Methodology:

Radiolabeled DOC kinetic experiments. To understand the mechanism of DOC compound uptake and to see whether species take up different DOC compounds passively or actively, we performed time-course experiments using [14C] labeled DOC compounds. Specific activities of the radiotracers were 52 µCi µmol-1 for [14C]acetate, 57 µCi µmol-1 for [14C]mannitol, and 160 µCi µmol-1 for [14C] glycerol, (PerkinElmer, Waltham, MA, USA).

Pulse-chase experiments. To test if the compounds that were taken up were assimilated within the cell, we performed pulse-chase experiments. This method first takes into account the cellular uptake of radiolabeled compound (“pulse”), that is then exposed to the same unlabeled compound (“cold chase”), at concentrations far above the labeled one. As an indicator of assimilation, [14C] labeled DOC compound would not be exchanged when “chased” with vastly-higher concentrations of the same unlabeled compound. Unassimilated compounds in intracellular pools, on the other hand, would be released from the cell as a new equilibrium between the intracellular and extracellular DOC concentrations is established (Balch, 1986).

Sampling and analytical procedures:

We prepared a solution of L1 medium and exponentially growing culture of each strain, with final concentrations of 5×104 cells L-1, and left them at their growth temperature in darkness for 24h to adapt. We divided the prepared solution in 45 mL aliquots to 24 vials for each compound and strain. To quadruplicate vials, we added 0.45 mL of unlabeled DOC compound from six stock solutions (1×10-6, 1×10-5, 1×10-4, 1×10-3, 1×10-2, and 1×10-1 mol L-1). The experiment started when we then added 0.02 mL of [14C] labeled DOC compound (2 µCi of added radioactivity) to each of 24 vials giving us final concentrations of organic compounds as stated in the table below. To one vial from those six concentration-quadruplicates we immediately added 1 mL of buffered formaldehyde to act as a killed control. We then incubated the 18 triplicates and six control vials at their growth temperature in darkness for up to 24h, with the sample timing to examine for linear uptake rates at 15 min, 1 h, 3 h, and 24 h. At each sampling, 5 mL of experimental culture were filtered onto a 0.4 µm pore-size, 25 mm diameter polycarbonate filter. We also filtered samples at 24 h for [14C]-microdiffusion analysis, which separates the POC fraction from the PIC fraction (Paasche & Brubak, 1994; Balch et al., 2000). Following the micro-diffusion step to separate acid-labile (PIC) versus acid-stabile (POC) fractions, each filter was then placed in the bottom of a clean scintillation vial, and scintillation cocktail was added (Balch et al., 2000). The radioactivity was measured using a Tri-Carb 3110TR liquid scintillation analyzer (PerkinElmer, Waltham, MA, USA). We calculated the net uptake velocity of [14C] labeled organic compounds using the equations of Parsons et al. (1984):

v = (Rn – Rf) × W / R × T

where v [mol L-1 h-1] is the net uptake rate, Rn [Bq] is the sample count, Rf [Bq] is the formalin-killed control count, and W [mol L-1] is the total concentration of the organic compound in the sample. R [Bq] is the total activity of the added compound to a sample and T [h] is the number of hours of incubation.

Following the radiolabeled DOC kinetic experiments, after 24h, we added the cold chase as 1 mL of 1 M of unlabeled compound to the remaining 25 mL in vials used in kinetics experiments. The addition of 1 mL of an organic compound could induce a substantial osmotic shock that could lead to short-term osmotic shrinkage of the protoplast before the entry of the organic substance raised the internal osmolarity to the external osmolarity.

We therefore measured particulate cellular radioactivity using the procedure described above at 5 min, 20 min, and 3 h post-chase, which provided three different time scales to evaluate how exchangeable the intracellular compounds were.