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Journal of Bacteriology, December 2006, p. 8005-8012, Vol. 188, No. 23
0021-9193/06/$08.00+0     doi:10.1128/JB.01101-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

A Member of the Second Carbohydrate Uptake Subfamily of ATP-Binding Cassette Transporters Is Responsible for Ribonucleoside Uptake in Streptococcus mutans

Alexander J. Webb and Arthur H. F. Hosie^*

Microbiology, King's College London Dental Institute, London, United Kingdom

Received 25 July 2006/ Accepted 12 September 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
Streptococcus mutans has a significant number of transporters of the ATP-binding cassette (ABC) superfamily. Members of this superfamily are involved in the translocation of a diverse range of molecules across membranes. However, the functions of many of these members remain unknown. We have investigated the role of the single S. mutans representative of the second subfamily of carbohydrate uptake transporters (CUT2) of the ABC superfamily. The genetic context of genes encoding this transporter indicates that it may have a role in ribonucleoside scavenging. Inactivation of rnsA (ATPase) or rnsB (solute binding protein) resulted in strains resistant to 5-fluorocytidine and 5-fluorouridine (toxic ribonucleoside analogues). As other ribonucleosides including cytidine, uridine, adenosine, 2-deoxyuridine, and 2-deoxycytidine protected S. mutans from 5-fluorocytidine and 5-fluorouridine toxicity, it is likely that this transporter is involved in the uptake of these molecules. Indeed, the rnsA and rnsB mutants were unable to transport [2-¹⁴C]cytidine or [2-¹⁴C]uridine and had significantly reduced [8-¹⁴C]adenosine uptake rates. Characterization of this transporter in wild-type S. mutans indicates that it is a high-affinity (K_m = 1 to 2 µM) transporter of cytidine, uridine, and adenosine. The inhibition of [¹⁴C]cytidine uptake by a range of structurally related molecules indicates that the CUT2 transporter is involved in the uptake of most ribonucleosides, including 2-deoxyribonucleosides, but not ribose or nucleobases. The characterization of this permease has directly shown for the first time that an ABC transporter is involved in the uptake of ribonucleosides and extends the range of substrates known to be transported by members of the ABC transporter superfamily.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The ATP-binding cassette (ABC) transporter superfamily is a large and ubiquitous protein group, with numerous examples associated with genetic disorders in humans or involved in bacterial pathogenesis (7, 11). It has been suggested that ABC transporters are promising targets for antimicrobial strategies (7, 31). Furthermore, as the members of this transporter superfamily are involved in diverse functions, a comprehensive characterization of those transporters present in any given species can provide a valuable insight into the lifestyle of an organism. Consequently, members of this transporter superfamily have been the subject of significant research for over 20 years. Although a few ABC transporters have been very well characterized, there is no experimental data for the majority of them in terms of their function and biological significance. Indeed, many ABC transporters have been identified only by genome annotation.

The basic structure of ABC transporters consists of four domains: two integral membrane domains and two ATPase subunits. The ATPase subunits of ABC transporters are characterized by an ABC signature motif conserved in such proteins. In eukaryotes, the domains of ABC transporters are typically combined to form one or two protein subunits containing both integral membrane and ATPase domains. However, in prokaryotes, the domains are frequently separate proteins. This is especially true of bacterial ABC transporters involved in uptake, which also require an additional solute binding protein. These protein subunits must associate to form an active ABC transporter (11).

Streptococcus mutans is one of the dominant etiological agents of dental caries (8). Carbohydrate uptake and metabolism by S. mutans are central to its pathogenesis, as the lactic acid produced as the end product of fermentation contributes to dental caries (8). However, many aspects of S. mutans physiology and carbohydrate metabolism are inadequately understood. The genome of S. mutans UA159 has been sequenced (1), and annotation indicates that it has over 60 complete members of the ABC transporter superfamily. By exploiting bioinformatic analysis and the transport classification scheme (http://www.tcdb.org), we have determined that three of the ABC transporters in S. mutans may be involved in carbohydrate uptake. Although the transport classification scheme groups proteins into subfamilies of homologous transporters (25), which can inform predictions of the function of individual transporters, this can sometimes be misleading. One of the three putative carbohydrate ABC transporters in S. mutans is a member of the second carbohydrate uptake transporter (CUT2) subfamily. In other species of bacteria, members of this subfamily are involved in the uptake of monosaccharides such as ribose (3) and xylose (6). However, the genetic context of the genes encoding the CUT2 transporter in S. mutans (Fig. 1) casts doubt on such a role in this species. The genes are situated in a locus with putative ribonucleoside metabolism enzymes rather than those associated with monosaccharide metabolism. It is therefore probable that the CUT2 ABC transporter in S. mutans is involved in ribonucleoside scavenging. However, none of the previously characterized ribonucleoside transporters belong to the ABC superfamily.

