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 The proteins from Bacillus subtilis (YpaA; C. Vogel, unpublished results) and Lactococcus lactis (RibU; Burgess et al., 2006) have been characterized and shown to be riboflavin transporters.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[Cecchini and Kearney, 1980; Gelfand et al., 1999; Kreneva et al., 2000; Vitreschak et al., 2002; C. Vogl et al., unpublished results) was first thought to be a riboflavin transporter because the regulatory region upstream of its structural gene included an RFN sequence which was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.



This family belongs to the BART Superfamily.

 

References:

Burgess, C.M., D.J. Slotboom, E.R. Geertsma, R.H. Duurkens, B. Poolman, and D. van Sinderen. (2006). The riboflavin transporter RibU in Lactococcus lactis: molecular characterization of gene expression and the transport mechanism. J. Bacteriol. 188: 2752-2760.

Cecchini, B. and E.B. Kearney. (1980). Uptake and binding of riboflavin by membrane vesicles of Bacillus subtilis. J. Supramol. Struct. 13: 93–100.

Duurkens, R.H., M.B. Tol, E.R. Geertsma, H.P. Permentier, D.J. Slotboom. (2007). Flavin binding to the high affinity riboflavin transporter RibU. J. Biol. Chem. 282: 10380-10386.

Gelfand, M.S., A.A. Mironov, J. Jomantas, Y.I. Kozlov, and A.D. Perumov. (1999). A conserved RNA structure element involved in the regulation of bacterial riboflavin synthesis genes. Trends Genet. 15: 439-442.

Gutiérrez-Preciado, A., A.G. Torres, E. Merino, H.R. Bonomi, F.A. Goldbaum, and V.A. García-Angulo. (2015). Extensive Identification of Bacterial Riboflavin Transporters and Their Distribution across Bacterial Species. PLoS One 10: e0126124.

Karpowich NK., Song JM., Cocco N. and Wang DN. (2015). ATP binding drives substrate capture in an ECF transporter by a release-and-catch mechanism. Nat Struct Mol Biol. 22(7):565-71.

Koser, S.A. (1968). Lactobacillus. In Vitamin Requirements of Bacteria and Yeasts (Thomas, C.C., ed.), Springfield, IL: pp. 340-345.

Kreneva, R.A., M.S. Gelfand, A.A. Mironov, I.A. Iomantus, I.I. Kozlov, A.S. Mironov, and D.A. Perumov. (2000). Inactivation of the ypaA gene in Bacillus subtilis: analysis of the resulting phenotypic expression. Russian J. Genet. 36: 972-974.

Ledesma, O.V., A. Ruiz Holgado, and G. Oliver. (1976). A synthetic medium for comparative nutritional studies of Lactobacilli. J. Appl. Bacteriol. 42: 123-133.

Light, S.H., L. Su, R. Rivera-Lugo, J.A. Cornejo, A. Louie, A.T. Iavarone, C.M. Ajo-Franklin, and D.A. Portnoy. (2018). A flavin-based extracellular electron transfer mechanism in diverse Gram-positive bacteria. Nature 562: 140-144.

Møretrø, T., B.F. Hagen, and L. Axelsson. (1998). A new, completely defined medium for meat lactobacilli. J. Appl. Microbiol. 85: 715-722.

Rodionov, D.A., P. Hebbeln, A. Eudes, J. ter Beek, I.A. Rodionova, G.B. Erkens, D.J. Slotboom, M.S. Gelfand, A.L. Osterman, A.D. Hanson, and T. Eitinger. (2009). A novel class of modular transporters for vitamins in prokaryotes. J. Bacteriol. 191: 42-51.

Vitreschak, A.G., D.A. Rodionov, A.A. Mironov, and M.S. Gelfand. (2002). Regulation of riboflavin biosynthesis and transport genes in bacteria by transcriptional and translational attenuation. Nucleic Acids Res. 30: 3141-3151.

Vogl, C., S. Grill, O. Schilling, J. Stülke, M. Mack, and J. Stolz. (2007). Characterization of riboflavin (vitamin B2) transport proteins from Bacillus subtilis and Corynebacterium glutamicum. J. Bacteriol. 189: 7367-7375.

