolopes has evolved independent sets of tissues capable of light detection, including a complex eye and a photophore or 'light organ', which houses the luminous bacterial symbiont Vibrio fischeri. As the eye and light organ originate from different embryonic tissues, we examined whether the eye-specification genes, pax6, eya, six, and dac, are shared by these two organs, and if so, whether they are regulated in the light organ by symbiosis. We obtained sequences of the four genes with PCR, confirmed orthology with phylogenetic analysis, and determined that each was expressed in the eye and light organ. With in situ hybridization (ISH), we localized the gene transcripts in developing embryos, comparing the patterns of expression in the two organs. The four transcripts localized to similar tissues, including those associated with the visual system ∼1/4 into embryogenesis (Naef stage 18) and the light organ ∼3/4 into embryogenesis (Naef stage 26). We used ISH and quantitative real-time PCR to examine transcript expression and differential regulation in postembryonic light organs in response to the following colonization conditions: wild-type, luminescent V. fischeri; a mutant strain defective in light production; and as a control, no symbiont. In ISH experiments light organs showed down regulation of the pax6, eya, and six transcripts in response to wild-type V. fischeri. Mutant strains also induced down regulation of the pax6 and eya transcripts, but not of the six transcript. Thus, luminescence was required for down regulation of the six transcript. We discuss these results in the context of symbiont-induced light-organ development. Our study indicates that the eye-specification genes are expressed in light-interacting tissues independent of their embryonic origin and are capable of responding to bacterial cues. These results offer evidence for evolutionary tinkering or the recruitment of eye development genes for use in a light-sensing photophore.

RevDate: 2017-02-20
CmpDate: 2013-10-24

Nakaguchi Y (2013)

Contamination by Vibrio parahaemolyticus and Its Virulent Strains in Seafood Marketed in Thailand, Vietnam, Malaysia, and Indonesia.

Tropical medicine and health, 41(3):95-102.

Infections by virulent strains of Vibrio parahaemolyticus are frequently reported in Southeast Asia. This is due to the frequent seafood contamination by virulent strains. In this study conducted from 2008 to 2011, seafood like fish, shrimp, squid, crab, and molluscan shellfish were purchased from provinces in Thailand and three Southeast Asian countries and examined for the prevalence of three genetic markers of V. parahaemolyticus (species-specific gene: toxR gene, virulence genes: tdh and trh genes). An enrichment culture of seafood was examined for these markers using PCR methods. Molluscan shellfish showed a high frequency of contamination in Thailand. The shellfish harvested from the Gulf of Thailand were significantly more contaminated with virulence genes than those from the Andaman Sea. The seafood purchased from three Southeast Asian countries was positive for the three markers of V. parahaemolytcus at differing frequencies. The virulence markers (tdh and trh markers) were frequently detected in molluscan shellfish from Vietnam (17.9 and 8.0%, respectively), Malaysia (11.1 and 16.7%), and Indonesia (9.1 and 13.6%). These data suggest that the molluscan shellfish sold in Southeast Asian markets are highly contaminated with virulent strains of V. parahaemolyticus.

RevDate: 2017-02-20
CmpDate: 2014-06-23

Koch EJ, Miyashiro T, McFall-Ngai MJ, et al (2014)

Features governing symbiont persistence in the squid-vibrio association.

Molecular ecology, 23(6):1624-1634.

