Quorum sensing inhibits interference competition among bacterial symbionts within a host.

The symbioses that animals form with bacteria play important roles in health and disease, but the molecular details underlying how bacterial symbionts initially assemble within a host remain unclear.1,2,3 The bioluminescent bacterium Vibrio fischeri establishes a light-emitting symbiosis with the Hawaiian bobtail squid Euprymna scolopes by colonizing specific epithelium-lined crypt spaces within a symbiotic organ called the light organ.4 Competition for these colonization sites occurs between different strains of V. fischeri, with the lancet-like type VI secretion system (T6SS) facilitating strong competitive interference that results in strain incompatibility within a crypt space.5,6 Although recent studies have identified regulators of this T6SS, how the T6SS is controlled as symbionts assemble in vivo remains unknown.7,8 Here, we show that T6SS activity is suppressed by N-octanoyl-L-homoserine lactone (C8 HSL), which is a signaling molecule that facilitates quorum sensing in V. fischeri and is important for efficient symbiont assembly.9,10 We find that this signaling depends on the quorum-sensing regulator LitR, which lowers expression of the needle subunit Hcp, a key component of the T6SS, by repressing transcription of the T6SS regulator VasH. We show that LitR-dependent quorum sensing inhibits strain incompatibility within the squid light organ. Collectively, these results provide new insights into the mechanisms by which regulatory networks that promote symbiosis also control competition among symbionts, which in turn may affect the overall symbiont diversity that assembles within a host.

A case study assessing the impact of mating frequency on the reproductive performance of the Hawaiian bobtail squid Euprymna scolopes.

BACKGROUND: The symbiosis between the Hawaiian bobtail squid Euprymna scolopes and bacterium Vibrio fischeri serves as a model for investigating the molecular mechanisms that promote the initial formation of animal-bacterial symbioses. Research with this system frequently depends on freshly hatched E. scolopes, but the husbandry factors that promote hatchling production in a mariculture facility remain underreported. Here we report on the reproductive performance of E. scolopes in response to decreased mating frequency. RESULTS: One animal cohort was maintained in a mariculture facility for 107 days, with females assigned to either a control group (mating once every 14 days) or an experimental group (mating once every 21 days). No differences between the groups were observed in survival, the number of egg clutches laid, or hatchling counts. Each group featured multiple females that were hyper-reproductive, i.e., they generated more than 8 egg clutches while in captivity. Examination of the distributions for daily hatchling counts of individual egg clutches revealed significant variation in the hatching patterns among clutches that was independent of mating frequency. Finally, an assessment of hatchling production showed that 93.5% of total hatchlings produced by the cohort were derived from egg clutches laid within the first 70 days. CONCLUSIONS: These results suggest a lower mating frequency does not impede hatchling production. Furthermore, the variation in hatchling production among egg clutches provides new insight into the reproductive performance of E. scolopes as a lab animal for microbiology research.

Two enhancer binding proteins activate σ54-dependent transcription of a quorum regulatory RNA in a bacterial symbiont.

To colonize a host, bacteria depend on an ensemble of signaling systems to convert information about the various environments encountered within the host into specific cellular activities. How these signaling systems coordinate transitions between cellular states in vivo remains poorly understood. To address this knowledge gap, we investigated how the bacterial symbiont Vibrio fischeri initially colonizes the light organ of the Hawaiian bobtail squid Euprymna scolopes. Previous work has shown that the small RNA Qrr1, which is a regulatory component of the quorum-sensing system in V. fischeri, promotes host colonization. Here, we report that transcriptional activation of Qrr1 is inhibited by the sensor kinase BinK, which suppresses cellular aggregation by V. fischeri prior to light organ entry. We show that Qrr1 expression depends on the alternative sigma factor σ54 and the transcription factors LuxO and SypG, which function similar to an OR logic gate, thereby ensuring Qrr1 is expressed during colonization. Finally, we provide evidence that this regulatory mechanism is widespread throughout the Vibrionaceae family. Together, our work reveals how coordination between the signaling pathways underlying aggregation and quorum-sensing promotes host colonization, which provides insight into how integration among signaling systems facilitates complex processes in bacteria.

