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

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, a significant 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

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 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

Cellular Levels and Binding of c-di-GMP Control Subcellular Localization and Activity of the Vibrio cholerae Transcriptional Regulator VpsT

The second messenger, cyclic diguanylate (c-di-GMP), regulates diverse cellular processes in bacteria. C-di-GMP is produced by diguanylate cyclases (DGCs), degraded by phosphodiesterases (PDEs), and receptors couple c-di-GMP production to cellular responses. In many bacteria, including Vibrio cholerae, multiple DGCs and PDEs contribute to c-di-GMP signaling, and it is currently unclear whether the compartmentalization of c-di-GMP signaling components is required to mediate c-di-GMP signal transduction. In this study we show that the transcriptional regulator, VpsT, requires c-di-GMP binding for subcellular localization and activity. Only the additive deletion of five DGCs markedly decreases the localization of VpsT, while single deletions of each DGC do not impact VpsT localization. Moreover, mutations in residues required for c-di-GMP binding, c-di-GMP-stabilized dimerization and DNA binding of VpsT abrogate wild type localization and activity. VpsT does not co-localize or interact with DGCs suggesting that c-di-GMP from these DGCs diffuses to VpsT, supporting a model in which c-di-GMP acts at a distance. Furthermore, VpsT localization in a heterologous host, Escherichia coli, requires a catalytically active DGC and is enhanced by the presence of VpsT-target sequences. Our data show that c-di-GMP signaling can be executed through an additive cellular c-di-GMP level from multiple DGCs affecting the localization and activity of a c-di-GMP receptor and furthers our understanding of the mechanisms of second messenger signaling

Temporal Variation In An Initial Marine Biofilm Community And Its Effect On Larval Settlement Of The Tubeworm Hydroides Elegans

Planktonic larvae of many invertebrates settle preferentially on surfaces covered by bacterial biofilms. The polychaete tubeworm Hydroides elegans is induced to settle by biofilms and is the primary colonizer of newly submerged surfaces in the succession of macrofouling invertebrates in Pearl Harbor, Hawai'i. This study examines culture-independent community composition, as well as densities of bacteria, and how these aspects of marine biofilms affect settlement preferences of H. elegans. Settlement assays of H. elegans were conducted on naturally formed biofilms of increasing age from Pearl Harbor, Hawai'i. Denaturing Gradient Gel Electrophoresis (DGGE) and epifluoresence microscopy were used to identify community composition and densities of bacterial biofilms. This study showed that increased densities of bacteria rather than dominant species composition are likely responsible for the primary colonization of submerged surfaces by H. elegans in Pearl Harbor

Identification and Characterization of OscR, a Transcriptional Regulator Involved in Osmolarity Adaptation in Vibrio cholerae▿ †

Vibrio cholerae is a facultative human pathogen. In its aquatic habitat and as it passes through the digestive tract, V. cholerae must cope with fluctuations in salinity. We analyzed the genome-wide transcriptional profile of V. cholerae grown at different NaCl concentrations and determined that the expression of compatible solute biosynthesis and transporter genes, virulence genes, and genes involved in adhesion and biofilm formation is differentially regulated. We determined that salinity modulates biofilm formation, and this response was mediated through the transcriptional regulators VpsR and VpsT. Additionally, a transcriptional regulator controlling an osmolarity adaptation response was identified. This regulator, OscR (osmolarity controlled regulator), was found to modulate the transcription of genes involved in biofilm matrix production and motility in a salinity-dependent manner. oscR mutants were less motile and exhibited enhanced biofilm formation only under low-salt conditions

VpsT Does Not Interact with CdgA or CdgH Directly.

<p>(A) Representative epifluorescence micrographs of wild-type <i>V. cholerae</i> expressing GFP-VpsT, CdgA-GFP or CdgH-GFP fusion proteins. Marker is 2 µm. (B) Subcellular fractionation of <i>V. cholerae</i> strains containing <i>vpsT</i>, <i>cdgA</i> or <i>cdgH</i> tagged with an HA epitope in their native chromosomal loci. Western immunoblot was performed on cellular fractions representing whole cell (WC), cytoplasmic (C) and total membrane (M) fractions. HA-tagged proteins were detected using a polyclonal anti-HA antibody. <i>gfp</i> was constitutively expressed from a chromosomal locus. GFP was detected using monoclonal anti-GFP antibody and is used as a cytoplasmic fraction control. OmpU was detected using a polyclonal anti-OmpU antibody and is used as a total membrane fraction control. One representative experiment of three biological replicates is shown. (C) Bacterial two-hybrid analysis of VpsT, CdgA and CdgH. Reconstitution of CyaA, indicative of protein-protein interaction, was detected by β-galactosidase activity on LB plates containing ampicillin (100 µg/ml), kanamycin (50 µg/ml), IPTG (500 µM) and X-gal (40 µg/ml). Plates were incubated at 30°C for 48 h.</p

The Subcellular Localization of VpsT is Dependent on c-di-GMP Binding and DNA Binding Residues.

<p>(A) The expression of a chromosomal <i>vpsL</i> promoter-<i>lacZ</i> fusion was measured in wild type (Wt) or Δ<i>vpsT</i> strains containing pBAD vector alone, or pBAD containing <i>gfp</i> fused to wild type and mutated versions of <i>vpsT</i> using β-galactosidase assays. A R134A mutation disrupts c-di-GMP binding and an I141E mutation abolishes c-di-GMP-dependent dimerization. H193A lies in the DNA binding domain of VpsT. (B) Subcellular localization of GFP, GFP-VpsT or GFP-VpsT containing the indicated point mutations, expressed in <i>V. cholerae</i> Δ<i>vpsT</i>. Representative epifluorescence micrographs are shown. Marker is 2 µm. (C) Single-cell quantification of subcellular fluorescence localization. The number of spots per cell is shown as a histogram for Δ<i>vpsT</i> strains expressing GFP, GFP-VpsT or GFP-VpsT containing the indicated point mutations. Data are acquired from at least 3 independent experiments and quantification was performed on at least 150 cells per treatment.</p

VpsT Localization in a Heterologous Host Depends on c-di-GMP.

<p>(A) Representative epifluorescence micrographs of <i>E. coli</i> strains expressing GFP or GFP-VpsT and containing a pKNT25 compatible plasmid or pKNT25 harboring <i>cdgA</i>, <i>cdgA</i>^G287A, <i>vpsL</i> promoter (<i>vpsLp</i>), <i>cdgA</i> and <i>vpsLp</i> or <i>adrA</i> from <i>S. typhimurium</i>. Marker is 2 µm. (B) Single-cell quantification of subcellular fluorescence localization. The number of spots per cell is shown as a histogram for <i>E. coli</i> strains containing the indicated plasmids. Data are acquired from at least 3 independent experiments and quantification was performed on at least 150 cells per treatment. (C) Representative motility phenotypes of <i>E. coli</i> expressing the indicated plasmids grown on soft agar plates containing kanamycin and 10 µM IPTG at 37°C for 12 h.</p