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

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

Salinity and Temperature Effects on Physiological Responses of <Emphasis Type="Italic">Vibrio fischeri</Emphasis> from Diverse Ecological Niches

Vibrio fischeri is a bioluminescent bacterial symbiont of sepiolid squids (Cephalopoda: Sepiolidae) and monocentrid fishes (Actinopterygii: Monocentridae). V. fischeri exhibit competitive dominance within the allopatrically distributed squid genus Euprymna, which have led to the evolution of V. fischeri host specialists. In contrast, the host genus Sepiola contains sympatric species that is thought to have given rise to V. fischeri that have evolved as host generalists. Given that these ecological lifestyles may have a direct effect upon the growth spectrum and survival limits in contrasting environments, optimal growth ranges were obtained for numerous V. fischeri isolates from both free-living and host environments. Upper and lower limits of growth were observed in sodium chloride concentrations ranging from 0.0% to 9.0%. Sepiola symbiotic isolates possessed the least variation in growth throughout the entire salinity gradient, whereas isolates from Euprymna were the least uniform at <2.0% NaCl. V. fischeri fish symbionts (CG101 and MJ101) and all free-living strains were the most dissimilar at >5.0% NaCl. Growth kinetics of symbiotic V. fischeri strains were also measured under a range of salinity and temperature combinations. Symbiotic V. fischeri ES114 and ET101 exhibited a synergistic effect for salinity and temperature, where significant differences in growth rates due to salinity existed only at low temperatures. Thus, abiotic factors such as temperature and salinity have differential effects between free-living and symbiotic strains of V. fischeri, which may alter colonization efficiency prior to infection.     

Gene-Swapping Mediates Host Specificity among Symbiotic Bacteria in a Beneficial Symbiosis

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

Effect of the Squid Host on the Abundance and Distribution of Symbiotic Vibrio fischeri in Nature

Euprymna scolopes, a Hawaiian species of bioluminescent squid, harbors Vibrio fischeri as its specific light organ symbiont. The population of symbionts grew inside the adult light organ with an average doubling time of about 5 h, which produced an excess of cells that were expelled into the surrounding seawater on a diurnal basis at the beginning of each period of daylight. These symbionts, when expelled into the ambient seawater, maintain or slightly increase their numbers for at least 24 h. Hence, locations inhabited by their hosts periodically receive a daily input of symbiotic V. fischeri cells and, as a result, become significantly enriched with these bacteria. As estimated by hybridization with a species-specific luxA gene probe, the typical number of V. fischeri CFU, both in the water column and in the sediments of E. scolopes habitats, was as much as 24 to 30 times that in similar locations where squids were not observed. In addition, the number of symbiotic V. fischeri CFU in seawater samples that were collected along a transect through Kaneohe Bay, Hawaii, decreased as a function of the distance from a location inhabited by E. scolopes. These findings constitute evidence for the first recognized instance of the abundance and distribution of a marine bacterium being driven primarily by its symbiotic association with an animal host.     

Alterations in the Proteome of the Euprymna scolopes Light Organ in Response to Symbiotic Vibrio fischeri

During the onset of the cooperative association between the Hawaiian sepiolid squid Euprymna scolopes and the marine luminous bacterium Vibrio fischeri, the anatomy and morphology of the host's symbiotic organ undergo dramatic changes that require interaction with the bacteria. This morphogenetic process involves an array of tissues, including those in direct contact with, as well as those remote from, the symbiotic bacteria. The bacteria induce the developmental program soon after colonization of the organ, although complete morphogenesis requires 96 h. In this study, to determine critical time points, we examined the biochemistry underlying bacterium-induced host development using two-dimensional polyacrylamide gel electrophoresis. Specifically, V. fischeri-induced changes in the soluble proteome of the symbiotic organ during the first 96 h of symbiosis were identified by comparing the protein profiles of symbiont-colonized and uncolonized organs. Both symbiosis-related changes and age-related changes were analyzed to determine what proportion of the differences in the proteomes was the result of specific responses to interaction with bacteria. Although no differences were detected over the first 24 h, numerous symbiosis-related changes became apparent at 48 and 96 h and were more abundant than age-related changes. In addition, many age-related protein changes occurred 48 h sooner in symbiotic animals, suggesting that the interaction of squid tissue with V. fischeri cells accelerates certain developmental processes of the symbiotic organ. These data suggest that V. fischeri-induced modifications in host tissues that occur in the first 24 h of the symbiosis are independent of marked alterations in the patterns of abundant proteins but that the full 4-day morphogenetic program requires significant alteration of the host soluble proteome.     

