Genomic features of a? multicellular filamentous macrobacterium from the “Humboldt Sulfuretum”

The sublittoral Humboldt Sulfuretum (HS) as part of the Humboldt Eastern Boundary Upwelling Ecosystem extends below the oxygen/deficient waters of the Peru­Chile Undercurrent between ca. 6°S and ca. 36°S. According to early studies this type of benthic habitat appears to have been dominant in early Earth ocean. Featured by sulfur compounds, the HS is a particularly fitting biotope for filamentous and spherical macro­ and mega­bacteria, capable of sulfide oxidation through nitrate and/or oxygen. We describe genomic features of slender, non­vacuolated marine benthic bacterial macro­filaments here named “?Candidatus Venteria ishoeyensis” ("CVi") of the newly proposed family Venteriaceae in the Thiotrichales. Thus, the potential nitrate­reducing, sulphur-oxidizing metabolism, P and C acquisition, CRISPR ­Cas, and the presence of gene clusters potentially involved in the biosynthesis of secondary metabolites were assessed. Filaments of "CVi" were collected in central Chile's sublittoral HS (ca. 36°S), their DNA extracted, amplified, and sequenced through a Roche 454 GS FLX platform. Filaments were isolated by micromanipulation from freshly collected samples and their whole genome amplified during two separate sessions (December 2008 and February 2009). Firstly, samples were diluted in sterile filtered seawater to reduce cell densities. This step was followed by isolation by micromanipulation and washing/cleaning of the selected filament in sterile filtered seawater. Amplification was performed using the GenomiPhi HY kit (GE Healthcare) by the 7 alkaline lysis method and was terminated after 6 hr at 30°C. The WGA products were diluted 2­ fold in TE­buffer (stored at ­20°C) and a 20­fold dilution was prepared as working solution for PCR analysis and quantification. The purity of the amplified DNA was examined by PCR/sequencing of the 16S rRNA gene using universal primers. Analysis of the 16S rDNAsequences confirmed that the filaments MDA1, MDA2 and MDA5 contained identical species, and two of them were selected for genome analysis by sequencing of 3kb mate-pair libraries and one single­ end library through the Roche 454 GS FLX platform of sequencing. The genome was constructed through the de novo assembly using Celera and the annotation was carried out using Prokka.