Figure 1

Characterization of RibM from Streptomyces davawensis as a riboflavin transporter. (A) The gene ribM from S. davawensis was overexpressed in E. coli BL21 using the plasmid pNCO113ribM (grey triangles). An Escherichia coli strain containing the empty vector pNCO113 (black circles) served as a control. E. coli strains were grown in riboflavin-free M9 medium. The cells were collected by centrifugation, suspended in transport buffer (50 mM K₂HPO₄/KH₂PO₄, 50 mM MgCl₂, pH 7.0) and the experiment was started by adding [¹⁴C]riboflavin to a final concentration of 1.6 μM. Samples were filtered, washed with water and the radioactivity was determined by liquid scintillation counting. "1 OD cells" means "the amount of cells that when present in 1 ml will lead to an OD₆₀₀ reading of 1.0". (B) Uptake experiments were performed as described for (A) with 2.2 μM [¹⁴C]riboflavin in the presence of a 10-fold excess of riboflavin (ribM+RF), flavin mononucleotide (ribM+FMN), flavin adenine dinucleotide (ribM+FAD), roseoflavin (ribM+RoF) or carbonyl cyanide m-chlorophenylhydrazone (ribM+CCCP; 130 μM). The uptake activity in the absence of competitors was 4.8 pmol pmol riboflavin × OD cells^-1 min^-1 (ribM). In the presence of CCCP uptake activity was 4.3 pmol riboflavin × OD cells^-1 min^-1. "1 OD cells" means "the amount of cells that when present in 1 ml will lead to an OD₆₀₀ reading of 1.0".