4, PBS) with final suspension in distilled water. Samples were negatively stained with an equal volume of 2% phosphotungstic acid (pH 7.0) and mounted on a formvar/carbon reinforced 200-mesh copper grid. Grids were examined at 80 kV under a FEI Tecnai G2 electron microscope click here equipped with AMT camera. Metabolic

characterization Bacterial cells from log phase culture grown in BMV with glucose and 10% bovine serum were collected by centrifugation (10,000 × g, 10 minutes), washed twice in isotonic saline and resuspended in isotonic saline to a density of 5–6 using McFarland standard. The API-ZYM test (bioMerieux) was performed per manufacturer’s instructions. The enzyme β-glucosidase (0.2 g/L, Sigma) was used as an internal control. Volatile fatty acid quantification To determine volatile fatty acid production, 9.9 mL of BMV medium with glucose and 10% bovine serum was inoculated with 100 μl of 1 × 108 growing bacterial cells/ml and incubated at 37°C for 72–96 hrs. The culture was then centrifuged to remove cellular material and the supernatant

prepared for gas-phase liquid chromatography as previously described [33–35]. Uninoculated medium was used as a control. Hydrogen sulfide production 100 μl containing 1 × 108 bacterial cells/ml from log phase cultures were inoculated into 9.9 ml BMV BI 6727 cell line and cultured for 72 hours. Hydrogen sulfide was assayed by using the lead acetate test as previously described [36]. DNA sequencing and analysis DNA from isolate 4A was extracted from 100 mL growing broth cultures using DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA) as per manufacturer’s instructions. Sequencing reactions were based upon Roche FLX-Titanium and Titanium + chemistry (Roche/454 Life Sciences, Branford, CT 06405; http://​www.​454.​com) as well as Illumina chemistry (Illumina, Inc., San Diego, CA

92122; http://​www.​illumina.​com). Genomic DNA was processed according to manufacturer’s instructions for preparation of DNA libraries. Whole genome random libraries were prepared and sequenced using the Illumina HiSeq 2000 and a Roche GS-FLX + instrument. In addition, genomic DNA Lepirudin was used to prepare paired-end libraries of 2Kb and 8Kb according to Roche protocols and was sequenced using the Roche GS-FLX + instrument and Titanium sequencing chemistries. Sequencing data from each of the methodologies was used to perform a de novo assembly using both the MIRA assembler [37] and the Roche gsAssembler (Newbler) version 2.6, (Roche/454 Life Sciences, Branford, CT 06405, USA; http://​www.​454.​com) Mauve Genome Alignment software was employed to compare assemblies and optimize the resulting de novo assembly. The draft genome assembly consisted of 42 contigs in 14 scaffolds and a total of 3,027,773 bp assembled (Newbler) from a combined coverage of greater than 90×. This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession AQCF00000000.