Community structure and abundance of ACC deaminase containing bacteria in soils with 16S-PICRUSt2 inference or direct acdS gene sequencing

Authors: Manter, Daniel; Hamm, Alison - Ali; Deel, Heather

Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/24/2023
Publication Date: 6/19/2023
Citation: Manter, D.K., Hamm, A.K., Deel, H.L. 2023. Community structure and abundance of ACC deaminase containing bacteria in soils with 16S-PICRUSt2 inference or direct acdS gene sequencing. Journal of Microbiological Methods. 211. Article e106740. https://doi.org/10.1016/j.mimet.2023.106740.
DOI: https://doi.org/10.1016/j.mimet.2023.106740

Interpretive Summary: Bacteria can reduce plant ethylene levels and increase root development and elongation resulting in increased resiliency to drought and other plant stressors. Although these bacteria are ubiquitous in the soil, non-culture-based methods for their enumeration and identification are not well developed. In this study we compare two culture-independent approaches for identifying these bacteria: direct sequencing of the acdS gene and phylogenetic construction of 16S rRNA amplicon libraries with the PICRUSt2 tool. Both methods were highly correlated and detected site differences in dryland wheat in eastern Colorado. The 16S-PICRUSt2 method paints a broader picture of the biological and biochemical function of the soil microbiome compared to direct acdS sequencing; however, phylogenetic analysis based on 16S gene relatedness may not reflect that of the functional gene of interest. This 16S-PICRUSt2 method can also be used to target any functional gene with a known KEGG ontology making it a more robust and cheaper method to target bacterial genes of interest without the need to develop a new qPCR assay.

Technical Abstract: 1-aminocyclopropane-1-carboxylate deaminase (ACCD+) bacteria can reduce plant ethylene levels and increase root development and elongation resulting in increased resiliency to drought and other plant stressors. Although these bacteria are ubiquitous in the soil, non-culture-based methods for their enumeration and identification are not well developed. In this study we compare two culture-independent approaches for identifying ACCD+. First, quantitative PCR (qPCR) and direct acdS sequencing with newly designed gene-specific primers; and second, phylogenetic construction of 16S rRNA amplicon libraries with the PICRUSt2 tool. Using soils from eastern Colorado, we showed complementary yet differing results in ACCD+ bacteria abundance and community structure responding to water availability. Across all sites, gene abundances estimated from qPCR with the acdS gene-specific primers and phylogenetic reconstruction using PICRUSt2 were significantly correlated. However, PICRUSt2 identified members of the Acidobacteria, Proteobacteria, and Bacteroidetes phyla as ACCD bacteria, whereas the acdS primers amplified only members of the Proteobacteria phyla. Despite these differences, both measures showed that bacterial abundance of ACCD decreased as soil water content decreased along a PET gradient at three sites in eastern Colorado. One major advantage of using 16S sequencing and PICRUSt2 in metagenomic studies is the ability to get a potential functional profile of all known KEGG enzymes within the bacterial community of a single soil sample. The 16S-PICRUSt2 method paints a broader picture of the biological and biochemical function of the soil microbiome compared to direct acdS sequencing; however, phylogenetic analysis based on 16S gene relatedness may not reflect that of the functional gene of interest.