Lactobacillus casei folT and thiT were amplified using Taq DNA polymerase (Invitrogen, Carlsbad, CA) with L. casei ATCC 334 genomic DNA as the template and ligated into vector pNZ8048. The resulting constructs were used as templates for the amplification of fragments containing the nisinA promoter, folT or thiT, and a terminator using Pfu Ultra DNA polymerase (Invitrogen). Amplicons were ligated into vector pIL252 between the BamHI and XhoI sites. The ecfAA′T operon was amplified using Pfu Ultra DNA polymerase and inserted between the NcoI and SstI sites of vector pNZ8048. The recombinant plasmids were cloned into Lactococcus lactis strain NZ9000. Cells were grown at 30°C in supplemented M17 medium (Difco), and for expression, nisin (0.1% [vol/vol] of a culture supernatant of the nisin A-producing strain NZ9700) was added.

Bacillus subtilis ypaA (ribU), ybaF (ecfT), yuaJ (thiT), and yceI (niaP) disruption strains were obtained from the joint Japanese and European Bacillus subtilis consortium (40). For the roseoflavin inhibition assay, B. subtilis cells were grown overnight at 40°C in chemically defined medium containing glucose (4 g/liter), tryptophan (50 mg/liter), glutamine (2 g/liter), K₂HPO₄ (10 g/liter), KH₂PO₄ (6 g/liter), sodium citrate (1 g/liter), MgSO₄ (0.2 g/liter), K₂SO₄ (2 g/liter), FeCl₃ (4 mg/liter), and MnSO₄ (0.2 mg/liter) in the presence of 0.5 mg/liter erythromycin (pMUTIN2 marker). These cultures were diluted ~10- to 20-fold to yield the same cell density (optical density at 600 nm [OD₆₀₀] of 0.05) in fresh medium and grown in duplicate in the absence or presence of roseoflavin (250 μM).

For [³H]5-formyltetrahydrofolate and [³H]thiamine uptake assays, L. lactis cells were washed once with cold phosphate-buffered saline (PBS) and resuspended in PBS at an OD₆₀₀ of 20. Assays were performed at 30°C with stirring. Cells (500 μl) were preincubated for 5 min with glucose or 2-deoxyglucose (25 mM final concentration). Assays were started by adding 500 μl of PBS containing 2.2 μM [³H]5-formyltetrahydrofolate (specific activity of 0.4 μCi/nmol) or 2.3 μM [³H]thiamine (0.33 μCi/nmol) to the mixture. At intervals, 150-μl aliquots were passed through a Whatman cellulose nitrate membrane filter (0.45 μm). Filters were washed twice with 2 ml of ice-cold PBS, and cell-bound radioactivity was quantitated by liquid scintillation counting. For [³H]riboflavin uptake assays, Bacillus subtilis cells were cultivated in mineral salts medium at 37°C with vigorous shaking. At an OD₆₀₀ of 0.5, [³H]riboflavin was added (16.6 nM final concentration; 588,000 dpm). Timed aliquots were mixed with 2 ml ice-cold 50 mM potassium phosphate buffer (pH 7.0), and cells were harvested immediately onto membrane filters (see above). The filters were washed twice with 2 ml of ice-cold buffer and dried. Radioactivity was determined by liquid scintillation counting.

Leuconostoc mesenteroides strain ATCC 8293 folT, panT, and ribU were amplified and inserted between the NcoI and BglII sites of pARCV (an expression vector containing an ampicillin resistance marker) (29). The resulting plasmids, pLmFolT, pLmPanT, and pLmRibU, encode the respective membrane proteins with a C-terminal FLAG tag. The ecfAA′T operon was likewise amplified and inserted into a variant of pARCV that harbored a streptomycin resistance gene and 10 histidine codons upstream of the insertion site. Plasmid pLmEcf encodes His₁₀-EcfA (with a deca-His tag at the N terminus), EcfA′, and EcfT-FLAG (with a C-terminal FLAG tag). An ampicillin resistance-conferring variant of pLmEcf (pLmEcf-Amp) was constructed by the insertion of the ecfAA′T fragment from pLmEcf into pARCV.

