Evolutionary history influences the microbiomes of a female symbiotic reproductive organ in cephalopods.

Many female squids and cuttlefishes have a symbiotic reproductive organ called the accessory nidamental gland (ANG) that hosts a bacterial consortium involved with egg defense against pathogens and fouling organisms. While the ANG is found in multiple cephalopod families, little is known about the global microbial diversity of these ANG bacterial symbionts. We used 16S rRNA gene community analysis to characterize the ANG microbiome from different cephalopod species and assess the relationship between host and symbiont phylogenies. The ANG microbiome of 11 species of cephalopods from four families (superorder: Decapodiformes) that span seven geographic locations was characterized. Bacteria of class Alphaproteobacteria, Gammaproteobacteria, and Flavobacteriia were found in all species, yet analysis of amplicon sequence variants by multiple distance metrics revealed a significant difference between ANG microbiomes of cephalopod families (weighted/unweighted UniFrac, Bray-Curtis, P = 0.001). Despite being collected from widely disparate geographic locations, members of the family Sepiolidae (bobtail squid) shared many bacterial taxa including (~50%) Opitutae (Verrucomicrobia) and Ruegeria (Alphaproteobacteria) species. Furthermore, we tested for phylosymbiosis and found a positive correlation between host phylogenetic distance and bacterial community dissimilarity (Mantel test r = 0.7). These data suggest that closely related sepiolids select for distinct symbionts from similar bacterial taxa. Overall, the ANGs of different cephalopod species harbor distinct microbiomes and thus offer a diverse symbiont community to explore antimicrobial activity and other functional roles in host fitness.IMPORTANCEMany aquatic organisms recruit microbial symbionts from the environment that provide a variety of functions, including defense from pathogens. Some female cephalopods (squids, bobtail squids, and cuttlefish) have a reproductive organ called the accessory nidamental gland (ANG) that contains a bacterial consortium that protects eggs from pathogens. Despite the wide distribution of these cephalopods, whether they share similar microbiomes is unknown. Here, we studied the microbial diversity of the ANG in 11 species of cephalopods distributed over a broad geographic range and representing 15-120 million years of host divergence. The ANG microbiomes shared some bacterial taxa, but each cephalopod species had unique symbiotic members. Additionally, analysis of host-symbiont phylogenies suggests that the evolutionary histories of the partners have been important in shaping the ANG microbiome. This study advances our knowledge of cephalopod-bacteria relationships and provides a foundation to explore defensive symbionts in other systems.

Symbiotic organs shaped by distinct modes of genome evolution in cephalopods.

Microbes have been critical drivers of evolutionary innovation in animals. To understand the processes that influence the origin of specialized symbiotic organs, we report the sequencing and analysis of the genome of Euprymna scolopes, a model cephalopod with richly characterized host-microbe interactions. We identified large-scale genomic reorganization shared between E. scolopes and Octopus bimaculoides and posit that this reorganization has contributed to the evolution of cephalopod complexity. To reveal genomic signatures of host-symbiont interactions, we focused on two specialized organs of E. scolopes: the light organ, which harbors a monoculture of Vibrio fischeri, and the accessory nidamental gland (ANG), a reproductive organ containing a bacterial consortium. Our findings suggest that the two symbiotic organs within E. scolopes originated by different evolutionary mechanisms. Transcripts expressed in these microbe-associated tissues displayed their own unique signatures in both coding sequences and the surrounding regulatory regions. Compared with other tissues, the light organ showed an abundance of genes associated with immunity and mediating light, whereas the ANG was enriched in orphan genes known only from E. scolopes Together, these analyses provide evidence for different patterns of genomic evolution of symbiotic organs within a single host.

Ambient pH Alters the Protein Content of Outer Membrane Vesicles, Driving Host Development in a Beneficial Symbiosis.

