The effect of thermal stress on beneficial symbiosis, in the face of rapid climate change, remains poorly understood. We investigate this using the model system,Euprymna scolopes(Es), the Hawaiian bobtail squid, and its bioluminescent symbiont,Vibrio fischeri(Vf), which enables the squid to camouflage itself through counter-illumination. Successful colonization of the squid by Vf must occur hours after hatching and is mediated by fluid flow due to respiration within the squid mantle cavity. To study this process, we develop a mathematical model using the method of regularized stokeslets to simulate the flow and resulting bacterial trajectories within the squid. We explore how thermal stress, mediated by physiological changes in respiration, ciliary dynamics and internal geometry, affects early colonization by analysing the time bacteria spend in regions crucial to the establishment of symbioses in these simulations. A variance-based sensitivity analysis of physiologically relevant parameters on these metrics demonstrated that changes in the breath cycle significantly affect and reduce the time bacteria spend in the critical zone within the squid, hindering colonization.

Recent studies have shown that symbiotic bacteria can have drastic effects on host neurobiology, but few simple, accessible models currently exist in which to study these interactions. Hawaiian bobtail squid ( Euprymna scolopes ) participate in a binary symbiosis with the bacterium Vibrio fischeri , a population of which resides in a specialized hindgut-derived organ called the light organ. Upon colonization by V. fischeri , the light organ undergoes transcriptional changes that suggest neurons are among the cell types impacted by the initiation of symbiosis, but the nascent light organ's innervation has remained uncharacterized. The light organ-associated nervous system (LONS) in hatchling E. scolopes is a remarkably complex segment of the peripheral nervous system. The LONS is largely plexiform and originates from two primary nerves connected by a local commissure. The abundance of synapsin-like immunoreactivity indicates that the lobe plexus is highly interconnected. We also highlight a small number of serotonin-like immunoreactive neurites innervating the anterior appendages that are poised to be directly impacted by symbiont-driven post-embryonic development. Finally, we present evidence that a limited but morphologically diverse population of neurons reside within the light organ and are often located near internal symbiont-interacting structures. Our results show that the LONS is structurally and molecularly complex and exhibits traits characteristic of an immature nervous system, suggesting that it may undergo substantial post-embryonic refinement. This initial characterization of the LONS provides a foundation from which to investigate how beneficial bacterial symbionts affect host peripheral neurobiology in a tractable model system.

All plants and animals form symbiotic associations with microbes, yet many of the underlying mechanisms associated with these interactions remain uncharacterized. There are inherent challenges to studying the cellular and metabolic interactions between eukaryotes and their microbial symbionts, thus new methodologies that enable the discovery of symbiotic processes are continually needed. Here, we explored the use of magnetic nanoparticles (MNPs) as a tool to track aspects of the host innate immune response to symbionts under both ex vivo and in vivo conditions. The symbiotic association between the Hawaiian bobtail squid Euprymna scolopes and its bioluminescent partner Vibrio fischeri was used as a model to explore the potential of MNPs as non-toxic, manipulable agents to investigate aquatic symbiotic associations. Results suggest that host cells can be effectively labeled with MNPs under ex vivo conditions and that the particles can be visualized and tracked within the host animal in vivo using magnetic particle imaging. Proteomic and metabolomic analyses also revealed minimal changes to the host innate immune cells after uptake of MNPs in the presence and absence of V. fischeri. Together, these results suggest that MNPs have minimal biochemical impact on the host cells and can serve as an effective tool to explore aquatic symbiotic interactions.

Bacterial outer membrane vesicles (OMVs) and the cargo they carry are increasingly recognized as a means of communication between microbial symbionts and the cells of their host. However, few studies have focused on the biochemical and molecular mechanisms underlying OMV signaling during symbiosis onset and development. We show here that SypC, an OMV protein of the bioluminescent symbiont Vibrio fischeri, is taken up by cells of the squid host Euprymna scolopes where it assumes a new function, i.e., the facilitation of symbiont-induced light-organ morphogenesis. SypC is a Wza-like outer membrane protein found in host-associated Vibrionaceae and is essential for V. fischeri biofilm formation. Colonization or direct treatment with V. fischeri OMVs triggers host development, which was reduced or delayed if the host is instead exposed to a ∆sypC mutant or ∆sypC OMVs. RNA-seq analyses comparing light organs colonized by either the mutant or its parent revealed differential expression of host genes associated with immune responses and tissue morphogenesis. In immunocytochemical imaging, SypC-bearing OMVs were taken up by the host's macrophage-like cells near the light-organ crypts, revealing the mechanism by which SypC travels through tissue to trigger morphogenesis. Taken together, the data provide evidence that in addition to its role in biofilm formation and colonization, SypC has a second function promoting the induction of symbiotic-tissue development. These findings provide a critical piece of a puzzle whereby a rich array of host and symbiont molecules work in concert to orchestrate normal symbiont colonization and host development within the first hours to days of symbiosis.