E. coli BL21 cells containing pLacI-RARE2 (encoding a Lac repressor and the tRNAs for rare codons) were used as the host for the heterologous production of L. mesenteroides proteins. Cells harboring pLmFolT, pLmPanT, pLmRibU, or pLmEcf-Amp individually or pLmEcf in combination with pLmFolT, pLmPanT, or pLmRibU were grown in 2 liters of Luria-Bertani broth supplemented with the appropriate antibiotics and 1 mM isopropyl-β,d-thiogalactopyranoside at 37°C with shaking to an OD₅₇₈ of ~2, harvested by centrifugation, washed in 35 mM sodium-potassium phosphate buffer (pH 7.0), resuspended in the same buffer containing a mixture of protease inhibitors (Roche), and disrupted by three passages through a French pressure cell. Membranes were pelleted by ultracentrifugation, resuspended and homogenized in 50 mM Tris-HCl (pH 8.0), and solubilized by agitation for 1 h in the presence of a solution containing 2% (wt/vol) dodecyl-β,d-maltoside, 5% (vol/vol) glycerol, 300 mM NaCl, and protease inhibitors at 4°C. Nonsolubilized material was pelleted by ultracentrifugation. Imidazole was added to the supernatant to a final concentration of 20 mM, and the solution (10 ml) was mixed with 0.5 ml Ni-nitrilotriacetic acid Superflow resin (Qiagen) and incubated for 30 min at 4°C with rotation. After transfer to an empty column, the resin was washed with 50 mM Tris-HCl (pH 7.5) containing 0.05% dodecyl-β,d-maltoside, 5% glycerol, 300 mM NaCl, and 100 mM imidazole. Bound protein was eluted with 4 ml of this buffer containing 500 mM imidazole. The protein solution was concentrated eightfold by Amicon concentrators (30-kDa cutoff), and samples (~10 μg protein) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were blotted onto nitrocellulose membranes and probed with anti-penta-His (Qiagen) or anti-FLAG (Sigma) antibodies by standard Western blotting protocols. For peptide mass fingerprint analysis, slices were excised from gels and incubated overnight in trypsin solution. Filtered samples were subjected to high-performance liquid chromatography-coupled electrospray ionization time-of-flight mass spectrometry using an Agilent 1100 system. Upon ionization, masses (m/z) were determined in the range of between 20 and 3,500, and peptides were matched using the BIOCONFIRM software package (Agilent).

The uptake of the transition metals cobalt and nickel, essential components of various enzymes, is mediated by transporters of several different types (6). Previous comparative genomic analyses identified the homologous CbiMNQO and NikMNQO transport systems as being the most widespread transporters for cobalt and nickel ions, respectively (29). The metal specificities were predicted using a combination of the identification of specific regulatory elements, namely, B₁₂ riboswitches (B₁₂ elements) and binding sites for the nickel-dependent repressor (NikR), and the analysis of colocalization with genes encoding nickel-dependent enzymes or enzymes involved in B₁₂ biosynthesis. The predicted specificities were experimentally confirmed by metal uptake assays upon the recombinant expression of the cbiMNQO and nikMNQO operons (29). In these transport systems, the transmembrane “Q” proteins (T component) and the “O” proteins (A components) energize their cognate metal-specific “MN” modules (S components). Thus, these systems belong to group I of the ECF transporter class. The “M” components constitute a unique family of integral membrane proteins with two separate subfamilies correlating with specificity for cobalt and nickel, respectively.

A biotin uptake function for the BioY protein family was previously predicted in the comparative genomic reconstruction of the biotin regulon governed by BirA (31). The widespread biotin repressor BirA and two specialized transcriptional factors from different families, BioR in alphaproteobacteria (28) and BioQ in the Actinobacteria (27), control the biotin synthesis genes and/or the biotin transporter gene bioY. In addition to the evidence from multiple regulons, bioY clusters with biotin synthesis genes in some genomes. On the other hand, BirA-regulated bioY genes occur in some organisms without the biotin synthesis pathway. The collective evidence from coregulation, colocalization, and cooccurrence experiments strongly implied that BioY proteins are transporters involved in biotin uptake. Members of the BioY family belong to either the group I transporters that are accompanied by the dedicated AT module BioMN (44 cases, belonging mostly to the Proteobacteria and Actinobacteria) or the group II transporters that depend on the shared EcfAA′T module (81 cases, belonging mostly to the Firmicutes). Some bioY genes (10 cases, mostly in the Archaea but also in Desulfitobacterium sp.) are located adjacent to ecfAA′T gene cassettes. A detailed phylogenetic analysis of the BioY family was performed recently. In a series of biochemical experiments, BioY from Rhodobacter capsulatus was identified as being a biotin capture and transport protein, which is converted into a high-affinity biotin transporter in the presence of its cognate BioMN module (14).

