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Extensive Identification of Bacterial Riboflavin Transporters and Their Distribution across Bacterial Species.

Gutiérrez-Preciado A, Torres AG, Merino E, Bonomi HR, Goldbaum FA, García-Angulo VA - PLoS ONE (2015)

Bottom Line: Two new putative riboflavin transporters were identified: RibZ in Clostridium and RibV in Mesoplasma florum.Our results indicate that some species possess ancestral riboflavin transporters, while others possess transporters that appear to have evolved recently.Moreover, our data suggest that unidentified riboflavin transporters also exist.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, México.

ABSTRACT
Riboflavin, the precursor for the cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide, is an essential metabolite in all organisms. While the functions for de novo riboflavin biosynthesis and riboflavin import may coexist in bacteria, the extent of this co-occurrence is undetermined. The RibM, RibN, RfuABCD and the energy-coupling factor-RibU bacterial riboflavin transporters have been experimentally characterized. In addition, ImpX, RfnT and RibXY are proposed as riboflavin transporters based on positional clustering with riboflavin biosynthetic pathway (RBP) genes or conservation of the FMN riboswitch regulatory element. Here, we searched for the FMN riboswitch in bacterial genomes to identify genes encoding riboflavin transporters and assessed their distribution among bacteria. Two new putative riboflavin transporters were identified: RibZ in Clostridium and RibV in Mesoplasma florum. Trans-complementation of an Escherichia coli riboflavin auxotroph strain confirmed the riboflavin transport activity of RibZ from Clostridium difficile, RibXY from Chloroflexus aurantiacus, ImpX from Fusobacterium nucleatum and RfnT from Ochrobactrum anthropi. The analysis of the genomic distribution of all known bacterial riboflavin transporters revealed that most occur in species possessing the RBP and that some bacteria may even encode functional riboflavin transporters from two different families. Our results indicate that some species possess ancestral riboflavin transporters, while others possess transporters that appear to have evolved recently. Moreover, our data suggest that unidentified riboflavin transporters also exist. The present study doubles the number of experimentally characterized riboflavin transporters and suggests a specific, non-accessory role for these proteins in riboflavin-prototrophic bacteria.

No MeSH data available.


Related in: MedlinePlus

Complementation of a riboflavin E. coli auxotroph with the candidate riboflavin transporter proteins.E. coli ∆ribB::cat and derivatives bearing plasmids encoding the indicated riboflavin transporter genes and negative control were grown at 37°C on modified M9 medium containing the indicated riboflavin concentrations. After 21 hours of growth, the O.D.600nm was assessed. For comparative purposes, the WT strain was cultivated in M9 medium without riboflavin.
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pone.0126124.g001: Complementation of a riboflavin E. coli auxotroph with the candidate riboflavin transporter proteins.E. coli ∆ribB::cat and derivatives bearing plasmids encoding the indicated riboflavin transporter genes and negative control were grown at 37°C on modified M9 medium containing the indicated riboflavin concentrations. After 21 hours of growth, the O.D.600nm was assessed. For comparative purposes, the WT strain was cultivated in M9 medium without riboflavin.

Mentions: To determine whether the identified candidates are riboflavin transporters, we cloned ribZ from C. difficile, the ribXY operon from Chloroflexus aurantiacus, impX from F. nucleatum and rfnT from O. anthropi into plasmids and evaluated their ability to complement the growth of an E. coli riboflavin auxotrophic ∆ribB strain. This complementation assay excluded ribV from M. florum because UGA, the triplet codon for the tryptophan residue in Mycoplasma [32], encodes a stop codon in E. coli. RibV possesses 5 tryptophan residues that would result in a truncated protein if the M. florum gene were expressed in E. coli. As previously reported [16], the E. coli ∆ribB is unable to grow in M9 minimal media in the absence of riboflavin because this strain bears a deletion in the gene encoding 3,4-dihydroxy-2-butanone-4-phosphate synthase, essential for riboflavin biosynthesis (Fig 1). Low riboflavin media (2.5 μM) did not support the growth of this strain, reflecting the lack of riboflavin transporters in E. coli. Hence, this strain requires large amounts of exogenous riboflavin (500 μM) to support adequate growth. The four plasmids encoding the candidate riboflavin transporters, pRibXY, pRibZ, pImpX and pRfnT, restored the growth of E. coli ∆ribB in low riboflavin in a similar fashion to that observed with the plasmid encoding RibN from O. anthropi, a characterized riboflavin transporter [16]. By contrast, the negative control plasmid pLpfA failed to promote the growth of the strain at low riboflavin concentration. Neither of the plasmids promoted the growth of the strain in media without riboflavin, demonstrating the riboflavin dependence of the rescue phenotype. Thus, these results indicate that RibXY, RibZ, ImpX and RfnT facilitate the uptakte of riboflavin into bacterial cells.


