The marine bryozoan, Bugula neritina, is the source of the bryostatins, a family of macrocyclic lactones with anticancer activity. Bryostatins have long been suspected to be bacterial products. B. neritina harbors the uncultivated gamma proteobacterial symbiont "Candidatus Endobugula sertula." In this work several lines of evidence are presented that show that the symbiont is the most likely source of bryostatins. Bryostatins are complex polyketides similar to bacterial secondary metabolites synthesized by modular type I polyketide synthases (PKS-I). PKS-I gene fragments were cloned from DNA extracted from the B. neritina-"E. sertula" association, and then primers specific to one of these clones, KSa, were shown to amplify the KSa gene specifically and universally from total B. neritina DNA. In addition, a KSa RNA probe was shown to bind specifically to the symbiotic bacteria located in the pallial sinus of the larvae of B. neritina and not to B. neritina cells or to other bacteria. Finally, B. neritina colonies grown in the laboratory were treated with antibiotics to reduce the numbers of bacterial symbionts. Decreased symbiont levels resulted in the reduction of the KSa signal as well as the bryostatin content. These data provide evidence that the symbiont E. sertula has the genetic potential to make bryostatins and is necessary in full complement for the host bryozoan to produce normal levels of bryostatins. This study demonstrates that it may be possible to clone bryostatin genes from B. neritina directly and use these to produce bryostatins in heterologous host bacteria.
Many bacterial plasmids and chromosomes rely on ParA ATPases for proper positioning within the cell and for efficient segregation to daughter cells. Here we demonstrate that the F-plasmid-partitioning protein SopA polymerizes into filaments in an ATP-dependent manner in vitro, and that the filaments elongate at a rate that is similar to that of plasmid separation in vivo. We show that SopA is a dynamic protein within the cell, undergoing cycles of polymerization and depolymerization, and shuttling back and forth between nucleoprotein complexes that are composed of the SopB protein bound to sopC-containing plasmids (SopB͞sopC). The dynamic behavior of SopA is critical for Sop-mediated plasmid DNA segregation; mutations that lock SopA into a static polymer in the cell inhibit plasmid segregation. We show that SopA colocalizes with SopB͞sopC in the cell and that SopB͞sopC nucleates the assembly of SopA and is required for its dynamic behavior. When SopA is polymerized in vitro in the presence of SopB and sopCcontaining DNA, SopA filaments emanate from the plasmid DNA in radial asters. We propose a mechanism in which plasmid separation is driven by the polymerization of SopA, and we speculate that the radial assembly of SopA polymers is responsible for positioning plasmids both before and after segregation.ParA ͉ chromosome segregation ͉ cytoskeleton ͉ plasmid ͉ Escherichia coli
The bryozoans Bugula neritina and Bugula simplex harbor bacteria in the pallial sinuses of their larvae as seen by electron microscopy. In B. neritina, the bacterial symbiont has been characterized as a gammaproteobacterium, "Candidatus Endobugula sertula." "Candidatus E. sertula" has been implicated as the source of the bryostatins, polyketides that provide chemical defense to the host and are also being tested for use in human cancer treatments. In this study, the bacterial symbiont in B. simplex larvae was identified by 16S rRNA-targeted PCR and sequencing as a gamma-proteobacterium closely related to and forming a monophyletic group with "Candidatus E. sertula." In a fluorescence in situ hybridization, a 16S ribosomal DNA probe specific to the B. simplex symbiont hybridized to long rod-shaped bacteria in the pallial sinus of a B. simplex larva. The taxonomic status "Candidatus Endobugula glebosa" is proposed for the B. simplex larval symbiont. Degenerate polyketide synthase (PKS) primers amplified a gene fragment from B. simplex that closely matched a PKS gene fragment from the bryostatin PKS cluster. PCR surveys show that the symbiont and this PKS gene fragment are consistently and uniquely associated with B. simplex. Bryostatin activity assays and chemical analyses of B. simplex extracts reveal the presence of compounds similar to bryostatins. Taken together, these findings demonstrate a symbiosis in B. simplex that is similar and evolutionarily related to that in B. neritina.Bugula is a bryozoan genus with a cosmopolitan distribution (15, 34). Members of this genus are colonial organisms that reproduce sexually by means of releasing free-swimming lecithotrophic (nonfeeding) larvae, which can settle, metamorphose, and develop into new colonies. One species, B. neritina, is a common fouling organism in temperate and tropical waters (34). By using transmission electron microscopy, Woollacott observed that the larvae of B. neritina harbored rod-shaped bacteria in the pallial sinus, a circular invagination of the larval surface, which is effectively sealed from the surrounding seawater (38). These extracellular symbionts were a monoculture of a gamma-proteobacterium, "Candidatus Endobugula sertula" (14), which has not been cultivated to date and has been implicated in the production of a family of polyketide metabolites called bryostatins (8). There are 20 known bryostatins in this family (21,25), and while they all share a common macrocyclic bryopyran skeleton, they can be separated into two groups, chemotype O and M (9). The O chemotype is only found in B. neritina from California waters deeper than 10 m; the M bryostatins are found in all B. neritina populations that have "Candidatus E. sertula" (9, 22). Bryostatins 10 and 20 confer chemical defense to B. neritina by deterring fish from eating its larvae (20,21). In addition to the ecological role of bryostatins, bryostatin 1 is being tested as an anticancer drug in humans (13,37). Bryostatin 1 in humans modulates protein kinase C (PKC) activity (13), and t...
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