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

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

New rfp- and pES213-Derived Tools for Analyzing Symbiotic Vibrio fischeri Reveal Patterns of Infection and lux Expression In Situ

Genetically altered or tagged Vibrio fischeri strains can be observed in association with their mutualistic host Euprymna scolopes, providing powerful experimental approaches for studying this symbiosis. Two limitations to such in situ analyses are the lack of suitably stable plasmids and the need for a fluorescent tag that can be used in tandem with green fluorescent protein (GFP). Vectors previously used in V. fischeri contain the p15A replication origin; however, we found that this replicon is not stable during growth in the host and is retained by fewer than 20% of symbionts within a day after infection. In contrast, derivatives of V. fischeri plasmid pES213 were retained by ~99% of symbionts even 3 days after infection. We therefore constructed pES213-derived shuttle vectors with a variety of selectable and visual markers. To include a visual tag that can be used in conjunction with GFP, we compared seven variants of the DsRed2 red fluorescent protein (RFP): mRFP1, tdimer2(12), DsRed.T3, DsRed.T4, DsRed.M1, DsRed.T3_S4T, and DsRed.T3(DNT). The last variant was brightest, displaying >20-fold more fluorescence than DsRed2 in V. fischeri. RFP expression did not detectably affect the fitness of V. fischeri, and cells were readily visualized in combination with GFP-expressing cells in mixed infections. Interestingly, even when inocula were dense enough that most E. scolopes hatchlings were infected by two strains, there was little mixing of the strains in the light organ crypts. We also used constitutive RFP in combination with the luxICDABEG promoter driving expression of GFP to visualize the spatial and temporal induction of this bioluminescence operon during symbiotic infection. Our results demonstrate the utility of pES213-based vectors and RFP for in situ experimental approaches in studies of the V. fischeri-E. scolopes symbiosis.     

The Inductive Role of Bacterial Symbionts in the Morphogenesis of a Squid Light Organ

SYNOPSIS. The association of the sepiolid squid Euprymna scolopeswith its marine luminous bacterial symbiont Vibrio fischeriis an emerging model system to study the initiation and developmentof bacterial symbioses in higher animals, in particular theinfluence of bacteria on the ontogenic development of symbiotic-specifichost tissues. Experiments comparing the development of juvenilesquid infected with symbiotic V. fischeri with that of uninfectedjuveniles suggest postembryonic development of the light organrequires cell-cell interactions with the bacterial symbionts.The presence of symbiotic bacteria induces specific morphologicalchanges by affecting such fundamental processes as cell deathand cell differentiation. The surface of the juvenile organis largely composed of ciliated cells that appear to facilitateinfection of the light organ. These cells begin to undergo celldeath within hours of infection with symbiotic V. fischeri.Within three days the epithelial cells that form the bacteriacontainingcrypts of the light organ increase in size; these cells do notappear mitotically active, and may represent a terminally differentiatedstate. The light organs of uninfected juvenile E. scolopes,however, do not exhibit any of these early postembryonic developmentalevents but remain in a state of arrested morphogenesis.     