The candidate riboflavin transporter gene ypaA (ribU) was first identified by comparative genomic analysis of the riboflavin biosynthesis regulons governed by RFN (for riboflavin) regulatory elements (10, 21, 42). The RFN-regulated ribU genes were identified both in genomes with the de novo riboflavin synthesis genes and in those of riboflavin auxotrophs that lack the de novo synthesis pathway (e.g., some Streptococcus species). The RFN element serves as the receptor to form a metabolite-dependent riboswitch that directly binds flavin mononucleotide in the absence of proteins (44). The participation of RibU from Bacillus subtilis and Lactococcus lactis in riboflavin uptake was demonstrated by vitamin uptake assays (2, 43). The very high affinity of the RibU protein for riboflavin (K_d [dissociation constant] of 0.6 nM) (5) likely allows the cells to scavenge the vitamin from environments with low concentrations of riboflavin. The group II RibU transporters are widely distributed among the Firmicutes and the Thermotogales (63 cases). Additional RibU transporters attributed to group I were identified in some Archaea and Actinobacteria species, providing genomic evidence for their dependence on an energy-coupling module (Fig. ​(Fig.2).2). For instance, ribU in Bifidobacterium species forms an operon with a gene encoding a pair of ABC ATPase domains and a transmembrane domain that is homologous to T components of AT modules.

The candidate tryptophan transporter YhaG (TrpP) was previously identified as being a member of the tryptophan regulon governed by the TRAP attenuation protein in B. subtilis (37, 45). On the other hand, trpP orthologs in the Clostridia are regulated by tryptophan-specific T-box RNA structures (41). The involvement of trpP in tryptophan transport was confirmed by the sensitivity of the trpP mutant to growth-inhibiting levels of tryptophan analogs (37). TrpP transporters belong to either the group I transporters that are accompanied by the dedicated energy-coupling module (in methanogenic Archaea and in some of the Firmicutes) (Fig. ​(Fig.2)2) or the group II transporters that depend on the shared EcfAA′T module (14 cases, in the Firmicutes).

Previous comparative genomic analyses of the thiamine regulons governed by THI elements (thiamine-pyrophosphate riboswitches) resulted in the identification of several candidate thiamine-related transporters, including YuaJ/ThiT, YkoEDC, and ThiW (34). Analysis of the distribution of genes involved in thiamine metabolism in the Firmicutes has revealed that thiT is the only thiamine-regulated gene in several pathogens that have no thiamine biosynthesis pathway, suggesting that it is involved in thiamine uptake (34). The YkoEDC system was predicted to be involved in salvaging the thiamine precursor hydroxymethylpyrimidine, based on the positional clustering of the ykoEDC cassette with the thiaminase II gene tenA, and some other thiamine salvage genes (34). Genetic and microarray studies of B. subtilis mutants in the thiT and ykoD genes demonstrated that both mutations result in a derepression of thiamine-regulated genes, confirming the involvement of thiT and ykoEDC in the uptake of thiamine and its precursors (38). The thiamine-related transporter ThiW was predicted to be involved in the uptake of the thiazole precursor of thiamine based on the colocalization of the thiW with thiM genes, with the latter encoding the thiazole salvage enzyme hydroxyethylthiazole kinase (34). In this work, we used the occurrence profile of ThiT transporters, which are present only in the Firmicutes (34 cases), to attribute them to group II ECF transporters. Thiamine binding activity was verified experimentally for the L. casei ThiT protein (7). ThiW transporters belong to either the group I transporters that are accompanied by the dedicated AT module (in some members of the Archaea and Chloroflexi) (Fig. ​(Fig.2)2) or the group II transporters that depend on the shared EcfAA′T module (21 cases, mostly in members of the Firmicutes but also in Rubrobacter xylanophilus and Korarchaeum cryptofilum). YkoEDC transporters (group I; 44 cases) always have an integral membrane substrate capture component (YkoE), a dedicated AT module composed of duplicated ABC ATPase domains fused in a single protein (YkoD), and a transmembrane T component (YkoC).

