2.A.87 The Prokaryotic Riboflavin Transporter (P-RFT) Family
The RFT family consists of 5 TMS proteins of about 200 aas. The proteins from Bacillus subtilis (YpaA; C. Vogel, unpublished results) and Lactococcus lactis (RibU; Burgess et al., 2006) have been characterized and shown to be riboflavin transporters. Many results had suggested them to be riboflavin transporters before the recent definitive experiments were performed. The RFT family belongs to the BART superfamily (Mansour et al., 2007)
The YpaA protein of Bacillus subtilis [Cecchini and Kearney, 1980; Gelfand et al., 1999; Kreneva et al., 2000; Vitreschak et al., 2002; C. Vogl et al., unpublished results) was first thought to be a riboflavin transporter because the regulatory region upstream of its structural gene included an RFN sequence which was known to be important for the regulation of riboflavin biosynthetic gene expression in response to cytoplasmic riboflavin (Gelfand et al., 1999). Many putative riboflavin transporter genes, in other low G+C Gram-positive organisms, showing homology to ypaA, also have an RFN element in their regulatory region, strengthening the argument that these proteins are riboflavin transporters (Gelfand et al., 1999). Additionally, based on hydropathy plots (see Fig. 1A), YpaA, like RibU, has 5 putative transmembrane segments (TMSs) consistent with a role in transmembrane transport. Based on these observations, at least some RibU homologues appeared to be riboflavin transporters.
In 1976, Ledesma et al. demonstrated that several Lactobacillus species are dependent on external riboflavin for growth. This discovery was confirmed and extended by Møretrø et al. (1998). These findings suggested that lactobacilli cannot synthesize but can transport riboflavin (Koser, 1968). Recently, functional data showed that RibU of Lactococcus lactis is a riboflavin transporter (Burgess et al., 2006). The protein is a 5 TMS protein homologous to the YpaA protein of Bacillus subtilis (see below). In contrast to wild-type L. lactis, a ribU mutant could not take up or use exogenous riboflavin. The mutant also exhibited altered transcriptional control of the riboflavin biosynthetic operon, ribGBAH (Burgess et al., 2006). Competitive inhibition suggested that flavin mononucleotide (FMN) and the toxic riboflavin analogue, roseoflavin, might also be substrates, but this was not demonstrated. A uniport mechanism of action was suggested (Burgess et al., 2006).
Almost all members of the RFT family derive from Firmicutes, but some are from Euryarchaeota and from actinobacteria, and one is from Thermotoga maritima. Surprisingly, no RFT family member is from a proteobacterium, and none of the Gram-negative bacterial subdivisions except Thermotogales is represented.
The majority of the organisms represented have a single RFT family member, but several have two, and one organism, Clostridium acetobutylicum, has three. All of the bacteria with 2 or 3 paralogues are firmicutes: Clostridia, Lactobacilli, Lactococci, Streptococci, Pediococcus pentosaceus and Thermoanaerobacter tengcongensis.
The transport reaction catalyzed by functionally characterized riboflavin (RF) transporters is:
RF (out) RF (in)
The energy-coupling mechanism, if any, is unknown.