Klebsiella pneumoniae makes up 85% of carbapenem-resistant Enterobacteriaceae, bacteria that have become an urgent threat to public health. K. pneumoniae is largely transmitted in healthcare settings, where inpatients and outpatients are often anesthetized with the widely used anesthetic induction agent propofol. Recent evidence obtained from rodent infection models indicates that propofol exposure can dramatically increase host susceptibility to microbial infections. Given that intensive care patients who are at a greater risk for K. pneumoniae lung infections are often given propofol during their hospitalization, we investigated the outcome of K. pneumoniae infections in mice briefly sedated with either propofol or ketamine/xylazine as a control. Propofol-sedated mice experienced more rapid dissemination from the lungs to secondary sites of infection, and their lungs exhibited more severe pathology. Based on these observations, we investigated bacterial factors involved in infection and dissemination in mice with propofol or ketamine/xylazine sedation using a high-throughput insertion sequencing (INSeq) approach. We identified numerous novel potential virulence factors together with previously identified gene products, confirming the validity of our screen. We further characterized a mutant lacking the phospholipid retrograde trafficking chaperone MlaC and found that the degree of mutant attenuation was dependent upon the sedation method. These results highlight the importance of sedative choice when studying hospital-acquired microbial infections and suggest that sedation can influence the outcome of K. pneumoniae infection and dissemination in animal models.

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.

The animal symbiont Vibrio fischeri has served as a model organism for molecular processes underlying bacterial group behaviors, including quorum sensing and biofilm development. Here, using a genetic approach to identify negative regulators of biofilm formation in V. fischeri, we identified a membrane-bound RNA-binding protein, RtmR (VF_2432), that acts as an inhibitor of the symbiosis polysaccharide (SYP) biofilm. Membrane localization of the protein seems to be required for protein stability, as truncation of the transmembrane helices led to an inability to detect the protein. The conserved RNP1 and RNP2 motifs in RtmR's cytoplasmic RNA recognition motif (RRM) domain are required for function, and we demonstrate binding to RNA substrates. Identification of RtmR RNA ligands was conducted with a CLIP-seq approach that revealed a large interactome. One transcript identified was that of the biofilm regulatory histidine kinase RscS. We found that RtmR biofilm inhibition depends on RscS activity and that RtmR negatively regulates levels of RscS. Overall, this work characterizes a novel type of bacterial RNA-binding protein.

ABSTRACTFunctional studies of host-microbe interactions benefit from natural model systems that enable exploration of molecular mechanisms at the host-microbe interface. BioluminescentVibrio fischericolonize 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 vivobiofilms, 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 theE. berryilight organ symbiosis parallels known processes inE. scolopes. However, the nature of theE. berryilight organ, including its microbial constituency and specificity for microbial partners, have not been examined. In this report, we isolate bacteria fromE. berryianimals and tank water. Assays of bacterial behaviors required in the host, as well as host responses to bacterial colonization, illustrate largely parallel phenotypes inE. berryiandE. scolopeshatchlings. This study revealsE. berryito be a valuable comparative model to complement studies inE. 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. TheEuprymna scolopes-Vibrio fischerisystem 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 forin vivobiofilm formation in development and specificity of an animal’s microbiome.Euprymna berryiis 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 identifiesE. berryias a strong symbiosis model host due to features that are conserved with those isE. scolopes, which will enable extension of functional studies in bobtail squid symbioses.

Microbial metabolomics studies are a common approach for identifying microbial strains that have a capacity to produce new chemistries both in vitro and in situ. A limitation to applying microbial metabolomics to the discovery of new chemical entities is the rediscovery of known compounds, or "known unknowns." One factor contributing to this rediscovery is that the majority of laboratories use one ionization source─electrospray ionization (ESI)─to conduct metabolomics studies. Although ESI is an efficient, widely adopted ionization method, its widespread use may contribute to the reidentification of known metabolites. Here, we present the use of a dielectric barrier discharge ionization (DBDI) for microbial metabolomics applications through the use of soft ionization chemical reaction in-transfer (SICRIT). Additionally, we compared SICRIT to ESI using two different Vibrio species: Vibrio fischeri, a symbiotic marine bacterium, and Vibrio cholerae, a pathogenic bacterium. Overall, we found that the SICRIT source ionizes a different set of metabolites than ESI, and it has the ability to ionize lipids more efficiently than ESI in the positive mode. This work highlights the value of using more than one ionization source for the detection of metabolites.

Vibrio fischeri is a model mutualist for studying molecular processes affecting microbial colonization of animal hosts. We present a detailed protocol for a barcode sequencing (BarSeq) approach that combines targeted gene deletion with short-read sequencing technology to enable studies of mixed bacterial populations. This protocol includes wet lab steps to plan and produce the deletions, approaches to scale up mutant generation, protocols to prepare and conduct the strain competition, library preparation for sequencing on an Illumina iSeq 100 instrument, and data analysis with the barseq python package. Aspects of this protocol could be readily adapted for tagging wild-type V. fischeri strains with a neutral barcode for examination of population dynamics or BarSeq analyses in other species. (c) 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Production of the erm-bar DNABasic Protocol 2: Generation of a targeted and barcoded deletion strain of V. fischeriAlternate Protocol: Parallel generation of multiple barcode-tagged V. fischeri deletion strainsBasic Protocol 3: Setting up mixed populations of barcode-tagged strainsBasic Protocol 4: Performing a competitive growth assayBasic Protocol 5: Amplicon library preparation and equimolar poolingBasic Protocol 6: Sequencing on Illumina iSeq 100Basic Protocol 7: BarSeq data analysis

Mobile clustered regularly interspaced palindromic repeats interference (Mobile-CRISPRi) is an established method for bacterial gene expression knockdown. The deactivated Cas9 protein and guide RNA are isopropyl β-D-1-thiogalactopyranoside inducible, and all components are integrated into the chromosome via Tn7 transposition. Here, we optimized methods specific for applying Mobile-CRISPRi in multiple Vibrio species.