Outer membrane vesicles (OMVs) are continuously produced by Gram-negative bacteria and are increasingly recognized as ubiquitous mediators of bacterial physiology. In particular, OMVs are powerful effectors in interorganismal interactions, driven largely by their molecular contents. These impacts have been studied extensively in bacterial pathogenesis but have not been well documented within the context of mutualism. Here, we examined the proteomic composition of OMVs from the marine bacterium Vibrio fischeri, which forms a specific mutualism with the Hawaiian bobtail squid, Euprymna scolopes We found that V. fischeri upregulates transcription of its major outer membrane protein, OmpU, during growth at an acidic pH, which V. fischeri experiences when it transitions from its environmental reservoir to host tissues. We used comparative genomics and DNA pulldown analyses to search for regulators of ompU and found that differential expression of ompU is governed by the OmpR, H-NS, and ToxR proteins. This transcriptional control combines with nutritional conditions to govern OmpU levels in OMVs. Under a host-encountered acidic pH, V. fischeri OMVs become more potent stimulators of symbiotic host development in an OmpU-dependent manner. Finally, we found that symbiotic development could be stimulated by OMVs containing a homolog of OmpU from the pathogenic species Vibrio cholerae, connecting the role of a well-described virulence factor with a mutualistic element. This work explores the symbiotic effects of OMV variation, identifies regulatory machinery shared between pathogenic and mutualistic bacteria, and provides evidence of the role that OMVs play in animal-bacterium mutualism.IMPORTANCE Beneficial bacteria communicate with their hosts through a variety of means. These communications are often carried out by a combination of molecules that stimulate responses from the host and are necessary for development of the relationship between these organisms. Naturally produced bacterial outer membrane vesicles (OMVs) contain many of those molecules and can stimulate a wide range of responses from recipient organisms. Here, we describe how a marine bacterium, Vibrio fischeri, changes the makeup of its OMVs under conditions that it experiences as it goes from its free-living lifestyle to associating with its natural host, the Hawaiian bobtail squid. This work improves our understanding of how bacteria change their signaling profile as they begin to associate with their beneficial partner animals.

Symbiotic bacteria associated with a bobtail squid reproductive system are detectable in the environment, and stable in the host and developing eggs.

Female Hawaiian bobtail squid, Euprymna scolopes, have an accessory nidamental gland (ANG) housing a bacterial consortium that is hypothesized to be environmentally transmitted and to function in the protection of eggs from fouling and infection. The composition, stability, and variability of the ANG and egg jelly coat (JC) communities were characterized and compared to the bacterial community composition of the surrounding environment using Illumina sequencing and transmission electron microscopy. The ANG bacterial community was conserved throughout hosts collected from the wild and was not affected by maintaining animals in the laboratory. The core symbiotic community was composed of Alphaproteobacteria and Opitutae (a class of Verrucomicrobia). Operational taxonomic units representing 94.5% of the average ANG abundance were found in either the seawater or sediment, which is consistent with the hypothesis of environmental transmission between generations. The bacterial composition of the JC was stable during development and mirrored that of the ANG. Bacterial communities from individual egg clutches also grouped with the ANG of the female that produced them. Collectively, these data suggest a conserved role of the ANG/JC community in host reproduction. Future directions will focus on determining the function of this symbiotic community, and how it may change during ANG development.

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

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

The role of the immune system in the initiation and persistence of the Euprymna scolopes--Vibrio fischeri symbiosis.

The squid-vibrio symbiosis is an experimental system being studied as a model of the chronic colonization of animal epithelia by bacterial partners. One principal question being asked with this model is: what is the role of the immune system in the dynamics of the onset and maintenance of the symbiotic state? This review focuses upon results of research to date, which have demonstrated that both cell-mediated and cell-free components of the innate immune system are involved in these processes.

Gene modelling and annotation for the Hawaiian bobtail squid, Euprymna scolopes.

Coleoid cephalopods possess numerous complex, species-specific morphological and behavioural adaptations, e.g., a uniquely structured nervous system that is the largest among the invertebrates. The Hawaiian bobtail squid (Euprymna scolopes) is one of the most established cephalopod species. With its recent publication of the chromosomal-scale genome assembly and regulatory genomic data, it also emerges as a key model for cephalopod gene regulation and evolution. However, the latest genome assembly has been lacking a native gene model set. Our manuscript describes the generation of new long-read transcriptomic data and, made using this combined with a plethora of publicly available transcriptomic and protein sequence data, a new reference annotation for E. scolopes.