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FIG. 1. Map of the locus containing rnsBACD and ribonucleoside-metabolizing enzymes. The locations of the binding sites of primers listed in Table 2 are indicated by the upper lines and labels in this diagram. Gene names are indicated below the arrows indicating the direction of transcription. The mRNA transcript, as determined by RT-PCR, is represented by the bold line.

Uptake or scavenging of ribonucleosides from the environment is a means of obtaining purine or pyrimidine compounds for nucleic acid synthesis and the sugar moieties as a source of carbon and energy. Each of the ribonucleoside transporters previously characterized in prokaryotes are secondary coupled systems of the concentrative nucleoside transporter family or the nucleoside:H⁺ symporter family (25). In Escherichia coli, NupC and NupG are involved in the uptake of deoxycytidine, cytidine, adenosine, thymidine, and uridine; NupG additionally transports guanosine (10, 19). Xanthosine is transported by a concentrative nucleoside transporter system in E. coli, XapB (28). In Bacillus subtilis, the gene nupC encodes the main uptake system for uridine (27). However, no orthologues of these transporters are apparent in S. mutans, and there have been no previous reports of ribonucleoside uptake in this species.

We have mutated the genes encoding components of the CUT2 ABC transporter in S. mutans. Phenotypic analysis indicates that this transporter is indeed involved in the uptake of purine ribonucleosides (adenosine, guanosine, inosine, 2-deoxyadenosine, and 2-deoxyguanosine) and pyrimidine ribonucleosides (cytidine, uridine, thymidine, deoxycytidine, and deoxyuridine). This is the first direct evidence of the uptake of such compounds by ABC transporters and therefore extends the range of substrates known to be transported by this important protein superfamily.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains, plasmids, and culture conditions. Bacterial strains and plasmids used in this study are detailed in Table 1. S. mutans strains were grown at 37°C anaerobically (80% N₂, 10% H₂, and 10% CO₂) on blood agar, on brain heart infusion (BHI) agar, or in BHI broth (Lab M, United Kingdom). Escherichia coli strains used for plasmid manipulation and maintenance were grown at 37°C aerobically on either Luria-Bertani (LB) agar or in LB broth. Oligonucleotides used in this study are detailed in Table 2. For S. mutans, cultures were supplemented with 800 µg/ml spectinomycin and 1 mg/ml kanamycin. For E. coli, cultures were supplemented with 50 µg/ml spectinomycin and 20 µg/ml kanamycin. Medium supplements, including 5-fluorocytidine and 5-fluorouridine, were purchased from Sigma Aldrich, United Kingdom.

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TABLE 1. Strains and plasmids used in this study

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TABLE 2. Oligonucleotide primers used in this study

Genetic modification of bacterial strains. Genetic manipulation of bacterial strains and general molecular techniques were carried out essentially as described previously (26). Transformation of S. mutans strains was carried out as previously described (20), occasionally with the addition of 1 µg/ml synthetic competence-stimulating peptide (SGSLSTFFRLFNRSFTQALGK; Sigma Aldrich, United Kingdom).

Insertional mutagenesis of genes of interest was performed essentially as described previously (15), with the specific cloning strategies outlined in Table 1. Briefly, strains containing rnsA::aphA3, rnsB::aphA3, and msmK::aphA3 were created by transforming S. mutans UA159 with the reamplified inserts from pKCL102, pKCL97, and pKCL98, respectively. Reverse transcriptase PCR (RT-PCR), using primers listed in Table 2 and represented in Fig. 1, confirmed that the insertion of the nonpolar aphA3 cassette did not impede the transcription of the downstream genes.