Strain-level variation among host-associated bacteria often determines host range and the extent to which colonization is beneficial, benign, or pathogenic. Vibrio fischeri is a beneficial symbiont of the light organs of fish and squid with known strain-specific differences that impact host specificity, colonization efficiency, and interbacterial competition. Here, we describe how the conserved global regulator, H-NS, has a strain-specific impact on a critical colonization behavior: biofilm formation. We isolated a mutant of the fish symbiont V. fischeri MJ11 with a transposon insertion in the hns gene. This mutant formed sticky, moderately wrinkled colonies on LBS plates, a condition not known to induce biofilm in this species. A reconstructed hns mutant displayed the same wrinkled colony, which became smooth when hns was complemented in trans, indicating the hns disruption is causal for biofilm formation in MJ11. Transcriptomes revealed differential expression for the syp biofilm locus in the hns mutant, relative to the parent, suggesting biofilm may in part involve SYP polysaccharide. However, enhanced biofilm in the MJ11 hns mutant was not sufficient to allow colonization of a non-native squid host. Finally, moving the hns mutation into other V. fischeri strains, including the squid symbionts ES114 and ES401, and seawater isolate PP3, revealed strain-specific biofilm phenotypes: ES114 and ES401 hns mutants displayed minimal biofilm phenotypes while PP3 hns mutant colonies were more wrinkled than the MJ11 hns mutant. These findings together define H-NS as a novel regulator of V. fischeri symbiotic biofilm and demonstrate key strain specificity in that role. Importance:This work, which shows how H-NS has strain-specific impacts on biofilm in Vibrio fischeri, underscores the importance of studying multiple strains, even when examining highly conserved genes and functions. Our observation that knocking out a conserved regulator can result in a wide range of biofilm phenotypes, depending on the isolate, serves as a powerful reminder that strain-level variation is common and worthy of exploration. Indeed, uncovering the mechanisms of strain-specific phenotypic differences is essential to understand drivers of niche differentiation and bacterial evolution. Thus, it is important to carefully match the number and type of strains used in a study with the research question to accurately interpret and extrapolate the results beyond a single genotype. The additional work required for multi-strain studies is often worth the investment of time and resources, as it provides a broader view of the complexity of within-species diversity in microbial systems.

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.

Cyclic diguanylate (c-di-GMP) is a near-ubiquitous signaling molecule that regulates the motility-to-sessility transition in many bacterial species. Among the phenotypes influenced by c-di-GMP are biofilm formation, motility, cell cycle, and virulence. The hallmark phenotypes regulated by c-di-GMP-biofilm formation and motility-are key determinants of host-bacterial interactions. A large body of research has identified the roles of c-di-GMP in regulating phenotypes in culture. While numerous studies have investigated roles for c-di-GMP during the establishment and maintenance of pathogenic host-bacterial associations, considerably less attention has been devoted to defining the roles of c-di-GMP during beneficial and commensal associations. This review describes the known roles of c-di-GMP in regulating phenotypes that contribute to host colonization, with a focus on knowledge gaps and future prospects for examining c-di-GMP during beneficial colonization.

The symbiotic relationship between the bioluminescent bacterium Vibrio fischeri and the bobtail squid Euprymna scolopes serves as a valuable system to investigate bacterial growth and peptidoglycan (PG) synthesis within animal tissues. To better understand the growth dynamics of V. fischeri in the crypts of the light -emitting organ of its juvenile host, we showed that, after the daily dawn -triggered expulsion of most of the population, the remaining symbionts rapidly proliferate for similar to 6 h. At that point the population enters a period of extremely slow growth that continues throughout the night until the next dawn. Further, we found that PG synthesis by the symbionts decreases as they enter the slow -growing stage. Surprisingly, in contrast to the most mature crypts (i.e., Crypt 1) of juvenile animals, most of the symbiont cells in the least mature crypts (i.e., Crypt 3) were not expelled and, instead, remained in the slow -growing state throughout the day, with almost no cell division. Consistent with this observation, the expression of the gene encoding the PG -remodeling enzyme, L,D-transpeptidase (LdtA), was greatest during the slowly growing stage of Crypt 1 but, in contrast, remained continuously high in Crypt 3. Finally, deletion of the ldtA gene resulted in a symbiont that grew and survived normally in culture, but was increasingly defective in competing against its parent strain in the crypts. This result suggests that remodeling of the PG to generate additional 3-3 linkages contributes to the bacterium's fitness in the symbiosis, possibly in response to stresses encountered during the very slow -growing stage.

Microbial biofilms represent an important lifestyle for bacteria and are dynamic three-dimensional structures. Cyclic dimeric guanosine monophosphate (c-di-GMP) is a ubiquitous signaling molecule that is known to be tightly regulated with biofilm processes. While measurements of global levels of c-di-GMP have proven valuable toward understanding the genetic control of c-di-GMP production, there is a need for tools to observe the local changes of c-di-GMP production in biofilm processes. We have developed a label-free method for the direct detection of c-di-GMP in microbial colony biofilms using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). We applied this method to the enteric pathogen Vibrio cholerae, the marine symbiont V. fischeri, and the opportunistic pathogen Pseudomonas aeruginosa PA14 and detected spatial and temporal changes in c-di-GMP signal that accompanied genetic alterations in factors that synthesize and degrade the compound. We further demonstrated how this method can be simultaneously applied to detect additional metabolites of interest from a single sample.