Maturation state of colonization sites promotes symbiotic resiliency in the Euprymna scolopes-Vibrio fischeri partnership.

BACKGROUND: Many animals and plants acquire their coevolved symbiotic partners shortly post-embryonic development. Thus, during embryogenesis, cellular features must be developed that will promote both symbiont colonization of the appropriate tissues, as well as persistence at those sites. While variation in the degree of maturation occurs in newborn tissues, little is unknown about how this variation influences the establishment and persistence of host-microbe associations. RESULTS: The binary symbiosis model, the squid-vibrio (Euprymna scolopes-Vibrio fischeri) system, offers a way to study how an environmental gram-negative bacterium establishes a beneficial, persistent, extracellular colonization of an animal host. Here, we show that bacterial symbionts occupy six different colonization sites in the light-emitting organ of the host that have both distinct morphologies and responses to antibiotic treatment. Vibrio fischeri was most resilient to antibiotic disturbance when contained within the smallest and least mature colonization sites. We show that this variability in crypt development at the time of hatching allows the immature sites to act as a symbiont reservoir that has the potential to reseed the more mature sites in the host organ when they have been cleared by antibiotic treatment. This strategy may produce an ecologically significant resiliency to the association. CONCLUSIONS: The data presented here provide evidence that the evolution of the squid-vibrio association has been selected for a nascent organ with a range of host tissue maturity at the onset of symbiosis. The resulting variation in physical and chemical environments results in a spectrum of host-symbiont interactions, notably, variation in susceptibility to environmental disturbance. This "insurance policy" provides resiliency to the symbiosis during the critical period of its early development. While differences in tissue maturity at birth have been documented in other animals, such as along the infant gut tract of mammals, the impact of this variation on host-microbiome interactions has not been studied. Because a wide variety of symbiosis characters are highly conserved over animal evolution, studies of the squid-vibrio association have the promise of providing insights into basic strategies that ensure successful bacterial passage between hosts in horizontally transmitted symbioses. Video Abstract.

The type-VI secretion system of the beneficial symbiont Vibrio fischeri.

The mutualistic symbiosis between the Hawaiian bobtail squid Euprymna scolopes and the marine bacterium Vibrio fischeri is a powerful experimental system for determining how intercellular interactions impact animal-bacterial associations. In nature, this symbiosis features multiple strains of V. fischeri within each adult animal, which indicates that different strains initially colonize each squid. Various studies have demonstrated that certain strains of V. fischeri possess a type-VI secretion system (T6SS), which can inhibit other strains from establishing symbiosis within the same host habitat. The T6SS is a bacterial melee weapon that enables a cell to kill adjacent cells by translocating toxic effectors via a lancet-like apparatus. This review describes the progress that has been made in understanding the factors that govern the structure and expression of the T6SS in V. fischeri and its effect on the symbiosis.

"Failure To Launch": Development of a Reproductive Organ Linked to Symbiotic Bacteria.

