Moisture effects on the active prokaryotic communities in a saline soil unraveled by 18O-informed metagenomics

Purpose Water availability influences soil ecosystem functioning by shaping soil microbial community composition and regulating metabolic rate. These moisture effects are mirrored in taxonomic and functional attributes of actively growing microorganisms. Methods Stable isotope probing (SIP) technique with 18 O-H 2 O as the labeling substrate was used to capture the active microbiome in a saline soil under different moisture conditions. The SIP microcosms were adjusted to 20%, 60%, or 100% of the soil water holding capacity (WHC) with 18 O- or 16 O-H 2 O and were incubated for 4 weeks. The bacterial communities in these microcosms were examined by amplicon sequencing of 16S rRNA genes. The 18 O-labeled DNA was recovered by ultracentrifugation and fractionation. Shotgun metagenomic sequencing was performed with the labeled DNA to explore the moisture effects on functional attributes of these active microbes. Results Isotopes ( 16 O and 18 O) had a negligible effect on soil bacterial communities compared with soil moisture. Taxonomic alignment of both total and 18 O-labeled microbiomes revealed contrasting tendencies for Actinobacteria and Proteobacteria along the moisture gradient. Archaeal components became more abundant at 60% WHC and were dominated by Euryarchaeota. Functional annotation of the active microbiomes revealed broad responses in genes associated with osmotic regulation, membrane transport, cell motility, iron acquisition, phages and prophages to moisture manipulation. In particular, the considerable changes in protein biosynthesis genes suggested an altered reproductive strategy of the dominant taxa at different WHC levels. Conclusion These findings indicate taxonomic and functional effects of soil moisture on active prokaryotes in a saline soil, and demonstrate the potential of combined 18 O-water SIP and metagenomics in achieving a holistic understanding of the active soil microbiome.