Publications

4416

ABSTRACT 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. IMPORTANCE The 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.

El objetivo del estudio fue detectar especies del género Rickettsia en garrapatas de la especie Amblyomma tigrinum colectadas sobre carnívoros domésticos y en sangre de caninos domésticos de la provincia de San Luis (Argentina). Entre 2013 y 2015 se colectaron 56 garrapatas adultas de la especie A. tigrinum sobre caninos y felinos domésticos, y se obtuvieron 65 muestras sanguíneas de caninos. Tres garrapatas resultaron positivas mediante la amplificación de un fragmento del espacio intergénico 23S-5S ARNr del género Rickettsia, lográndose secuenciar uno de los productos positivos. La muestra positiva secuenciada también resultó positiva por PCRs de los fragmentos de los genes gltA y ompA. Las secuencias obtenidas resultaron tener una identidad del 100 % de identidad con “Candidatus Rickettsia andeanae”. Todas las muestras sanguíneas resultaron negativas. “Ca. R. andeanae” no ha sido asociada con enfermedad en humanos o animales, sin embargo, es necesario realizar nuevas investigaciones para lograr un mayor conocimiento del riesgo potencial de transmisión de rickettsiosis en la región.

Abstract Tenericutes are a unique class of bacteria that lack a cell wall and are typically parasites or commensals of eukaryotic hosts. Environmental 16S rDNA surveys have identified a number of tenericute clades in diverse environments, introducing the possibility that these Tenericutes may represent non-host-associated, free-living microorganisms. Metagenomic sequencing of deep-sea methane seep sediments resulted in the assembly of two genomes from a Tenericutes-affiliated clade currently known as ‘NB1-n’ (SILVA taxonomy) or ‘RF3’ (Greengenes taxonomy). Metabolic reconstruction revealed that, like cultured members of the Mollicutes, these ‘NB1-n’ representatives lack a tricarboxylic acid cycle and instead use anaerobic fermentation of simple sugars for substrate level phosphorylation. Notably, the genomes also contained a number of unique metabolic features including hydrogenases and a simplified electron transport chain containing an RNF complex, cytochrome bd oxidase and complex I. On the basis of the metabolic potential predicted from the annotated genomes, we devised an anaerobic enrichment media that stimulated the growth of these Tenericutes at 10 °C, resulting in a mixed culture where these organisms represented ~60% of the total cells by targeted fluorescence in situ hybridization (FISH). Visual identification by FISH confirmed these organisms were not directly associated with Eukaryotes and electron cryomicroscopy of cells in the enrichment culture confirmed an ultrastructure consistent with the defining phenotypic property of Tenericutes, with a single membrane and no cell wall. On the basis of their unique gene content, phylogenetic placement and ultrastructure, we propose these organisms represent a novel class within the Tenericutes, and suggest the names Candidatus ‘Izimaplasma sp. HR1’ and Candidatus ‘Izimaplasma sp. HR2’ for the two genome representatives.