Animals typically form symbiotic relationships with bacteria that contribute to their physiology and behaviors. The ability to genetically modify these bacterial symbionts is important for investigating the molecular mechanisms that promote symbiosis establishment and maintenance. However, the molecular tools developed for laboratory-adapted strains may fail when applied to non-canonical strains. Here, we report a method to expand the use of Tn7 site-specific transposon-insertion mutagenesis in Vibrio fischeri, which is the bioluminescent bacterial symbiont of the Hawaiian bobtail squid Euprymna scolopes. In this protocol, the laboratory-adapted strain ES114 is used as a surrogate strain for introducing genetic information into the attTn7 insertion site. Genomic DNA extracted from the resulting strain is used as template for transformation of another strain, in which natural transformation is induced. As a proof of principle, this approach is used to complement an rpoN mutant with an IPTG-inducible rpoN construct in trans.

Many animals have multiple organs or tissues that are colonized by diverse microbiota. The female Hawaiian bobtail squid, Euprymna scolopes, has two organs with distinct symbiotic communities: the accessory nidamental gland (ANG) and the light organ (LO). The ANG hosts a bacterial consortium, whereas the LO has a binary relationship with Vibrio fischeri, housed in extracellular crypt spaces as part of the central core (CC). To understand how the host maintains distinct symbiotic communities, we used transcriptomics to identify immune-related genes that are uniquely and similarly expressed in the ANG and LO-CC compared to organs without a known microbiota. Genes such as peptidoglycan recognition proteins EsPGRP2 and EsPGRP3, cathepsin-Z, alkaline phosphatase, and acidic phospholipase exhibited significant upregulation in the symbiotic organs compared to other tissues like gills, skin, mantle, optic lobe, ovaries, and brain. Moreover, EsPGRP2 displayed distinct localization patterns within the ANG, inversely correlating with bacterial presence, whereas the protein was colocalized with V. fischeri in the LO-CC. Notably, 10 different galaxins (EsGal) were uniquely highly expressed in both the ANG and LO-CC, with EsGal1 messenger RNA predominantly localized to the LO-CC epithelium, while EsGal2 and EsGal3 were primarily found in the epithelia of ANG tubules. Furthermore, antimicrobial assays using partial peptides derived from EsGal1 and EsGal2 showed varying and distinct patterns of inhibitory activity for these peptides. In summary, our findings identify similar immune gene families expressed across functionally distinct symbiotic organs in E. scolopes, suggesting that common immunomodulatory factors may maintain distinct symbiotic niches in the host.

Cephalopods play a central ecological role across all oceans and depths. However, under the current climate crisis, their physiology and behaviour are impacted, and we are beginning to comprehend the effects of environmental stressors at a molecular level. Here, we study the Hawaiian bobtail squid (Euprymna scolopes), known for its specific binary symbiosis with the bioluminescent bacterium Vibrio fischeri acquired post-hatching. We aim to understand the response (i.e. developmental and molecular) of E. scolopes after embryogenetic exposure to different conditions: (i) standard conditions (control), (ii) increased CO2 (∆pH 0.4 units), (iii) warming (+3°C), or (iv) a combination of the two treatments. We observed a decrease in hatching success across all treatments relative to the control, and elevated temperature shortened the developmental time. Using transcriptomics, we identified modulation in metabolic pathways and energy production, at the expense of development under increased CO2. In addition to finding differentially expressed genes related to RNA editing, we also identified several splicing events linked to phenotypic plasticity in response to increased CO2 and temperature. The data also suggest that the initiation of the symbiosis may be negatively affected by these environmental drivers of change in the biosphere, although the animal may counter these via coping mechanisms.

