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 (...
BackgroundRiboflavin is the precursor of important redox cofactors such as flavin mononucleotide (FMN) and flavin adenine dinucleotide, required for several biological processes. Vibrio cholerae, a pathogenic bacterium responsible for the cholera disease, possesses the ability to biosynthesize de novo as well as to uptake riboflavin through the riboflavin biosynthetic pathway (RBP) and the RibN importer, respectively. The intra-organism relationship between riboflavin biosynthesis and uptake functions has not been studied.ResultsThis work determined the transcriptional organization of RBP genes and ribN in V. cholerae through reverse transcription polymerase chain reaction and analyzed their expression when growing with or without extracellular riboflavin using real time PCR. The RBP is organized in three transcriptional units, the major one containing ribD, ribE, ribA and ribH together with genes involved in functions not directly related to riboflavin biosynthesis such as nrdR and nusB. In addition, two independent monocistronic units contain ribA2 and ribB, the later conserving a putative FMN riboswitch. The ribN gene is encoded in operon with a gene coding for a predicted outer membrane protein and a gene encoding a protein with a glutaredoxin domain. Regulation analysis showed that among these transcriptional units, only ribB is negatively regulated by riboflavin and that its repression depends on the RibN riboflavin importer. Moreover, external riboflavin highly induced ribB transcription in a ΔribN strain. Also, a genomic database search found a negative correlation between the presence of nrdR and nusB and the FMN riboswitch in bacterial RBP operons.ConclusionsGrowing in the presence of riboflavin downregulates only a single element among the transcriptional units of riboflavin supply pathways. Thus, endogenous riboflavin biosynthesis seems to be negatively regulated by extracellular riboflavin through its specific effect on transcription of ribB in V. cholerae.Electronic supplementary materialThe online version of this article (doi:10.1186/s13099-017-0159-z) contains supplementary material, which is available to authorized users.
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