Glyphosate as a Source of Phosphorus for Bacteria: Selection of an Enrichment Culture for Glyphosate Degradation, and Analysis of the C-P Lyase Pathway

Abstract

Glyphosate is one of the world’s most widely used herbicides, the active ingredient in Roundup as well as many generic herbicide brands. Though it is one of the safer herbicides, its ubiquity necessitates further study of the pathways that bacteria can use to degrade it. This study investigates the ability of bacteria to break the strong carbon-phosphorus bond of glyphosate. The carbon-phosphorus bond has the highest activation energy of the bonds within glyphosate, and breaking this bond is therefore a rate-limiting step of complete glyphosate mineralization. By optimizing a defined media and supplementing with glyphosate as the only source of phosphorus, the ability of bacterial species to grow on glyphosate as the sole source of phosphorus can be tested and compared to their growth when easily accessible ortho-phosphate is provided. This defined media was used to create an enrichment culture to study which species are selected for by glyphosate as the sole source of phosphorus, and compare them to cultures selected on easily accessible ortho-phosphate. Glyphosate as sole source of phosphorus selected for a different suite of species, especially those in the Rhizobiaceae family, than ortho-phosphate selected for. The Rhizobiaceae genus Agrobacterium was found to contain particularly good degraders of glyphosate, and a deletion and replacement experiment revealed that the C-P lyase pathway is required for glyphosate degradation in an Agrobacterium pusense isolate. Genome and growth comparisons between A. pusense, A. tumefaciens, and A. fabrum isolates revealed them all to be able to degrade glyphosate, and that they share very similar C-P lyase operons, despite having significant heterogeneity in their genomes. Additionally, a commercial enzyme linked immunosorbent assay was used to quantify glyphosate degradation. This work adds support to the theory that C-P lyase is required for glyphosate degradation in Agrobacterium, and sets the stage for additional research into glyphosate degradation pathways by bacteria.