Zhang P., Wang J. and Shi Y. (2010). Structure and mechanism of the S component of a bacterial ECF transporter. Nature. 468(7324):717-20.

Zhang, M., Z. Bao, Q. Zhao, H. Guo, K. Xu, C. Wang, and P. Zhang. (2014). Structure of a pantothenate transporter and implications for ECF module sharing and energy coupling of group II ECF transporters. Proc. Natl. Acad. Sci. USA 111: 18560-18565.

Examples:

TC#NameOrganismal TypeExample
2.A.87.1.1The riboflavin uptake transporter, RibU (may also transport roseoflavin and flavin mononucleotide (FMN)) (Burgess et al., 2006). Substrates noted above bind RibU with nM (Vitamin B2 with 0.6 nM) affinity with 1:1 stoichiometry; FAD does not bind (Duurkens et al., 2007).Bacteria and ArchaeaRibU of Lactococcus lactis (AAY43361)
 
2.A.87.1.2

Riboflavin transporter RibU (Riboflavin ECF transporter S component RibU or YpaA). Regulated by FMN riboswitch (Rodionov et al. 2009). RibU-mediated riboflavin uptake was sensitive to protonophores and reduced in the absence of glucose, demonstrating that the protein requires metabolic energy for substrate translocation (Vogl et al. 2007).

Firmicutes

RibU of Bacillus subtilis (P50726)

 
2.A.87.1.3

Riboflavin ECF Transporter, RibU. The substrate-binding component (S) has been solved by x-ray crystallography (Zhang et al., 2010).

Bacteria

RibU of Staphylococcus aureus (D3EWM9)

 
2.A.87.1.4

The riboflavin transporter of 203 aas and 5 TMSs, RibU or EcfS. Mediates riboflavin and maybe also FMN, FAD and roseoflavin uptake (and efflux) (Light et al. 2018). Interacts with the energy-coupling factor (ECF) module, EcfAA'T, which energizes transport of a number of different substrates, each with its own EcfS subunit.  Does not use an extracytoplasmic receptor (Karpowich et al. 2015). It provides extracellur FAD for an extracellular electron transfer process in firmicutes (Light et al. 2018).

Firmicutes

Riboflavin transporter of Listeria monocytogenes

 
Examples:

TC#NameOrganismal TypeExample
2.A.87.2.1

Pantothenate transporter PanT (Pantothenic acid ECF transporter S component PanT).  A similar pantothenate transporter, also called PanT, has been characterized in Lactobacillus brevis (Zhang et al. 2014).

Bacteria

PanT of Leuconostoc mesenteroides (Q03YI6)

 

 
2.A.87.2.3

Putative Thiamine Transporter, ThiA (Duf165; YuaJ) (187 aas; 6 TMSs)

Bacteria

ThiA of Streptococcus mitis (F9LXW4)

 
Examples:

TC#NameOrganismal TypeExample
2.A.87.3.1

Uncharacterized protein of 172 aas and 5 TMSs

Firmicutes

UP of Clostridium kluyveri

 
2.A.87.3.2

Putative heptaprenyl diphosphate synthase component I of 173 aas and 5 TMSs. 

Firmicutes

P-RFT family member of Clostridium leptum

 
2.A.87.3.3

Uncharacterized protein of 169 aas and 5 TMSs.

Tenericutes

UP of Acholeplasma laidlawii

 
Examples:

TC#NameOrganismal TypeExample
2.A.87.4.1

Riboflavin uptake transporter of 228 aas and 5 TMSs (Gutiérrez-Preciado et al. 2015).

RibV of Mesoplasma florum

 
2.A.87.4.2

Putative riboflavin transporter of 227 aas and 5 TMSs (Gutiérrez-Preciado et al. 2015).

RibV of Mycoplasma capricolum

 
2.A.87.4.3

Uncharacterized protein, probably a riboflavin transporter of 234 aas and 5 TMSs.

UP of Spiroplasma citri