Growth experiments in cocultures.

Cocultures of P. tricornutum with strains KarMa and M1, respectively, were set up in 100-ml Erlenmeyer flasks containing 50 ml SW-f/2 medium containing MMA or NH₄Cl (each 2 mM) instead of NaNO₃ as the nitrogen source. Cocultures were incubated in the same way described above for monocultures of P. tricornutum. For inoculation of cocultures, main cultures of the individual microorganisms were set up as described above. Appropriate volumes of the bacterial main cultures were harvested by centrifugation (8,000 × g, 5 min, room temperature) and added to the coculture medium to an OD₆₀₀ of 0.01. In parallel, appropriate volumes of the diatom main culture were harvested by centrifugation (16,000 × g, 10 min, room temperature) and added to the coculture medium to a chlorophyll concentration of 0.02 μg ml⁻¹. Cocultures containing Donghicola sp. strain KarMa tagged with the gene for the green fluorescent protein (GFP) were also supplemented with 20 μg ml⁻¹ gentamicin. Growth of P. tricornutum in cocultures was measured by chlorophyll fluorescence and by microscopic cell counts using aliquots taken at regular intervals from the growing cultures. To measure chlorophyll fluorescence, 200-μl aliquots were transferred to a microtiter plate, which was placed into an imaging device as described previously (28). For counting of cell numbers, aliquots were transferred to a microscopic Thoma cell counting chamber; each sample was evaluated three times in six-count quadrats. Bacterial growth in coculture was determined as described above for monocultures.