CRISPRi (Clustered Regularly Interspaced Palindromic Repeats interference) is a gene knockdown method that uses a deactivated Cas9 protein (dCas9) that binds a specific gene target locus dictated by an encoded guide RNA (sgRNA) to block transcription. Mobile-CRISPRi is a suite of modular vectors that enable CRISPRi knockdowns in diverse bacteria by integrating IPTG-inducible dcas9 and sgRNA genes into the genome using Tn7 transposition. Here, we show that the Mobile-CRISPRi system functions robustly and specifically in multiple Vibrio species: Vibrio cholerae, Vibrio fischeri, Vibrio vulnificus, Vibrio parahaemolyticus, and Vibrio campbellii. We demonstrate efficacy by targeting both essential and non-essential genes that function to produce defined, measurable phenotypes: bioluminescence, quorum sensing, cell division, and growth arrest. We anticipate that Mobile-CRISPRi will be used in Vibrio species to systematically probe gene function and essentiality in various behaviors and native environments.IMPORTANCEThe genetic manipulation of bacterial genomes is an invaluable tool in experimental microbiology. The development of CRISPRi (Clustered Regularly Interspaced Palindromic Repeats interference) tools has revolutionized genetics in many organisms, including bacteria. Here, we optimized the use of Mobile-CRISPRi in five Vibrio species, each of which has significant impacts on marine environments and organisms that include squid, shrimp, shellfish, finfish, corals, and multiple of which pose direct threats to human health. The Mobile-CRISPRi technology is easily adaptable, moveable from strain to strain, and enables researchers to selectively turn off gene expression. Our experiments demonstrate Mobile-CRISPRi is effective and robust at repressing gene expression of both essential and non-essential genes in Vibrio species.

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.

ABSTRACTBeneficial microbial symbionts that are horizontally acquired by their animal hosts undergo a lifestyle transition from free-living in the environment to associating with host tissues. In the model symbiosis between the Hawaiian bobtail squid and its microbial symbiont Vibrio fischeri, one mechanism used to make this transition during host colonization is the formation of biofilm-like aggregates in host mucosa. Previous work identified factors that are sufficient to induce V. fischeri biofilm formation, yet much remains unknown regarding the breadth of target genes induced by these factors. Here, we probed two widely used in vitro models of biofilm formation to identify novel regulatory pathways in the squid symbiont V. fischeri ES114. We discovered a shared set of 232 genes that demonstrated similar patterns in expression in both models. These genes comprise multiple exopolysaccharide loci that are upregulated and flagellar motility genes that are downregulated, with a consistent decrease in measured swimming motility. Furthermore, we identified genes regulated downstream of the key sensor kinase RscS that are induced independent of the response regulator SypG. Our data suggest that transcriptional regulator VpsR plays a strong role in expression of at least a subset of these genes. Overall, this study adds to our understanding of the genes involved in V. fischeri biofilm regulation while revealing new regulatory pathways branching from previously characterized signaling networks.IMPORTANCEThe V. fischeri-squid system provides an opportunity to study biofilm development both in the animal host and in culture-based biofilm models that capture key aspects of in vivo signaling. In this work, we report the results of the transcriptomic profiling of two V. fischeri biofilm models followed by phenotypic validation and examination of novel signaling pathway architecture. Remarkable consistency between the models provides a strong basis for future studies using either approach or both. A subset of the factors identified by the approaches were validated in the work, and the body of transcriptomic data provides a number of leads for future studies in culture and during animal colonization.

ABSTRACT 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.

