Phosphorus Release and Regeneration Following Laboratory Lysis of Bacterial Cells

Abstract

The availability of phosphorus limits primary production in large regions of the oceans, and marine microbes use a variety of strategies to overcome this limitation. One strategy is the production of alkaline phosphatase (APase), which allows hydrolysis of larger dissolved organic phosphorus (DOP) compounds in the periplasm or at the cell surface for transport of orthophosphate into the cell. Cell lysis, driven by grazing and viral infection, releases phosphorus-containing cell components, along with active enzymes that could persist after lysis. The importance of this continued enzymatic activity for orthophosphate regeneration is unknown. We used three model bacteria - Escherichia coli K-12 MG1655, Synechococcus sp. WH7803, and Prochlorococcus sp. MED4 - to assess the impact of continued APase activity after cell lysis, via lysozyme treatment, on orthophosphate regeneration. Direct release of orthophosphate scaled with cell size and was reduced under phosphate-starved conditions where APase activity continued for days after lysis. All lysate incubations showed post-lysis orthophosphate generation suggesting phosphatases other than APase maintain activity. Rates of DOP hydrolysis and orthophosphate remineralization varied post-lysis among strains. Escherichia coli K-12 MG1655 rates of remineralization were 0.6 and 1.2 amol cell^-1hr^-1 under deplete and replete conditions; Synechococcus WH7803 lysates ranged from 0.04 up to 0.3 amol cell^-1hr^-1 during phosphorus deplete and replete conditions, respectively, while in Prochlorococcus MED4 lysates, rates were stable at 0.001 amol cell^-1hr^-1 in both conditions. The range of rates of hydrolysis and regeneration underscores the taxonomic and biochemical variability in the process of nutrient regeneration and further highlights the complexity of quantitatively resolving the major fluxes within the microbial loop.

Keywords: lysis; microbial loop; nutrient cycle; phosphorus; regeneration.

FIGURE 1

Total dissolved phosphorus and soluble reactive phosphorus release from cell cultures following lysis. (A) Synechococcus WH7803 TDP release; (B) Synechococcus WH7803 fraction of TDP as SRP; (C) Prochlorococcus MED4 TDP release; (D) Prochlorococcus MED4 fraction TDP as SRP. (E) SRP regeneration in E. coli incubations normalized to cell density. Open symbols represent lysates from P-deplete cells, filled symbols represent lysates from P-replete cells. All points represent the mean of triplicate measurements and error bars the standard deviation. Synechococcus WH7803 and Prochlorococcus MED 4 phosphorus replete and deplete conditions are statistically significant in the figures above; p < 0.01 and p < 0.1 after initial release, respectively.

FIGURE 2

Alkaline phosphatase activity following cell lysis. (A) E. coli; (B) Synechococcus WH7803; (C) Prochlorococcus MED4. Filled circles, cells grown in P-replete conditions; open circles, cells grown in P-deplete conditions. DiFMUP concentrations for incubations were 10 μM and all enzymatic rates are determined as maximum activities, where substrate is not deplete. For each time point, triplicate lysate incubations were measured, and the analytical error bars for each measurement are within the size of the symbols. Replete and deplete phosphorus conditions are statistically significant for the all the strains above; p < 0.01.

FIGURE 3

Proteinase K treatment of purified enzyme and lysate: (Top) Change in soluble reactive phosphorus (SRP) concentration during incubation of a 1 mM glycerophosphate solution with calf purified APase under three conditions: (1) open circles represent APase enzyme with no proteinase K present; (2) filled circles represent APase with Proteinase K present, but no temperature activation of the enzyme; (3) filled squares represent incubations where APase is incubated with Proteinase K activated at 55°C. (Bottom) Change in SRP concentration during similar incubations in which calf APase is replaced by E. coli lysate in the incubations, again spiked to 1 mM glycerophosphate. Open circles represent incubations with only E. coli lysate, filled circles represent E. coli lysate incubated with unactivated proteinase K, and filled squares represent incubations with E. coli lysate and Proteinase K activated at 55°C. Purified APase showed a two order of magnitude reduction in activity when proteinase K was activated at 55°C. When incubated at room temperature, unactivated proteinase K had no observable effect on diminishing APase activity (top). Proteinase K had no effect on APase activity in fresh E. coli lysate even when it was heat activated at 55°C (bottom). Analytical errror bars are within the size of the symbol. Differences between unactivated and activated Proteinase K treatments are statistically significant P < 0.01. There is no statistically significant difference between the E. coli lysate conditions.

FIGURE 4

Hydrolysis of model DOP compounds in E. coli lysates: E. coli MG1655 P-Deplete and P-Replete lysates incubated with a range of DOP compounds (1 mM DOP concentration in lysate solution at beginning of incubation) were monitored for [SRP] over time. Model DOP compounds used include glycerophosphate (GYP), 5’adenosine monophosphate (5’AMP), ribonucleic acid (RNA), and pyrophosphate (Pyro-P). For each time point, triplicate lysate incubations were measured, and the analytical error bars for each measurement are within the size of the symbols. SRP release in GYP incubations is the only treatment above where p-conditions represent statistical differences in SRP regenerated; p < 0.01.

FIGURE 5

Hydrolysis of GYP during incubations with pure culture lysate: Figures represent pure culture lysates, E. coli K-12 MG1655 (top), Synechococcus WH7803 (middle) and Prochlorococcus MED4 (bottom) in which the lysate was spiked to a 1mM initial glycerophosphate concentration and was monitored for at least 96hrs. For each time point, triplicate lysate incubations were measured, and the analytical error bars for each measurement are within the size of the symbols. Each phosphorus treatment is statistically significant for the strains above; p < 0.01.