Draft genome sequence of Exiguobacterium sp. strain MMG028 isolated from a salt marsh.

Here, we report the draft genome sequence of Exiguobacterium sp. strain MMG028, isolated from Rose Creek, San Diego, CA, USA, assembled and analyzed by undergraduate students participating in a marine microbial genomics course. A genomic comparison suggests that MMG028 is a novel species, providing a resource for future microbiology and biotechnology investigations.

Linking bacterial tetrabromopyrrole biosynthesis to coral metamorphosis.

An important factor dictating coral fitness is the quality of bacteria associated with corals and coral reefs. One way that bacteria benefit corals is by stimulating the larval to juvenile life cycle transition of settlement and metamorphosis. Tetrabromopyrrole (TBP) is a small molecule produced by bacteria that stimulates metamorphosis with and without attachment in a range of coral species. A standing debate remains, however, about whether TBP biosynthesis from live Pseudoalteromonas bacteria is the primary stimulant of coral metamorphosis. In this study, we create a Pseudoalteromonas sp. PS5 mutant lacking the TBP brominase gene, bmp2. Using this mutant, we confirm that the bmp2 gene is critical for TBP biosynthesis in Pseudoalteromonas sp. PS5. Mutation of this gene ablates the bacterium's ability in live cultures to stimulate the metamorphosis of the stony coral Porites astreoides. We further demonstrate that expression of TBP biosynthesis genes is strongest in stationary and biofilm modes of growth, where Pseudoalteromonas sp. PS5 might exist within surface-attached biofilms on the sea floor. Finally, we create a modular transposon plasmid for genomic integration and fluorescent labeling of Pseudoalteromonas sp. PS5 cells. Our results functionally link a TBP biosynthesis gene from live bacteria to a morphogenic effect in corals. The genetic techniques established here provide new tools to explore coral-bacteria interactions and could help to inform future decisions about utilizing marine bacteria or their products for coral restoration.

A modular plasmid toolkit applied in marine bacteria reveals functional insights during bacteria-stimulated metamorphosis.

A conspicuous roadblock to studying marine bacteria for fundamental research and biotechnology is a lack of modular synthetic biology tools for their genetic manipulation. Here, we applied, and generated new parts for, a modular plasmid toolkit to study marine bacteria in the context of symbioses and host-microbe interactions. To demonstrate the utility of this plasmid system, we genetically manipulated the marine bacterium Pseudoalteromonas luteoviolacea, which stimulates the metamorphosis of the model tubeworm, Hydroides elegans. Using these tools, we quantified constitutive and native promoter expression, developed reporter strains that enable the imaging of host-bacteria interactions, and used CRISPR interference (CRISPRi) to knock down a secondary metabolite and a host-associated gene. We demonstrate the broader utility of this modular system for testing the genetic tractability of marine bacteria that are known to be associated with diverse host-microbe symbioses. These efforts resulted in the successful conjugation of 12 marine strains from the Alphaproteobacteria and Gammaproteobacteria classes. Altogether, the present study demonstrates how synthetic biology strategies enable the investigation of marine microbes and marine host-microbe symbioses with potential implications for environmental restoration and biotechnology. IMPORTANCE Marine Proteobacteria are attractive targets for genetic engineering due to their ability to produce a diversity of bioactive metabolites and their involvement in host-microbe symbioses. Modular cloning toolkits have become a standard for engineering model microbes, such as Escherichia coli, because they enable innumerable mix-and-match DNA assembly and engineering options. However, such modular tools have not yet been applied to most marine bacterial species. In this work, we adapt a modular plasmid toolkit for use in a set of 12 marine bacteria from the Gammaproteobacteria and Alphaproteobacteria classes. We demonstrate the utility of this genetic toolkit by engineering a marine Pseudoalteromonas bacterium to study their association with its host animal Hydroides elegans. This work provides a proof of concept that modular genetic tools can be applied to diverse marine bacteria to address basic science questions and for biotechnology innovations.

Future research directions of the model marine tubeworm Hydroides elegans and synthesis of developmental staging of the complete life cycle.