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The global pathogen Vibrio cholerae undergoes cycles of biofilm formation and dispersal in the environment and the human host. Little is understood about biofilm dispersal. Here, we show that MbaA, a periplasmic polyamine sensor, and PotD1, a polyamine importer, regulate V. cholerae biofilm dispersal. Spermidine, a commonly produced polyamine, drives V. cholerae dispersal, whereas norspermidine, an uncommon polyamine produced by vibrios, inhibits dispersal. Spermidine and norspermidine differ by one methylene group. Both polyamines control dispersal via MbaA detection in the periplasm and subsequent signal relay. Our results suggest that dispersal fails in the absence of PotD1 because endogenously produced norspermidine is not reimported, periplasmic norspermidine accumulates, and it stimulates MbaA signaling. These results suggest that V. cholerae uses MbaA to monitor environmental polyamines, blends of which potentially provide information about numbers of ‘self’ and ‘other’. This information is used to dictate whether or not to disperse from biofilms. Introduction Bacteria frequently colonize environmental habitats and infection sites by forming surface-attached multicellular communities called biofilms. Participating in the biofilm lifestyle allows bacteria to collectively acquire nutrients and resist threats (Flemming et al., 2016). By contrast, the individual free-swimming state allows bacteria to roam. The global pathogen Vibrio cholerae undergoes repeated rounds of clonal biofilm formation and disassembly, and both biofilm formation and biofilm exit are central to disease transmission as V. cholerae alternates between the marine niche and the human host (Conner et al., 2016; Gallego-Hernandez et al., 2020; Tamayo et al., 2010). The V. cholerae biofilm lifecycle occurs in three stages. First, a founder cell attaches to a substate; second, through cycles of growth, division, and extracellular matrix secretion, the biofilm matures; and finally, when the appropriate environmental conditions are detected, cells leave the biofilm through a process termed dispersal (Bridges et al., 2020). Bacterial cells liberated through dispersal can depart their current environment and repeat the lifecycle in a new niche, such as in a new host (Guilhen et al., 2017). In general, the initial stages of the biofilm lifecycle, from attachment through maturation, have been well studied in V. cholerae and other bacterial species, whereas the dispersal phase remains underexplored. Components underlying biofilm dispersal are beginning to be revealed. For example, in many bacterial species, the Lap system that is responsible for cell attachments during biofilm formation is also involved in dispersal (Boyd et al., 2012; Christensen et al., 2020; Gjermansen et al., 2005; Kitts et al., 2019; Newell et al., 2011). To uncover mechanisms controlling V. cholerae biofilm dispersal, we recently developed a brightfield microscopy assay that allows us to follow the entire biofilm lifecycle (Bridges and Bassler, 2019). We combined this assay with mutagenesis and high-content imaging to identify mutants that failed to properly disperse (Bridges et al., 2020). Our screen revealed genes encoding proteins that fall into three functional groups: signal transduction, matrix degradation, and cell motility. Our hypothesis is that these classes of components act in sequence to drive biofilm dispersal. First, the cues that induce dispersal are detected by the signal transduction proteins. Second, activation of matrix digestion components occurs. Finally, cell motility engages and permits cells to swim away from the disassembling biofilm. The majority of the genes identified in the screen encoded signal transduction proteins. Elsewhere, we characterized one of the signaling systems identified in the screen, a new two-component phospho-relay that we named DbfS-DbfR (for Dispersal of Biofilm Sensor and Dispersal of Biofilm Regulator) that controls biofilm dispersal (Bridges et al., 2020). Of the remaining signaling proteins identified in the screen, four are proteins involved in regulating production/degradation of, or responding to, the second messenger molecule cyclic diguanylate (c-di-GMP). c-di-GMP regulates biofilm formation in many bacteria including V. cholerae in which low c-di-GMP levels correlate with motility and high c-di-GMP levels promote surface attachment and matrix production, and thus, biofilm formation (Conner et al., 2017). c-di-GMP is synthesized by enzymes that contain catalytic GGDEF domains that have diguanylate cyclase activity (Conner et al., 2017). c-di-GMP is degraded by proteins with EAL or HD-GYP domains that possess phosphodiesterase activity. V. cholerae encodes >50 proteins harboring one or both of these domains, underscoring the global nature of c-di-GMP signaling in this pathogen. The activities of these enzymes are often regulated by environmental stimuli through ligand binding sensory domains; however, in most cases, the identities of ligands for GGDEF- and EAL-containing enzymes are unknown (Römling et al., 2013). Of the c-di-GMP regulatory proteins identified in our dispersal screen, only one, the hybrid GGDEF/EAL protein, MbaA, which has previously been shown to regulate V. cholerae biofilm formation (Bomchil et al., 2003), has a defined environmental stimulus: MbaA responds to the ubiquitous family of small molecules called polyamines, aliphatic cations containing two or more amine groups (Miller-Fleming et al., 2015). Our dispersal screen also identified PotD1, a periplasmic binding protein that mediates polyamine import and that has also been shown to control V. cholerae biofilm formation (Cockerell et al., 2014; McGinnis et al., 2009). Thus, identification of these two genes in our screen suggested that beyond roles in biofilm formation, polyamine detection could be central to biofilm dispersal. In the present work, we explore this new possibility. Polyamines are essential small molecules derived from amino acids and were likely produced by the last universal common ancestor (Michael, 2018; Miller-Fleming et al., 2015). Different organisms make distinct blends of polyamines (Michael, 2018), and they are involved in biological processes ranging from stress responses to signal transduction to protein synthesis to siderophore production (Karatan et al., 2005; McGinnis et al., 2009; Michael, 2018). In some cases, mechanisms underlying polyamine function are known. For example, the eukaryotic translation initiation factor 5A is modified with hypusine that is derived from the polyamine spermidine (Puleston et al., 2019). In most cases, however, the mechanisms by which polyamines control physiology remain mysterious (Miller-Fleming et al., 2015). Polyamines are known to be involved in biofilm development in many bacterial species (Hobley et al., 2014; Karatan and Michael, 2013; Michael, 2018; Nesse et al., 2015; Patel et al., 2006). Intriguingly, the biofilm alterations that occur in response to a given polyamine vary among species. In V. cholerae, norspermidine, a rare polyamine produced by Vibrionaceae and select other organisms, promotes biofilm formation (Karatan et al., 2005; Lee et al., 2009). In contrast, the nearly ubiquitous polyamine spermidine, which differs from norspermidine only by a