Developmental processes in animals are influenced by colonization and/or signaling from microbial symbionts. Here, we show that bacteria from the environment are linked to development of a symbiotic organ that houses a bacterial consortium in female Hawaiian bobtail squid, Euprymna scolopes. In addition to the well-characterized light organ association with the bioluminescent bacterium Vibrio fischeri, female E. scolopes house a simple bacterial community in a reproductive organ, the accessory nidamental gland (ANG). In order to understand the influences of bacteria on ANG development, squid were raised in the laboratory under conditions where exposure to environmental microorganisms was experimentally manipulated. Under conditions where hosts were exposed to depleted environmental bacteria, ANGs were completely absent or stunted, a result independent of the presence of the light organ symbiont V. fischeri. When squid were raised in the laboratory with substrate from the host's natural environment containing the native microbiota, normal ANG development was observed, and the bacterial communities were similar to wild-caught animals. Analysis of the bacterial communities from ANGs and substrates of wild-caught and laboratory-raised animals suggests that certain bacterial groups, namely, the Verrucomicrobia, are linked to ANG development. The ANG community composition was also experimentally manipulated. Squid raised with natural substrate supplemented with a specific ANG bacterial strain, Leisingera sp. JC1, had high proportions of this strain in the ANG, suggesting that once ANG development is initiated, specific strains can be introduced and subsequently colonize the organ. Overall, these data suggest that environmental bacteria are required for development of the ANG in E. scolopes. IMPORTANCE Microbiota have profound effects on animal and plant development. Hosts raised axenically or without symbionts often suffer negative outcomes resulting in developmental defects or reduced organ function. Using defined experimental conditions, we demonstrate that environmental bacteria are required for the formation of a female-specific symbiotic organ in the Hawaiian bobtail squid, Euprymna scolopes. Although nascent tissues from this organ that are involved with bacterial recruitment formed initially, the mature organ failed to develop and was absent or severely reduced in sexually mature animals that were not exposed to microbiota from the host's natural environment. This is the first example of complete organ development relying on exposure to symbiotic bacteria in an animal host. This study broadens the use of E. scolopes as a model organism for studying the influence of beneficial bacteria on animal development.

Ciliated epithelia are key elements in the recruitment of bacterial partners in the squid-vibrio symbiosis.

The Hawaiian bobtail squid, Euprymna scolopes, harvests its luminous symbiont, Vibrio fischeri, from the surrounding seawater within hours of hatching. During embryogenesis, the host animal develops a nascent light organ with ciliated fields on each lateral surface. We hypothesized that these fields function to increase the efficiency of symbiont colonization of host tissues. Within minutes of hatching from the egg, the host's ciliated fields shed copious amounts of mucus in a non-specific response to bacterial surface molecules, specifically peptidoglycan (PGN), from the bacterioplankton in the surrounding seawater. Experimental manipulation of the system provided evidence that nitric oxide in the mucus drives an increase in ciliary beat frequency (CBF), and exposure to even small numbers of V. fischeri cells for short periods resulted in an additional increase in CBF. These results indicate that the light-organ ciliated fields respond specifically, sensitively, and rapidly, to the presence of nonspecific PGN as well as symbiont cells in the ambient seawater. Notably, the study provides the first evidence that this induction of an increase in CBF occurs as part of a thus far undiscovered initial phase in colonization of the squid host by its symbiont, i.e., host recognition of V. fischeri cues in the environment within minutes. Using a biophysics-based mathematical analysis, we showed that this rapid induction of increased CBF, while accelerating bacterial advection, is unlikely to be signaled by V. fischeri cells interacting directly with the organ surface. These overall changes in CBF were shown to significantly impact the efficiency of V. fischeri colonization of the host organ. Further, once V. fischeri has fully colonized the host tissues, i.e., about 12-24 h after initial host-symbiont interactions, the symbionts drove an attenuation of mucus shedding from the ciliated fields, concomitant with an attenuation of the CBF. Taken together, these findings offer a window into the very first interactions of ciliated surfaces with their coevolved microbial partners.

Vibrio fischeri Possesses Xds and Dns Nucleases That Differentially Influence Phosphate Scavenging, Aggregation, Competence, and Symbiotic Colonization of Squid.

