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Journal of Bacteriology, April 2006, p. 2752-2760, Vol. 188, No. 8
0021-9193/06/$08.00+0     doi:10.1128/JB.188.8.2752-2760.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

The Riboflavin Transporter RibU in Lactococcus lactis: Molecular Characterization of Gene Expression and the Transport Mechanism

Catherine M. Burgess,1 Dirk Jan Slotboom,2 Eric R. Geertsma,2 Ria H. Duurkens,2 Bert Poolman,2 and Douwe van Sinderen1,3*

Department of Microbiology and Biosciences Institute, National University of Ireland Cork, Western Road, Cork, Ireland,1 Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands,2 Alimentary Pharmabiotic Centre, Biosciences Institute, National University of Ireland Cork, Western Road, Cork, Ireland3

Received 12 October 2005/ Accepted 6 February 2006

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.

* Corresponding author. Mailing address: Alimentary Pharmabiotic Centre, Department of Microbiology and Biosciences Institute, National University of Ireland Cork, Western Road, Cork, Ireland. Phone: 353214901365. Fax: 353214903101. E-mail: d.vansinderen{at}ucc.ie.

Journal of Bacteriology, April 2006, p. 2752-2760, Vol. 188, No. 8
0021-9193/06/$08.00+0     doi:10.1128/JB.188.8.2752-2760.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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