The candidate folate transporter FolT was identified using comparative genomic analyses of the folate biosynthesis subsystem and the previously published amino acid composition and molecular mass data (16) for the L. casei folate binding protein (7). The folT genes in several Mycoplasma genomes and in Streptococcus suis cluster on the chromosome with the folC gene, encoding the bifunctional enzyme dihydrofolate synthase/folylpolyglutamate synthase, which can add a polyglutamyl tail to folate molecules. The folT gene is widely distributed in the Firmicutes (55 cases) and is also present in a single Thermotogales species; thus, it occurs only in genomes encoding the shared energy-coupling module. Therefore, FolT was predicted to function as a group II ECF transporter. Folate binding activity was verified experimentally for the L. casei FolT protein (7).

The candidate pantothenate transporter PanT was identified in this study by genome context analysis of the coenzyme A biosynthesis subsystem in the SEED database. Many free-living bacteria are capable of de novo coenzyme A biosynthesis via the precursor pantothenate, whereas numerous pathogens are dependent on exogenous pantothenate. In many cases, the panT gene is colocalized on the chromosome with various pantothenate salvage genes including the phosphopantothenoylcysteine decarboxylase gene coaB, the phosphopantothenoylcysteine synthetase gene coaC, and the pantothenate kinase gene coaX. PanT was found mostly in members of the Firmicutes that require pantothenate for growth (Lactobacillales, Streptococcus, pathogenic members of the Clostridia, and Mycoplasma); thus, it functions presumably as a group II transporter. In contrast, the panT genes in R. xylanophilus and Oenococcus oeni cluster with genes encoding A and T components of the dedicated energy-coupling module (Fig. ​(Fig.2),2), and consequently, the corresponding PanT transporters were attributed to group I.

The candidate queuosine precursor transporters QrtT and QueT were first predicted in this work using the observed coregulation of the qrtT and queT genes by PreQ₁ riboswitches that sense the modified nucleobase PreQ₁ (7-aminomethyl-7-deazaguanine) and control the expression of queuosine synthesis genes (22, 35). The QrtT transporters belong to either the group I transporters that are accompanied by the dedicated AT module QrtUVW (16 cases, in members of the Enterobacteria, Actinobacteria, and Thermotogales) or the group II transporters that depend on the shared EcfAA′T module (8 cases, in the Firmicutes). The QueT transporters were found to belong mostly to group II (41 cases, in the Firmicutes but also in R. xylanophilus, Petrotoga mobilis, and two Archaea species). However, in a single genome of Staphylothermus marinus, the group I QueT transporter is accompanied by the dedicated energy-coupling module (Fig. ​(Fig.22).

The candidate coenzyme B₁₂-regulated transporter CblT was identified in a previous comparative genomic analysis of the B₁₂ regulon in bacteria (33). Orthologs of the cblT gene in members of the Firmicutes are regulated by B₁₂ riboswitches and colocalized with the cobalamin biosynthesis genes (cbi). Analysis of distributions of genes from the B₁₂ synthesis pathway suggested a possible role of CblT in the uptake of dimethylbenzimidazole (33), a compound attached to the cobinamide precursor to form cobalamin. Here, we attributed most of the CblT transporters in the Firmicutes to group II (11 cases). In a single species of the Firmicutes that lacks the shared energy-coupling cassette (Desulfitobacterium sp.), CblT is accompanied by a dedicated energy-coupling module (Fig. ​(Fig.2),2), thus constituting a group I transporter.

The CbrT transporters belong to either the group I transporters that are accompanied by the dedicated AT module CbrUV (18 cases, in members of the Actinobacteria and some members of the Firmicutes) or the group II transporters that depend on the shared EcfAA′T module (11 cases, in the Firmicutes). The genomic colocalization of cbrT with the cobalamin adenosyltransferase (pduO) and adenosylcobalamin-dependent ribonucleotide reductase (nrdJ) genes in four Lactobacillus species suggests the involvement of CbrT in cobalamin uptake. In addition, the cbrT gene of L. reuteri belongs to a large gene cluster encoding the B₁₂ biosynthesis pathway and the B₁₂-dependent pathway of propanediol utilization. With the exception of L. reuteri, other analyzed Lactobacillus genomes lack all known genes involved in adenosylcobalamin biosynthesis and salvage. Genomic analysis of four Lactobacillus species that possess the nrdJ-cbrT-pduO gene cluster found NrdJ as a single known B₁₂-dependent enzyme, providing an explanation for the observed colocalization of the genes involved in the salvage and utilization of cobalamin.