Cells of Vibrio fischeri colonize the light organ of Euprymna scolopes, providing the squid bioluminescence in exchange for nutrients and protection. The bacteria encounter DNA-rich mucus throughout their transition to a symbiotic lifestyle, leading us to hypothesize a role for nuclease activity in the colonization process. In support of this, we detected abundant extracellular nuclease activity in growing cells of V. fischeri. To discover the gene(s) responsible for this activity, we screened a V. fischeri transposon mutant library for nuclease-deficient strains. Interestingly, only one strain, whose transposon insertion mapped to nuclease gene VF_1451, showed complete loss of nuclease activity in our screens. A database search revealed that VF_1451 is homologous to the nuclease-encoding gene xds in Vibrio cholerae. However, V. fischeri strains lacking xds eventually revealed slight nuclease activity on plates after 72 h. This led us to hypothesize that a second secreted nuclease, identified through a database search as VF_0437, a homolog of V. cholerae dns, might be responsible for the residual nuclease activity. Here, we show that Xds and/or Dns are involved in essential aspects of V. fischeri biology, including natural transformation, aggregation, and phosphate scavenging. Furthermore, strains lacking either nuclease were outcompeted by the wild type for squid colonization. Understanding the specific role of nuclease activity in the squid colonization process represents an intriguing area of future research. IMPORTANCE From soil and water to host-associated secretions such as mucus, environments that bacteria inhabit are awash in DNA. Extracellular DNA (eDNA) is a nutritious resource that microbes dedicate significant energy to exploit. Calcium binds eDNA to promote cell-cell aggregation and horizontal gene transfer. eDNA hydrolysis impacts construction of and dispersal from biofilms. Strategies in which pathogens use nucleases to avoid phagocytosis or disseminate by degrading host secretions are well documented; significantly less is known about nucleases in mutualistic associations. This study describes the role of nucleases in the mutualism between V. fischeri and its squid host, Euprymna scolopes. We find that nuclease activity is an important determinant of colonization in V. fischeri, broadening our understanding of how microbes establish and maintain beneficial associations.

Prevalence and diversity of type VI secretion systems in a model beneficial symbiosis.

The type VI secretion system (T6SS) is widely distributed in diverse bacterial species and habitats where it is required for interbacterial competition and interactions with eukaryotic cells. Previous work described the role of a T6SS in the beneficial symbiont, Vibrio fischeri, during colonization of the light organ of Euprymna scolopes squid. However, the prevalence and diversity of T6SSs found within the distinct symbiotic structures of this model host have not yet been determined. Here, we analyzed 73 genomes of isolates from squid light organs and accessory nidamental glands (ANGs) and 178 reference genomes. We found that the majority of these bacterial symbionts encode diverse T6SSs from four distinct classes, and most share homology with T6SSs from more distantly related species, including pathogens of animals and humans. These findings indicate that T6SSs with shared evolutionary histories can be integrated into the cellular systems of host-associated bacteria with different effects on host health. Furthermore, we found that one T6SS in V. fischeri is located within a genomic island with high genomic plasticity. Five distinct genomic island genotypes were identified, suggesting this region encodes diverse functional potential that natural selection can act on. Finally, analysis of newly described T6SSs in roseobacter clade ANG isolates revealed a novel predicted protein that appears to be a fusion of the TssB-TssC sheath components. This work underscores the importance of studying T6SSs in diverse organisms and natural habitats to better understand how T6SSs promote the propagation of bacterial populations and impact host health.

A peptidoglycan-recognition protein orchestrates the first steps of symbiont recruitment in the squid-vibrio symbiosis.

In symbioses established through horizontal transmission, evolution has selected for mechanisms that promote the recruitment of symbionts from the environment. Using the binary association between the Hawaiian bobtail squid, Euprymna scolopes, and its symbiont, Vibrio fischeri, we explored the first step of symbiont enrichment around sites where V. fischeri cells will enter host tissues. Earlier studies of the system had shown that, within minutes of hatching in natural seawater, ciliated epithelia of the nascent symbiotic tissue secrete a layer of mucus in response to exposure to the cell-wall biomolecule peptidoglycan (PGN) from non-specific bacterioplankton. We hypothesized that a peptidoglycan recognition protein, EsPGRP4, is the receptor that mediates host mucus secretion by sensing the environmental PGN; earlier studies of this protein family had shown that this is the only member predicted to behave as a membrane receptor. Immunocytochemistry localized EsPGRP4 to the superficial ciliated fields of the juvenile organ. We found that production of EsPGRP4 increased over the first 48 h after hatching if the light organ remained uncolonized. When colonized by V. fischeri, the levels of the protein in light-organ tissue remained similar to that of hatchling organs. Pharmacologically curing the initially colonized light organ with antibiotics resulted in return of EsPGRP4 production to levels similar to light organs that had remained uncolonized since hatching. Furthermore, we found that preincubation of the tissues with an EsPGRP4 antibody decreased light organ mucus production and colonization. These findings provide evidence of an innate mechanism that underlies a crucial first step in the horizontal recruitment of bacterial symbionts.