Transcriptional patterns in both host and bacterium underlie a daily rhythm of anatomical and metabolic change in a beneficial symbiosis.

Mechanisms for controlling symbiont populations are critical for maintaining the associations that exist between a host and its microbial partners. We describe here the transcriptional, metabolic, and ultrastructural characteristics of a diel rhythm that occurs in the symbiosis between the squid Euprymna scolopes and the luminous bacterium Vibrio fischeri. The rhythm is driven by the host's expulsion from its light-emitting organ of most of the symbiont population each day at dawn. The transcriptomes of both the host epithelium that supports the symbionts and the symbiont population itself were characterized and compared at four times over this daily cycle. The greatest fluctuation in gene expression of both partners occurred as the day began. Most notable was an up-regulation in the host of >50 cytoskeleton-related genes just before dawn and their subsequent down-regulation within 6 h. Examination of the epithelium by TEM revealed a corresponding restructuring, characterized by effacement and blebbing of its apical surface. After the dawn expulsion, the epithelium reestablished its polarity, and the residual symbionts began growing, repopulating the light organ. Analysis of the symbiont transcriptome suggested that the bacteria respond to the effacement by up-regulating genes associated with anaerobic respiration of glycerol; supporting this finding, lipid analysis of the symbionts' membranes indicated a direct incorporation of host-derived fatty acids. After 12 h, the metabolic signature of the symbiont population shifted to one characteristic of chitin fermentation, which continued until the following dawn. Thus, the persistent maintenance of the squid-vibrio symbiosis is tied to a dynamic diel rhythm that involves both partners.

Emergence of novel genomic regulatory regions associated with light-organ development in the bobtail squid.

Light organs (LO) with symbiotic bioluminescent bacteria are hallmarks of many bobtail squid species. These organs possess structural and functional features to modulate light, analogous to those found in coleoid eyes. Previous studies identified four transcription factors and modulators (SIX, EYA, PAX6, DAC) associated with both eyes and light organ development, suggesting co-option of a highly conserved gene regulatory network. Using available topological, open chromatin, and transcriptomic data, we explore the regulatory landscape around the four transcription factors as well as genes associated with LO and shared LO/eye expression. This analysis revealed several closely associated and putatively co-regulated genes. Comparative genomic analyses identified distinct evolutionary origins of these putative regulatory associations, with the DAC locus showing a unique topological and evolutionarily recent organization. We discuss different scenarios of modifications to genome topology and how these changes may have contributed to the evolutionary emergence of the light organ.

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

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

Cephalopod-omics: Emerging Fields and Technologies in Cephalopod Biology.

Few animal groups can claim the level of wonder that cephalopods instill in the minds of researchers and the general public. Much of cephalopod biology, however, remains unexplored: the largest invertebrate brain, difficult husbandry conditions, and complex (meta-)genomes, among many other things, have hindered progress in addressing key questions. However, recent technological advancements in sequencing, imaging, and genetic manipulation have opened new avenues for exploring the biology of these extraordinary animals. The cephalopod molecular biology community is thus experiencing a large influx of researchers, emerging from different fields, accelerating the pace of research in this clade. In the first post-pandemic event at the Cephalopod International Advisory Council (CIAC) conference in April 2022, over 40 participants from all over the world met and discussed key challenges and perspectives for current cephalopod molecular biology and evolution. Our particular focus was on the fields of comparative and regulatory genomics, gene manipulation, single-cell transcriptomics, metagenomics, and microbial interactions. This article is a result of this joint effort, summarizing the latest insights from these emerging fields, their bottlenecks, and potential solutions. The article highlights the interdisciplinary nature of the cephalopod-omics community and provides an emphasis on continuous consolidation of efforts and collaboration in this rapidly evolving field.

Characterization of a Vibrio fischeri aminopeptidase and evidence for its influence on an early stage of squid colonization.

