Background
Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), the main derivatives of riboflavin, are cofactors for enzymes mediating many redox reactions in the cell [
1]. Globally, up to 17% of enzymes dependent on cofactors use flavins [
2]. Flavoenzymes are involved in a mixture of biological processes such as vitamin, fat and carbohydrate metabolism, photosensitization and oxidative stress response. In bacteria, flavins
per se are also involved in extracellular processes such as assimilatory iron reduction, extracellular respiratory chain and symbiotic interactions [
1,
3‐
6].
There are two ways bacteria can obtain riboflavin, one of them being the riboflavin biosynthetic pathway (RBP) and the other the riboflavin importer systems. The RBP uses guanosine-5-triphosphate (GTP) and ribulose 5-phosphate as precursors for the biosynthesis of riboflavin. Generally, the RBP consists of five enzymes, GTP cyclohydrolase II (RibA), a bifunctional pyrimidine deaminase/reductase (RibD), 3,4-dihydroxy-2-butanone-4-phosphate (3,4-DHBP) synthase (RibB), 6,7-dimethyl-8-ribityllumazine (lumazine) synthase (RibH) and riboflavin synthase (RibE) [
1,
7,
8]. To date, nine riboflavin transport systems have been identified in bacteria. This include the energy-coupling factor system-RibU, RfuABCD and RibXYZ, which are members of the ATP binding cassette family of transporters, and the RfnT, RibM, RibN, RibZ and RibV transporters [
9‐
17]. The transcriptional organization of the RBP genes largely differ among bacteria species. While some species arrange the full pathway genes in a single operon, others have the genes scattered in different transcriptional units along the chromosome. In addition, operons with RBP genes may also contain genes not directly related to riboflavin biosynthesis. Particularly, the global regulators
nrdR and
nusB, encoding a transcriptional repressor of nucleotide reductases and other genes and a factor of the bacterial antiterminator complex, respectively, have been found genetically associated to RBP in various species [
14,
16,
18‐
22]. In some bacteria, RBP and riboflavin importer genes conserve the FMN riboswitch [
9,
12,
15,
16,
23‐
25]. This is a genetic element encoded in RNA leader regions, which downregulates transcription and/or translation by adopting alternative expression-permissive or expression-repressive secondary structures in response to FMN binding [
25,
26]. Thus, many riboflavin supply genes seem to be regulated in response to intracellular flavin levels.
Despite the fact that some bacteria lack the RBP and fulfill their riboflavin demands through riboflavin uptake, in many organisms the RBP and riboflavin importer genes coexist [
10,
15,
27,
28]. Recently, a search on a set of fully sequenced bacterial genomes showed that most bacteria with a riboflavin importer also encode the RBP. Moreover, some species conserve two different families of riboflavin importers besides the RBP [
12]. Additionally, bacteria may encode duplicated or multiplicated orthologs of some RBP enzymes. The complexity of bacterial riboflavin supply pathways seem to respond to species-specific riboflavin needs. For example, a duplicated RibH ortholog in
Brucella abortus is specifically associated to survival inside the host and the
ribBA gene of
Sinorhizobium meliloti is specialized in the production of riboflavin targeted for secretion [
29,
30]. This has led to the hypothesis that riboflavin biosynthesis has a modular structure in bacteria [
30]. Although it is possible that riboflavin importers substitute for the RBP in riboflavin prototrophs when environmental riboflavin is available, the way intraspecies riboflavin provision pathways coordinate to accomplish flavin requirements in bacteria has been scarcely studied.
Vibrio cholerae is an aquatic gammaproteobacteria that causes cholera, a human pandemic disease characterized by acute watery diarrhea, which can lead to death in a short term if untreated. Normally thriving in sea and estuarine waters, the environmental cycle of
V. cholerae includes biofilm formation in biotic and abiotic surfaces and the entrance into the metabolically quiescent viable but non-culturable state under unfavorable conditions [
31]. After human consumption,
V. cholera expresses a series of virulence factors in human intestinal tract, most notably the cholera toxin and the toxin-coregulated pilus. These factors contribute to host colonization and diarrhea development [
32].
Vibrio cholerae is a riboflavin prototroph and it also has the ability to scavenge riboflavin through the RibN riboflavin importer [
14,
33]. Given the wide range of conditions comprising its life cycle, it is likely that
V. cholerae faces variable riboflavin concentrations. This feature makes it an interesting species to study the interconnections between riboflavin biosynthesis and uptake. The present work determined the transcriptional organization of the RBP and
ribN genes in
V. cholerae. In addition, to gain insights into the cues governing the interrelation between riboflavin biosynthesis and transport, we investigated the effect of extracellular riboflavin on the expression of the transcriptional units encoding the riboflavin supply pathways.
