HT-SIP: a semi-automated stable isotope probing pipeline identifies cross-kingdom interactions in the hyphosphere of arbuscular mycorrhizal fungi

Background: Linking the identity of wild microbes with their ecophysiological traits and environmental functions is a key ambition for microbial ecologists. Of many techniques that strive for this goal, Stable-isotope probing-SIP-remains among the most comprehensive for studying whole microbial communities in situ. In DNA-SIP, actively growing microorganisms that take up an isotopically heavy substrate build heavier DNA, which can be partitioned by density into multiple fractions and sequenced. However, SIP is relatively low throughput and requires significant hands-on labor. We designed and tested a semi-automated, high-throughput SIP (HT-SIP) pipeline to support well-replicated, temporally resolved amplicon and metagenomics experiments. We applied this pipeline to a soil microhabitat with significant ecological importance-the hyphosphere zone surrounding arbuscular mycorrhizal fungal (AMF) hyphae. AMF form symbiotic relationships with most plant species and play key roles in terrestrial nutrient and carbon cycling. Results: Our HT-SIP pipeline for fractionation, cleanup, and nucleic acid quantification of density gradients requires one-sixth of the hands-on labor compared to manual SIP and allows 16 samples to be processed simultaneously. Automated density fractionation increased the reproducibility of SIP gradients compared to manual fractionation, and we show adding a non-ionic detergent to the gradient buffer improved SIP DNA recovery. We applied HT-SIP to C-13-AMF hyphosphere DNA from a (CO2)-C-13 plant labeling study and created metagenome-assembled genomes (MAGs) using high-resolution SIP metagenomics (14 metagenomes per gradient). SIP confirmed the AMF Rhizophagus intraradices and associated MAGs were highly enriched (10-33 atom% C-13), even though the soils'overall enrichment was low (1.8 atom% C-13). We assembled 212 C-13-hyphosphere MAGs; the hyphosphere taxa that assimilated the most AMF-derived C-13 were from the phyla Myxococcota, Fibrobacterota,Verrucomicrobiota, and the ammonia-oxidizing archaeon genus Nitrososphaera. Conclusions: Our semi-automated HT-SIP approach decreases operator time and improves reproducibility by targeting the most labor-intensive steps of SIP-fraction collection and cleanup. We illustrate this approach in a unique and understudied soil microhabitat-generating MAGs of actively growing microbes living in the AMF hyphosphere (without plant roots). The MAGs' phylogenetic composition and gene content suggest predation, decomposition, and ammonia oxidation may be key processes in hyphosphere nutrient cycling.

Automated summary

The study introduced a semi-automated, high-throughput SIP pipeline to support well-replicated experiments in soil microhabitats, showcasing how this approach can improve operational efficiency and reproducibility in studying actively growing microbes within AMF hyphosphere. This method generated a series of metagenome-assembled genomes (MAGs) from C-13-labeled AMF hyphosphere DNA, revealing insights into key processes involved in nutrient cycling within the hyphosphere.