Vibrio fischeri cells are the sole colonists of a specialized light organ in the mantle cavity of the sepiolid squid Euprymna scolopes. The process begins when the bacteria aggregate in mucus secretions outside the light organ. The cells eventually leave the aggregate, enter the light organ, and encounter a rich supply of peptides. The need to dissociate from mucus and presumably utilize peptides led us to hypothesize that protease activity is integral to the colonization process. Protease activity associated with whole cells of Vibrio fischeri strain ES114 was identified as the product of a putative cell membrane-associated aminopeptidase (PepN). To characterize this activity, the aminopeptidase was cloned, overexpressed, and purified. Initial steady-state kinetic studies revealed that the aminopeptidase has broad activity, with a preference for basic and hydrophobic side chains and k(cat) and K(m) values that are lower and smaller, respectively, than those of Escherichia coli PepN. A V. fischeri mutant unable to produce PepN is significantly delayed in its ability to colonize squid within the first 12 h, but eventually it establishes a wild-type colonization level. Likewise, in competition with the wild type for colonization, the mutant is outcompeted at 12 h postinoculation but then competes evenly by 24 h. Also, the PepN-deficient strain fails to achieve wild-type levels of cells in aggregates, suggesting an explanation for the initial colonization delay. This study provides a foundation for more studies on PepN expression, localization, and role in the early stages of squid colonization.

Diversity and partitioning of bacterial populations within the accessory nidamental gland of the squid Euprymna scolopes.

Microbial consortia confer important benefits to animal and plant hosts, and model associations are necessary to examine these types of host/microbe interactions. The accessory nidamental gland (ANG) is a female reproductive organ found among cephalopod mollusks that contains a consortium of bacteria, the exact function of which is unknown. To begin to understand the role of this organ, the bacterial consortium was characterized in the Hawaiian bobtail squid, Euprymna scolopes, a well-studied model organism for symbiosis research. Transmission electron microscopy (TEM) analysis of the ANG revealed dense bacterial assemblages of rod- and coccus-shaped cells segregated by morphology into separate, epithelium-lined tubules. The host epithelium was morphologically heterogeneous, containing ciliated and nonciliated cells with various brush border thicknesses. Hemocytes of the host's innate immune system were also found in close proximity to the bacteria within the tubules. A census of 16S rRNA genes suggested that Rhodobacterales, Rhizobiales, and Verrucomicrobia bacteria were prevalent, with members of the genus Phaeobacter dominating the consortium. Analysis of 454-shotgun sequencing data confirmed the presence of members of these taxa and revealed members of a fourth, Flavobacteria of the Bacteroidetes phylum. 16S rRNA fluorescent in situ hybridization (FISH) revealed that many ANG tubules were dominated by members of specific taxa, namely, Rhodobacterales, Verrucomicrobia, or Cytophaga-Flavobacteria-Bacteroidetes, suggesting symbiont partitioning to specific host tubules. In addition, FISH revealed that bacteria, including Phaeobacter species from the ANG, are likely deposited into the jelly coat of freshly laid eggs. This report establishes the ANG of the invertebrate E. scolopes as a model to examine interactions between a bacterial consortium and its host.

Emergence of novel cephalopod gene regulation and expression through large-scale genome reorganization.

Coleoid cephalopods (squid, cuttlefish, octopus) have the largest nervous system among invertebrates that together with many lineage-specific morphological traits enables complex behaviors. The genomic basis underlying these innovations remains unknown. Using comparative and functional genomics in the model squid Euprymna scolopes, we reveal the unique genomic, topological, and regulatory organization of cephalopod genomes. We show that coleoid cephalopod genomes have been extensively restructured compared to other animals, leading to the emergence of hundreds of tightly linked and evolutionary unique gene clusters (microsyntenies). Such novel microsyntenies correspond to topological compartments with a distinct regulatory structure and contribute to complex expression patterns. In particular, we identify a set of microsyntenies associated with cephalopod innovations (MACIs) broadly enriched in cephalopod nervous system expression. We posit that the emergence of MACIs was instrumental to cephalopod nervous system evolution and propose that microsyntenic profiling will be central to understanding cephalopod innovations.

Draft genome of Phaeobacter gallaeciensis ANG1, a dominant member of the accessory nidamental gland of Euprymna scolopes.