For complementation, rnsB was amplified by PCR using primers P205 and P209 (Table 2), and the 1.2-kb product cloned as described in Table 1 to generate pKCL146 with rnsB downstream of the constitutive S. mutans lactate dehydrogenase (ldh) promoter. The maintenance of this plasmid and the no-insert equivalent control (pKCL14) in S. mutans was confirmed by reisolating plasmid DNA from the transformed strains (KCL75 to KCL80).

Evaluation of operon structure by RT-PCR. To show that the CUT2 transporter is cotranscribed with the adjacent ribonucleoside-metabolizing enzymes, RT-PCR was carried out. The template RNA was obtained from strains UA159, KCL39, and KCL41 using the Ribo Pure Bacteria kit (Ambion, United Kingdom), and the RNA was transcribed into cDNA using the Retroscript kit (Ambion, United Kingdom) according to the instructions provided by the manufacturer. Subsequent PCRs were performed with the primers listed in Table 2. The locations of the primer binding sites are shown in Fig. 1.

Solute uptake by S. mutans strains. Uptake of [2-¹⁴C]cytidine, [2-¹⁴C]uridine, and [8-¹⁴C]adenosine (Sigma Aldrich, United Kingdom) was assayed by the rapid filtration method essentially as described previously for Rhizobium leguminosarum (12). S. mutans cells were grown in semidefined medium, which was adapted from a previously described defined medium (acid minimal salts medium, pH 7) (12, 22) excluding EDTA and with the following supplements added after autoclaving: 10 mM glucose, 0.025% cysteine, 33 mM sodium acetate, 20 mM sodium carbonate, 0.2% Casamino Acids, and 1x supplement solution. The 1,000x stock of supplement solution contained 0.04% riboflavin, 0.01% folic acid, 0.01% 4-aminobenzoic acid, 0.2% nicotinamide, and 0.08% pyridoxamine. Exponentially growing S. mutans cells were harvested by centrifugation when the optical density at 620 nm was approximately 0.5 and washed with an equal volume of 50 mM potassium phosphate buffer (pH 7.2) supplemented with 1 mM MgCl₂. The cells were resuspended in this buffer to an approximate optical density at 620 nm of 1. Prior to their use in transport assays, the cells were incubated for 1 h at 37°C to deplete any remaining extracellular ribonucleosides. The cells (approximately 0.8 ml per 1-ml assay mixture) were energized by the addition of 10 mM glucose 20 min prior to initiating uptake. Uptake assays were performed with a final concentration of 25 µM and 0.125 µCi [¹⁴C]solute. Samples (100 µl) were removed at 0, 1, 2, and 3 min, immediately filtered through glass fiber filter paper (Whatman GF/F), and rinsed with 10 ml phosphate buffer. Ready Safe liquid scintillation cocktail (Beckman Coulter) was added to the filters, and the amount of radioactivity retained on the filters was determined by using a scintillation counter. By using a bicinchoninic acid protein assay (Sigma Aldrich, United Kingdom), we previously determined that suspensions of S. mutans with an optical density of 1 are equivalent to 0.238 mg protein ml^–1. This figure was used to convert the radioactivity counts to nmol solute mg protein^–1. As the increase of incorporated [¹⁴C]ribonucleosides was linear over at least the first 3 min (see Fig. 4), this time period was used to calculate the presented uptake rates. For uptake competition assays, competing solutes were added to a final concentration of 1 mM. Only the initial rate of uptake (0 to 1 min) was used to calculate the K_m and V_max from rates obtained using a range of solute concentrations. All transport assays were performed on at least three independent cultures.

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FIG. 4. Uptake of ribonucleosides by S. mutans. The uptake of [2-14C]cytidine (A and black bars in D), [2-14C]uridine (B and light gray bars in D), and [8-14C]adenosine (C and dark gray bars in D) was determined with 25 µM 0.125 µCi 14C-solute as described in Materials and Methods. As the accumulation in wild-type S. mutans (solid lines in A, B, and C) increased over at least the first 3 min, this period was used to calculate the rates shown in D. KCL39 (dotted lines in A, B, and C) and KCL41 (dashed lines in A, B, and C) are mutated (aphA3 insertion) in rnsB and rnsA, respectively. The complementing plasmid, pKCL146, contained rnsB downstream from the ldh promoter, and pKCL14 was the same plasmid without this insert (Table 1).

