The riboflavin transporter RibU in Lactococcus lactis: molecular characterization of gene expression and the transport mechanism

Abstract

This study describes the characterization of the riboflavin transport protein RibU in the lactic acid bacterium Lactococcus lactis subsp. cremoris NZ9000. RibU is predicted to contain five membrane-spanning segments and is a member of a novel transport protein family, not described in the Transport Classification Database. Transcriptional analysis revealed that ribU transcription is downregulated in response to riboflavin and flavin mononucleotide (FMN), presumably by means of the structurally conserved RFN (riboflavin) element located between the transcription start site and the start codon. An L. lactis strain carrying a mutated ribU gene exhibits altered transcriptional control of the riboflavin biosynthesis operon ribGBAH in response to riboflavin and FMN and does not consume riboflavin from its growth medium. Furthermore, it was shown that radiolabeled riboflavin is not taken up by the ribU mutant strain, in contrast to the wild-type strain, directly demonstrating the involvement of RibU in riboflavin uptake. FMN and the toxic riboflavin analogue roseoflavin were shown to inhibit riboflavin uptake and are likely to be RibU substrates. FMN transport by RibU is consistent with the observed transcriptional regulation of the ribGBAH operon by external FMN. The presented transport data are consistent with a uniport mechanism for riboflavin translocation and provide the first detailed molecular and functional analysis of a bacterial protein involved in riboflavin transport.

FIG. 1.

Alignment of RibU homologues from various bacterial strains. The positions of the predicted TMSs are shaded and numbered I to V. The region between TMSs IV and V is predicted to be membrane spanning in both L. lactis RibU proteins, while in the other aligned sequences a large part of this region is absent and not predicted to be traversing the membrane.

FIG. 2.

Primer extension (PE) analysis of P_ribU run alongside a sequencing ladder. The deduced −35 and −10 boxes are indicated by boldface in the sequence displayed on the right side of this figure. The bent arrow indicates the identified transcription start site. The identified RFN element is underlined. The assumed ribosomal binding site is boxed, and the ribU start codon is in boldface.

FIG. 3.

Transcriptional analysis of ribU. (A) Northern hybridization analysis of ribU in NZ9000 and CB010. Lane 1, NZ9000 RNA from CDM; lane 2, NZ9000 RNA from CDM plus 5 μM riboflavin; lane 3, CB010 RNA from CDM; lane 4, CB010 RNA from CDM plus 5 μM riboflavin. An RNA size ladder (in kilobases) is indicated on the left. The size of the transcripts is indicated to the right. (B) β-Galactosidase activities of NZ9000 containing pPTPLribU grown in CDM or CDM plus 5 μM riboflavin are represented by circles and inverted triangles, respectively. β-Galactosidase activities produced by CB010 containing pPTPLribU grown in CDM or CDM plus 5 μM riboflavin are represented by squares and diamonds, respectively. The dashed line indicates growth of the strains plotted on a semilog scale.

FIG. 4.

Analysis of growth and riboflavin levels of NZ9000 and NZ9000ΔribU in GM17. The solid lines represent log OD₆₀₀, and the dashed lines represent riboflavin levels as measured by HPLC in the cell-free supernatant following growth. Data obtained using NZ9000 are shown with black solid circles, and those obtained using NZ9000ΔribU are depicted with empty inverted triangles.

FIG. 5.

P_ribGBAH activity in NZ9000 and NZ9000ΔribU in various media. The solid circles represent NZ9000, and the empty inverted triangles represent NZ9000ΔribU. The solid lines represent growth (on a semilog scale), and the dashed lines represent β-galactosidase activity. (A) CDM; (B) CDM plus 5 μM riboflavin; (C) CDM plus 5 μM FMN; (D) CDM plus 50 μM riboflavin.

FIG. 6.

Uptake of [³H]riboflavin in whole cells. Cells concentrated to an OD₆₀₀ of 10 were energized for 5 min with glucose, and uptake was started by the addition of 1 μM [³H]riboflavin. At given time points, the uptake was stopped with ice-cold buffer, the samples were filtered, and radioactivity was counted. Black circles represent NZ9000, open circles represent NZ9000ΔribA, and black inverted triangles represent NZ9000ΔribU. Open triangles represent NZ9000 cells that were deenergized with 2-deoxyglucose.

FIG. 7.

Competition of riboflavin uptake in whole cells (NZ9000) by FMN and roseoflavin. For the uptake assays, no additions, 0.5 to 10 μM roseoflavin, or 1 to 60 μM FMN was supplemented concomitantly with [³H]riboflavin. The concentration of [³H]riboflavin in each experiment was 1 μM. Initial uptake rates were determined from the riboflavin uptake after 4 min. One hundred percent corresponds to a riboflavin uptake rate of 5 pmol/mg of protein/min.

FIG. 8.

(A) Effect of the proton motive force on riboflavin uptake in whole cells of L. lactis NZ9000. Shown is the uptake of radiolabeled riboflavin and alanine (inset) in cells deenergized with 2-deoxyglucose (inverted triangles) and in deenergized cells in which an artificial proton motive force was applied (circles). The expected level of radiolabeled riboflavin inside the cells when no accumulation would take place (the concentration inside equals the concentration in the uptake buffer) is indicated by the dashed line. (B) Chase of internalized radiolabeled riboflavin from NZ9000 cells with unlabeled riboflavin. Uptake of [³H]riboflavin was performed as for Fig. 6 (black circles). After 30 min, unlabeled riboflavin at a final concentration of 70 μM was added, and at the given time points the samples were filtered and radioactivity was counted (white circles).