Discussion
Results showed that riboflavin provision pathways in
V. cholerae are encoded in four transcriptional elements. The main RBP operon includes the genes
ribD,
ribE,
ribA-COG3236 and
ribH, together with genes involved in proline biosynthesis (
proBA), thiamin biosynthesis (
thiL) and a phosphatidylglycerophosphatase (
pgpA) in addition to
nusB and
nrdR. In spite of its large upstream intergenic region,
ribH and downstream genes are part of the operon. Noteworthy, this region conserves a truncated riboswitch. This may hint to a relatively recent integration of this segment to the operon. Two other RBP components,
ribB and
ribA2 were found to be encoded monocistronically. The conservation of a FMN riboswitch only in
ribB suggested a differential regulation for the RBP components
a priori. Later results showed a negative correlation between
nrdR and
nusB and the FMN riboswitch in RBP genes. This is an interesting finding as it starts to define patterns in the apparently highly variable ways bacteria transcriptionally arrange their RBP genes [
28]. Finally, RibN was found encoded in operon with a putative outer membrane protein (VCA1008) and a protein with a domain of unknown function fused to a glutaredoxin-like domain (VCA1011). Glutaredoxins are enzymes involved in redox homeostasis in bacteria [
40]. Thus, it is possible that imported riboflavin is related to functions of VCA1008 and VCA1011.
Riboflavin needs in bacteria are highly diverse and seem to be quite dependent on species specific traits. While some bacteria may fulfill all of its riboflavin needs through the RBP and others rely solely on riboflavin uptake, many have conserved both functions. The selection constraints driving this phenomenon are not known. It has been pointed out that riboflavin biosynthesis requires more metabolic energy than its transport inside the cell [
41]. Hence, activation of riboflavin uptake and biosynthesis halt when growing in nutrient rich environments would help save energy in a prototroph species. This could be the case for
V. cholerae. Growing on riboflavin rich media downregulated
ribB. Noteworthy, RibB activity is the only one lacking on the otherwise full RBP encoded in the main riboflavin biosynthetic operon. Thus, it seems that the extracellular availability of riboflavin diminishes endogenous riboflavin biosynthesis at the level of 3,4-DHBP synthesis. Nonetheless,
ribB transcription was not completely abolished. This may reflect that some
ribB expression is still afforded in the presence of extracellular riboflavin or that the
ribB FMN riboswitch also acts at the translational level to fully shut down expression, as recently shown for the FMN riboswitch in
ribB from
Escherichia coli [
25]. It is intriguing why constitutive expression of the rest of the RBP enzymes is maintained in the presence of extracellular riboflavin. One explanation for this may be the participation of these enzymes in other metabolic pathways. Indeed, RibA has been involved in folate biosynthesis in
Chlamydia trachomatis [
42] and it has been suggested a participation of RibD and RibA in toxoflavin biosynthesis in
Pseudomonas protegens [
43].
RibN was necessary for external riboflavin to repress ribB. Moreover, extracellular riboflavin highly induced the expression of ribB after deletion of ribN. Importantly, this result may imply that the presence of extracellular riboflavin triggers riboflavin-dependent traits inside the cell. In WT strain, these emergent riboflavin demands may be fulfilled through the RibN uptake activity. However, the ∆ribN strain may suffer a high reduction of intracellular flavin levels in such conditions, which could account for ribB overexpression.
Overall, results indicate that V. cholerae riboflavin provision pathways components are encoded in four transcriptional units. These units are differentially regulated by riboflavin, which may putatively reduce riboflavin biosynthesis through repression of RibB transcription. Although the transcription of the ribN riboflavin importer gene was not affected, the presence of riboflavin may trigger responses that increase the intracellular flavin requirements. Further experimental work is needed to effectively determine if endogenous riboflavin biosynthesis is diminished when growing in the presence of riboflavin, and to assess the bacterial physiological traits activated in response to it.
Authors’ contributions
ISC performed reverse transcriptase and real time PCR experiments, discussed results and helped writing the paper. AT constructed the ribN mutant and discussed results. AFF helped in RNA extractions, discussed results and helped preparing the manuscript. VAG conceived the study, analyzed results, searched the ProOpDB and wrote the manuscript. All authors read and approved the final manuscript.