A sensitive fluorescent assay for measuring carbon-phosphorus lyase activity in aquatic systems

In the oligotrophic ocean where inorganic phosphate (P-i) concentrations are low, microorganisms supplement their nutrient requirements with phosphorus (P) extracted from dissolved organic matter (DOM). Most P in DOM is bound as phosphate esters, which are hydrolyzed by phosphoesterases to P-i. However, a large fraction of DOM-P occurs as phosphonates, reduced organophosphorus compounds with a C-P bond that do not yield P-i through simple ester hydrolysis alone. Phosphonates require an additional step that cleaves the C-P bond and oxidizes P(III) to P(V) to yield P-i. Most phosphonates are metabolized by the C-P lyase pathway, which cleaves C-P bonds and oxidizes phosphonates to P-i, enabling microbial assimilation. While the activity of common phosphoesterases such as alkaline phosphatase and phosphodiesterase can be measured by a fluorescent assay, a comparable method to assess C-P lyase activity (CLA) in natural water samples does not exist. To address this, we synthesized a dansyl-labeled phosphonate compound, and measured its hydrolysis by C-P lyase using high performance liquid chromatography. We found that laboratory cultures of marine bacteria expressing the C-P lyase pathway are able to hydrolyze the dansyl phosphonate, while bacteria expressing other phosphonate degradation pathways do not. Finally, we performed several field tests of the assay to measure water column profiles of CLA at Sta. ALOHA in the North Pacific Subtropical Gyre. Activity was elevated near the deep chlorophyll maximum suggesting high levels of phosphonate degradation in that region.

Automated summary

In oligotrophic oceans, microorganisms supplement their nutrient requirements by extracting phosphorus from dissolved organic matter. While most phosphorus is in the form of phosphate esters hydrolyzed to P-i, a portion exists as phosphonates that require an additional step for cleavage.