Identification and Characterization of RibN, a Novel Family of Riboflavin Transporters from Rhizobium leguminosarum and Other Proteobacteria

Abstract

bRhizobia are symbiotic bacteria able to invade and colonize the roots of legume plants, inducing the formation of nodules, where bacteria reduce atmospheric nitrogen (N 2 ) to ammonia (NH 3 ). Riboflavin availability influences the capacity of rhizobia to survive in the rhizosphere and to colonize roots. In this study, we identified the RL1692 gene of Rhizobium leguminosarum downstream of a flavin mononucleotide (FMN) riboswitch. RL1692 encodes a putative transmembrane permease with two EamA domains. The presence of an FMN riboswitch regulating a transmembrane protein is usually observed in riboflavin transporters, suggesting that RL1692 may be involved in riboflavin uptake. The product of RL1692, which we named RibN, is conserved in members of the alpha-, beta-, and gammaproteobacteria and shares no significant identity with any riboflavin transporter previously identified. In this work, we show that RibN is localized in the membrane cellular fraction and its expression is downregulated by riboflavin. By heterologous expression in a Brucella abortus mutant auxotrophic for riboflavin, we demonstrate that RibN possesses flavin transport activity. Similarly, we also demonstrate that RibN orthologues from Ochrobactrum anthropi and Vibrio cholerae (which lacks the FMN riboswitch) are able to transport riboflavin. An R. leguminosarum ribN null mutant exhibited lower nodule occupancy levels in pea plants during symbiosis assays. Thus, we propose that RibN and its homologues belong to a novel family of riboflavin transporters. This work provides the first experimental description of riboflavin transporters in Gram-negative bacteria. R hizobium leguminosarum bv. viciae is a soil Gram-negative bacterium that infects and establishes a symbiotic relationship with its host, Pisum sativum (the pea plant). R. leguminosarum is a member of the rhizobial group, which is phylogenetically diverse and includes 12 genera and about 70 species of alpha-and betaproteobacteria. Rhizobia induce nitrogen-fixing nodules in the roots of legume plants. The order Rhizobiales is included in the class Alphaproteobacteria, where several rhizobia can be found along with the mammal zoonotic pathogens Brucella spp. (1, 2).Riboflavin is the precursor of the cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which are the typical cofactors of flavoproteins. Flavoproteins are essential for multiple cellular processes, including energy production, redox reactions, light emission, biosynthesis, and DNA repair (3, 4). Riboflavin availability can influence the onset of rhizobial symbiotic interactions. A Rhizobium trifolii strain auxotrophic for riboflavin requires the addition of riboflavin to the plant growth medium in order to attain effective symbiosis with red clover plants, and the effectiveness of the nodulation of this strain in other clover cultivars relates to the flavin content in nodules (5). Also, the addition of riboflavin to the rhizosphere can increase the colonization of alfalfa roots by Sinorhizobium meliloti (...

The ribB FMN riboswitch from Escherichia coli operates at the transcriptional and translational level and regulates riboflavin biosynthesis

“…In light of our earlier flavoproteome analysis , it is remarkable that E. coli has multiple targets for this antibiotic. Recently, the first riboflavin transporter from a Gram‐negative bacterium was identified , raising the hope that roseoflavin or flavin analogs in general could become useful for also treating infections with Gram‐negative bacteria. Our studies with E. coli certainly provide a solid basis for the development of novel antibacterials with novel targets.…”

Metabolic Response of Clostridium ljungdahlii to Oxygen Exposure

“…Flavins used by this protein may be transported into the cell by the putative membrane protein encoded by CLJU_c19430, which contains two EamA domains like the characterized riboflavin-transporting transmembrane permeases (RibN) from Ochrobactrum anthropi and Vibrio cholerae (90). A blastx query of the putative epimerase gene (CLJU_c19420) in this oxygen-induced gene cluster revealed that it had higher homology to a phenazine biosynthesis-like protein than its annotated function as an epimerase.…”

Rare variants of the FMN riboswitch class in Clostridium difficile and other bacteria exhibit altered ligand specificity

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