Abstract A critical challenge in multifunctional room‐temperature phosphorescent (RTP) hydrogels is the balance between the persistent afterglow and the editable phosphorescent information. Inspired by the Hawaiian bobtail squid ( Euprymna scolopes ), which modulates bioluminescence through symbiotic bacterial storage and release, a biomimetic RTP hydrogel system is introduced that overcomes this limitation. The hydrogel, synthesized via polymerization of acrylic acid (AA) with diethylenetriamine (DETA), followed by Ca 2+ coordination with COO − in polyacrylic acid (PAA) chains, exhibits remarkable phosphorescence (afterglow > 6.5 s) and three synergistic functionalities: 1) Dynamic information re‐editability via water erasing and salt printing for real‐time information programming; 2) Thermomechanical responsiveness enabling reversible stiffness modulation and thermal concealment of photonic information; 3) Self‐healing through dynamic hydrogen bonding, facilitating dynamic reconfiguration of luminescent patterns. This RTP hydrogel establishes a robust platform for the edition of information, which emphasizes the potential of bioinspired strategies for advancing intelligent materials in bioimaging, flexible displayers, and information encryption.

D-glu is a key component of peptidoglycan (PG) and is essential for growth in most bacteria. To assess constraints on PG evolution and bacterial requirements for D-glu, we sought to artificially evolve PG biosynthesis, leading to either replacement of D-glu in the PG peptide or alternative pathways to D-glu incorporation. We previously found that suppression of D-glu auxotrophy in a murI racD mutant of Vibrio fischeri grown on lysogeny broth salts (LBS) medium was rare but could be accomplished by mutation of bsrF, with restoration of wild-type PG structure. Here, we selected nine additional prototrophic suppressors of the same murI racD mutant from 1010 colony-forming units (CFU) plated on LBS supplemented with ~2.7 mM D-gln. Each suppressor had a mutation in gltS, which encodes a putative sodium:glutamate symporter. Increased copy numbers of mutant gltS alleles enabled growth on unsupplemented LBS and resulted in PG containing D-glu. Examination of media components suggests that D-gln supplementation had inadvertently added ~14 μM D-glu, and that LBS itself contains ~1.4 μM D-glu. The mutations in gltS enabled growth with similarly low D-glu concentrations, but also increased sensitivity to homocysteic acid, suggesting more promiscuous transport. Surprisingly, we discovered that expression of mutant gltS in the auxotroph leads to incorporation of lysine into PG, in addition to canonical D-glu. When seawater is supplemented with D-glu, this V. fischeri mutant still colonized Euprymna scolopes and triggered PG-induced morphogenesis. Our results shed light on glutamate transport, highlight trade-offs in GltS structure and function, and reveal an unusual PG modification.IMPORTANCED-glu is an important building block in the peptidoglycan (PG) component of the bacterial cell wall, and its endogenous production is considered essential in most bacteria, even when grown in complex media. In Vibrio fischeri, in trans expression of mutant GltS symporters allows D-glu auxotrophic strains to grow on lysogeny broth salts (LBS) medium without exogenous D-glu, although there is a fitness trade-off of increased sensitivity to homocysteic acid. Our finding that LBS contains sufficient D-glu to support robust growth highlights the undervalued importance of D-amino acid transport and the ubiquity of D-amino acids. Moreover, the discovery of D-lysine in the PG peptide is an unusual PG modification that warrants further study.

Functional studies of host-microbe interactions benefit from natural model systems that enable the exploration of molecular mechanisms at the host-microbe interface. Bioluminescent Vibrio fischeri colonize the light organ of the Hawaiian bobtail squid, Euprymna scolopes, and this binary model has enabled advances in understanding host-microbe communication, colonization specificity, in vivo biofilms, intraspecific competition, and quorum sensing. The hummingbird bobtail squid, Euprymna berryi, can be generationally bred and maintained in lab settings and has had multiple genes deleted by CRISPR approaches. The prospect of expanding the utility of the light organ model system by producing multigenerational host lines led us to determine the extent to which the E. berryi light organ symbiosis parallels known processes in E. scolopes. However, the nature of the E. berryi light organ, including its microbial constituency and specificity for microbial partners, has not been examined. In this report, we isolated bacteria from E. berryi animals and tank water. Assays of bacterial behaviors required in the host, as well as host responses to bacterial colonization, illustrate largely parallel phenotypes in E. berryi and E. scolopes hatchlings. This study reveals E. berryi to be a valuable comparative model to complement studies in E. scolopes.IMPORTANCEMicrobiome studies have been substantially advanced by model systems that enable functional interrogation of the roles of the partners and the molecular communication between those partners. The Euprymna scolopes-Vibrio fischeri system has contributed foundational knowledge, revealing key roles for bacterial quorum sensing broadly and in animal hosts, for bacteria in stimulating animal development, for bacterial motility in accessing host sites, and for in vivo biofilm formation in development and specificity of an animal's microbiome. Euprymna berryi is a second bobtail squid host, and one that has recently been shown to be robust to laboratory husbandry and amenable to gene knockout. This study identifies E. berryi as a strong symbiosis model host due to features that are conserved with those of E. scolopes, which will enable the extension of functional studies in bobtail squid symbioses.