single methylene group, is not produced at substantial levels by V. cholerae and it represses V. cholerae biofilm formation (Figure 1; Karatan et al., 2005; Lee et al., 2009; McGinnis et al., 2009). Likewise, the polyamine spermine, typically made by eukaryotes, also represses V. cholerae biofilm formation (Sobe et al., 2017). Both spermidine and spermine are abundant in the human intestine (Benamouzig et al., 1997; McEvoy and Hartley, 1975; Osborne and Seidel, 1990). These results have led to the hypothesis that V. cholerae detects norspermidine as a measure of ‘self’ and spermidine and spermine as measures of ‘other’ to assess the species composition of the vicinal community (Sobe et al., 2017; Wotanis et al., 2017). V. cholerae funnels that information internally to determine whether or not to make a biofilm (Sobe et al., 2017; Wotanis et al., 2017). V. cholerae produces intracellular norspermidine; however, norspermidine has not been detected in cell-free culture fluids of laboratory-grown strains (Parker et al., 2012). Thus, if norspermidine does indeed enable V. cholerae to take a census of ‘self,’ it is apparently not via a canonical quorum-sensing-type mechanism. Figure 1 Download asset Open asset Polyamine sensing in V. cholerae. Schematic showing the previously proposed polyamine detection and import mechanisms in V. cholerae. Norspermidine (Nspd, triangles) promotes biofilm formation and spermidine (Spd, circles) represses biofilm formation. The primarily eukaryotic polyamine spermine (not pictured) also signals through the NspS-MbaA pathway. See text for details. OM: outer membrane; IM: inner membrane. As mentioned above, information contained in extracellular polyamines is transduced internally by MbaA, which is embedded in the inner membrane. MbaA contains a periplasmic domain that interacts with the periplasmic binding protein NspS. nspS is located immediately upstream of mbaA in the chromosome (Cockerell et al., 2014; Karatan et al., 2005; Young et al., 2021). MbaA also possesses cytoplasmic GGDEF (SGDEF in MbaA) and EAL (EVL in MbaA) domains (Figure 1). Genetic evidence suggests that when NspS is bound to norspermidine, the complex associates with MbaA and biofilm formation is promoted (Cockerell et al., 2014). Specifically, NspS interaction with MbaA inhibits MbaA phosphodiesterase activity, leading to increased c-di-GMP levels, and in turn, elevated biofilm formation (Figure 1, left panel) (Cockerell et al., 2014). It is proposed that Apo-NspS and NspS bound to spermidine or spermine do not bind to MbaA, and thus, under this condition, biofilm formation is reduced (Cockerell et al., 2014; Sobe et al., 2017). In this case, MbaA phosphodiesterase activity is not inhibited, which leads to decreased c-di-GMP levels and repression of biofilm formation (Figure 1, right panel). Currently, it is not known whether or not MbaA has diguanylate cyclase activity. The purified MbaA cytoplasmic domain functions as a phosphodiesterase in vitro (Cockerell et al., 2014). In a proposed second regulatory mechanism, norspermidine and spermidine are thought to control biofilm formation via import through the inner membrane ABC transporter, PotABCD1 (McGinnis et al., 2009). Following internalization, via an undefined cytoplasmic mechanism, polyamines are proposed to modulate biofilm formation (Figure 1; McGinnis et al., 2009). This hypothesis was based on the finding that elimination of PotD1, which is required for import of norspermidine and spermidine, resulted in elevated biofilm formation. Importantly, the studies reporting the MbaA and PotD1 findings used quorum-sensing-deficient V. cholerae strains (Joelsson et al., 2006) that likely cannot disperse from biofilms. Furthermore, the end-point biofilm assays used could not differentiate between enhanced biofilm formation and the failure to disperse. Here, we combine real-time biofilm lifecycle measurements, mutagenesis, a reporter of cytoplasmic c-di-GMP levels, and measurements of intra- and extracellular polyamine concentrations to define the roles that norspermidine and spermidine play in controlling the V. cholerae biofilm lifecycle. In wildtype V. cholerae, exogenous norspermidine and spermidine inversely alter cytoplasmic c-di-GMP levels and they exert their effects through the NspS-MbaA circuit. Norspermidine promotes biofilm formation and suppresses biofilm dispersal. Spermidine represses biofilm formation and promotes biofilm dispersal. Both the MbaA SGDEF and EVL domains are required for polyamine control of the biofilm lifecycle. We provide evidence that MbaA synthesizes c-di-GMP in the presence of norspermidine and degrades c-di-GMP in the presence of spermidine and that is what drives V. cholerae to form and disperse from biofilms, respectively. When MbaA is absent, V. cholerae is unable to alter the biofilm lifecycle in response to extracellular polyamines. We demonstrate that polyamine internalization via PotD1 is not required to promote V. cholerae entrance or exit from biofilms, but rather, periplasmic detection of polyamines by MbaA is the key regulatory step. Specifically, our results suggest that the ΔpotD1 mutant fails to disperse because it is unable to reimport self-secreted norspermidine. The consequence is that excess periplasmic norspermidine accumulates, is detected by MbaA, and leads to production of c-di-GMP and suppression of biofilm dispersal. Collectively, our work reveals the mechanisms by which V. cholerae detects and transduces the information contained in polyamine signals into modulation of its biofilm lifecycle. We propose that the polyamine sensing mechanisms revealed in this study allow V. cholerae to distinguish relatives from competitors and potentially the presence of predators in the vicinity and, in response, modify its biofilm lifecycle to appropriately colonize territory or disperse from an existing community. Results The polyamine signaling proteins MbaA and PotD1 regulate biofilm dispersal by changing c-di-GMP levels Our combined mutagenesis-imaging screen identified the inner membrane polyamine sensor, MbaA, and the periplasmic polyamine binding protein PotD1 as essential for proper V. cholerae biofilm dispersal, motivating us to explore the mechanisms underlying these effects. To probe polyamine signaling across the full biofilm lifecycle, we used our established brightfield imaging assay. In the case of WT V. cholerae, peak biofilm biomass is reached at ~8–9 hr of growth, and subsequently, dispersal occurs, and is completed by ~12 hr post inoculation (Figure 2A, B). Deletion of mbaA caused a mild biofilm dispersal defect with no detectable difference in peak biofilm biomass compared to WT, a less than 1 hr delay in the onset of dispersal, and 27% biomass remaining at 16 hr (Figure 2A, B). Expression of mbaA from an ectopic locus in the ΔmbaA mutant complemented the biofilm dispersal defect (Figure 2—figure supplement 1A). Thus, MbaA is required for WT V. cholerae biofilm dispersal. The ΔpotD1 mutant exhibited a 60% greater peak biofilm biomass than WT and nearly all of the biomass remained at 16 hr, indicating that PotD1 both represses biofilm formation and promotes biofilm dispersal (Figure 2A, B). Introduction of potD1 at an ectopic locus in the ΔpotD1 mutant drove premature biofilm dispersal and reduced overall biofilm formation (Figure 