Primer design to assess bacterial degradation of glyphosate and other phosphonates

Abstract

Phosphonates are organic phosphorous (P) compounds frequently detected in the environment due to a very stable C[sbnd]P bond that render them relatively recalcitrant. Glyphosate [N-phosphonomethyl glycine] is the most widely used and best-known synthetic phosphonate, and one of the most concerning herbicides in the world today. Microbial degradation of glyphosate and organophosphonates in general, is the main dissipation mechanism operating in most environments. One microbial metabolic pathway in this process is the C[sbnd]P lyase pathway, entailing an enzymatic complex encoded by about 14 genes (the Phn operon). Our goal was to develop a quantitative polymerase chain reaction (qPCR) assay for a key enzyme, the C[sbnd]P lyase that breaks down the C[sbnd]P bond, via quantification of the codifying phnJ gene. The primers designed in this study fulfill the requirements for a successful qPCR assay, with high efficiency and sensitivity, as well as specific detection of the target sequence in a wide range of taxonomic groups. This is, to our knowledge, the first report of primers designed to target phnJ in both pure cultures and metagenomic DNA from different environmental sources. Direct quantification of phnJ may be a cost-effective proxy to determine glyphosate degradation potential in different matrixes.