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

Abstract

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.

Chemical controls on the cycling and reactivity of marine dissolved organic matter

“…CLA was slightly elevated in the surface (114.0 ± 0.9 pmol P L -1 d -1 ) and decreased to a broad minimum in the subsurface around 100 meters (105.5 ± 0.6 pmol P L -1 d -1 ). These rates are low, but consistent with previously measured rates of CLA in the NPSG (6.1-931.0 pmol P L -1 d -1 ; Granzow et al, 2021). The high-resolution sampling around the DMC conducted on the PARAGON I expedition showed that CLA was highly variable in that region.…”

“…Several studies have found CLA to be somewhat independent of Pi concentration and have suggested that C-P lyase may be utilized as a mechanism for extracting labile C and N from organic matter (Benitez-Nelson et al, 2004;Chin et al, 2018). These conclusions are supported by the high CLA rates observed in seawater collected from the mesopelagic, although it is unclear what effects pressure and temperature modulation during incubation have on those results (Granzow et al, 2021). Recently, a phosphate insensitive cosmopolitan phosphomonoesterase produced by Bacteroidetes was found to be associated with phytoplankton and POM and was hypothesized to be used in C acquisition from phosphorylated compounds (Lidbury et al, 2022).…”

“…Additionally, the APA assay specifically measures phosphate ester hydrolysis by particle-associated AP, while the CLA assay measures C-P lyase-associated hydrolysis in whole water samples. No CLA is detected in cell lysate, or in filtered seawater samples, however, suggesting that most or all C-P lyase is cell or particle associated, allowing for a more rigorous comparison of the two enzyme assays (Granzow et al, 2021; It is important to recognize that macronutrient balance may not be the only control on DOP remineralization. Several studies have found CLA to be somewhat independent of Pi concentration and have suggested that C-P lyase may be utilized as a mechanism for extracting labile C and N from organic matter (Benitez-Nelson et al, 2004;Chin et al, 2018).…”

“…Chapter 4 describes the development of a fluorescent assay to measure CLA in the ocean. Profiles collected in the North Pacific Subtropical Gyre (NPSG) on three field expeditions found that CLA displayed a sharp maximum concurrent with the deep chlorophyll maximum (DCM) (Granzow et al, 2021). This result was unexpected as Pi concentrations are lower in the surface than near the DCM, and it was expected that phosphate limitation would drive phosphonate remineralization in the same way that APA is promoted under low P conditions (Duhamel et al, 2010).…”

“…n-DPPh and n-DP eluted at 7 and 11 min, respectively. SeeGranzow et al (2021) for a complete description of the CLA analysis.…”