Characterization of riboflavin (vitamin B₂) transport proteins from Bacillus subtilis and Corynebacterium glutamicum

Lehrstuhl für Zellbiologie und Pflanzenphysiologie, Universität Regensburg, Universitätsstrasse 31, 93040 Regensburg, Germany, Institut für Technische Mikrobiologie, Hochschule Mannheim, Windeckstrasse 110, 68163 Mannheim, Germany, Institut für Mikrobiologie und Genetik, Abteilung für Allgemeine Mikrobiologie, Grisebachstr. 8, 37077 Göttingen, Germany

^* To whom correspondence should be addressed. Email: juergen.stolz{at}wzw.tum.de.

	Abstract

Riboflavin (Vitamin B₂) is the direct precursor of the flavin cofactors FMN and FAD, essential components of cellular biochemistry. In this work we investigated the unrelated proteins YpaA from Bacillus subtilis and PnuX from Corynebacterium glutamicum for a role in riboflavin uptake. Based on the regulation of the corresponding genes by a riboswitch mechanism, both proteins have been predicted to be involved in flavin metabolism. Moreover, their primary structures suggested that these proteins integrate into the cytoplasmic membrane. We provide experimental evidence that YpaA is a plasma-membrane protein with five transmembrane domains and cytoplasmic C-terminus. In B. subtilis, riboflavin uptake was increased when ypaA was over-expressed and abolished when ypaA was deleted. Riboflavin uptake activity and the abundance of the YpaA protein were also increased when riboflavin auxotrophic mutants were grown in limiting amounts of riboflavin. YpaA-mediated riboflavin uptake was sensitive to protonophors and reduced in the absence of glucose, demonstrating that the protein requires metabolic energy for substrate translocation. In addition, we demonstrate that PnuX from C. glutamicum also is a riboflavin transporter. Transport by PnuX was not energy-dependant and had high apparent affinity for riboflavin (K_m=11±6 µM). Roseoflavin, a toxic riboflavin analog, appears to be a substrate of PnuX and YpaA. We propose to designate the gene names ribU for ypaA and ribM for pnuX to reflect that the encoded proteins function in riboflavin uptake and that the genes have different phylogenetic origins.