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

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

By their own devices: invasive Argentine ants have shifted diet without clear aid from symbiotic microbes

The functions and compositions of symbiotic bacterial communities often correlate with host ecology. Yet cause–effect relationships and the order of symbiont vs. host change remain unclear in the face of ancient symbioses and conserved host ecology. Several groups of ants exemplify this challenge, as their low‐nitrogen diets and specialized symbioses appear conserved and ancient. To address whether nitrogen‐provisioning symbionts might be important in the early stages of ant trophic shifts, we studied bacteria from the Argentine ant, Linepithema humile – an invasive species that has transitioned towards greater consumption of sugar‐rich, nitrogen‐poor foods in parts of its introduced range. Bacteria were present at low densities in most L. humile workers, and among those yielding quality 16S rRNA amplicon sequencing data, we found just three symbionts to be common and dominant. Two, a Lactobacillus and an Acetobacteraceae species, were shared between native and introduced populations. The other, a Rickettsia, was found only in two introduced supercolonies. Across an eight‐year period of trophic reduction in one introduced population, we found no change in symbionts, arguing against a relationship between natural dietary change and microbiome composition. Overall, our findings thus argue against major changes in symbiotic bacteria in association with the invasion and trophic shift of L. humile. In addition, genome content from close relatives of the identified symbionts suggests that just one can synthesize most essential amino acids; this bacterium was only modestly abundant in introduced populations, providing little support for a major role of nitrogen‐provisioning symbioses in Argentine ant's dietary shift.     

The pathogenic actinobacterium Rhodococcus equi: what's in a name?

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

An ancient but promiscuous host–symbiont association between Burkholderia gut symbionts and their heteropteran hosts

Here, we investigated 124 stinkbug species representing 20 families and 5 superfamilies for their Burkholderia gut symbionts, of which 39 species representing 6 families of the superfamilies Lygaeoidea and Coreoidea were Burkholderia-positive. Diagnostic PCR surveys revealed high frequencies of Burkholderia infection in natural populations of the stinkbugs, and substantial absence of vertical transmission of Burkholderia infection to their eggs. In situ hybridization confirmed localization of the Burkholderia in their midgut crypts. In the lygaeoid and coreoid stinkbugs, development of midgut crypts in their alimentary tract was coincident with the Burkholderia infection, suggesting that the specialized morphological configuration is pivotal for establishment and maintenance of the symbiotic association. The Burkholderia symbionts were easily isolated as pure culture on standard microbiological media, indicating the ability of the gut symbionts to survive outside the host insects. Molecular phylogenetic analysis showed that the gut symbionts of the lygaeoid and coreoid stinkbugs belong to a β-proteobacterial clade together with Burkholderia isolates from soil environments and Burkholderia species that induce plant galls. On the phylogeny, the stinkbug-associated, environmental and gall-forming Burkholderia strains did not form coherent groups, indicating host–symbiont promiscuity among these stinkbugs. Symbiont culturing revealed that slightly different Burkholderia genotypes often coexist in the same insects, which is also suggestive of host–symbiont promiscuity. All these results strongly suggest an ancient but promiscuous host–symbiont relationship between the lygaeoid/coreoid stinkbugs and the Burkholderia gut symbionts. Possible mechanisms as to how the environmentally transmitted promiscuous symbiotic association has been stably maintained in the evolutionary course are discussed.     

Gut symbiotic bacteria in the cabbage bugs <Emphasis Type="Italic">Eurydema rugosa</Emphasis> and <Emphasis Type="Italic">Eurydema dominulus</Emphasis> (Heteroptera: Pentatomidae)

The cabbage bugs Eurydema rugosa Motschulsky and Eurydema dominulus (Scopoli) (Heteroptera: Pentatomidae: Strachiini) possess a number of crypts in a posterior region of the midgut, which are filled with bacterial symbiont cells. Here we characterized the gut symbionts of Eurydema stinkbugs using molecular phylogenetic and histological techniques. Specific gammaproteobacteria were consistently identified from the posterior midgut of E. rugosa representing nine populations and E. dominulus representing six populations, respectively. The bacterial 16S rRNA gene sequences were identical within the species but slightly different (98.2% sequence identity) between the species. Molecular phylogenetic analysis revealed that the Eurydema symbionts formed a well-defined monophyletic group in the Gammaproteobacteria. The symbionts were phylogenetically distinct from the gut symbionts of the stinkbug families Acanthosomatidae, Plataspidae, Parastrachiidae, Scutelleridae, and other pentatomid species, suggesting multiple evolutionary origins of the gut symbiotic bacteria among diverse stinkbugs. In situ hybridization confirmed that the symbiont is located in the cavity of the midgut crypts. Aposymbiotic insects of E. rugosa, which were produced by egg surface sterilization, were viable but suffered retarded growth, reduced body weight, and abnormal body color, suggesting the biological importance of the symbiont for the host.     