Extensive Identification of Bacterial Riboflavin Transporters and Their Distribution across Bacterial Species.

Gutiérrez-Preciado A, Torres AG, Merino E, Bonomi HR, Goldbaum FA, García-Angulo VA - PLoS ONE (2015)

Complementation of a riboflavin E. coli auxotroph with the candidate riboflavin transporter proteins.E. coli ∆ribB::cat and derivatives bearing plasmids encoding the indicated riboflavin transporter genes and negative control were grown at 37°C on modified M9 medium containing the indicated riboflavin concentrations. After 21 hours of growth, the O.D.600nm was assessed. For comparative purposes, the WT strain was cultivated in M9 medium without riboflavin.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4418817&req=5

pone.0126124.g001: Complementation of a riboflavin E. coli auxotroph with the candidate riboflavin transporter proteins.E. coli ∆ribB::cat and derivatives bearing plasmids encoding the indicated riboflavin transporter genes and negative control were grown at 37°C on modified M9 medium containing the indicated riboflavin concentrations. After 21 hours of growth, the O.D.600nm was assessed. For comparative purposes, the WT strain was cultivated in M9 medium without riboflavin.
Mentions: To determine whether the identified candidates are riboflavin transporters, we cloned ribZ from C. difficile, the ribXY operon from Chloroflexus aurantiacus, impX from F. nucleatum and rfnT from O. anthropi into plasmids and evaluated their ability to complement the growth of an E. coli riboflavin auxotrophic ∆ribB strain. This complementation assay excluded ribV from M. florum because UGA, the triplet codon for the tryptophan residue in Mycoplasma [32], encodes a stop codon in E. coli. RibV possesses 5 tryptophan residues that would result in a truncated protein if the M. florum gene were expressed in E. coli. As previously reported [16], the E. coli ∆ribB is unable to grow in M9 minimal media in the absence of riboflavin because this strain bears a deletion in the gene encoding 3,4-dihydroxy-2-butanone-4-phosphate synthase, essential for riboflavin biosynthesis (Fig 1). Low riboflavin media (2.5 μM) did not support the growth of this strain, reflecting the lack of riboflavin transporters in E. coli. Hence, this strain requires large amounts of exogenous riboflavin (500 μM) to support adequate growth. The four plasmids encoding the candidate riboflavin transporters, pRibXY, pRibZ, pImpX and pRfnT, restored the growth of E. coli ∆ribB in low riboflavin in a similar fashion to that observed with the plasmid encoding RibN from O. anthropi, a characterized riboflavin transporter [16]. By contrast, the negative control plasmid pLpfA failed to promote the growth of the strain at low riboflavin concentration. Neither of the plasmids promoted the growth of the strain in media without riboflavin, demonstrating the riboflavin dependence of the rescue phenotype. Thus, these results indicate that RibXY, RibZ, ImpX and RfnT facilitate the uptakte of riboflavin into bacterial cells.

Bottom Line: Two new putative riboflavin transporters were identified: RibZ in Clostridium and RibV in Mesoplasma florum.Our results indicate that some species possess ancestral riboflavin transporters, while others possess transporters that appear to have evolved recently.Moreover, our data suggest that unidentified riboflavin transporters also exist.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, México.

ABSTRACT
Riboflavin, the precursor for the cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide, is an essential metabolite in all organisms. While the functions for de novo riboflavin biosynthesis and riboflavin import may coexist in bacteria, the extent of this co-occurrence is undetermined. The RibM, RibN, RfuABCD and the energy-coupling factor-RibU bacterial riboflavin transporters have been experimentally characterized. In addition, ImpX, RfnT and RibXY are proposed as riboflavin transporters based on positional clustering with riboflavin biosynthetic pathway (RBP) genes or conservation of the FMN riboswitch regulatory element. Here, we searched for the FMN riboswitch in bacterial genomes to identify genes encoding riboflavin transporters and assessed their distribution among bacteria. Two new putative riboflavin transporters were identified: RibZ in Clostridium and RibV in Mesoplasma florum. Trans-complementation of an Escherichia coli riboflavin auxotroph strain confirmed the riboflavin transport activity of RibZ from Clostridium difficile, RibXY from Chloroflexus aurantiacus, ImpX from Fusobacterium nucleatum and RfnT from Ochrobactrum anthropi. The analysis of the genomic distribution of all known bacterial riboflavin transporters revealed that most occur in species possessing the RBP and that some bacteria may even encode functional riboflavin transporters from two different families. Our results indicate that some species possess ancestral riboflavin transporters, while others possess transporters that appear to have evolved recently. Moreover, our data suggest that unidentified riboflavin transporters also exist. The present study doubles the number of experimentally characterized riboflavin transporters and suggests a specific, non-accessory role for these proteins in riboflavin-prototrophic bacteria.

No MeSH data available.


Related in: MedlinePlus