RibR, a possible regulator of theBacillus subtilisriboflavin biosynthetic operon,in vivointeracts with the 5′-untranslated leader ofribmRNA

Abstract

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.

Genetic Control of Biosynthesis and Transport of Riboflavin and Flavin Nucleotides and Construction of Robust Biotechnological Producers

“…6. The carboxy terminus of the B. subtilis ribR monofunctional RF kinase binds to the FMN riboswitch, but this binding is partially lost in cells bearing mutations in the RFN element (177). The data suggest that riboswitch regulation using FMN as an effector can be supplemented with the action of monofunctional RF kinase, thus acting as a regulatory protein.…”

“…The structures of S. pombe and human RF kinases showed a novel family of phosphoryl-transferring enzymes (26,211). The role of monofunctional B. subtilis RF kinase in FMN synthesis in vivo has yet to be clarified (177,457,458). In addition to monofunctional RF kinases, bifunctional RF kinase/FAD synthetase is the main enzyme involved in eubacterial flavin nucleotide biosynthesis (101,280,286,320).…”

The Bifunctional Flavokinase/Flavin Adenine Dinucleotide Synthetase from Streptomyces davawensis Produces Inactive Flavin Cofactors and Is Not Involved in Resistance to the Antibiotic Roseoflavin

“…In contrast, both enzymatic activities were purified separately from eukaryotic organisms (2,34). B. subtilis is the only known organism containing a second cryptic monofunctional flavokinase (RibR) (17,38,39). There is evidence that RibR may be involved in regulation of the rib biosynthetic operon (17).…”

Characterization of Riboflavin (Vitamin B2) Transport Proteins fromBacillus subtilisandCorynebacterium glutamicum

“…Consistent with this function, mutations that cause riboflavin overproduction map at various positions within the RFN element (14,21) or in ribC, the gene encoding a bifunctional flavokinase/FAD synthetase (6,26). Moreover, the B. subtilis genome encodes a monofunctional flavokinase (ribR), but the in vivo contribution of this protein to the generation of FMN remains unclear (15,41).…”

“…Consistent with this function, mutations that cause riboflavin overproduction map at various positions within the RFN element (14, 21) or in ribC, the gene encoding a bifunctional flavokinase/FAD synthetase (6, 26). Moreover, the B. subtilis genome encodes a monofunctional flavokinase (ribR), but the in vivo contribution of this protein to the generation of FMN remains unclear (15,41).Although a requirement for riboflavin is very rare among bacteria, it is known that riboflavin is an essential growth factor for Enterococcus faecalis, Streptococcus pyogenes, Listeria monocytogenes, and some lactobacilli (22). This biosynthetic deficiency correlates with the absence of riboflavin biosynthetic genes in the genomes of these organisms (46).…”