Dominance of Vibrio fischeri in Secreted Mucus outside the Light Organ of Euprymna scolopes: the First Site of Symbiont Specificity

Previous studies of the Euprymna scolopes-Vibrio fischeri symbiosis have demonstrated that, during colonization, the hatchling host secretes mucus in which gram-negative environmental bacteria amass in dense aggregations outside the sites of infection. In this study, experiments with green fluorescent protein-labeled symbiotic and nonsymbiotic species of gram-negative bacteria were used to characterize the behavior of cells in the aggregates. When hatchling animals were exposed to 10³ to 10⁶ V. fischeri cells/ml added to natural seawater, which contains a mix of approximately 10⁶ nonspecific bacterial cells/ml, V. fischeri cells were the principal bacterial cells present in the aggregations. Furthermore, when animals were exposed to equal cell numbers of V. fischeri (either a motile or a nonmotile strain) and either Vibrio parahaemolyticus or Photobacterium leiognathi, phylogenetically related gram-negative bacteria that also occur in the host's habitat, the symbiont cells were dominant in the aggregations. The presence of V. fischeri did not compromise the viability of these other species in the aggregations, and no significant growth of V. fischeri cells was detected. These findings suggested that dominance results from the ability of V. fischeri either to accumulate or to be retained more effectively within the mucus. Viability of the V. fischeri cells was required for both the formation of tight aggregates and their dominance in the mucus. Neither of the V. fischeri quorum-sensing compounds accumulated in the aggregations, which suggested that the effects of these small signal molecules are not critical to V. fischeri dominance. Taken together, these data provide evidence that the specificity of the squid-vibrio symbiosis begins early in the interaction, in the mucus where the symbionts aggregate outside of the light organ.     

Does host outcrossing disrupt compatibility with heritable symbionts?

Vertically transmitted microbes are common in macro‐organisms and can enhance host defense against environmental stress. Because vertical transmission couples host and symbiont lineages, symbionts may become specialized to host species or genotypes. Specialization and contrasting reproductive modes of symbiotic partners could create incompatibilities between inherited symbionts and novel host genotypes when hosts outcross or hybridize. Such incompatibilities could manifest as failed colonization or poor symbiont growth in host offspring that are genetically dissimilar from their maternal host. Moreover, outcrossing between host species could influence both host and symbiont reproductive performance. We tested these hypotheses by manipulating outcrossing between populations and species of two grasses, Elymus virginicus and E. canadensis, that host vertically transmitted fungal endophytes (genus Epichloё). In both greenhouse and field settings, we found that host–symbiont compatibility was robust to variation in host genetic background, spanning within‐population, between‐population and between‐species crosses. Symbiont transmission into the F₁ generation was generally high and weakly affected by host outcrossing. Furthermore, endophytes grew equally well in planta regardless of host genetic background and transmitted at high frequencies into the F₂ generation. However, outcrossing, especially inter‐specific hybridization, reduced reproductive fitness of the host, and thereby the symbiont. Our results challenge the hypothesis that host genetic recombination, which typically exceeds that of symbionts, is a disruptive force in heritable symbioses. Instead, symbionts may be sufficiently generalized to tolerate ecologically realistic variation in host outcrossing.     