High Levels of Cyclic Diguanylate Interfere with Beneficial Bacterial Colonization.

During colonization of the Hawaiian bobtail squid (Euprymna scolopes), Vibrio fischeri bacteria undergo a lifestyle transition from a planktonic motile state in the environment to a biofilm state in host mucus. Cyclic diguanylate (c-di-GMP) is a cytoplasmic signaling molecule that is important for regulating motility-biofilm transitions in many bacterial species. V. fischeri encodes 50 proteins predicted to synthesize and/or degrade c-di-GMP, but a role for c-di-GMP regulation during host colonization has not been investigated. We examined strains exhibiting either low or high levels of c-di-GMP during squid colonization and found that while a low-c-di-GMP strain had no colonization defect, a high c-di-GMP strain was severely impaired. Expression of a heterologous c-di-GMP phosphodiesterase restored colonization, demonstrating that the effect is due to high c-di-GMP levels. In the constitutive high-c-di-GMP state, colonizing V. fischeri exhibited reduced motility, altered biofilm aggregate morphology, and a regulatory interaction where transcription of one polysaccharide locus is inhibited by the presence of the other polysaccharide. Our results highlight the importance of proper c-di-GMP regulation during beneficial animal colonization, illustrate multiple pathways regulated by c-di-GMP in the host, and uncover an interplay of multiple exopolysaccharide systems in host-associated aggregates. IMPORTANCE There is substantial interest in studying cyclic diguanylate (c-di-GMP) in pathogenic and environmental bacteria, which has led to an accepted paradigm in which high c-di-GMP levels promote biofilm formation and reduce motility. However, considerably less focus has been placed on understanding how this compound contributes to beneficial colonization. Using the Vibrio fischeri-Hawaiian bobtail squid study system, we took advantage of recent genetic advances in the bacterium to modulate c-di-GMP levels and measure colonization and track c-di-GMP phenotypes in a symbiotic interaction. Studies in the animal host revealed a c-di-GMP-dependent genetic interaction between two distinct biofilm polysaccharides, Syp and cellulose, that was not evident in culture-based studies: elevated c-di-GMP altered the composition and abundance of the in vivo biofilm by decreasing syp transcription due to increased cellulose synthesis. This study reveals important parallels between pathogenic and beneficial colonization and additionally identifies c-di-GMP-dependent regulation that occurs specifically in the squid host.

Impact of transit time on the reproductive capacity of Euprymna scolopes as a laboratory animal.

BACKGROUND: The Hawaiian bobtail squid Euprymna scolopes hosts various marine bacterial symbionts, and these symbioses have served as models for the animal-microbe relationships that are important for host health. Within a light organ, E. scolopes harbors populations of the bacterium Vibrio fischeri, which produce low levels of bioluminescence that the squid uses for camouflage. The symbiosis is initially established after a juvenile squid hatches from its egg and acquires bacterial symbionts from the ambient marine environment. The relative ease with which a cohort of wild-caught E. scolopes can be maintained in a mariculture facility has facilitated over 3 decades of research involving juvenile squid. However, because E. scolopes is native to the Hawaiian archipelago, their transport from Hawaii to research facilities often represents a stress that has the potential to impact their physiology. RESULTS: Here, we describe animal survival and reproductive capacity associated with a cohort of squid assembled from two shipments with markedly different transit times. We found that the lower juvenile squid counts generated by animals with the longer transit time were not due to the discrepancy in shipment but instead to fewer female squid that produced egg clutches at an elevated rate, which we term hyper-reproductivity. We find that hyper-reproductive females were responsible for 58% of the egg clutches laid. CONCLUSIONS: The significance of these findings for E. scolopes biology and husbandry is discussed, thereby providing a platform for future investigation and further development of this cephalopod as a valuable lab animal for microbiology research.