Growing Apart: Global Warming Severely Impacts the Symbiosis of the Hawaiian Bobtail Squid and Bioluminescent Bacteria.

The Vibrio fischeri—Euprymna scolopes symbiosis has become a powerful animal—microbe model system to examine the genetic underpinnings of symbiont development and regulation. Although there has been a number of elegant bacterial genetic technologies developed to examine this symbiosis, there is still a need to develop more sophisticated methodologies to better understand complex regulatory pathways that lie within the association. Therefore, we have developed a suite of CRISPR interference (CRISPRi) vectors for inducible repression of specific V. fischeri genes associated with symbiotic competence. The suite utilizes both Tn7-integrating and shuttle vector plasmids that allow for inducible expression of CRISPRi dCas9 protein along with single-guide RNAs (sgRNA) modules. We validated this CRISPRi tool suite by targeting both exogenous (an introduced mRFP reporter) and endogenous genes (luxC in the bioluminescence producing lux operon, and flrA, the major regulatory gene controlling flagella production). The suite includes shuttle vectors expressing both single and multiple sgRNAs complementary to the non-template strand of multiple targeted genetic loci, which were effective in inducible gene repression, with significant reductions in targeted gene expression levels. V. fischeri cells harboring a version of this system targeting the luxC gene and suppressing the production of luminescence were used to experimentally validate the hypothesis that continuous luminescence must be produced by the symbiont in order to maintain the symbiosis at time points longer than the known 24-h limit. This robust new CRISPRi genetic toolset has broad utility and will enhance the study of V. fischeri genes, bypassing the need for gene disruptions by standard techniques of allelic knockout-complementation-exchange and the ability to visualize symbiotic regulation in vivo.

Functional studies of host-microbe interactions benefit from natural model systems that enable exploration of molecular mechanisms at the host-microbe interface. Bioluminescent Vibrio fischeri colonize the light organ of the Hawaiian bobtail squid, Euprymna scolopes, and this binary model has enabled advances in understanding host-microbe communication, colonization specificity, in vivo biofilms, intraspecific competition, and quorum sensing. The hummingbird bobtail squid, Euprymna berryi, can be generationally bred and maintained in lab settings and has had multiple genes deleted by CRISPR approaches. The prospect of expanding the utility of the light organ model system by producing multigenerational host lines led us to determine the extent to which the E. berryi light organ symbiosis parallels known processes in E. scolopes. However, the nature of the E. berryi light organ, including its microbial constituency and specificity for microbial partners, have not been examined. In this report, we isolate bacteria from E. berryi animals and tank water. Assays of bacterial behaviors required in the host, as well as host responses to bacterial colonization, illustrate largely parallel phenotypes in E. berryi and E. scolopes hatchlings. This study reveals E. berryi to be a valuable comparative model to complement studies in E. scolopes.

Under the current climate crisis, marine heatwaves (MHW) are expected to intensify and become more frequent in the future, leading to adverse effects on marine life. Here, we aimed to investigate the impact of environmental warming on the symbiotic relationship between the Hawaiian bobtail squid (Euprymna scolopes) and the bioluminescent bacterium Vibrio fischeri. We exposed eggs of E. scolopes to three different temperatures during embryogenesis, namely: (i) 25°C (yearly average), (ii) 27°C (summer maximum) or (iii) 30°C (category IV MHW), followed by a colonisation assay under the same conditions. Decreased hatching success and reduced developmental time were observed across warmer conditions compared to 25°C. Moreover, exposure to the category IV MHW led to a significant decrease in survival after 48 h. With increasing temperature, bobtail squids required more bacteria in the surrounding seawater for successful colonisation. When colonised, the regression of the light organ's appendages was not dependent on temperature, but the opposite was found in non-colonised bobtail squids. Furthermore, the capacity for crypt 3 formation in the squid's light organ, which is crucial for enhancing resilience under stress, also declined with warming conditions. This study emphasises the critical need to study the dynamics of microbial symbiosis under the projected conditions for the ocean of tomorrow.