Experimental studies of the interaction between host and symbiont in a maturing symbiotic organ have presented a challenge for most animal-bacterial associations. Advances in the rearing of the host squid Euprymna scolopes have enabled us to explore the relationship between a defect in symbiont light production and late-stage development (e.g. symbiont persistence and tissue morphogenesis) by experimental colonization with specific strains of the symbiont Vibrio fischeri. During the first 4 weeks postinoculation of juvenile squid, the population of wild-type V. fischeri increased 100-fold; in contrast, a strain defective in light production (Δlux) colonized normally the first day, but exhibited an exponential decline to undetectable levels over subsequent weeks. Co-colonization of organs by both strains affected neither the trajectory of colonization by wild type nor the decline of Δlux levels. Uninfected animals retained the ability to be colonized for at least 2 weeks posthatch. However, once colonized by the wild-type strain for 5 days, a subsequent experimentally induced loss of the symbionts could not be followed by a successful recolonization, indicating the host's entry into a refractory state. However, animals colonized by the Δlux before the loss of their symbionts were receptive to recolonization. Analyses of animals colonized with either a wild-type or a Δlux strain revealed slight, if any, differences in the developmental regression of the ciliated light-organ tissues that facilitate the colonization process. Thus, some other feature(s) of the Δlux strain's defect also may be responsible for its inability to persist, and its failure to induce a refractory state in the host.

RevDate: 2013-12-06
CmpDate: 2013-10-29

Cohen J (2013)

Great presenters: lighting up the auditorium.

Science (New York, N.Y.), 342(6154):78.

RevDate: 2017-02-20
CmpDate: 2014-03-10

Kåhrström CT (2013)

Symbiosis: breaking the ice with your host.

Nature reviews. Microbiology, 11(10):663.

RevDate: 2017-02-20
CmpDate: 2014-03-18

Verma SC, T Miyashiro (2013)

Quorum sensing in the squid-Vibrio symbiosis.

International journal of molecular sciences, 14(8):16386-16401 pii:ijms140816386.

Quorum sensing is an intercellular form of communication that bacteria use to coordinate group behaviors such as biofilm formation and the production of antibiotics and virulence factors. The term quorum sensing was originally coined to describe the mechanism underlying the onset of luminescence production in cultures of the marine bacterium Vibrio fischeri. Luminescence and, more generally, quorum sensing are important for V. fischeri to form a mutualistic symbiosis with the Hawaiian bobtail squid, Euprymna scolopes. The symbiosis is established when V. fischeri cells migrate via flagella-based motility from the surrounding seawater into a specialized structure injuvenile squid called the light organ. The cells grow to high cell densities within the light organ where the infection persists over the lifetime of the animal. A hallmark of a successful symbiosis is the luminescence produced by V. fischeri that camouflages the squid at night by eliminating its shadow within the water column. While the regulatory networks governing quorum sensing are critical for properly regulating V. fischeri luminescence within the squid light organ, they also regulate luminescence-independent processes during symbiosis. In this review, we discuss the quorum-sensing network of V. fischeri and highlight its impact at various stages during host colonization.

RevDate: 2017-02-20
CmpDate: 2014-03-10

Kremer N, Philipp EE, Carpentier MC, et al (2013)

Initial symbiont contact orchestrates host-organ-wide transcriptional changes that prime tissue colonization.

Cell host & microbe, 14(2):183-194.

Upon transit to colonization sites, bacteria often experience critical priming that prepares them for subsequent, specific interactions with the host; however, the underlying mechanisms are poorly described. During initiation of the symbiosis between the bacterium Vibrio fischeri and its squid host, which can be observed directly and in real time, approximately five V. fischeri cells aggregate along the mucociliary membranes of a superficial epithelium prior to entering host tissues. Here, we show that these few early host-associated symbionts specifically induce robust changes in host gene expression that are critical to subsequent colonization steps. This exquisitely sensitive response to the host's specific symbiotic partner includes the upregulation of a host endochitinase, whose activity hydrolyzes polymeric chitin in the mucus into chitobiose, thereby priming the symbiont and also producing a chemoattractant gradient that promotes V. fischeri migration into host tissues. Thus, the host responds transcriptionally upon initial symbiont contact, which facilitates subsequent colonization.

RevDate: 2016-10-19
CmpDate: 2014-03-10

Wernegreen JJ (2013)

First impressions in a glowing host-microbe partnership.

Cell host & microbe, 14(2):121-123.