Bioluminescence in the symbiotic squid Euprymna scolopes is controlled by a daily biological rhythm

In most symbioses between animals and luminous bacteria it has been assumed that the bacterial symbionts luminesce continuously, and that the control of luminescent output by the animal is mediated through elaborate accessory structures, such as chromatophores and muscular shutters that surround the host light organ. However, we have found that while in the light organ of the sepiolid squid Euprymna scolopes, symbiotic cells of Vibrio fischeri do not produce a continuously uniform level of luminescence, but instead exhibit predictable cyclic fluctuations in the amount of light emitted per cell. This daily biological rhythm exhibits many features of a circadian pattern, and produces an elevated intensity of symbiont luminescence in juvenile animals during the hours preceding the onset of ambient darkness. Comparisons of the specific luminescence of bacteria in the intact light organ with that of newly released bacteria support the existence of a direct host regulation of the specific activity of symbiont luminescence that does not require the intervention of accessory tissues. A model encompassing the currently available evidence is proposed for the control of growth and luminescence activity in the E. scolopes/V. fischeri light organ symbiosis.Abbreviations CFU colony-forming-unit - LD light-dark     

srDna-based taxonomic affinities of algal symbionts from a planktonic foraminifer and a solitary radiolarian

Planktonic sarcodines (acantharia, radiolaria, and planktonic foraminifera) are oceanic amoeboid protozoa that often harbor a variety of microalgae as intracellular symbionts. The identity and function of these endosymbiotic algae have intrigued and perplexed biologists for more than a century. The most conspicuous and well‐studied symbiotic algae of planktonic foraminifera and radiolaria are dinoflagellates, but a variety of nondinoflagellate taxa have also been reported. Ultrastructural features have been used to characterize some of these nondinoflagellate algae, but rarely has this led to clear taxonomic affiliations. We analyzed the nuclear small subunit ribosomal DNA (srDNA) isolated from the symbionts of the spinose planktonic foraminiferan Globigerinella siphonifera d'Orbigny (=Globigerinella aequilateralis Brady) and a solitary radiolarian (Spongodrymus sp. Haeckel) in order to determine the identity of these symbionts. The small coccoid algae isolated from G. siphonifera correspond to the Type I symbionts described by Faber et al. (1988) . Phylogenetic analysis of the srDNA sequences places these symbionts within the prymnesiophyte (haptophyte) lineage, closer to Prymnesium Conrad than to Phaeocystis Lagerheim. To our knowledge, this is the first confirmed case of a symbiotic prymnesiophyte. In addition, we were able to examine the level of sequence heterogeneity between symbionts isolated from different individuals of a single host species. The three isolates in this study had srDNA sequences that were almost identical, indicating that the three were all of the same species. Very green symbiotic algae were isolated from three solitary radiolaria identified as species of Spongodrymus. The symbiont srDNA sequences from the three individual hosts were identical to each other, again implicating a single species of algae in that symbiotic association. These symbionts are prasinophytes most closely related to the clade containing Tetaselmis convolutae Norris, Hori et Chihara. Tetraselmis convolutae is the algal symbiont of the marine flatworm, Convolutae roscoffensis Graff.     

The response regulator SypE controls biofilm formation and colonization through phosphorylation of the syp‐encoded regulator SypA in Vibrio fischeri

Bacteria utilize multiple regulatory systems to modulate gene expression in response to environmental changes, including two‐component signalling systems and partner‐switching networks. We recently identified a novel regulatory protein, SypE, that combines features of both signalling systems. SypE contains a central response regulator receiver domain flanked by putative kinase and phosphatase effector domains with similarity to partner‐switching proteins. SypE was previously shown to exert dual control over biofilm formation through the opposing activities of its terminal effector domains. Here, we demonstrate that SypE controls biofilms in Vibrio fischeri by regulating the activity of SypA, a STAS (sulphate transporter and anti‐sigma antagonist) domain protein. Using biochemical and genetic approaches, we determined that SypE both phosphorylates and dephosphorylates SypA, and that phosphorylation inhibits SypA's activity. Furthermore, we found that biofilm formation and symbiotic colonization required active, unphosphorylated SypA, and thus SypA phosphorylation corresponded with a loss of biofilms and impaired host colonization. Finally, expression of a non‐phosphorylatable mutant of SypA suppressed both the biofilm and symbiosis defects of a constitutively inhibitory SypE mutant strain. This study demonstrates that regulation of SypA activity by SypE is a critical mechanism by which V. fischeri controls biofilm development and symbiotic colonization.