BACKGROUND: The biofouling marine tube worm, Hydroides elegans, is an indirect developing polychaete with significance as a model organism for questions in developmental biology and the evolution of host-microbe interactions. However, a complete description of the life cycle from fertilization through sexual maturity remains scattered in the literature, and lacks standardization. RESULTS AND DISCUSSION: Here, we present a unified staging scheme synthesizing the major morphological changes that occur during the entire life cycle of the animal. These data represent a complete record of the life cycle, and serve as a foundation for connecting molecular changes with morphology. CONCLUSIONS: The present synthesis and associated staging scheme are especially timely as this system gains traction within research communities. Characterizing the Hydroides life cycle is essential for investigating the molecular mechanisms that drive major developmental transitions, like metamorphosis, in response to bacteria.

Linking bacterial tetrabromopyrrole biosynthesis to coral metamorphosis.

An important factor dictating coral fitness is the quality of bacteria associated with corals and coral reefs. One way that bacteria benefit corals is by stimulating the larval to juvenile life cycle transition of settlement and metamorphosis. Tetrabromopyrrole (TBP) is a small molecule produced by bacteria that stimulates metamorphosis in a range of coral species. A standing debate remains, however, about whether TBP biosynthesis from live Pseudoalteromonas bacteria is the primary stimulant of coral metamorphosis. In this study, we create a Pseudoalteromonas sp. PS5 mutant lacking the TBP brominase gene, bmp2 . Using this mutant, we confirm that the bmp2 gene is critical for TBP biosynthesis in Pseudoalteromonas sp. PS5. Mutation of this gene ablates the bacterium's ability in live cultures to stimulate the metamorphosis of the stony coral Porites astreoides . We further demonstrate that expression of TBP biosynthesis genes is strongest in stationary and biofilm modes of growth, where Pseudoalteromonas sp. PS5 might exist within surface-attached biofilms on the sea floor. Finally, we create a modular transposon plasmid for genomic integration and fluorescent labeling of Pseudoalteromonas sp. PS5 cells. Our results functionally link a TBP biosynthesis gene from live bacteria to a morphogenic effect in corals. The genetic techniques established here provide new tools to explore coral-bacteria interactions and could help to inform future decisions about utilizing marine bacteria or their products for restoring degraded coral reefs.

A modular plasmid toolkit applied in marine Proteobacteria reveals functional insights during bacteria-stimulated metamorphosis.

A conspicuous roadblock to studying marine bacteria for fundamental research and biotechnology is a lack of modular synthetic biology tools for their genetic manipulation. Here, we applied, and generated new parts for, a modular plasmid toolkit to study marine bacteria in the context of symbioses and host-microbe interactions. To demonstrate the utility of this plasmid system, we genetically manipulated the marine bacterium Pseudoalteromonas luteoviolacea , which stimulates the metamorphosis of the model tubeworm, Hydroides elegans . Using these tools, we quantified constitutive and native promoter expression, developed reporter strains that enable the imaging of host-bacteria interactions, and used CRISPR interference (CRISPRi) to knock down a secondary metabolite and a host-associated gene. We demonstrate the broader utility of this modular system for rapidly creating and iteratively testing genetic tractability by modifying marine bacteria that are known to be associated with diverse host-microbe symbioses. These efforts enabled the successful transformation of twelve marine strains across two Proteobacteria classes, four orders and ten genera. Altogether, the present study demonstrates how synthetic biology strategies enable the investigation of marine microbes and marine host-microbe symbioses with broader implications for environmental restoration and biotechnology.

Draft Genome Sequence of Nereida sp. Strain MMG025, Isolated from Giant Kelp.

Here, we report the draft genome sequence of Nereida sp. strain MMG025, isolated from the surface of giant kelp and assembled and analyzed by undergraduate students participating in a marine microbial genomics course. A genomic comparison suggests that MMG025 is a novel species, providing a resource for future microbiology and biotechnology investigations.

Diacylglycerol, PKC and MAPK signaling initiate tubeworm metamorphosis in response to bacteria.