Despite the clear significance of beneficial animal-microbe associations, mechanisms underlying their initiation and establishment are rarely understood. In this issue of Cell Host & Microbe, Kremer et al. (2013) reveal that first contact within the squid-vibrio symbiosis triggers profound molecular and chemical changes that are crucial for bacterial colonization.

RevDate: 2016-10-19
CmpDate: 2014-03-31

Brennan CA, Mandel MJ, Gyllborg MC, et al (2013)

Genetic determinants of swimming motility in the squid light-organ symbiont Vibrio fischeri.

MicrobiologyOpen, 2(4):576-594.

Bacterial flagellar motility is a complex cellular behavior required for the colonization of the light-emitting organ of the Hawaiian bobtail squid, Euprymna scolopes, by the beneficial bioluminescent symbiont Vibrio fischeri. We characterized the basis of this behavior by performing (i) a forward genetic screen to identify mutants defective in soft-agar motility, as well as (ii) a transcriptional analysis to determine the genes that are expressed downstream of the flagellar master regulator FlrA. Mutants with severe defects in soft-agar motility were identified due to insertions in genes with putative roles in flagellar motility and in genes that were unexpected, including those predicted to encode hypothetical proteins and cell division-related proteins. Analysis of mutants for their ability to enter into a productive symbiosis indicated that flagellar motility mutants are deficient, while chemotaxis mutants are able to colonize a subset of juvenile squid to light-producing levels. Thirty-three genes required for normal motility in soft agar were also downregulated in the absence of FlrA, suggesting they belong to the flagellar regulon of V. fischeri. Mutagenesis of putative paralogs of the flagellar motility genes motA, motB, and fliL revealed that motA1, motB1, and both fliL1 and fliL2, but not motA2 and motB2, likely contribute to soft-agar motility. Using these complementary approaches, we have characterized the genetic basis of flagellar motility in V. fischeri and furthered our understanding of the roles of flagellar motility and chemotaxis in colonization of the juvenile squid, including identifying 11 novel mutants unable to enter into a productive light-organ symbiosis.

RevDate: 2017-02-20
CmpDate: 2015-12-03

Altura MA, Heath-Heckman EA, Gillette A, et al (2013)

The first engagement of partners in the Euprymna scolopes-Vibrio fischeri symbiosis is a two-step process initiated by a few environmental symbiont cells.

Environmental microbiology, 15(11):2937-2950.

We studied the Euprymna scolopes-Vibrio fischeri symbiosis to characterize, in vivo and in real time, the transition between the bacterial partner's free-living and symbiotic life styles. Previous studies using high inocula demonstrated that environmental V. fischeri cells aggregate during a 3 h period in host-shed mucus along the light organ's superficial ciliated epithelia. Under lower inoculum conditions, similar to the levels of symbiont cells in the environment, this interaction induces haemocyte trafficking into these tissues. Here, in experiments simulating natural conditions, microscopy revealed that at 3 h following first exposure, only ∼ 5 V. fischeri cells aggregated on the organ surface. These cells associated with host cilia and induced haemocyte trafficking. Symbiont viability was essential and mutants defective in symbiosis initiation and/or production of certain surface features, including the Mam7 protein, which is implicated in host cell attachment of V. cholerae, associated normally with host cilia. Studies with exopolysaccharide mutants, which are defective in aggregation, suggest a two-step process of V. fischeri cell engagement: association with host cilia followed by aggregation, i.e. host cell-symbiont interaction with subsequent symbiont-symbiont cell interaction. Taken together, these data provide a new model of early partner engagement, a complex model of host-symbiont interaction with exquisite sensitivity.

RevDate: 2013-06-07
CmpDate: 2013-06-12

Pennisi E (2013)

Mysteries of development. How do microbes shape animal development?.

Science (New York, N.Y.), 340(6137):1159-1160.

RevDate: 2017-02-20
CmpDate: 2013-08-16

Kåhrström CT (2013)

Symbiosis: Bacteria seize control of the clock.

Nature reviews. Microbiology, 11(6):362.