Phaeobacter gallaeciensis strain ANG1 represents the dominant member of the bacterial consortium within the reproductive accessory nidamental gland (ANG) of the squid Euprymna scolopes. We present a 4.59-Mb assembly of its genome, which may provide clues as to how it benefits its host.

Isolation and characterization of a protochordate histocompatibility locus.

Histocompatibility--the ability of an organism to distinguish its own cells and tissue from those of another--is a universal phenomenon in the Metazoa. In vertebrates, histocompatibility is a function of the immune system controlled by a highly polymorphic major histocompatibility complex (MHC), which encodes proteins that target foreign molecules for immune cell recognition. The association of the MHC and immune function suggests an evolutionary relationship between metazoan histocompatibility and the origins of vertebrate immunity. However, the MHC of vertebrates is the only functionally characterized histocompatibility system; the mechanisms underlying this process in non-vertebrates are unknown. A primitive chordate, the ascidian Botryllus schlosseri, also undergoes a histocompatibility reaction controlled by a highly polymorphic locus. Here we describe the isolation of a candidate gene encoding an immunoglobulin superfamily member that, by itself, predicts the outcome of histocompatibility reactions. This is the first non-vertebrate histocompatibility gene described, and may provide insights into the evolution of vertebrate adaptive immunity.

A lasting symbiosis: how the Hawaiian bobtail squid finds and keeps its bioluminescent bacterial partner.

For more than 30 years, the association between the Hawaiian bobtail squid, Euprymna scolopes, and the bioluminescent bacterium Vibrio fischeri has been studied as a model system for understanding the colonization of animal epithelia by symbiotic bacteria. The squid-vibrio light-organ system provides the exquisite resolution only possible with the study of a binary partnership. The impact of this relationship on the partners' biology has been broadly characterized, including their ecology and evolutionary biology as well as the underlying molecular mechanisms of symbiotic dynamics. Much has been learned about the factors that foster initial light-organ colonization, and more recently about the maturation and long-term maintenance of the association. This Review synthesizes the results of recent research on the light-organ association and also describes the development of new horizons for E. scolopes as a model organism that promises to inform biology and biomedicine about the basic nature of host-microorganism interactions.

Development of the Accessory Nidamental Gland and Associated Bacterial Community in the Hawaiian Bobtail Squid, Euprymna scolopes.

AbstractThe Hawaiian bobtail squid, Euprymna scolopes, has a female reproductive organ called the accessory nidamental gland that contains a symbiotic bacterial consortium. These bacteria are deposited from the accessory nidamental gland into the squid's egg cases, where the consortium prevents microbial fouling. The symbiont community is environmentally transmitted and conserved across host populations, yet little is known about how the organ develops and is colonized by bacteria. In order to understand accessory nidamental gland development in E. scolopes, we characterized the gland during maturation by using histology and confocal and transmission electron microscopy. We found that an epithelial field formed first about four weeks after hatching, followed by the proliferation of numerous pores during what we hypothesize to be the initiation of bacterial recruitment (early development). Microscopy revealed that these pores were connected to ciliated invaginations that occasionally contained bacteria. During mid development, these epithelial fields expanded, and separate colonized tubules were observed below the epithelial layer that contained the pores and invaginations. During late development, the superficial epithelial fields appeared to regress as animals approached sexual maturity and were never observed in fully mature adults (about 2-3 months post-hatching), suggesting that they help facilitate bacterial colonization of the accessory nidamental gland. An analysis of 16S rRNA gene diversity in accessory nidamental glands from females of varying size showed that the bacterial community changed as the host approached sexual maturity, increasing in community evenness and shifting from a Verrucomicrobia-dominated to an Alphaproteobacteria-dominated consortium. Given the host's relationship with the well-characterized light organ symbiont Vibrio fischeri, our work suggests that the accessory nidamental gland of E. scolopes may have similar mechanisms to recruit bacteria from the environment. Understanding the developmental and colonization processes of the accessory nidamental gland will expand the use of E. scolopes as a model organism for studying bacterial consortia in marine symbioses.