Analysis of rnsA and rnsB conservation in clinical isolates of S. mutans. Fifty-six S. mutans clinical isolates from a range of different countries and continents were screened for rnsA and rnsB by PCR. The number of strains and geographical locations where the strains were isolated were as follows: 31 from United Kingdom, 3 from South Africa, 1 from Sweden, 4 from the United States, 11 from Turkey, and 6 from Hong Kong. Genomic DNA was isolated from two independent cultures of each isolate by using a Genelute bacterial genomic DNA kit (Sigma Aldrich, United Kingdom) for gram-positive bacteria according to instructions provided by the manufacturer and was used as a template for the PCR screen using primers P151 with P152 and P153 with P154 and an annealing temperature of 58°C.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Identification of a putative ribonucleoside transporter of the CUT2 ABC transporter subfamily in S. mutans. One of the ABC transporters identified in the genome sequence of S. mutans has the greatest amino acid sequence similarity to the CUT2 subfamily of ABC transporters (transport classification no. 3.A.1.2) (25). This CUT2 transporter comprises the genes SMU.1118, SMU.1119, SMU.1120, and SMU.1121, which, due to the findings of this study, we propose to be renamed rnsBACD (ribonucleoside uptake genes B, A, C, and D). We have assigned the letters to the different genes in accordance with the corresponding nomenclature of the well-characterized ribose transporter (3). Thus, SMU.1120, the ATPase or ABC domain, becomes RnsA; SMU.1121c, the solute binding protein, becomes RnsB; and the two integral membrane proteins SMU.1119c and SMU.1118c become RnsC and RnsD, respectively. Consistent with its classification as a member of the CUT2 subfamily, RnsA consists of a fused heterodimer of ATPase domains. It is most likely that RnsA interacts with a membrane complex consisting of a heterodimer of RnsC and RnsD. The lipoprotein, RnsB, completes the ABC transporter complex.

Although rnsA does have significant similarity to ABC domains of CUT2 transporters in general, it does not show particular similarity to any one of the previously characterized ATPases in the transport classification database, indicating that it may represent a unique subclass within this subfamily of ABC transporters. Analysis of the region surrounding rnsBACD indicated that adjacent genes were involved in the metabolism of ribonucleosides (Fig. 1). The genes present were cdd, encoding a putative cytidine deaminase; deoC, encoding a deoxyribose aldolase (9); and pdp, encoding a putative pyrimidine phosphorylase. We searched the available bacterial genome sequences for putative orthologues of rnsBACD and noticed that frequently, but not exclusively, orthologues were also adjacent to genes encoding putative enzymes involved in ribonucleoside metabolism. For example, in Sinorhizobium meliloti 1021, SMc04125 to SMc04127 are putative orthologues of rnsACD, and they are in a genetic locus with cdd, pdp, and deoA. Similarly, in Streptomyces coelicolor A3, the similar SC04883 to SC04888 genes are adjacent to cdd and pdp. It is unlikely that this synteny is coincidental, but rather, it suggests that the RnsBACD locus is involved in ribonucleoside metabolism.

RT-PCR of the rnsBACD locus. Although the genetic context indicated an association between the ribonucleoside metabolism genes and rnsBACD, it is not apparent whether they are part of the same operon. Therefore, using the primers listed in Table 2 and represented in Fig. 1, we performed RT-PCRs to determine which genes are cotranscribed. RT-PCR products were obtained with primers that amplify the region between SMU.1125c and pdp (P66/P112), the region between pdp and deoC (P115/P116), the region between deoC and cdd (P98/P117), the region between cdd and rnsB (P113/P114), the region between rnsB and rnsA (P118/P90), an internal fragment of rnsA (a positive control) (P89/P90), the region between rnsA and rnsC (P119/P88), and the region between rnsC and rnsD (P120/P124). The positive RT-PCR result obtained with these primer combinations indicates that the transporter and metabolic genes are indeed cotranscribed as one mRNA transcript, as indicated in Fig. 1. No RT-PCR products were observed with primers specific for the region between coaA and SMU.1125c (P51/P52) and rnsD and naoX (P120/P124). Therefore, the operon is flanked by coaA and naoX, and the promoter for this operon must lie between coaA and SMU.1125c. This cotranscription of rnsBACD with the ribonucleoside metabolic genes is further circumstantial evidence that they are involved in related processes, probably ribonucleoside scavenging.