External environmental cues can have significant impacts on the timing and outcomes of animal development. For the swimming larvae of many marine invertebrates, the presence of specific surface-bound bacteria are important cues that help larvae identify a suitable location on the sea floor for metamorphosis and adult life. While metamorphosis in response to bacteria occurs in diverse animals from across the animal tree of life, we know little about the signal transduction cascades stimulated at the onset of metamorphosis upon their interaction with bacteria. The metamorphosis of a model tubeworm, Hydroides elegans, is triggered by the bacterium Pseudoalteromonas luteoviolacea which produces a stimulatory protein called Mif1. In this work, we define three key nodes in a signaling cascade promoting Hydroides metamorphosis in response to Mif1. Using metabolomic profiling, we find that the stimulation of Hydroides larvae by P. luteoviolacea leads to an increase in diacylglycerol during the initiation of metamorphosis, and that Mif1 is necessary for this upregulation. Genomic and pharmacological examination suggests that diacylglycerol triggers a phosphotransferase signaling cascade involving Protein Kinase C (PKC) and Mitogen-Activated Protein Kinase (MAPK), to induce Hydroides metamorphosis. Additionally, Mif1 activates the expression of two nuclear hormone receptors, HeNHR1 and HeNHR2 in the cerebral ganglia of Hydroides larvae. Our results define a post-translational signal transduction pathway mediating bacteria-stimulated metamorphosis in a model invertebrate animal.

Draft Genome Sequences of 10 Bacteria from the Marine Pseudoalteromonas Group.

Here, we report the draft genome sequences of 10 marine Pseudoalteromonas bacteria that were isolated, assembled, and annotated by undergraduate students participating in a marine microbial genomics course. Genomic comparisons suggest that 7 of the 10 strains are novel isolates, providing a resource for future marine microbiology investigations.

Bacteria-Stimulated Metamorphosis: an Ocean of Insights from Investigating a Transient Host-Microbe Interaction.

Recent research on host-microbe interactions has focused on intimate symbioses. Yet transient interactions, such as the stimulation of animal metamorphosis by bacteria, can have significant impacts on each partner. During these short-lived interactions, swimming animal larvae identify a desirable location on the seafloor and undergo metamorphosis into a juvenile based on the presence of specific bottom-dwelling bacteria. While this phenomenon is critical for seeding new animals to establish or maintain benthic ecosystems, there is an ocean of fundamental questions that remain unanswered. Here, I propose an updated model of how bacteria stimulate animal metamorphosis based on evidence that bacteria inject a stimulatory protein that prompts tubeworm metamorphosis. I consider what we hope to learn about stimulatory bacterial products, how animals recognize these products, and the consequences for both partners. Finally, I provide examples of how studying an enigmatic host-microbe interaction can serve as an engine for scientific discovery.

Draft Genome Sequences of Two Bacteria from the Roseobacter Group.

Here, we report the draft genome sequences of strains HS012 and HS039, which were isolated from cnidarian polyps that had recently undergone metamorphosis. Genomic analyses place these strains within the Phaeobacter and Leisingera genera, members of the Roseobacter group.

The Influence of Bacteria on Animal Metamorphosis.

The swimming larvae of many marine animals identify a location on the seafloor to settle and undergo metamorphosis based on the presence of specific surface-bound bacteria. While bacteria-stimulated metamorphosis underpins processes such as the fouling of ship hulls, animal development in aquaculture, and the recruitment of new animals to coral reef ecosystems, little is known about the mechanisms governing this microbe-animal interaction. Here we review what is known and what we hope to learn about how bacteria and the factors they produce stimulate animal metamorphosis. With a few emerging model systems, including the tubeworm Hydroides elegans, corals, and the hydrozoan Hydractinia, we have begun to identify bacterial cues that stimulate animal metamorphosis and test hypotheses addressing their mechanisms of action. By understanding the mechanisms by which bacteria promote animal metamorphosis, we begin to illustrate how, and explore why, the developmental decision of metamorphosis relies on cues from environmental bacteria.

Genetic examination of the marine bacterium Pseudoalteromonas luteoviolacea and effects of its metamorphosis-inducing factors.

Pseudoalteromonas luteoviolacea is a globally distributed marine bacterium that stimulates the metamorphosis of marine animal larvae, an important bacteria-animal interaction that can promote the recruitment of animals to benthic ecosystems. Recently, different P. luteoviolacea isolates have been shown to produce two stimulatory factors that can induce tubeworm and coral metamorphosis; Metamorphosis-Associated Contractile structures (MACs) and tetrabromopyrrole (TBP) respectively. However, it remains unclear what proportion of P. luteoviolacea isolates possess the genes encoding MACs, and what phenotypic effect MACs and TBP have on other larval species. Here, we show that 9 of 19 sequenced P. luteoviolacea genomes genetically encode both MACs and TBP. While P. luteoviolacea biofilms producing MACs stimulate the metamorphosis of the tubeworm Hydroides elegans, TBP biosynthesis genes had no effect under the conditions tested. Although MACs are lethal to larvae of the cnidarian Hydractinia symbiologicarpus, P. luteoviolacea mutants unable to produce MACs are capable of stimulating metamorphosis. Our findings reveal a hidden complexity of interactions between a single bacterial species, the factors it produces and two species of larvae belonging to different phyla.

