Dataset: soxB Sequences
Deployment: AT18-08

Diffuse fluids were collected from newly discovered snowblower vents at Axial Seamount in late July 2011 with the ROV Jason II using the hydrothermal fluid and particle sampler (Butterfield et al., 2004). White and orange flocculent materials were collected on the subsequent University Of Washington Visions' 11 cruise, in support of the Regional Scale Nodes component of the Ocean Observatories Initiative in August 2011. White flocculent material was collected from the orifice of the Subway snowblower vent on dives R1467 (White Floc 1) and R1472 (White Floc 2) and orange flocculent material was collected on the seafloor distal to Marker 33 during dive R1472 where it coated freshly deposited basalt. All of the fluid and floc samples analyzed in this study are from a small area in the south rift zone at the southeastern edge of Axial Caldera, with the exception of background seawater which was collected outside of the caldera.

Total genomic DNA was extracted from Sterivex filters as previously described (Sogin et al., 2006) with the minor modifications described by Akerman et al. (2013). Total genomic DNA was extracted from 20 to 30mg of wet flocculent material using a MoBio UltraClean® Soil DNA Isolation Kit.

We targeted the amplification of the soxB gene in Epsilonproteobacteria to assess the diversity of sulfur oxidizers in white floc vs. orange floc samples. The soxB gene was amplified in one white flocculent sample (White Floc 2) and in the orange flocculent sample, using the newly designed primers and conditions described by Akerman et al. (2013). The PCR reaction mixture consisted of 1X buffer (Promega), 4 mM MgCl2, 0.2 mM of each deoxynucleoside triphosphate (dNTP), 0.6 uM of each primer, 1U GoTaq polymerase (Promega), 1 ul DNA template, and DEPC H2O to 25 ul. Thermocycling conditions on an Eppendorf thermal cycler consisted of an initial denaturation step at 94 degrees C for 3 min, followed by 35 cycles of 94 degrees C for 30s, 46 degrees C for 45 s, and 72 degrees C for 1 min, followed by a final extension at 72 degrees C for 5 min. The primers used in this study were sox527F (5'-TGGTWGGWCAYTGGGAATTTA-3') and sox1198R (5'-AGAANGTATCTCKYTTATAAAG-3'). These primers target the genera Sulfurovum, Sulfurimonas, and Nitratiruptor. The soxB gene in members of the Epsilonproteobacterial genera Arcobacter and Nitratifractor are more like sequences in Gammaproteobacteria and are not expected to amplify with this primer set. Successfully amplified soxB PCR products were cleaned with a Qiagen MinElute PCR purification kit and run on a 0.8% agarose gel. Bands in the expected size range were gel excised, purified with the MinElute kit, cloned, and sequenced as previously described (Huber et al., 2009).

Related references:
Meyer, J.L., Akerman, N.H., Proskurowski, G. and J.A. Huber. 2013. Microbiological characterization of post-eruption "snowblower" vents at Axial Seamount, Juan de Fuca Ridge. Frontiers in Microbiology. 4:153. doi: 10.3389/fmicb.2013.00153