A role for RnsBACD in the sensitivity of S. mutans strains to 5-fluorocytidine and 5-fluorouridine. 5-Fluoropyrimidine analogues are powerful reagents for analyzing pyrimidine metabolism (13, 17). To inhibit growth, 5-fluorocytidine and 5-fluorouridine, toxic ribonucleoside derivatives, must be taken into the bacterium and converted by salvage enzymes to the corresponding nucleotides (21). Therefore, we investigated the effect of 5-fluorocytidine and 5-fluorouridine on S. mutans strains. Wild-type S. mutans was sensitive to both 5-fluorocytidine and 5-fluorouridine (50 µg/ml), but strains lacking a functional RnsBACD transporter were significantly less susceptible (Fig. 2). This phenotype is specific to the insertional inactivation of the CUT2 ABC transporter, as S. mutans strains with an identical insertion in an unrelated ABC transport gene, msmK, did not have altered sensitivity to 5-fluorocytidine and 5-fluorouridine. Similarly, complementation of the rnsB mutant (KCL39), which restored sensitivity to 5-fluorocytidine and 5-fluorouridine to levels comparable to those observed in the wild-type strain (data not shown), confirmed that the increased resistance to 5-fluorocytidine and 5-fluorouridine was due specifically to the inactivation of rnsB and not any secondary mutation. Furthermore, this complementation confirms that the insertion of the nonpolar aphA3 cassette did not prevent the expression of downstream transporter genes. These data indicate that RnsBACD is involved in the uptake of 5-fluorocytidine and 5-fluorouridine into S. mutans.

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FIG. 2. Sensitivity of S. mutans strains to 5-fluorocytidine and 5-fluorouridine. Strains were grown in BHI broth (black bars), BHI broth plus 50 µg/ml 5-fluorocytidine (gray bars), or BHI broth plus 50 µg/ml 5-fluorouridine (clear bars). The optical density at 620 nm was determined after 18 h of growth.

Specificity of ribonucleoside uptake by RnsBACD. Although the data presented in Fig. 2 indicate that RnsBACD could transport 5-fluorocytidine and 5-fluorouridine, it is extremely unlikely that these are the natural substrates for this transporter. Therefore, to determine the specificity of this transporter, we investigated whether related molecules could block 5-fluorocytidine and 5-fluorouridine uptake and hence toxicity. A range of ribonucleosides protected S. mutans from the toxic effects. Cytidine and uridine, and, to a lesser extent, 2-deoxyadenosine, 2-deoxygaunosine, 2-deoxycytidine, 2-deoxyuridine, thymidine, guanosine, adenosine, and inosine, protected S. mutans from 5-fluorocytidine toxicity (Fig. 3A). Similarly, 500 µM cytidine and thymidine, and, to a lesser extent, uridine, inhibited 5-fluorouridine toxicity (Fig. 3B), and at higher concentrations (5 mM), cytidine, uridine, guanosine, inosine, adenosine, 2-deoxyguanosine, 2-deoxyuridine, 2-deoxycytidine, thymidine, and 2-deoxyadenosine each protected S. mutans from 5-fluorouridine (Fig. 3C). In contrast, glucose, ribose, and none of the nucleobases tested conferred protection from the toxicity of 5-fluorocytidine and 5-fluorouridine. The protective effect of the ribonucleoside derivatives is best explained as competitive inhibition of 5-fluorocytidine and 5-fluorouridine uptake. This indicates that these ribonucleoside derivatives interact with RnsBACD and may be transported by this permease.

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FIG. 3. Effect of competing solutes on 5-fluorocytidine and 5-fluorouridine toxicity. S. mutans cells were grown in BHI broth with 50 µM 5-fluorocytidine (FC) (A) or 50 µM 5-fluorouridine (FU) (B and C) with 500 µM competing substrates (A and B) or 5 mM competing substrates (C). The optical density at 620 nm was determined after 18 h of growth. An asterisk () indicates data that are significantly different (t test, P < 0.01) from the equivalent 5-fluorocytidine- or 5-fluorouridine-only control (with no competing solute).

