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, 5 (6), 386-92

YeeO From Escherichia Coli Exports Flavins

Affiliations

YeeO From Escherichia Coli Exports Flavins

Michael J McAnulty et al. Bioengineered.

Abstract

Multidrug and toxic compound extrusion (MATE) proteins help maintain cellular homeostasis by secreting metabolic wastes. Flavins may occur as cellular waste products, with their production and secretion providing potential benefit for industrial applications related to biofuel cells. Here we find that MATE protein YeeO from Escherichia coli exports both flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Significant amounts of flavins were trapped intracellularly when YeeO was produced indicating transport limits secretion of flavins. Wild-type E. coli secreted 3 flavins (riboflavin, FMN, and FAD), so E. coli likely produces additional flavin transporters.

Keywords: FAD, Flavin adenine dinucleotide; FMN, Flavin mononucleotide; IPTG, Isopropyl β-D-1-thiogalactopyranoside; LB, Lysogeny Broth; M9-glc, M9 minimal media supplemented with 0.4% glucose; M9-glc-caa, M9 minimal media supplemented with 0.4% glucose and 0.4% casamino acids; MATE, multidrug and toxic efflux; PVDF, Polyvinylidene fluoride; flavin; flavin adenine dinucleotide; flavin mononucleotide; transporter.

Figures

Figure 1.
Figure 1.
Chemical structures of the 3 flavins in study. The chemical structures of riboflavin, FMN, and FAD are shown.
Figure 2.
Figure 2.
Flavin secretion by E. coli BW25113 and C43(DE3). Cultures were grown in M9-glc for 20 hours before flavins in culture supernatants were analyzed by HPLC. Two independent cultures were tested for each strain, and error bars represent one standard deviation.
Figure 3.
Figure 3.
Enhanced flavin secretion upon YeeO production. Cultures were grown in M9-glc-caa and induced with (A) 1 mM IPTG, or (B) 0.1 mM IPTG for 6 hours before flavins in culture supernatants were analyzed by HPLC. BW/pCA24N represents BW25113/pCA24N and BW/YeeO represents BW25113/pCA24N-yeeO. Two independent cultures were tested for each strain, and error bars represent one standard deviation.
Figure 4.
Figure 4.
Intracellular flavin profile resulting from YeeO production. Cultures were grown in M9-glc-caa and induced with 1 mM IPTG for 6 hours before flavins in sonicated cell pellets were analyzed by HPLC. BW/pCA24N represents BW25113/pCA24N and BW/YeeO represents BW25113/pCA24N-yeeO. Two independent cultures were tested for each strain, and error bars represent one standard deviation.
Figure 5.
Figure 5.
YeeO elongates cells. Cultures were grown in M9-glc-caa and induced with 1 mM IPTG for 6 hours before visualizing by phase contrast microscopy. Empty plasmid represents BW25113/pCA24N as control, and YeeO as BW25113/pCA24N-yeeO. Scale bar indicates 10 μm.
Figure 6.
Figure 6.
Representative microscopy images of cells producing GFP-tagged YeeO. (A) Cultures were grown in M9-glc-caa and induced with 1 mM IPTG for 6 hours, and (B) cultures were grown in LB and induced with 1 mM IPTG for 2 hours. All samples were visualized by phase contrast and epi-fluorescence, and overlay images are indicated. GFP represents BW25113/pCA24N-gfp, YeeO represents BW25113/pCA24N-yeeO-gfp, and OppA represents BW25113/pCA24N-oppA-gfp. Scale bar indicates 10 μm.

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