ABSTRACT
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 (SopB/sopC). 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 SopB/sopC in the cell and that SopB/sopC nucleates the assembly of SopA and is required for its dynamic behavior. When SopA is polymerized in vitro in the presence of SopB and sopC-containing 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.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Chromosome Segregation , Chromosomes, Bacterial/metabolism , DNA, Bacterial/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Cell Nucleus/genetics , Cell Nucleus/metabolism , Chromosomes, Bacterial/genetics , DNA, Bacterial/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Mutation/genetics , Plasmids/genetics , Protein Binding , Protein Structure, QuaternaryABSTRACT
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 gamma-proteobacterium, "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.
Subject(s)
Bryozoa/microbiology , Gammaproteobacteria/classification , Symbiosis , Amino Acid Sequence , Animals , Bryostatins , Gammaproteobacteria/genetics , Gammaproteobacteria/growth & development , In Situ Hybridization, Fluorescence , Lactones/metabolism , Larva/microbiology , Macrolides , Molecular Sequence Data , Multienzyme Complexes/genetics , Multienzyme Complexes/metabolism , Phylogeny , Polymerase Chain Reaction , SeawaterABSTRACT
This review discusses approaches to identify, clone, and express bioactive metabolite genes from symbionts of marine invertebrates. Criteria for proving symbiotic origin of bioactive metabolites are presented, followed by a comprehensive, practically-oriented overview of techniques to be applied. The Bugula neritina/Endobugula sertula association is used as a primary example, but other symbioses are discussed. Thirty-six compounds are presented and 111 references are cited.