Uptake of radiolabeled uridine, cytidine, and adenosine by RnsBACD. To confirm that RnsBACD is involved in the uptake of ribonucleosides, we performed uptake assays with [2-¹⁴C]cytidine, [2-¹⁴C]uridine, and [8-¹⁴C]adenosine. Wild-type S. mutans can accumulate each of these radioisotopes with V_max values of 8.5 ± 1.6, 6.7 ± 0.9, and 7.11 ± 0.52 nmol^–1 mg protein^–1 min^–1, respectively. The linear accumulation of the radiolabeled ribonucleosides over a significant period (Fig. 4) is typical of uptake rather than extracellular binding. Furthermore, when cells were not energized with glucose prior to starting the transport assay, no uptake was observed (data not shown), confirming that the observed increase in labeled ribonucleosides is active uptake. If either rnsB (KCL39) or rnsA (KCL41) was disrupted by mutation, the uptake of cytidine and uridine was negligible and the uptake of adenosine was considerably reduced (Fig. 4). Uptake rates for these solutes were restored to near-wild-type levels in the rnsB complemented strain (Fig. 4D). This confirms that at the concentrations tested, RnsBACD is the only transporter for cytidine and uridine and the dominant adenosine transporter in S. mutans. The affinity of RnsBACD for each of these ribonucleosides was high, with apparent K_m values of 1.1 ± 0.5 µM, 1.7 ± 0.5 µM, and 1.9 ± 0.3 µM for cytidine, uridine, and adenosine, respectively. The residual uptake of adenosine (Fig. 4C and D) indicates that there is at least one other transporter of this ribonucleoside in this species. However, the apparent affinity of this alternative adenosine transporter is substantially higher than that observed for RnsBACD, perhaps indicating that it is a secondary coupled transporter. Certainly, at the concentrations required to determine the affinity of RnsBACD for adenosine, no uptake was observed in the rnsA mutant (data not shown).

Inhibition of cytidine uptake by competing solutes. To obtain an indication of the range of solutes transported by RnsBACD, [2-¹⁴C]cytidine uptake was carried out in the presence of 1 mM (40-fold excess) competing solutes (Fig. 5). It is evident that most of the ribonucleosides and all of the 2-deoxyribonucleosides tested inhibit cytidine uptake at the concentrations tested. Thymidine was able to compete with cytidine but to a lesser degree than other ribonucleosides, perhaps indicating that RnsBACD has a lower affinity for thymidine. However, the ribonucleoside xanthosine was not able to inhibit [2-¹⁴C]cytidine uptake. Ribose and the tested nucleobases also had no effect on [2-¹⁴C]cytidine uptake. Therefore, RnsBACD has an apparent specificity for most ribonucleosides, including 2-deoxyribonucleosides. The data suggest that the preferred solute specificity of RnsBACD is as follows: cytidine, uridine, adenosine, guanosine, and inosine > 2-deoxycytidine, 2-deoxyuridine, 2-deoxyadenosine, and 2-deoxyguanosine > thymidine.

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FIG. 5. Inhibition of [2-14C]cytidine uptake by competing solutes. Uptake of 25 µM (0.03 µCi) [2-14C]cytidine was determined as described in Materials and Methods. Competing solutes were added to a final concentration of 1 mM.

Distribution of rnsBACD in a range of clinical S. mutans strains. Fifty-six clinical isolates of S. mutans from disparate geographical locations, including the United States, Sweden, South Africa, Hong Kong, Turkey, and the United Kingdom, were screened for rnsB and rnsA using PCR. Each of these genes was amplified from DNA purified from all the S. mutans isolates tested. Therefore, rnsBACD is apparently part of the core genome of S. mutans.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The data presented provide conclusive evidence that RnsBACD, a member of the CUT2 subfamily of ABC transporters, is required for the uptake of ribonucleosides in S. mutans. The genetic context of the genes encoding this transporter and cotranscription with ribonucleoside metabolism enzymes suggested that it was involved in ribonucleoside scavenging. Inactivation of the genes encoding the ATPase or the solute binding protein resulted in strains that were less susceptible to the toxic ribonucleosides 5-fluorocytidine and 5-fluorouridine. Furthermore, these strains had considerably reduced rates of [2-¹⁴C]cytidine, [2-¹⁴C]uridine, and [8-¹⁴C]adenosine uptake. The broad range of substrates that inhibit 5-flurocytidine and 5-flurouridine toxicity and [2-¹⁴C]cytidine uptake indicates that RnsBACD can transport most ribonucleosides, including 2-deoxyribonucleosides. In contrast, xanthosine, ribose, and nucleobases did not inhibit 5-flurocytidine and 5-flurouridine toxicity and [2-¹⁴C]cytidine uptake, so it is unlikely that these substrates are transported by this permease. These data are the first direct evidence of uptake of ribonucleosides by a member of the ABC transporter superfamily.

