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

Abstract

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 13C-AMF hyphosphere DNA from a 13CO2 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% 13C), even though the soils’ overall enrichment was low (1.8 atom% 13C). We assembled 212 13C-hyphosphere MAGs; the hyphosphere taxa that assimilated the most AMF-derived 13C 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.

A standardized quantitative analysis strategy for stable isotope probing metagenomics

“…Conversely, individual assemblies can outperform co-assemblies in samples where high levels of microdiversity impede contig formation (46-48). Here, we found that co-assembly of all density fractions generated the most medium- and high-quality MAGs, which agrees with two recent SIP metagenomics studies (23, 24). However, we also found that merging binning results from individual fraction assemblies and larger co-assemblies via MAG de-replication provided more medium- and high-quality MAGs than did co-assembly alone.…”

“…DNA-SIP has been an established method in microbial ecology for many years and has primarily relied on 16S rRNA gene sequencing to identify active taxa (16, 30, 39, 40) (14). With decreases in sequencing costs and increases in compute capacity, DNA-SIP studies can now utilize shotgun metagenomic sequencing to establish links between population genomes and in situ activities (22-24, 41-43). In addition, automated sample preparation substantially increases the potential scale of SIP metagenomic studies and allows for more biological replication (24).…”

“…With decreases in sequencing costs and increases in compute capacity, DNA-SIP studies can now utilize shotgun metagenomic sequencing to establish links between population genomes and in situ activities (22-24, 41-43). In addition, automated sample preparation substantially increases the potential scale of SIP metagenomic studies and allows for more biological replication (24). However, the growth of SIP metagenomics also depends on adapting analysis tools to work with shotgun metagenomic data and validating their performance.…”

“…Comparing assembly strategies for SIP metagenomic data was a key goal of our study. Previous SIP studies have used different strategies, including assembling unfractionated DNA, assembling individual SIP fractions, and co-assembling several fractions (22-24, 44, 45). However, it was not clear which assembly strategy produces the most medium- and high-quality MAGs.…”

“…We refer to this general approach as “SIP metagenomics” from here on to distinguish it from traditional 16S rRNA-based DNA-SIP. Some recent studies have applied the qSIP approach to shotgun sequencing data to estimate the isotopic enrichment of soil metagenome assembled genomes (MAGs) (22-24). While these represent exciting advancements in the field, SIP metagenomics faces challenges related to data analysis and interpretation.…”