What makes a bacterium pathogenic? Since the early days of germ theory, researchers have categorized bacteria as pathogens or non-pathogens, those that cause harm and those that do not, but this binary view is not always accurate. Vibrio fischeri is an exclusive mutualistic symbiont found within the light organs of Hawaiian bobtail squid. This symbiotic interaction requires V. fischeri to utilize a range of behaviors and produce molecules that are often associated with pathogenicity. This juxtaposition of employing "pathogenic" behaviors for a symbiotic relationship led the field to focus on how V. fischeri establishes a beneficial association with its host. In this study, we observe that V. fischeri induces mortality in zebrafish embryos and Artemia nauplii. Non-lethal doses of V. fischeri lead to zebrafish growth delays and phenotypes indicative of disease. Our data also provide evidence that the conserved type VI secretion system on chromosome I (T6SS1) plays a role in the V. fischeri-induced mortality of zebrafish embryos and Artemia nauplii. These results support the hypothesis that the V. fischeri T6SS1 is involved in eukaryotic cell interactions. Despite its traditional view as a beneficial symbiont, we provide evidence that V. fischeri is capable of harming aquatic organisms, indicating its potential to be pathogenic toward non-symbiotic hosts.IMPORTANCEVibrio fischeri is best known for its beneficial partnership with the Hawaiian bobtail squid, where it uses molecular tools often associated with disease-causing bacteria. Our research shows that V. fischeri can also cause harm, killing zebrafish embryos and brine shrimp larvae. We pinpoint one of V. fischeri's two type VI secretion systems (T6SS1) as a key factor in this pathogenicity. These findings reveal that V. fischeri is not strictly a mutualistic microbe but can act like a pathogen under certain conditions. This broadens our understanding of how V. fischeri could interact with different hosts and offers new insights into the dual roles bacteria can play in nature.

Many plants and animals house symbiotic microorganisms in specialized tissues or organs. Here, we used multidimensional in situ imaging techniques to illuminate how host organ structure and bacterial microbiogeography contribute to the symbiotic function of an organ in the Hawaiian bobtail squid, Euprymna scolopes. Along with the well-studied light organ, female E. scolopes harbor a community of bacteria in the accessory nidamental gland (ANG). The ANG is a dense network of epithelium-lined tubules, some of which are dominated by a single bacterial taxon. These bacteria are deposited into squid eggs, where they defend the developing embryos from harmful biofouling. This study used a combination of imaging techniques to visualize different dimensions of the ANG and its bacterial communities. Imaging entire organs with light sheet microscopy revealed that the ANG is a composite tissue of individual, non-intersecting tubules that each harbor their own bacterial population. The organ is bisected, with tubules converging toward two points at the posterior end. At these points, tubules empty into a space where bacteria can mix with squid jelly to be deposited onto eggs. Observations of the symbiotic community correlated bacterial taxa with cell morphology and revealed that tubule populations varied: some tubules contained populations of mixed taxa, whereas others contained only one bacterial genus. Together, these data shed light on how bacterial populations interact within the ANG and how the host uses physical structure to maintain and employ a symbiotic bacterial population in a defensive context.IMPORTANCESequence-based microbiome studies have revealed much about how hosts interact with communities of symbiotic microbiota but often lack a spatial understanding of how microbes relate to each other and the host in which they reside. This study uses a combination of microscopy techniques to reveal how the structure of a symbiotic organ in the female bobtail squid, Euprymna scolopes, houses diverse, beneficial bacterial populations and deploys them for egg defense. These findings suggest that spatial partitioning may be key to harboring a diverse population of antimicrobial-producing bacteria and establishing a foundation for further understanding how host structures mediate symbiotic interactions.

Animals typically form symbiotic relationships with bacteria that contribute to their physiology and behaviors. The ability to genetically modify these bacterial symbionts is important for investigating the molecular mechanisms that promote symbiosis establishment and maintenance. However, the molecular tools developed for laboratory-adapted strains may fail when applied to non-canonical strains. Here, we report a method to expand the use of Tn7 site-specific transposon-insertion mutagenesis in Vibrio fischeri, which is the bioluminescent bacterial symbiont of the Hawaiian bobtail squid Euprymna scolopes. In this protocol, the laboratory-adapted strain ES114 is used as a surrogate strain for introducing genetic information into the attTn7 insertion site. Genomic DNA extracted from the resulting strain is used as template for transformation of another strain, in which natural transformation is induced. As a proof of principle, this approach is used to complement an rpoN mutant with an IPTG-inducible rpoN construct in trans.