2—figure supplement 1B). The differences in severity between the dispersal phenotypes of the ΔmbaA and ΔpotD1 mutants are noteworthy because these strains behaved similarly in the previous end-point biofilm formation assays (McGinnis et al., 2009). Figure 2 with 2 supplements see all Download asset Open asset Polyamine signaling regulates V. cholerae biofilm dispersal. (A) Representative images of the designated V. cholerae strains at 16 hr. (B) Quantitation of biofilm biomass over time measured by time-lapse microscopy for WT V. cholerae and the designated mutants. In all cases, N = 3 biological and N = 3 technical replicates, ± SD (shaded). a.u.: arbitrary unit. (C) Relative c-di-GMP reporter signals for the indicated strains. Values are expressed as the percentage difference relative to the WT strain. N = 4 biological replicates. Each black bar shows the sample mean. Unpaired t-tests were performed for statistical analysis, with p values denoted as *p<0.05; ***p<0.001. (D) The corresponding PvpsL-lux outputs for the strains and growth conditions in (B). For vpsL-lux measurements, N = 3 biological replicates, ± SD (shaded). RLU: relative light units. MbaA has previously been shown to possess phosphodiesterase activity in vitro (Cockerell et al., 2014), thus, we reasoned that MbaA functions by altering cytoplasmic c-di-GMP levels, the consequence of which is changes in expression of genes encoding biofilm components. As far as we are aware, no connection has yet been made between PotD1 and cytoplasmic c-di-GMP levels. To determine if the observed biofilm dispersal defects in the ΔmbaA and ΔpotD1 mutants track with altered c-di-GMP levels, we compared the relative cytoplasmic c-di-GMP levels in the WT, ΔmbaA, and ΔpotD1 strains. To do this, we employed a fluorescent reporter construct in which expression of turboRFP is controlled by two c-di-GMP-responsive riboswitches (Zhou et al., 2016). The TurboRFP signal is normalized to constitutively produced AmCyan encoded on the same plasmid. Previous studies have demonstrated a linear relationship between reporter output and c-di-GMP levels measured by mass spectrometry (Zhou et al., 2016). Indeed, the reporter showed that deletion of mbaA caused only a moderate increase in cytoplasmic c-di-GMP (8% higher than WT), while the ΔpotD1 mutant produced 39% more signal than WT (Figure 2C). Thus, MbaA, as expected, mediates changes in c-di-GMP levels, and moreover PotD1-mediated import of polyamines also influences cytoplasmic c-di-GMP concentrations. In V. cholerae, increased cytoplasmic c-di-GMP levels are associated with elevated extracellular matrix production (called VPS for vibrio polysaccharide) and, in turn, increased biofilm formation. Using a PvpsL-lux promoter fusion that reports on the major matrix biosynthetic operon, we previously showed that matrix gene expression decreases as cells transition from the biofilm to the planktonic state, suggesting that repression of matrix production genes correlates with biofilm dispersal. We wondered how the increased c-di-GMP levels present in the ΔmbaA and ΔpotD1 mutants impinged on vpsL expression. Using the vpsL-lux reporter, we found that the light production patterns mirrored the severities of the dispersal phenotypes and the magnitudes of changes in cytoplasmic c-di-GMP levels: the ΔmbaA mutant had a light production profile similar to WT, while the ΔpotD1 mutant produced 10-fold more light than WT throughout growth (Figure 2D). These results indicate that the ΔmbaA mutant makes normal levels and the ΔpotD1 mutant produces excess matrix. As mentioned in the Introduction, our screen revealed three additional genes in c-di-GMP signaling pathways involved in V. cholerae biofilm dispersal. While not the focus of the current work, we performed preliminary characterization by deleting these genes and making measurements of the mutants' biofilm lifecycle phenotypes, assessing them for changes in cytoplasmic c-di-GMP levels, and quantifying their matrix production profiles (Figure 2—figure supplement 2A–D). Norspermidine and spermidine inversely regulate V. cholerae biofilm dispersal Above, we show that polyamine signaling proteins are required for normal V. cholerae biofilm dispersal. Previous studies demonstrated that norspermidine and spermidine have opposing effects on biofilm formation in end-point assays (Karatan et al., 2005; McGinnis et al., 2009). We wondered if and how these two polyamines affect each stage of the biofilm lifecycle – biofilm formation and biofilm dispersal. To investigate their roles, we assayed the biofilm lifecycle in WT V. cholerae and our mutants following exogenous administration of norspermidine and spermidine, alone and in combination. Addition of 100 µM norspermidine strongly promoted biofilm formation and completely prevented biofilm dispersal while 100 µM spermidine dramatically reduced biofilm formation and promoted premature biofilm dispersal (Figure 3A, Video 1). vpsL-lux expression increased by >10-fold following norspermidine treatment and decreased >10-fold following spermidine treatment (Figure 3B). These results show that norspermidine and spermidine have opposing activities with regard to biofilm formation and dispersal: norspermidine drives biofilm formation by inducing matrix production, and this prevents biofilm dispersal and spermidine, by suppressing matrix production prevents biofilm formation and drives biofilm dispersal. Figure 3 with 1 supplement see all Download asset Open asset Periplasmic detection of polyamines controls V. cholerae biofilm dispersal. (A) Quantitation of biofilm biomass over time measured by time-lapse microscopy following addition of water (Ctrl), 100 µM norspermidine, or 100 µM spermidine to WT V. cholerae. (B) Light output from the PvpsL-lux reporter for the treatments in (A) over the growth curve. (C) c-di-GMP reporter output at the indicated polyamine concentrations for WT V. cholerae. Relative reporter signal (% difference) is displayed as a heatmap (teal and purple represent the lowest and highest reporter output, respectively). (D) As in (A) for the ΔmbaA mutant. (E) As in (C) for the ΔmbaA mutant. (F) As in (C) for the ΔnspS mutant. Biofilm biomass data are represented as means normalized to the peak biofilm biomass of the Ctrl condition. In all biofilm biomass measurements, N = 3 biological and N = 3 technical replicates, ± SD (shaded). a.u.: arbitrary unit. In vpsL-lux measurements, N = 3 biological replicates, ± SD (shaded). RLU: relative light units. For the c-di-GMP reporter assays, values are expressed as the percentage difference relative to the untreated WT strain, allowing comparisons to be made across all heatmaps in all figures in this article. The same color bar applies to all heatmaps in this article. For each condition, N = 3 biological replicates. Numerical values and associated SDs are available in Supplementary file 1. Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Representative time-lapse images of the biofilm lifecycles of the WT, ΔmbaA, ΔpotD1, and ΔmbaA ΔpotD1 V. cholerae strains following treatment with water (Ctrl), 100 µM norspermidine (Nspd), or 100 µM spermidine (Spd). We wondered by what mechanism the information encoded in exogenous polyamines was translated into alterations in matrix gene expression and subsequent changes in biofilm dispersal. We reasoned that MbaA and/or PotD1-mediated changes in cytoplasmic c-di-GMP levels could be responsible. To test this possibility, we assessed how changes in cytoplasmic c-di-GMP levels tracked with changes in extracellular polyamine levels by measuring the c-di-GMP reporter output in response to supplied mixtures of norspermidine and spermidine (Figure 3C). In the heatmaps, teal and purple represent the lowest and highest values of reporter output, respectively. When provided alone and above a concentration of 10 µM, norspermidine and spermidine strongly increased and decreased, respectively, c-di-GMP reporter activity. At the limit, relative to the WT, norspermidine increased the c-di-GMP reporter output by ~60% while spermidine reduced reporter output by ~25%. Notably, consistent with previous end-point biofilm assays, when norspermidine was present above 50 µM, it overrode the effect of exogenous addition of spermidine, irrespective of spermidine concentration (Figure 3C; McGinnis et al., 2009). Thus, V. cholerae cytoplasmic c-di-GMP levels are responsive to extracellular blends of polyamines. When norspermidine is abundant, V. cholerae produces high levels of c-di-GMP, matrix expression is increased, and biofilms do not disperse. In contrast, when spermidine is abundant and norspermidine is absent or present below a threshold concentration, V. cholerae c-di-GMP levels drop, matrix production is repressed, and biofilm dispersal occurs. MbaA transduces external polyamine information internally to control biofilm dispersal Our data show that both the MbaA periplasmic polyamine sensor and the PotD1 polyamine importer are required for biofilm dispersal. Our next goal was to distinguish the contribution of periplasmic detection from cytoplasmic import of polyamines to the biofilm lifecycle. We began with periplasmic polyamine detection, mediated by NspS together with MbaA. We supplied norspermidine or spermidine to the ΔmbaA mutant and monitored biofilm biomass over time. To our surprise, the ΔmbaA mutant was impervious to the addition of either polyamine as both polyamines caused only a very modest reduction in overall biofilm biomass, and dispersal timing resembled the untreated ΔmbaA control (Figure 3D, Video 1). Consistent with this result, titration of the polyamines alone and in combination onto the ΔmbaA strain carrying the c-di-GMP reporter did not substantially alter reporter output (Figure 3E). These results show that the dynamic response of WT to polyamines requires MbaA. We suspect that the minor reduction in biofilm production that occurred in the ΔmbaA mutant when supplied polyamines is due to non-specific effects, perhaps via interaction of polyamines with negatively charged matrix components. We next investigated the role of the polyamine periplasmic binding protein NspS that transmits polyamine information to MbaA. In the absence of its partner polyamine binding protein, NspS, MbaA is thought to function as a constitutive phosphodiesterase (Cockerell et al., 2014). Consistent with this model, deletion of nspS in an otherwise WT strain reduced overall peak biofilm biomass by 65% and dispersal initiated 4 hr prior to when dispersal begins in the WT (Figure 3—figure supplement 1). In the ΔnspS mutant, c-di-GMP levels were lower than in the WT as judged by the c-di-GMP reporter (Figure 3F), showing that MbaA is locked as a constitutive phosphodiesterase. Exogenous addition of polyamines had no effect on c-di-GMP levels (Figure 3F). Together, these findings demonstrate that the WT V. cholerae response to polyamines is controlled by the NspS-MbaA polyamine sensing circuit. Both the MbaA EVL and SGDEF domains are required for MbaA to detect polyamines We wondered how the putative MbaA phosphodiesterase and diguanylate cyclase enzymatic activities contribute to the V. cholerae responses to norspermidine and spermidine. The cytoplasmic domain of MbaA exhibits phosphodiesterase but not diguanylate cyclase activity in vitro (Cockerell et al., 2014). We reasoned that, when an intact regulatory domain is present, MbaA could possess both enzymatic activities, with the activity of each domain inversely controlled by NspS binding. To probe the role of each domain, we introduced inactivating point mutations in the catalytic motifs. To ensure that our mutations did not destabilize MbaA, we first fused MbaA to 3xFLAG and introduced the gene encoding the fusion onto the chromosome at the mbaA locus. Tagging did not alter MbaA control over the biofilm lifecycle (Figure 4—figure supplement 1A, Video 2). To inactivate the MbaA phosphodiesterase activity, we substituted the conserved catalytic residue E553 with alanine, converting EVL to AVL (referred to as EVL* henceforth). This change did not alter MbaA-3xFLAG abundance (Figure 4—figure supplement 1B). V. cholerae harboring MbaAEVL* exhibited an increase in biofilm biomass and a strong biofilm dispersal defect (Figure 4A), and only a modest response to exogenous polyamines, with norspermidine eliciting some inhibition of biofilm dispersal and spermidine driving a small reduction in overall biofilm biomass (Figure 4B, Video 2). Treatment with norspermidine did increase c-di-GMP levels in V. cholerae carrying MbaAEVL* as judged by the reporter output (Figure 4C), albeit not to the level of WT (Figure 3C), suggesting that the V. cholerae mbaAEVL* mutant, which is incapable of c-di-GMP degradation, retains the capacity to synthesize some c-di-GMP via the MbaA SGDEF domain. In contrast, the V. cholerae mbaAEVL* strain displayed little reduction in c-di-GMP levels in response to spermidine, presumably because it lacks the phosphodiesterase activity required to degrade c-di-GMP (Figure 4C). Thus, despite the fact that the purified cytoplasmic domain functioned only as a phosphodiesterase in vitro, our results suggest that, in vivo, MbaA is capable of synthesizing c-di-GMP in the presence of norspermidine. To validate this prediction, we altered the conserved catalytic residues D426 and E427 to alanine residues, yielding the inactive MbaA SGAAF variant (referred to as SGDEF*). These substitutions did not affect protein levels (Figure 4—figure supplement 1B). In every regard, the mbaASGDEF* mutant behaved identically to the ΔmbaA mutant. The V. cholerae mbaASGDEF* mutant exhibited a modest biofilm dispersal defect (Figure 4A) and biofilm biomass was reduced in response to exogenous norspermidine and spermidine (Figure 4D, Video 2). Furthermore, addition of polyamines to the mbaASGDEF* mutant harboring the c-di-GMP reporter did not drive substantial changes in reporter output (Figure 4E). These results show that the MbaA SGDEF domain is indispensable for the MbaA response to polyamines. Figure 4 with 1 supplement see all Download asset Open asset Both the MbaA SGDEF and EVL domains are required for regulation of V. cholerae biofilm dispersal. (A) Quantitation of biofilm biomass over time for the V. cholerae strains carrying mbaA-3xFLAG, mbaAEVL*−3xFLAG, and mbaASGDEF*−3xFLAG. (B) Quantitation of biofilm biomass over time measured by time-lapse microscopy for V. cholerae carrying mbaAEVL*−3xFLAG following addition of water (Ctrl), 100 µM norspermidine, or 100 µM spermidine. (C) c-di-GMP reporter output at the indicated polyamine concentrations for V. cholerae carrying mbaAEVL*−3xFLAG. Relative reporter signal

