An official website of the United States government
Abstract We optimized a high throughput method to quantify turnover rates of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) in marine microbes from simultaneous measures of the respective stocks and phosphorylation rates. We combined a microbial adenylate extraction method using boiling 20 mM Tris buffer with purification and analysis by high pressure liquid chromatography optimized to quantify these intracellular adenylate concentrations in marine microbes. Additionally, we incorporated radiolabeled phosphate (32Pi) incubations to quantify phosphorus (P) uptake rates and the phosphorylation rates for these adenylate compounds in microbial cells. With this method, we can directly assess the variations in microbial growth rates, metabolic turnover rates, energy charge, and adenylate storage. We applied and validated this method application with environmental samples from Biscayne Bay, Florida, and quantified adenylate turnover times of 12, 15, and 73 min, for ATP, ADP, and AMP, respectively. Future incorporation of this method into experiments and geographic surveys across marine environments will allow for direct assessments of changes in microbial metabolic activity in relation to other ecological variables.
more »
« less
Dissolved organic phosphorus bond-class utilization by Synechococcus
Abstract Dissolved organic phosphorus (DOP) contains compounds with phosphoester, phosphoanhydride, and phosphorus–carbon bonds. While DOP holds significant nutritional value for marine microorganisms, the bioavailability of each bond-class to the widespread cyanobacterium Synechococcus remains largely unknown. This study evaluates bond-class specific DOP utilization by Synechococcus strains from open and coastal oceans. Both strains exhibited comparable growth rates when provided phosphate, a phosphoanhydride [3-polyphosphate and 45-polyphosphate], or a DOP compound with both phosphoanhydride and phosphoester bonds (adenosine 5′-triphosphate). Growth rates on phosphoesters [glucose-6-phosphate, adenosine 5′-monophosphate, bis(4-methylumbelliferyl) phosphate] were variable, and neither strain grew on selected phosphorus–carbon compounds. Both strains hydrolyzed 3-polyphosphate, then adenosine 5′-triphosphate, and lastly adenosine 5′-monophosphate, exhibiting preferential enzymatic hydrolysis of phosphoanhydride bonds. The strains’ exoproteomes contained phosphorus hydrolases, which combined with enhanced cell-free hydrolysis of 3-polyphosphate and adenosine 5′-triphosphate under phosphate deficiency, suggests active mineralization of phosphoanhydride bonds by these exoproteins. Synechococcus alkaline phosphatases presented broad substrate specificities, including activity toward the phosphoanhydride 3-polyphosphate, with varying affinities between strains. Collectively, these findings underscore the potentially significant role of compounds with phosphoanhydride bonds in Synechococcus phosphorus nutrition and highlight varied growth and enzymatic responses to molecular diversity within DOP bond-classes, thereby expanding our understanding of microbially mediated DOP cycling in marine ecosystems.
more »
« less
- PAR ID:
- 10532628
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- FEMS Microbiology Ecology
- Volume:
- 100
- Issue:
- 9
- ISSN:
- 1574-6941
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Three carboxamidequinoline ligands were synthesized and their complexes with Eu 3+ were used for recognition and detection of organic/inorganic phosphates in water. The signal transduction process is based on an “On–Off–On” switch in the fluorescence signal utilizing changes in the intramolecular charge transfer (ICT). The fluorescence emission of ligands is quenched upon exposure to the Eu 3+ (Off signal). Following the addition of the phosphate analytes the ligand–Eu 3+ complex disassembles, which results in the regeneration of the original emission of the ligand (On signal). In general, the Eu 3+ complexes show higher affinity towards adenosine 5′-triphosphate (ATP) and lower affinity to other phosphates, namely adenosine 5′-diphosphate (ADP), adenosine 5′-monophosphate (AMP), pyrophosphate (H 2 P 2 O 7 2− , PPi), and dihydrogenphosphate (H 2 PO 4 − , Pi).more » « less
-
A misconception among biology students is that breaking bonds in adenosine triphosphate (ATP) releases energy. This misconception may be related to imprecise representations of chemical bonding in common diagrams of ATP hydrolysis. We interviewed 33 undergraduate students and randomly assigned them to interpret a figure of ATP hydrolysis that either emphasized bond breaking in the reactants or the formation of new bonds in the products. Students who saw the figure emphasizing bond breaking were more likely to incorrectly classify ATP hydrolysis as endergonic, while students who saw the figure explicitly illustrating bond formation were more likely to use chemically-sound reasoning to describe the reaction.more » « less
-
Abstract A mechanistic understanding of dissolved organic phosphorus (DOP) utilization, and its role in the marine P cycle, requires knowledge of DOP molecular composition. In this study, a recently developed approach coupling electrodialysis and reverse osmosis with solution31P‐NMR analysis was used to examine DOP composition within a tidally dominated salt‐marsh estuary (North Inlet, South Carolina) over seasonal and tidal time frames. The isolation technique allowed for near complete recovery of the DOP pool (90% ± 13%;n= 12) with six broad compound classes quantified: phosphonates, phosphomonoesters, phosphodiesters, pyrophosphate, di‐ and tri‐phosphate nucleotides (nucleoPα), and polyphosphate. Our results indicate that phosphomonoesters (ca. 61%) and phosphodiesters (ca. 31%) comprise the majority of the DOP pool, with relatively small contributions from pyrophosphates (ca. 4%), phosphonates (ca. 2%), nucleoPα(ca. 1%), and polyphosphates (ca. 1%). The study found no significant differences in DOP composition or concentration between tidal stages, despite significant tidal changes in dissolved organic nitrogen (DON):DOP stoichiometry. Significant seasonal variation was observed, with higher concentrations of phosphonates, nucleoPα, and monophosphates and lower phosphomonoester concentrations in Fall relative to all other seasons. We hypothesize that these seasonal variations reflect the balance between specific compound class seasonal production, lability, and local P demands associated with marine vs. terrestrial sources. Our results indicate that DOP composition exists at a dynamic equilibrium that is strongly conserved across diverse marine environments.more » « less
-
Abstract In oligotrophic ocean regions, dissolved organic phosphorus (DOP) plays a prominent role as a source of phosphorus (P) to microorganisms. An important bioavailable component of DOP is phosphonates, organophosphorus compounds with a carbon‐phosphorus (C‐P) bond, which are ubiquitous in high molecular weight dissolved organic matter (HMWDOM). In addition to being a source of P, the degradation of phosphonates by the bacterial C‐P lyase enzymatic pathway causes the release of trace hydrocarbon gases relevant to climate and atmospheric chemistry. In this study, we investigated the roles of phosphate and phosphonate cycling in the production of methane (CH4) and ethylene (C2H4) in the western North Atlantic Ocean, a region that features a transition in phosphate concentrations from coastal to open ocean waters. We observed an inverse relationship between phosphate and the saturation state of CH4and C2H4in the water column, and between phosphate and the relative abundance of the C‐P lyase marker genephnJ. In phosphate‐depleted waters, methylphosphonate and 2‐hydroxyethylphosphonate, the C‐P lyase substrates that yield CH4and C2H4, respectively, were readily degraded in proportions consistent with their abundance and bioavailability in HMWDOM and with the concentrations of CH4and C2H4in the water column. We conclude that phosphonate degradation through the C‐P lyase pathway is an important source and a common production pathway of CH4and C2H4in the phosphate‐depleted surface waters of the western North Atlantic Ocean and that phosphate concentration can be an important control on the saturation state of these gases in the upper ocean.more » « less