Evidence of Genomic Diversification in a Natural Symbiotic Population Within Its Host.

Planktonic cells of the luminous marine bacterium Vibrio fischeri establish themselves in the light-emitting organ of each generation of newly hatched Euprymna scolopes bobtail squid. A symbiont population is maintained within the 6 separated crypts of the organ for the ∼9-month life of the host. In the wild, the initial colonization step is typically accomplished by a handful of planktonic V. fischeri cells, leading to a species-specific, but often multi-strain, symbiont population. Within a few hours, the inoculating cells proliferate within the organ's individual crypts, after which there is evidently no supernumerary colonization. Nevertheless, every day at dawn, the majority of the symbionts is expelled, and the regrowth of the remaining ∼5% of cells provides a daily opportunity for the population to evolve and diverge, thereby increasing its genomic diversity. To begin to understand the extent of this diversification, we characterized the light-organ population of an adult animal. First, we used 16S sequencing to determine that species in the V. fischeri clade were essentially the only ones detectable within a field-caught E. scolopes. Efforts to colonize the host with a minor species that appeared to be identified, V. litoralis, revealed that, although some cells could be imaged within the organ, they were <0.1% of the typical V. fischeri population, and did not persist. Next, we determined the genome sequences of seventy-two isolates from one side of the organ. While all these isolates were associated with one of three clusters of V. fischeri strains, there was considerable genomic diversity within this natural symbiotic population. Comparative analyses revealed a significant difference in both the number and the presence/absence of genes within each cluster; in contrast, there was little accumulation of single-nucleotide polymorphisms. These data suggest that, in nature, the light organ is colonized by a small number of V. fischeri strains that can undergo significant genetic diversification, including by horizontal-gene transfer, over the course of ∼1500 generations of growth in the organ. When the resulting population of symbionts is expelled into seawater, its genomic mix provides the genetic basis for selection during the subsequent environmental dispersal, and transmission to the next host.

Aquaculture production of hatchling Hawaiian Bobtail Squid (Euprymna scolopes) is negatively impacted by decreasing environmental microbiome diversity.

AIMS: The Hawaiian Bobtail Squid (Euprymna scolopes) is a model organism for investigating host-symbiont relationships. The current scientific focus is on the microbiome within E. scolopes, while very little is known about the microbiome of the tanks housing E. scolopes. We examined the hypothesis that bacterial communities and geochemistry within the squid tank environment correlate with the production of viable paralarval squid. METHODS AND RESULTS: Total DNA was extracted from sediment and filtered water samples from 'productive' squid cohorts with high embryonic survival and paralarval hatching, 'unproductive' cohorts with low embryonic survival and paralarval hatching. As a control total DNA was extracted from environmental marine locations where E. scolopes is indigenous. Comparative analysis of the bacterial communities by the 16S rRNA gene was performed using next generation sequencing. Thirty-eight differentially abundant genera were identified in the adult tank waters. The majority of the sequences represented unclassified, candidate or novel genera. The characterized genera included Aquicella, Woeseia and Ferruginibacter, with Hyphomicrobium and Rhizohapis were found to be more abundant in productive adult tank water. In addition, nitrate and pH covaried with productive cohorts, explaining 67% of the bacterial populations. The lower abundance of nitrate-reducing bacteria in unproductive adult tank water could explain detected elevated nitrate levels. CONCLUSIONS: We conclude that microbiome composition and water geochemistry can negatively affect E. scolopes reproductive physiology in closed tank systems, ultimately impacting host-microbe research using these animals. SIGNIFICANCE AND IMPACT OF STUDY: These results identify the tight relationship between the microbiome and geochemistry to E. scolopes. From this study, it may be possible to design probiotic counter-measures to improve aquaculture conditions for E. scolopes.