The lifelong relationship between the Hawaiian bobtail squid Euprymna scolopes and its microbial symbiont Vibrio fischeri represents a simplified model system for studying microbiome establishment and maintenance. The bacteria colonize a dedicated symbiotic light organ in the squid, from which bacterial luminescence camouflages the host in a process termed counterillumination. The squid host hatches without its symbionts, which must be acquired from the ocean amidst a diversity of nonbeneficial bacteria, such that precise molecular communication is required for initiation of the specific relationship. Therefore it is likely there are specialized metabolites used in the light organ microenvironment to modulate these processes. To identify small molecules that may influence the establishment of this symbiosis, we used imaging mass spectrometry to analyze metabolite production in V. fischeri with altered biofilm production, which correlates directly to colonization capability in its host. "Biofilm-up" and "biofilm-down" mutants were compared to a wild-type strain, and ions that were more abundantly produced by the biofilm-up mutant were detected. Using a combination of structural elucidation and synthetic chemistry, one such signal was determined to be a diketopiperazine, cyclo(d-histidyl-l-proline). This diketopiperazine modulated luminescence in V. fischeri and, using imaging mass spectrometry, was directly detected in the light organ of the colonized host. This work highlights the continued need for untargeted discovery efforts in host-microbe interactions and showcases the benefits of the squid-Vibrio system for identification and characterization of small molecules that modulate microbiome behaviors.IMPORTANCE The complexity of animal microbiomes presents challenges to defining signaling molecules within the microbial consortium and between the microbes and the host. By focusing on the binary symbiosis between Vibrio fischeri and Euprymna scolopes, we have combined genetic analysis with direct imaging to define and study small molecules in the intact symbiosis. We have detected and characterized a diketopiperazine produced by strong biofilm-forming V. fischeri strains that was detectable in the host symbiotic organ, and which influences bacterial luminescence. Biofilm formation and luminescence are critical for initiation and maintenance of the association, respectively, suggesting that the compound may link early and later development stages, providing further evidence that multiple small molecules are important in establishing these beneficial relationships.