Animals often acquire their microbial symbionts from the environment, but the mechanisms underlying how specificity of the association is achieved are poorly understood. We demonstrate that the conserved proton pump, V-type ATPase (VHA), plays a key role in the establishment of the model light-organ symbiosis between the squid Euprymna scolopes and its bacterial partner, Vibrio fischeri. Recruitment of V. fischeri from the surrounding seawater is mediated by juvenile-specific ciliated fields on the organ’s surface. These epithelia produce acidic mucus containing antimicrobials with low-pH optima, creating a chemical environment fostering specific recruitment of V. fischeri. We provide evidence that this critical acidic landscape is created by activity of VHA. VHA inhibition abolished epithelial-cell acidity, resulting in increased mucus pH and inefficient symbiont colonization. Thus, VHA provides a mechanistic link between host modulation of microenvironmental acidity, immune function, and selection of microbial symbionts, a strategy for specificity that may govern other symbioses.

What makes a bacterium pathogenic? Since the early days of germ theory, researchers have categorized bacteria as pathogens or non-pathogens, those that cause harm and those that do not, but this binary view is not always accurate. Vibrio fischeri is an exclusive mutualistic symbiont found within the light organs of Hawaiian bobtail squid. This symbiotic interaction requires V. fischeri to utilize a range of behaviors and produce molecules that are often associated with pathogenicity. This juxtaposition of employing “pathogenic” behaviors for a symbiotic relationship led the field to focus on how V. fischeri establishes a beneficial association with its host. In this study, we observe that V. fischeri induces mortality in zebrafish embryos and Artemia nauplii. Non-lethal doses of V. fischeri leads to zebrafish growth delays and phenotypes indicative of disease. Our data also provide evidence that the conserved type VI secretion system on chromosome I (T6SS1) plays a role in the V. fischeri-induced mortality of zebrafish embryos and Artemia nauplii. These results support the hypothesis that the V. fischeri T6SS1 is involved in eukaryotic cell interactions. Despite its traditional view as a beneficial symbiont, we provide evidence that V. fischeri is capable of harming aquatic organisms, indicating its potential to be pathogenic toward non-symbiotic hosts.

As bacterial symbionts transition from a motile free-living state to a sessile biofilm state, they must coordinate behavior changes suitable to each lifestyle. Cyclic diguanylate (c-di-GMP) is an intracellular signaling molecule that can regulate this transition, and it is synthesized by diguanylate cyclase (DGC) enzymes and degraded by phosphodiesterase (PDE) enzymes. Generally, c-di-GMP inhibits motility and promotes biofilm formation. While c-di-GMP and the enzymes that contribute to its metabolism have been well studied in pathogens, considerably less focus has been placed on c-di-GMP regulation in beneficial symbionts. Vibrio fischeri is the sole beneficial symbiont of the Hawaiian bobtail squid (Euprymna scolopes) light organ, and the bacterium requires both motility and biofilm formation to efficiently colonize. c-di-GMP regulates swimming motility and cellulose exopolysaccharide production in V. fischeri. The genome encodes 50 DGCs and PDEs, and while a few of these proteins have been characterized, the majority have not undergone comprehensive characterization. In this study, we use protein overexpression to systematically characterize the functional potential of all 50 V. fischeri proteins. All 28 predicted DGCs and 10 of the 14 predicted PDEs displayed at least one phenotype consistent with their predicted function, and a majority of each displayed multiple phenotypes. Finally, active site mutant analysis of proteins with the potential for both DGC and PDE activities revealed potential activities for these proteins. This work presents a systems-level functional analysis of a family of signaling proteins in a tractable animal symbiont and will inform future efforts to characterize the roles of individual proteins during lifestyle transitions.IMPORTANCECyclic diguanylate (c-di-GMP) is a critical second messenger that mediates bacterial behaviors, and Vibrio fischeri colonization of its Hawaiian bobtail squid host presents a tractable model in which to interrogate the role of c-di-GMP during animal colonization. This work provides systems-level characterization of the 50 proteins predicted to modulate c-di-GMP levels. By combining multiple assays, we generated a rich understanding of which proteins have the capacity to influence c-di-GMP levels and behaviors. Our functional approach yielded insights into how proteins with domains to both synthesize and degrade c-di-GMP may impact bacterial behaviors. Finally, we integrated published data to provide a broader picture of each of the 50 proteins analyzed. This study will inform future work to define specific pathways by which c-di-GMP regulates symbiotic behaviors and transitions.