Generation of fluorescence-tagged cells of Donghicola sp. strain KarMa.

Strain KarMa was tagged with a stable version of the green fluorescence protein (GFP). For this, the gfp gene with restriction sites for SacI and XbaI (gfp forward, 5′-TTTTTTTGAATTCATCCCCGGGTACCTAGAATTA-3′; gfp reverse, 5′-TAATCTAGACAGGGTTTTCCCAGTCACGA-3′) was amplified from the vector pUCP18::gfp, cloned into the restriction sites of the vector pBBR1MCS-5 (38), and transformed into chemically competent cells of Escherichia coli strain S17-1 (39). The generated E. coli strain, S17-1(pBBR1MCS-5::gfp), was used for conjugation of Donghicola sp. strain KarMa. The biparental conjugation protocol as described in Piekarski et al. (40) was modified. The donor strain was incubated in LB medium containing 20 μg ml⁻¹ gentamicin at 37°C overnight. A main culture of strain KarMa was incubated until an OD₆₀₀ of 1 was reached. Both cultures were mixed with an OD₆₀₀ donor-to-recipient ratio of 1:2 in 500 μl SW-f/2 medium, and 100 μl of suspension was plated as droplets on LBS agar plates (1% [wt/vol] tryptone, 0.5% [wt/vol] yeast extract, 2% [wt/vol] NaCl, 50 mM Tris-HCl, with pH adjusted to 7.5 prior to autoclaving). After incubation for 6 h at 30°C, colony material was transferred to 4 ml SW-f/2 medium and mixed vigorously. Suspensions (50, 100, and 200 μl) were plated on SW-f/2 medium agar plates with 10 mM glucose and 2 mM MMA containing 20 μg ml⁻¹ gentamicin. Cells of strain KarMa carrying the plasmid were identified by detection of GFP fluorescence with the imager. Strain KarMa(pBBR1MCS-5::gfp) showed a similar growth pattern to the untagged wild type in single culture (see Fig. S1A in the supplemental material).