Identification of soil bacteria assimilating carbon from 13C-labelled wheat residue by RNA-based Stable Isotope Probing
To analyze the taxonomic composition of the soil bacterial community in selected density fractions, the V1-V3 region of the 16S rRNA gene (Escherichia coli positions 8 to 536) was chosen for amplification and subsequent pyrosequencing of the PCR products. The V1-V3 region was amplified with the primer set Ba27f and Ba518r containing the Roche 454 pyrosequencing adaptors (Ba27f: 5’-CGTATCGCCTCCCTCGCGCCATCAGAGAGTTTGATCCTGGCTCAG-3’, Ba518r_mod: 5’-CTATGCGCCTTGCCAGCCCGCTCAGCGTATTACCGCGGCTGCTGG-3’) in duplicates. Between the Roche 454 pyrosequencing adaptor and the forward primer, 10-bp-barcodes were added to allow the sorting of each sample from the mixed pyrosequencing outcome. PCR products were pooled and purified, quantification of the PCR products was performed using the Quant-iT dsDNA BR assay kit. Equal amounts of ten barcoded samples were pooled and the sequences of the partial 16S rRNA genes determined by employing the Roche GS-FLX 454 pyrosequencer.  13CGL is the pyrosequencing library of partial bacterial 16S rRNA sequences of the light gradient fraction (L, BD=1.794 g/ml) of the grassland (G) soil microcosm amended with 13C wheat roots (13C) and incubated for 72 hours.