Riboflavin (vitamin B 2 ) is the direct precursor of the flavin cofactors flavin mononucleotide and flavin adenine dinucleotide, 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 a cytoplasmic C terminus. In B. subtilis, riboflavin uptake was increased when ypaA was overexpressed 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 dependent and had high apparent affinity for riboflavin (K m 11 M). 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.Riboflavin consists of a ribityl side chain linked to an aromatic isoalloxazine ring structure. It is the precursor of the cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which both are essential components of cellular metabolism. Riboflavin is phosphorylated to give FMN by flavokinase (EC 2.7.1.26), and FMN is subsequently converted to FAD by FAD synthetase (EC 2.7.7.2). Whereas free riboflavin does not have biological activity, the mostly noncovalently bound flavin cofactors FMN and FAD are the active groups of a large number of flavoproteins. These are involved in a wide range of redox reactions and catalyze the dehydrogenation of metabolites, one-and two-electron transfer reactions from and to redox centers, and hydroxylation reactions (9). Flavins are also known to act as chromophores in photoreceptors, such as the plant blue light sensors cryptochrome and phototropin (reviewed in reference 3). Moreover, flavins are the ligands of dodecin, a recently identified flavoprotein that has the highest binding affinity to lumichrome, a lightinduced degradation product of riboflavin with an alloxazine ring structure lacking a ribityl side chain (13, 48).Whereas vertebrates can generate FMN and FAD from riboflavin, they lack the enzymes to synthesize riboflavin, making this compound a vitamin (vitamin B 2 ). In contrast, plants and most microorganisms are capable of ...

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BackgroundThe bacterium Bacillus subtilis, which is not a natural riboflavin overproducer, has been converted into an excellent production strain by classical mutagenesis and metabolic engineering. To our knowledge, the enhancement of riboflavin excretion from the cytoplasm of overproducing cells has not yet been considered as a target for (further) strain improvement. Here we evaluate the flavin transporter RibM from Streptomyces davawensis with respect to improvement of a riboflavin production strain.ResultsThe gene ribM from S. davawensis, coding for a putative facilitator of riboflavin uptake, was codon optimized (ribMopt) for expression in B. subtilis. The gene ribMopt was functionally introduced into B. subtilis using the isopropyl-β-thiogalactopyranoside (IPTG)-inducible expression plasmid pHT01: Northern-blot analysis of total RNA from IPTG treated recombinant B. subtilis cells revealed a ribMopt specific transcript. Western blot analysis showed that the his6-tagged heterologous gene product RibM was present in the cytoplasmic membrane. Expression of ribM in Escherichia coli increased [14C]riboflavin uptake, which was not affected by the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). Expression of ribMopt supported growth of a B. subtilis ΔribB::Ermr ΔribU::Kanr double mutant deficient in riboflavin synthesis (ΔribB) and also deficient with respect to riboflavin uptake (ΔribU). Expression of ribMopt increased roseoflavin (a toxic riboflavin analog produced by S. davawensis) sensitivity of a B. subtilis ΔribU::Kanr strain. Riboflavin synthesis by a model riboflavin B. subtilis production strain overproducing RibM was increased significantly depending on the amount of the inducer IPTG.ConclusionsThe energy independent flavin facilitator RibM could in principle catalyze riboflavin export and thus may be useful to increase the riboflavin yield in a riboflavin production process using a recombinant RibM overproducing B. subtilis strain (or any other microorganism).

We present substantial extensions to the Monte Carlo radiative transfer code tardis to perform spectral synthesis for type II supernovae. By incorporating a non-LTE ionization and excitation treatment for hydrogen, a full account of free-free and bound-free processes, a self-consistent determination of the thermal state and by improving the handling of relativistic effects, the improved code version includes the necessary physics to perform spectral synthesis for type II supernovae to high precision as required for the reliable inference of supernova properties. We demonstrate the capabilities of the extended version of tardis by calculating synthetic spectra for the prototypical type II supernova SN1999em and by deriving a new and independent set of dilution factors for the expanding photosphere method. We have investigated in detail the dependence of the dilution factors on photospheric properties and, for the first time, on changes in metallicity. We also compare our results with two previously published sets of dilution factors by Eastman et al. (1996) and by Dessart & Hillier (2005a), and discuss the potential sources of the discrepancies between studies.