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

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

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

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

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

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

Control of Competence in Vibrio fischeri.

Vibrio species, including the squid symbiont Vibrio fischeri, become competent to take up DNA under specific conditions. For example, V. fischeri becomes competent when grown in the presence of chitin oligosaccharides or upon overproduction of the competence regulatory factor TfoX. While little is known about the regulatory pathway(s) that controls V. fischeri competence, this microbe encodes homologs of factors that control competence in the well-studied V. cholerae To further develop V. fischeri as a genetically tractable organism, we evaluated the roles of some of these competence homologs. Using TfoX-overproducing cells, we found that competence depends upon LitR, the homolog of V. cholerae master quorum-sensing and competence regulator HapR, and upon homologs of putative pilus genes that in V. cholerae facilitate DNA uptake. Disruption of genes for negative regulators upstream of LitR, namely, the LuxO protein and the small RNA (sRNA) Qrr1, resulted in increased transformation frequencies. Unlike LitR-controlled light production, however, competence did not vary with cell density under tfoX overexpression conditions. Analogous to the case with V. cholerae, the requirement for LitR could be suppressed by loss of the Dns nuclease. We also found a role for the putative competence regulator CytR. Finally, we determined that transformation frequencies varied depending on the TfoX-encoding plasmid, and we developed a new dual tfoX and litR overexpression construct that substantially increased the transformation frequency of a less genetically tractable strain. By advancing the ease of genetic manipulation of V. fischeri, these findings will facilitate the rapid discovery of genes involved in physiologically relevant processes, such as biofilm formation and host colonization.IMPORTANCE The ability of bacteria to take up DNA (competence) and incorporate foreign DNA into their genomes (transformation) permits them to rapidly evolve and gain new traits and/or acquire antibiotic resistances. It also facilitates laboratory-based investigations into mechanisms of specific phenotypes, such as those involved in host colonization. Vibrio fischeri has long been a model for symbiotic bacterium-host interactions as well as for other aspects of its physiology, such as bioluminescence and biofilm formation. Competence of V. fischeri can be readily induced upon overexpression of the competence factor TfoX. Relatively little is known about the V. fischeri competence pathway, although homologs of factors known to be important in V. cholerae competence exist. By probing the importance of putative competence factors that control transformation of V. fischeri, this work deepens our understanding of the competence process and advances our ability to genetically manipulate this important model organism.

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

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

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

The recent recognition that many symbioses exhibit daily rhythms has encouraged research into the partner dialogue that drives these biological oscillations. Here we characterized the pivotal role of the versatile cytokine macrophage migration inhibitory factor (MIF) in regulating a metabolic rhythm in the model light-organ symbiosis between Euprymna scolopes and Vibrio fischeri As the juvenile host matures, it develops complex daily rhythms characterized by profound changes in the association, from gene expression to behavior. One such rhythm is a diurnal shift in symbiont metabolism triggered by the periodic provision of a specific nutrient by the mature host: each night the symbionts catabolize chitin released from hemocytes (phagocytic immune cells) that traffic into the light-organ crypts, where the population of V. fischeri cells resides. Nocturnal migration of these macrophage-like cells, together with identification of an E. scolopes MIF (EsMIF) in the light-organ transcriptome, led us to ask whether EsMIF might be the gatekeeper controlling the periodic movement of the hemocytes. Western blots, ELISAs, and confocal immunocytochemistry showed EsMIF was at highest abundance in the light organ. Its concentration there was lowest at night, when hemocytes entered the crypts. EsMIF inhibited migration of isolated hemocytes, whereas exported bacterial products, including peptidoglycan derivatives and secreted chitin catabolites, induced migration. These results provide evidence that the nocturnal decrease in EsMIF concentration permits the hemocytes to be drawn into the crypts, delivering chitin. This nutritional function for a cytokine offers the basis for the diurnal rhythms underlying a dynamic symbiotic conversation.