Methylthioadenosine is a degradation product of S-adenosylmethionine and recycled to form methionine. A group I transporter encoded by the mtaTUV gene cassette was identified in only three bacterial genomes and a single archaeal genome. The colocalization of mtaTUV with the mtnPNKA genes involved in methylthioadenosine salvage implicates MtaTUV in the uptake of external methylthioadenosine.

A group I transporter encoded by the mtsTUV gene cluster was identified in members of the Proteobacteria and the Firmicutes and in some members of the Archaea (33 cases) as an optional member of various methionine regulons, suggesting its involvement in the uptake of a methionine precursor. For example, the mtsTUV operon is regulated by the S-adenosylmethionine riboswitch (S box) in Bacillus cereus, by the methionine-specific T-box attenuator in Leuconostoc mesenteroides, and by the methionine regulator MtaR in Streptococcus species (32). In proteobacteria, the mtsTUV cassette was found only in Vibrio species, where it is predicted to be regulated by the methionine repressor MetJ, as it is preceded by candidate MetJ binding sites.

The group II transporters of the PdxT family were found only in the Firmicutes (11 cases) and are weakly similar (16 to 18% identity) to BioY, CblT, CbrT, and PanT. The colocalization of pdxT with the pyridoxine kinase gene pdxK and the absence of de novo pyridoxine synthesis in Lactobacillus and Streptococcus suggest a role for PdxT in pyridoxine uptake.

Most of the HmpT transporters belong to group II, which depends on the shared energy-coupling module (38 cases, in members of the Firmicutes and the Thermotogales and some members of the Archaea). The group I HmpT transporters accompanied by a dedicated AT module (Fig. ​(Fig.2)2) are present in two members of the Archaea and a single species of the Actinobacteria. Positional analysis of the hmpT genes suggests their likely involvement in the salvage of thiamine or its precursor hydroxymethylpyrimidine. In Streptococcus and Enterococcus species, hmpT is located in a possible operon with a paralog of the phosphomethylpyrimidine kinase gene thiD, whereas in the Archaea, it is colocalized with the thiamine-monophosphate kinase gene thiL.

The candidate niacin transporter NiaX was previously identified in the comparative genomics study of the niacin regulon controlled by NiaR (30). The NiaR-regulated niaX genes are present in Streptococcus and Enterococcus species that lack genes for de novo NAD biosynthesis, suggesting the involvement of NiaX in niacin uptake. Here, we attributed the NiaX transporters to the group II ECF transporters (18 cases) based on their limited distribution only in the Firmicutes and weak similarity (16 to 19% identity) to QrtT and RibU.

The group II transporters of the LipT family were identified only in three Phytoplasma species that possess the shared EcfAA′T module. The LipT proteins are weakly similar (16 to 19% identity) to ThiT, FolT, PdxT, RibU, and HmpT. The colocalization of lipT with the lipoate-protein ligase A gene lplA and the absence of the lipoate synthesis pathway in Phytoplasma suggest a role for LipT in lipoate uptake.

To validate our bioinformatic analysis, we tested three of its predictions, namely, that (i) the previously characterized riboflavin transporter RibU depends on the AT module; (ii) the S components FolT and ThiT are specific for folate and thiamine, respectively, and are AT module dependent; and (iii) the multiple S components of group II transporters (Fig. ​(Fig.1)1) share a common energy-coupling module (EcfAA′T).

The dependence of riboflavin transport on the S component (RibU) and the energy-coupling module was analyzed in B. subtilis knockout strains. [³H]Riboflavin transport required functional ribU (ypaA) and ecfT (ybaF) genes; the disruption of either gene abolished the uptake of the vitamin (Fig. ​(Fig.3).3). This result was corroborated by inhibitor studies with the toxic riboflavin analog roseoflavin, which enters cells via the riboflavin transporter (43); disrupting either ribU or ecfT reduced sensitivity to this inhibitor (see Fig. S1 in the supplemental material).