SIP 2.0: Multi-Substrate Isotope Labeling and Metagenomic Analysis of Active Soil Bacterial Communities.

The study of microbial diversity in natural environments has been transformed by the application of various molecular tools. This diversity represents an enormous reservoir of undiscovered organisms and potentially valuable enzymes. DNA stable-isotope probing (DNA-SIP) enables the characterization of uncultured and low-abundance microorganisms in environmental samples that actively metabolize particular substrates. Previous studies focused on individual environmental samples and substrates; our study expands SIP analysis to include multiple substrates and soils to strengthen substrate or environment-specific inferences. We performed DNA-SIP incubations with three disparate Canadian soils (tundra, temperate rainforest and agricultural) and five carbohydrates (glucose, cellobiose, xylose, arabinose and cellulose). The soils were amended with native carbon (¹²C) or stable-isotope labelled (¹³C) substrates. Samples were collected at defined time intervals (one, three and six weeks), followed by DNA extraction and cesium chloride density gradient ultracentrifugation. Denaturing gradient gel electrophoresis (DGGE) of all gradient fractions confirmed the recovery of labeled nucleic acids. Both “heavy” and “light” DNA fractions were characterized by Illumina sequencing of 16S rRNA genes from all treatments. Heavy DNA from similar 16S rRNA gene clusters were pooled and subjected to shotgun metagenomic sequencing to identify enzymes affiliated with degradation of these substrates. The results demonstrate unique heavy DNA patterns associated with all soils and substrates, indicating successful enrichment of active microorganisms. Multi-response permutation procedures (MRPP) analysis confirmed differences between soil type (A=0.1759; T=-20.41) as well as between heavy and light fractions for all soil types (A=0.4009; T=-28.32). Indicator species analysis revealed an important role for Actinomycetales and Rhizobiales in the heavy DNA of agricultural and temperate rainforest soils (Indicator values 90-100%; p=0.001 for all substrates); members of the Burkholderiales were predominant indicator species in heavy fractions from all soils (Indicator values 90-100%; p=0.001). Furthermore, heavy DNA from the tundra soil was dominated by Xanthomonadales and Sphingomonadales (Indicator values 95-99%; p=0.001) for all substrates. Many other OTUs were associated with the heavy DNA from SIP incubations, reflecting the high diversity of active organisms assimilating carbon from the ¹³C-labelled carbohydrates tested. Functional annotation of the metagenomic data reveals diverse glycosyl hydrolase (GH) gene representation within the pooled heavy DNA. Heavy DNA from cellulose-incubated samples was subjected to multiple-displacement amplification and cloned into a large-insert cosmid library with >80,000 clones and average insert sizes >30 kb. To our knowledge, this study represents the most comprehensive DNA-SIP experiment conducted to date and demonstrates the value of including multiple samples, time points and substrates for the characterization of complex microbial communities. Heavy DNA samples are compatible with functional metagenomics approaches for the recovery of novel glycosyl hydrolase genes for potential industrial applications.