Prokaryotes rely on a distant tubulin homolog, FtsZ, for assembling the cytokinetic ring essential for cell division, but are otherwise generally thought to lack tubulin-like polymers that participate in processes such as DNA segregation. Here we characterize a protein (TubZ) from the Bacillus thuringiensis virulence plasmid pBtoxis, which is a member of the tubulin/FtsZ GTPase superfamily but is only distantly related to both FtsZ and tubulin. TubZ assembles dynamic, linear polymers that exhibit directional polymerization with plus and minus ends, movement by treadmilling, and a critical concentration for assembly. A point mutation (D269A) that alters a highly conserved catalytic residue within the T7 loop completely eliminates treadmilling and allows the formation of stable polymers at a much lower protein concentration than the wild-type protein.When expressed in trans, TubZ(D269A) coassembles with wild-type TubZ and significantly reduces the stability of pBtoxis, demonstrating a direct correlation between TubZ dynamics and plasmid maintenance. The tubZ gene is in an operon with tubR, which encodes a putative DNA-binding protein that regulates TubZ levels. Our results suggest that TubZ is representative of a novel class of prokaryotic cytoskeletal proteins important for plasmid stability that diverged long ago from the ancient tubulin/FtsZ ancestor.[Keywords: FtsZ; tubulin; cytoskeleton] Supplemental material is available at http://www.genesdev.org.

Summary of recent advances Bryostatins are a family of protein kinase C modulators that have potential applications in biomedicine. Found in miniscule quantities in a small marine invertebrate, lack of supply has hampered their development. In recent years, bryostatins have been shown to have potent bioactivity in the central nervous system, an uncultivated marine bacterial symbiont has been shown to be the likely natural source of the bryostatins, the bryostatin biosynthetic genes have been identified and characterized, and bryostatin analogues with promising biological activity have been developed and tested. Challenges in development of bryostatins for biomedical and biotechnological application include the cultivation of the bacterial symbiont and heterologous expression of bryostatin biosynthesis genes. Continued exploration of the biology and the symbiotic origin of the bryostatins presents promising opportunities for discovery of additional bryostatins, and new functions for bryostatins.

The uncultured bacterial symbiont "Candidatus Endobugula sertula" is known to produce cytotoxic compounds called bryostatins, which protect the larvae of its host, Bugula neritina. The symbiont has never been successfully cultured, and it was thought that its genome might be significantly reduced. Here, we took a shotgun metagenomics and metatranscriptomics approach to assemble and characterize the genome of "Ca. Endobugula sertula." We found that it had specific metabolic deficiencies in the biosynthesis of certain amino acids but few other signs of genome degradation, such as small size, abundant pseudogenes, and low coding density. We also identified homologs to genes associated with insect pathogenesis in other gammaproteobacteria, and these genes may be involved in host-symbiont interactions and vertical transmission. Metatranscriptomics revealed that these genes were highly expressed in a reproductive host, along with bry genes for the biosynthesis of bryostatins. We identified two new putative bry genes fragmented from the main bry operon, accounting for previously missing enzymatic functions in the pathway. We also determined that a gene previously assigned to the pathway, bryS, is not expressed in reproductive tissue, suggesting that it is not involved in the production of bryostatins. Our findings suggest that "Ca. Endobugula sertula" may be able to live outside the host if its metabolic deficiencies are alleviated by medium components, which is consistent with recent findings that it may be possible for "Ca. Endobugula sertula" to be transmitted horizontally. IMPORTANCEThe bryostatins are potent protein kinase C activators that have been evaluated in clinical trials for a number of indications, including cancer and Alzheimer's disease. There is, therefore, considerable interest in securing a renewable supply of these compounds, which is currently only possible through aquaculture of Bugula neritina and total chemical synthesis. However, these approaches are labor-intensive and low-yielding and thus preclude the use of bryostatins as a viable therapeutic agent. Our genome assembly and transcriptome analysis for "Ca. Endobugula sertula" shed light on the metabolism of this symbiont, potentially aiding isolation and culturing efforts. Our identification of additional bry genes may also facilitate efforts to express the complete pathway heterologously. Marine invertebrates, such as tunicates and sponges, are known to harbor symbiotic communities of bacteria. Because these animals are sessile and have limited physical defenses, their microbiomes are thought to serve defensive functions, and bacterial symbionts in these invertebrates have been implicated in the production of many bioactive small molecules (1, 2). Such small molecules can possess therapeutically relevant activities, so there is great interest in studying the biosynthetic potential and symbiotic functions of these defensive microorganisms (1, 2). However, most symbionts (and most environmental bacteria in general [1,3,4]) are difficul...

Direct, untargeted sequencing of environmental samples (metagenomics) and de novo genome assembly enable the study of uncultured and phylogenetically divergent organisms. However, separating individual genomes from a mixed community has often relied on the differential-coverage analysis of multiple, deeply sequenced samples. In the metagenomic investigation of the marine bryozoan Bugula neritina, we uncovered seven bacterial genomes associated with a single B. neritina individual that appeared to be transient associates, two of which were unique to one individual and undetectable using certain “universal” 16S rRNA primers and probes. We recovered high quality genome assemblies for several rare instances of “microbial dark matter,” or phylogenetically divergent bacteria lacking genomes in reference databases, from a single tissue sample that was not subjected to any physical or chemical pre-treatment. One of these rare, divergent organisms has a small (593 kbp), poorly annotated genome with low GC content (20.9%) and a 16S rRNA gene with just 65% sequence similarity to the closest reference sequence. Our findings illustrate the importance of sampling strategy and de novo assembly of metagenomic reads to understand the extent and function of bacterial biodiversity.