DNeasy PowerLyzer Microbial Kit (50)

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Soils originated from the Great Lakes Bioenergy Research Center (GLBRC) located at the W.K. Kellogg Biological Station (KBS) Long-Term Ecological Research Site (Hickory Corners, MI, 42°24′N, 85°24′W). No-till soil samples under perennial switchgrass (Panicum virgatum) were collected from 0–15 cm depth over three days in May 2017. The upper 5 cm were discarded to reduce the influence of surface litter on the soil. Bulk soil was transported to the lab on ice packs, and passed through a 2 mm sieve. Phylogenetically-specific agar was used to enrich gram-negative and gram-positive bacteria, and fungi from whole soil using Yeast mannitol agar (YMA)25 to enrich the gram-negatives Bradyrhizobium and Rhodopseudomonas, Nutrient agar (NUA)26 for the gram-positive Bacillus, and Czapeck Dox agar (CDA)27 as synthetic media for fungal growth. After two weeks of incubating soil on the phylogenetically-specific agar, grown colonies were transferred to broth comprising of 50 mL M9 mineral medium28 and incubated at 21 °C. Once the stationary phase was reached, the cell solution was centrifuged at 10,000 rpm for 15 min (Eppendorf Centrifuge 5810 R, Eppendorf North America, New York, USA). For TEM imaging, 50 μg of each cell pellet was resuspended in 2 mL sterile ultra-pure water and used to image intact cells or disrupted using an ultra-sonication bath for 10 min (Branson 2800 CPX, Branson Ultrasonics, Danbury, USA) to image lysed cells. A whole mount approach was applied for imaging of the cells. For each sample, a 5 μL drop of the cell solution was applied to a 100-mesh Cu grid covered with formvar support film sputtered with carbon (Electron Microscopy Sciences, Hatfield, PA, USA). The material was allowed to adhere to the grid for 1 min before the liquid was gently blotted with a filter paper, and the material was negatively stained with a 5 μL drop of Nano-W (Nanoprobes, Yaphank, NY, USA). After 3 s, the excess liquid was removed by wicking and the sample was allowed to air dry. Samples were examined with a Tecnai T-12 TEM (FEI) with a LaB6 filament operating at 120 kV. Images were collected digitally with a 2 × 2 K UltraScan CCD (Gatan, Pleasanton, CA, USA). For each sample, at least 50 regions within the grid containing multiple cells were analyzed before collecting representative images at 6,500x magnification. 50 μg of the remaining cell pellet was used for DNA extraction with the DNeasy PowerLyzer Microbial Kit part #12255-50 (Qiagen, Venlo, Netherlands).

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