Nuclear magnetic resonance analysis of [1-13C]dimethylsulfoniopropionate (DMSP) and [1-13C]acrylate metabolism by a DMSP lyase-producing marine isolate of the alpha-subclass of Proteobacteria

Abstract

The prominence of the alpha-subclass of Proteobacteria in the marine bacterioplankton community and their role in dimethylsulfide (DMS) production has prompted a detailed examination of dimethylsulfoniopropionate (DMSP) metabolism in a representative isolate of this phylotype, strain LFR. [1-(13)C]DMSP was synthesized, and its metabolism and that of its cleavage product, [1-(13)C]acrylate, were studied using nuclear magnetic resonance (NMR) spectroscopy. [1-(13)C]DMSP additions resulted in the intracellular accumulation and then disappearance of both [1-(13)C]DMSP and [1-(13)C]beta-hydroxypropionate ([1-(13)C]beta-HP), a degradation product. Acrylate, the immediate product of DMSP cleavage, apparently did not accumulate to high enough levels to be detected, suggesting that it was rapidly beta-hydroxylated upon formation. When [1-(13)C]acrylate was added to cell suspensions of strain LFR it was metabolized to [1-(13)C]beta-HP extracellularly, where it first accumulated and was then taken up in the cytosol where it subsequently disappeared, indicating that it was directly decarboxylated. These results were interpreted to mean that DMSP was taken up and metabolized by an intracellular DMSP lyase and acrylase, while added acrylate was beta-hydroxylated on (or near) the cell surface to beta-HP, which accumulated briefly and was then taken up by cells. Growth on acrylate (versus that on glucose) stimulated the rate of acrylate metabolism eightfold, indicating that it acted as an inducer of acrylase activity. DMSP, acrylate, and beta-HP all induced DMSP lyase activity. A putative model is presented that best fits the experimental data regarding the pathway of DMSP and acrylate metabolism in the alpha-proteobacterium, strain LFR.

FIG. 1

DMSP and acrylate metabolism in cell suspensions of strain LFR cultured on either glucose (A) or acrylate (B). Substrates and products were measured in either the extracellular medium or gas phase following the addition of DMSP or acrylate (500 μM) to the cell suspension. The cells had an A₆₀₀ of ca. 0.5 and protein concentration of 0.12 mg · ml⁻¹. Symbols: ■, DMS production from added DMSP; ▴, extracellular acrylate resulting from DMSP cleavage; ●, uptake (or metabolism) of added acrylate.

FIG. 2

¹H NMR analysis of intracellular [1-¹³C]DMSP and its metabolite(s) following the addition of 5 mM [1-¹³C]DMSP to a cell suspension of strain LFR (acrylate-grown). Samples were taken at the indicated time intervals and prepared as described in Materials and Methods. The NMR spectra were acquired by using the same conditions and number of scans for each sample so that the peak intensity would represent an approximate concentration of each metabolite(s) relative to each sample. ¹H resonances centered at 3.38 and 2.65 ppm and 3.71 and 2.35 ppm were identified as those belonging to [1-¹³C]DMSP and [1-¹³C]β-HP, respectively.

FIG. 3

The gradient-enhanced ¹H{¹³C} HMQC spectrum of the cell extract taken at 0.5 h after the addition of 5 mM [1-¹³C]DMSP to a concentrated cell suspension of strain LFR. NMR parameters were exactly the same as those reported elsewhere (3).

FIG. 4

¹H NMR analysis of the extracellular medium following the addition of 5 mM [1-¹³C]acrylate to a concentrated cell suspension. Samples were taken at the various time intervals indicated; the cells were removed by centrifugation and the supernatant was assayed directly. The NMR spectra were acquired using the same conditions and number of scans for each sample so that the peak intensity would represent an approximate concentration of each metabolite relative to each sample. ¹H resonances at 5.55, 5.90, and 6.05 ppm and at 2.35 and 3.64 ppm were identified as those belonging to that of acrylate and β-HP, respectively.

FIG. 5

Gradient-enhanced ¹H{¹³C} HMQC spectrum of the cell extract (cytosol) taken at 10 min after the exposure of cell suspension to [1-¹³C]acrylate. NMR parameters are as reported previously (3). (Inset) Cytosolic concentration of β-HP following the addition of [1-¹³C]acrylate, as measured by the intensity of the α-C protons (3.71 ppm) of the 180-ppm ¹³C-labeled component from HMQC data (i.e., β-HP).

FIG. 6

A model comparing the metabolism of DMSP and acrylate in strain LFR and A. faecalis M3A. Abbreviations: DL, DMSP lyase; Ac*, constitutive levels of acrylase; Ac, inducible acrylase; BP, uptake/binding proteins; acrylate*, intracellular acrylate (assumed to be present, but not actually observed, presumably due to low pool size).