In the beginning: egg-microbe interactions and consequences for animal hosts.

Microorganisms are associated with the eggs of many animals. For some hosts, the egg serves as the ideal environment for the vertical transmission of beneficial symbionts between generations, while some bacteria use the egg to parasitize their hosts. In a number of animal groups, egg microbiomes often perform other essential functions. The eggs of aquatic and some terrestrial animals are especially susceptible to fouling and disease since they are exposed to high densities of microorganisms. To overcome this challenge, some hosts form beneficial associations with microorganisms, directly incorporating microbes and/or microbial products on or in their eggs to inhibit pathogens and biofouling. Other functional roles for egg-associated microbiomes are hypothesized to involve oxygen and nutrient acquisition. Although some egg-associated microbiomes are correlated with increased host fitness and are essential for successful development, the mechanisms that lead to such outcomes are often not well understood. This review article will discuss different functions of egg microbiomes and how these associations have influenced the biology and evolution of animal hosts. This article is part of the theme issue 'The role of the microbiome in host evolution'.

Hawaiian Bobtail Squid Symbionts Inhibit Marine Bacteria via Production of Specialized Metabolites, Including New Bromoalterochromides BAC-D/D'.

The Hawaiian bobtail squid, Euprymna scolopes, has a symbiotic bacterial consortium in the accessory nidamental gland (ANG), a female reproductive organ that protects eggs against fouling microorganisms. To test the antibacterial activity of ANG community members, 19 bacterial isolates were screened for their ability to inhibit Gram-negative and Gram-positive bacteria, of which two strains were inhibitory. These two antibacterial isolates, Leisingera sp. ANG59 and Pseudoalteromonas sp. JC28, were subjected to further genomic characterization. Genomic analysis of Leisingera sp. ANG59 revealed a biosynthetic gene cluster encoding the antimicrobial compound indigoidine. The genome of Pseudoalteromonas sp. JC28 had a 14-gene cluster with >95% amino acid identity to a known bromoalterochromide (BAC) cluster. Chemical analysis confirmed production of known BACs, BAC-A/A' (compounds 1a/1b), as well as two new derivatives, BAC-D/D' (compounds 2a/2b). Extensive nuclear magnetic resonance (NMR) analyses allowed complete structural elucidation of compounds 2a/2b, and the absolute stereochemistry was unambiguously determined using an optimized Marfey's method. The BACs were then investigated for in vitro antibacterial, antifungal, and nitric oxide (NO) inhibitory activity. Compounds 1a/1b were active against the marine bacteria Bacillus algicola and Vibrio fischeri, while compounds 2a/2b were active only against B. algicola Compounds 1a/1b inhibited NO production via lipopolysaccharide (LPS)-induced inflammation in RAW264.7 macrophage cells and also inhibited the pathogenic fungus Fusarium keratoplasticum, which, coupled with their antibacterial activity, suggests that these polyketide-nonribosomal peptides may be used for squid egg defense against potential pathogens and/or fouling microorganisms. These results indicate that BACs may provide Pseudoalteromonas sp. JC28 an ecological niche, facilitating competition against nonsymbiotic microorganisms in the host's environment.IMPORTANCE Animals that deposit eggs must protect their embryos from fouling and disease by microorganisms to ensure successful development. Although beneficial bacteria are hypothesized to contribute to egg defense in many organisms, the mechanisms of this protection are only recently being elucidated. Our previous studies of the Hawaiian bobtail squid focused on fungal inhibition by beneficial bacterial symbionts of a female reproductive gland and eggs. Herein, using genomic and chemical analyses, we demonstrate that symbiotic bacteria from this gland can also inhibit other marine bacteria in vitro One bacterial strain in particular, Pseudoalteromonas sp. JC28, had broad-spectrum abilities to inhibit potential fouling bacteria, in part via production of novel bromoalterochromide metabolites, confirmed via genomic annotation of the associated biosynthetic gene cluster. Our results suggest that these bacterial metabolites may contribute to antimicrobial activity in this association and that such defensive symbioses are underutilized sources for discovering novel antimicrobial compounds.