The riboflavin analog roseoflavin is an antibiotic produced by Streptomyces davawensis. Riboflavin transporters are responsible for roseoflavin uptake by target cells. Roseoflavin is converted to the flavin mononucleotide (FMN) analog roseoflavin mononucleotide (RoFMN) by flavokinase and to the flavin adenine dinucleotide (FAD) analog roseoflavin adenine dinucleotide (RoFAD) by FAD synthetase. In order to study the effect of RoFMN and RoFAD in the cytoplasm of target cells, Escherichia coli was used as a model. E. coli is predicted to contain 38 different FMN-or FAD-dependent proteins (flavoproteins). These proteins were overproduced in recombinant E. coli strains grown in the presence of sublethal amounts of roseoflavin. The flavoproteins were purified and analyzed with regard to their cofactor contents. It was found that 37 out of 38 flavoproteins contained either RoFMN or RoFAD. These cofactors have different physicochemical properties than FMN and FAD and were reported to reduce or completely abolish flavoprotein function. The Gram-positive bacterium S. davawensis JCM 4913 (1, 2) synthesizes the antibiotic roseoflavin, a structural riboflavin (vitamin B 2 ) analog (2, 3). For Bacillus subtilis, S. davawensis, and Corynebacterium glutamicum, it was shown that roseoflavin is taken up via riboflavin transporters (4-8). Moreover, it was found that roseoflavin is converted to the flavin cofactor analogs roseoflavin mononucleotide (RoFMN) and roseoflavin adenine dinucleotide (RoFAD) by flavokinases (EC 2.7.1.26) and FAD synthetases (EC 2.7.7.2) in vitro (9, 10) (Fig. 1A).RoFMN was reported to reduce expression of genes involved in riboflavin biosynthesis and/or transport in B. subtilis, Streptomyces coelicolor, and the human pathogen Listeria monocytogenes (10-13). These genes are all controlled by FMN riboswitches (14), regulatory elements which are negatively affected by RoFMN. This reduction of gene expression at least in part explains why roseoflavin acts as an antibiotic. For example, reduced expression of the FMN riboswitch-controlled riboflavin-biosynthetic genes ribEMAH in S. coelicolor (caused by RoFMN) led to a significantly decreased level of riboflavin synthase (RibE) activity (10) and consequently to a reduced supply of riboflavin. In another study, the addition of roseoflavin to riboflavin-auxotrophic L. monocytogenes led to reduced expression of the FMN riboswitch-controlled riboflavin transporter gene lmo1945 and to a reduced supply of riboflavin as well (11). In this study, an L. monocytogenes strain was described that contained a mutant FMN riboswitch which was not blocked by RoFMN. This strain constitutively transcribed lmo1945 and showed a significant decrease in roseoflavin sensitivity; however, it was still roseoflavin sensitive. We therefore concluded that additional targets for roseoflavin must be present in L. monocytogenes and very likely in other bacteria as well.Approximately 1 to 3% of all bacterial proteins depend on the riboflavin-derived cofactors FMN or FAD (15) and thus are plausi...

FMN riboswitches are genetic elements that, in many bacteria, control genes responsible for biosynthesis and/or transport of riboflavin (vitamin B 2 ). We report that the Escherichia coli ribB FMN riboswitch controls expression of the essential gene ribB coding for the riboflavin biosynthetic enzyme 3,4-dihydroxy-2-butanone-4-phosphate synthase (RibB; EC 4.1.99.12). Our data show that the E. coli ribB FMN riboswitch is unusual because it operates at the transcriptional and also at the translational level. Expression of ribB is negatively affected by FMN and by the FMN analog roseoflavin mononucleotide, which is synthesized enzymatically from roseoflavin and ATP. Consequently, in addition to flavoenzymes, the E. coli ribB FMN riboswitch constitutes a target for the antibiotic roseoflavin produced by Streptomyces davawensis.

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RibR is a minor cryptic flavokinase (EC 2.7.1.26) of the Gram-positive bacterium Bacillus subtilis with an unknown cellular function. The flavokinase activity appears to be localized to the N-terminal domain of the protein. Using the yeast three-hybrid system, it was shown that RibR specifically interacts in vivo with the nontranslated wild-type leader of the mRNA of the riboflavin biosynthetic operon. This interaction is lost partially when a leader containing known cis-acting deregulatory mutations in the so-called RFN element is tested. The RFN element is a sequence within the rib-leader mRNA reported to serve as a receptor for an FMN-dependent 'riboswitch'. In RibR itself, interaction was localized to the carboxy-terminate part of the protein, a segment of unknown function that does not show similarity to other proteins in the public databases. Analysis of a ribR-defective strain revealed a mild deregulation with respect to flavin (riboflavin, FMN and FAD) biosynthesis. The results indicate that the RNA-binding protein RibR may be involved in the regulation of the rib genes.

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