Complete Genome Sequences of Two Marine Biofilm Isolates, Leisingera sp. nov. Strains 201A and 204H, Novel Representatives of the Roseobacter Group.

Here, we report the complete-genome assemblies of biofilm isolates 201A and 204H. They possess six and seven plasmids, respectively, with a size ranging from 44 kb to 159 kb. Genomic comparisons place the two strains into one new species belonging to the genus Leisingera as novel representatives of the Roseobacter group.

A Distinct Contractile Injection System Gene Cluster Found in a Majority of Healthy Adult Human Microbiomes.

Many commensal bacteria antagonize each other or their host by producing syringe-like secretion systems called contractile injection systems (CIS). Members of the Bacteroidales family have been shown to produce only one type of CIS-a contact-dependent type 6 secretion system that mediates bacterium-bacterium interactions. Here, we show that a second distinct cluster of genes from Bacteroidales bacteria from the human microbiome may encode yet-uncharacterized injection systems that we term Bacteroidales injection systems (BIS). We found that BIS genes are present in the gut microbiomes of 99% of individuals from the United States and Europe and that BIS genes are more prevalent in the gut microbiomes of healthy individuals than in those individuals suffering from inflammatory bowel disease. Gene clusters similar to that of the BIS mediate interactions between bacteria and diverse eukaryotes, like amoeba, insects, and tubeworms. Our findings highlight the ubiquity of the BIS gene cluster in the human gut and emphasize the relevance of the gut microbiome to the human host. These results warrant investigations into the structure and function of the BIS and how they might mediate interactions between Bacteroidales bacteria and the human host or microbiome.IMPORTANCE To engage with host cells, diverse pathogenic bacteria produce syringe-like structures called contractile injection systems (CIS). CIS are evolutionarily related to the contractile tails of bacteriophages and are specialized to puncture membranes, often delivering effectors to target cells. Although CIS are key for pathogens to cause disease, paradoxically, similar injection systems have been identified within healthy human microbiome bacteria. Here, we show that gene clusters encoding a predicted CIS, which we term Bacteroidales injection systems (BIS), are present in the microbiomes of nearly all adult humans tested from Western countries. BIS genes are enriched within human gut microbiomes and are expressed both in vitro and in vivo Further, a greater abundance of BIS genes is present within healthy gut microbiomes than in those humans with with inflammatory bowel disease (IBD). Our discovery provides a potentially distinct means by which our microbiome interacts with the human host or its microbiome.

A contractile injection system stimulates tubeworm metamorphosis by translocating a proteinaceous effector.

The swimming larvae of many marine animals identify a location on the sea floor to undergo metamorphosis based on the presence of specific bacteria. Although this microbe-animal interaction is critical for the life cycles of diverse marine animals, what types of biochemical cues from bacteria that induce metamorphosis has been a mystery. Metamorphosis of larvae of the tubeworm Hydroides elegans is induced by arrays of phage tail-like contractile injection systems, which are released by the bacterium Pseudoalteromonas luteoviolacea. Here we identify the novel effector protein Mif1. By cryo-electron tomography imaging and functional assays, we observe Mif1 as cargo inside the tube lumen of the contractile injection system and show that the mif1 gene is required for inducing metamorphosis. Purified Mif1 is sufficient for triggering metamorphosis when electroporated into tubeworm larvae. Our results indicate that the delivery of protein effectors by contractile injection systems may orchestrate microbe-animal interactions in diverse contexts.

A Bacterial Phage Tail-like Structure Kills Eukaryotic Cells by Injecting a Nuclease Effector.

Many bacteria interact with target organisms using syringe-like structures called contractile injection systems (CISs). CISs structurally resemble headless bacteriophages and share evolutionarily related proteins such as the tail tube, sheath, and baseplate complex. In many cases, CISs mediate trans-kingdom interactions between bacteria and eukaryotes by delivering effectors to target cells. However, the specific effectors and their modes of action are often unknown. Here, we establish an ex vivo model to study an extracellular CIS (eCIS) called metamorphosis-associated contractile structures (MACs) that target eukaryotic cells. MACs kill two eukaryotic cell lines, fall armyworm Sf9 cells and J774A.1 murine macrophage cells, by translocating an effector termed Pne1. Before the identification of Pne1, no CIS effector exhibiting nuclease activity against eukaryotic cells had been described. Our results define a new mechanism of CIS-mediated bacteria-eukaryote interaction and are a step toward developing CISs as novel delivery systems for eukaryotic hosts.