RevDate: 2017-02-20
CmpDate: 2013-06-04

Kostic AD, Howitt MR, WS Garrett (2013)

Exploring host-microbiota interactions in animal models and humans.

Genes & development, 27(7):701-718.

The animal and bacterial kingdoms have coevolved and coadapted in response to environmental selective pressures over hundreds of millions of years. The meta'omics revolution in both sequencing and its analytic pipelines is fostering an explosion of interest in how the gut microbiome impacts physiology and propensity to disease. Gut microbiome studies are inherently interdisciplinary, drawing on approaches and technical skill sets from the biomedical sciences, ecology, and computational biology. Central to unraveling the complex biology of environment, genetics, and microbiome interaction in human health and disease is a deeper understanding of the symbiosis between animals and bacteria. Experimental model systems, including mice, fish, insects, and the Hawaiian bobtail squid, continue to provide critical insight into how host-microbiota homeostasis is constructed and maintained. Here we consider how model systems are influencing current understanding of host-microbiota interactions and explore recent human microbiome studies.

RevDate: 2016-10-19
CmpDate: 2013-09-30

Morris AR, KL Visick (2013)

Inhibition of SypG-induced biofilms and host colonization by the negative regulator SypE in Vibrio fischeri.

PloS one, 8(3):e60076.

Vibrio fischeri produces a specific biofilm to promote colonization of its eukaryotic host, the squid Euprymna scolopes. Formation of this biofilm is induced by the sensor kinase RscS, which functions upstream of the response regulator SypG to regulate transcription of the symbiosis polysaccharide (syp) locus. Biofilm formation is also controlled by SypE, a multi-domain response regulator that consists of a central regulatory receiver (REC) domain flanked by an N-terminal serine kinase domain and a C-terminal serine phosphatase domain. SypE permits biofilm formation under rscS overexpression conditions, but inhibits biofilms induced by overexpression of sypG. We previously investigated the function of SypE in controlling biofilm formation induced by RscS. Here, we examined the molecular mechanism by which SypE naturally inhibits SypG-induced biofilms. We found that SypE's N-terminal kinase domain was both required and sufficient to inhibit SypG-induced biofilms. This effect did not occur at the level of syp transcription. Instead, under sypG-overexpressing conditions, SypE inhibited biofilms by promoting the phosphorylation of another syp regulator, SypA, a putative anti-sigma factor antagonist. Inhibition by SypE of SypG-induced biofilm formation could be overcome by the expression of a non-phosphorylatable SypA mutant, indicating that SypE functions primarily if not exclusively to control SypA activity via phosphorylation. Finally, the presence of SypE was detrimental to colonization under sypG-overexpressing conditions, as cells deleted for sypE outcompeted wild-type cells for colonization when both strains overexpressed sypG. These results provide further evidence that biofilm formation is critical to symbiotic colonization, and support a model in which SypE naturally functions to restrict biofilm formation, and thus host colonization, to the appropriate environmental conditions.


ESP Quick Facts

ESP Origins

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.

ESP Support

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.

ESP Rationale

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.

ESP Goal

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.

ESP Usage

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.

ESP Content

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.

ESP Help

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.

ESP Plans

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.

Electronic Scholarly Publishing
21454 NE 143rd Street
Woodinville, WA 98077

E-mail: RJR8222 @

Papers in Classical Genetics

The ESP began as an effort to share a handful of key papers from the early days of classical genetics. Now the collection has grown to include hundreds of papers, in full-text format.

Digital Books

Along with papers on classical genetics, ESP offers a collection of full-text digital books, including many works by Darwin (and even a collection of poetry — Chicago Poems by Carl Sandburg).


ESP now offers a much improved and expanded collection of timelines, designed to give the user choice over subject matter and dates.


Biographical information about many key scientists.

Selected Bibliographies

Bibliographies on several topics of potential interest to the ESP community are now being automatically maintained and generated on the ESP site.

ESP Picks from Around the Web (updated 07 JUL 2018 )