Others have previously commented on the genetic association of ABC transporters with putative ribonucleoside metabolism enzymes (4), and some genome annotators have designated related genes as putative ribonucleoside transporters. However, as far as we know, there are no experimental data to support this. Further indirect evidence that an RnsBACD orthologue was involved in the transport of ribonucleosides was published as this paper was being prepared. Deka et al. (5) reported that PnrA, a lipoprotein in Treponema pallidum, binds purine ribonucleosides (adenosine, guanosine, inosine, 2-deoxyadenosine, and 2-deoxyguanosine) and, to a lesser extent, pyrimidine ribonucleosides (cytidine and uridine). Although such binding of substrates by a solute binding protein is strongly indicative that they are transported by the associated ABC transporter, this is not necessarily so, as it is known that the membrane components of ABC transporters contribute to their specificity (18, 32). Unfortunately, the genetic intransigence of T. pallidum hinders further characterization of pnrA or the adjacent ABC transporter genes. Nevertheless, the similarity of PnrA to RnsB, which we show is necessary for ribonucleoside uptake, is further evidence that PnrA is involved in the transport of these solutes. It does appear that the solute specificity of RnsBACD and the T. pallidum homologue may differ. For example, PnrA could not bind thymidine, whereas thymidine does inhibit cytidine uptake by RnsBACD, and although xanthosine does not inhibit cytidine uptake, it can be bound by PnrA. Although we are cautious in interpreting data from disparate experiments, they do indicate that the related transporters may have slightly different roles in these bacteria.

As mutants could be made and as they had no impaired growth in BHI or minimal medium (data not shown), rnsA and rnsB are clearly not essential for growth of S. mutans in vitro. However, these genes do appear to be part of the core S. mutans genome. None of the clinical isolates that we screened lacked either of these genes. Furthermore, rnsA orthologues are apparent in a range of bacteria. These bacteria include all Streptococcus species that have been completely sequenced but not in all strains. Orthologues are also present in other gram-positive bacteria, including Bacillus subtilis subsp. subtilis strain 168; in gram-negative bacteria, for example, Sinorhizobium meliloti 1021; and in Archaea, such as Halobacterium sp. strain NRC-1. Unfortunately, in many of the genome annotations, rnsBACD orthologues are designated ribose or galactose transporters. As RnsBACD is now known to be involved in ribonucleoside uptake, it is more prudent to consider the closely related transporters to be putative ribonucleoside transporters.

The most likely role of RnsBACD in S. mutans is in scavenging ribonucleosides. This is consistent with the high affinity for uptake observed for this transporter. Once the ribonucleosides are taken up, they could either be used as a source of nucleotides for nucleic acid synthesis or be degraded, with the sugar moiety being used as a carbon and energy source. However, as S. mutans is unable to ferment ribose (data not shown), it is more likely that the ribonucleosides salvaged from the extracellular environment are used for nucleotide synthesis. This is comparable to the situation in Lactococcus lactis reported previously (14).

Other members of the CUT2 subfamily of ABC transporters are known to transport monosaccharides (such as ribose [3], galactose [24], xylose [6], and rhamnose [23]), the autoinducer AI2 (29, 30), and erythritol (33). As ribonucleoside uptake has not been previously demonstrated in this group, or by any other ABC permease, this report extends the range of substrates known to be transported by ABC transporters.

 
This work was funded by King's College London Dental Institute and the University of London Central Research Fund.

We thank Karen Homer for critical discussion of the data and for her critical reading of the manuscript.

 
* Corresponding author. Mailing address: King's College London Dental Institute, Microbiology, Floor 28 Guy's Tower, King's College London, Guy's Campus, London SE1 9RT, United Kingdom. Phone: 44 (0)20 7188 1825. Fax: 44 (0)20 7188 3871. E-mail: arthur.hosie{at}kcl.ac.uk.

Published ahead of print on 22 September 2006.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Bacteriology, December 2006, p. 8005-8012, Vol. 188, No. 23
0021-9193/06/$08.00+0     doi:10.1128/JB.01101-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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