Stepwise metamorphosis of the tubeworm Hydroides elegans is mediated by a bacterial inducer and MAPK signaling.

Diverse animal taxa metamorphose between larval and juvenile phases in response to bacteria. Although bacteria-induced metamorphosis is widespread among metazoans, little is known about the molecular changes that occur in the animal upon stimulation by bacteria. Larvae of the tubeworm Hydroides elegans metamorphose in response to surface-bound Pseudoalteromonas luteoviolacea bacteria, producing ordered arrays of phage tail-like metamorphosis-associated contractile structures (MACs). Sequencing the Hydroides genome and transcripts during five developmental stages revealed that MACs induce the regulation of groups of genes important for tissue remodeling, innate immunity, and mitogen-activated protein kinase (MAPK) signaling. Using two MAC mutations that block P. luteoviolacea from inducing settlement or metamorphosis and three MAPK inhibitors, we established a sequence of bacteria-induced metamorphic events: MACs induce larval settlement; then, particular properties of MACs encoded by a specific locus in P. luteoviolacea initiate cilia loss and activate metamorphosis-associated transcription; finally, signaling through p38 and c-Jun N-terminal kinase (JNK) MAPK pathways alters gene expression and leads to morphological changes upon initiation of metamorphosis. Our results reveal that the intricate interaction between Hydroides and P. luteoviolacea can be dissected using genomic, genetic, and pharmacological tools. Hydroides' dependency on bacteria for metamorphosis highlights the importance of external stimuli to orchestrate animal development. The conservation of Hydroides genome content with distantly related deuterostomes (urchins, sea squirts, and humans) suggests that mechanisms of bacteria-induced metamorphosis in Hydroides may have conserved features in diverse animals. As a major biofouling agent, insight into the triggers of Hydroides metamorphosis might lead to practical strategies for fouling control.

Biofilm Formation and Detachment in Gram-Negative Pathogens Is Modulated by Select Bile Acids.

Biofilms are a ubiquitous feature of microbial community structure in both natural and host environments; they enhance transmission and infectivity of pathogens and provide protection from human defense mechanisms and antibiotics. However, few natural products are known that impact biofilm formation or persistence for either environmental or pathogenic bacteria. Using the combination of a novel natural products library from the fish microbiome and an image-based screen for biofilm inhibition, we describe the identification of taurine-conjugated bile acids as inhibitors of biofilm formation against both Vibrio cholerae and Pseudomonas aeruginosa. Taurocholic acid (1) was isolated from the fermentation broth of the fish microbiome-derived strain of Rhodococcus erythropolis and identified using standard NMR and MS methods. Screening of the twelve predominant human steroidal bile acid components revealed that a subset of these compounds can inhibit biofilm formation, induce detachment of preformed biofilms under static conditions, and that these compounds display distinct structure-activity relationships against V. cholerae and P. aeruginosa. Our findings highlight the significance of distinct bile acid components in the regulation of biofilm formation and dispersion in two different clinically relevant bacterial pathogens, and suggest that the bile acids, which are endogenous mammalian metabolites used to solubilize dietary fats, may also play a role in maintaining host health against bacterial infection.

Marine tubeworm metamorphosis induced by arrays of bacterial phage tail-like structures.

Many benthic marine animal populations are established and maintained by free-swimming larvae that recognize cues from surface-bound bacteria to settle and metamorphose. Larvae of the tubeworm Hydroides elegans, an important biofouling agent, require contact with surface-bound bacteria to undergo metamorphosis; however, the mechanisms that underpin this microbially mediated developmental transition have been enigmatic. Here, we show that a marine bacterium, Pseudoalteromonas luteoviolacea, produces arrays of phage tail-like structures that trigger metamorphosis of H. elegans. These arrays comprise about 100 contractile structures with outward-facing baseplates, linked by tail fibers and a dynamic hexagonal net. Not only do these arrays suggest a novel form of bacterium-animal interaction, they provide an entry point to understanding how marine biofilms can trigger animal development.