Global differential transcription profiles were obtained for growth of S. mutans in 13 different sugars using microarrays. The results achieved for all samples grown in the same sugar showed excellent reproducibility, as presented in Fig. for glucose- and fructose-grown samples. Nevertheless, selected microarray results were verified using RT-PCR, and these two data sets showed an excellent correlation (Fig. ).
Microarray results indicated that fructose was transported by the PTS in S. mutans
. In fact, previous studies indicated that fructose was transported into the cell via an inducible PTS and a constitutive PTS (11
). Wen and colleagues (40
) have characterized transcription of both transporters. Comparison of both published gene clusters and their flanking regions to the genome database (4
) revealed that the EIIs for these PTSs were designated as SMU.872 (EIIFru
, constitutive) and SMU.113 to -116 (inducible). The microarray results confirmed that SMU.872 was constitutively expressed (Fig. and ). However, our previous findings (V. Pham, G. Savic, N. McElwee, and D. Ajdic, presented at the Streptococcal Genetics meeting, Saint-Malo, France, 18 to 21 June, 2006) and current transcriptional analysis revealed a different fructose-induced operon (SMU.1956 to -1961) (Fig. and ). Recently published data (42
) concerning fructose metabolism confirmed our results. The previously reported fructose inducible operon (SMU.113 to -116) (40
) showed very low transcription in fructose-grown cells. Furthermore, this operon exhibited low expression in every condition tested in this study. It is not completely clear why this operon was not transcribed in the presence of fructose. One explanation could be that there is a mutation in the promoter region that hinders operon transcription in the tested strain. Alternatively, this may be not a fructose EII but an EII for some unknown sugar.
Growth in sucrose is inherently difficult due to dextran (glucan) production. Strain UA159 formed visible aggregates when grown in the presence of sucrose, and consequently it was difficult to obtain an accurate optical density of the culture. Since the growth rate of the culture and the number of the cells in each sample compared were critical for microarrays, we could not perform microarray experiments following growth in sucrose. To address this question, we have analyzed sucrose transport in UA159 grown in a culture supplemented with extracellular endodextranase. This enzyme catalyzes the endohydrolysis at the α-1,6-glycosidic bond of dextran (glucan) and releases short isomaltosaccharides consisting of three to five glucose molecules. S. mutans
is also known to produce an extracellular endodextranase that cleaves dextran (12
). The activity of this enzyme is apparently well synchronized with the production of other enzymes involved in extracellular sugar metabolism, resulting in the accumulation of dextrans if sucrose is available. However, when the cells were grown in the presence of a high concentration of dextranase, dextran was cleaved and aggregation of S. mutans
Induction of EIIFru/Man
following growth in sucrose was expected because sucrose serves as a substrate for fructosyltransferase (13
) and fructanase (8
). These extracellular enzymes release the fructose moiety from sucrose, which is then taken up by its specific PTS. Although fructose is the most probable substrate for this PTS, we cannot rule out the possibility that this PTS also transports sucrose.
It is not completely clear why there was decreased transcription of the maltodextrin ABC transporter (SMU.1568 to -1571) in the presence of sucrose and dextranase, since one would expect that this condition would favor production of isomaltosaccharides. The high concentration of dextranase added to the cultures possibly lowered the concentration of longer isomaltosaccharides (maltodextrins). Therefore, longer isomaltosaccharides might be a substrate for this ABC transporter, and their lower concentration in the culture might result in its decreased transcription. Alternatively, since the control samples (glucose- and dextranase-grown cultures) did not contain products of hydrolyzed glucans, it is possible that this transporter exhibits some specificity for glucose.
It was surprising to find a decreased transcription of the raffinose ABC transporter (SMU.878 to -882) upon growth in sucrose given that it was previously reported that this transporter takes up multiple sugars, including sucrose, in strain LT11 (17
). Our microarray data demonstrated that the transcription of the raffinose transporter is very low in sucrose compared to any other sugar tested (Fig. ). This result suggested that neither the raffinose nor maltodextrin ABC transporter was involved in sucrose transport under the conditions tested. Thus, sucrose is not an inducer of the two ABC operons. However, it is possible that upon their induction by the specific substrates, these transporters may play a minor role in sucrose transport.
Several transporters in S. mutans
take up sucrose, and among them is the best-characterized, high-affinity sucrose PTS. The gene for EIISuc
is located in an operon that also includes the gene for sucrose-6-phosphate hydrolase (30
). Therefore, sucrose is converted to sucrose-6-phosphate during transport into the cell and is subsequently hydrolyzed to glucose-6-phosphate and fructose. A BLAST search of the nucleotide sequence for the EIISuc
) revealed that this gene was designated SMU.1841 in the genome database (4
). The scrA
gene was consistently highly transcribed under every condition tested in this study. However, the RT-PCR data revealed increased transcription of scrA
in the presence of sucrose (Fig. ), suggesting that even highly expressed EIIScr
could be further induced. RT-PCR data also revealed increased transcription of SMU.872 (encoding EIIFru
) and decreased transcription of SMU.2047 (encoding EIIMal/Glu
) in the presence of sucrose. EIIFru
is a part of the PTS that has been characterized as a constitutive fructose transporter (11
), so it was not surprising to find further increase of transcription of the EIIFru
gene in sucrose, since the fructose moiety of this disaccharide can be released extracellularly. Alternatively, the possibility exists that EIIFru
might transport sucrose. It was intriguing to find a slight transcriptional decrease of the EIIMal/Glu
gene. This PTS could also be involved in maltodextrin transport, and its lower transcription might be due to a decreased concentration of longer isomaltosaccharides in the culture grown with dextranase.
Several studies have suggested the existence of a second inducible sucrose PTS (25
). We did not detect the induction of any PTS, except for those for fructose uptake, following S. mutans
growth in sucrose. It has been suggested that the second PTS for sucrose was the trehalose PTS in S. mutans
). We did not detect induction of the trehalose PTS in the presence of sucrose. Furthermore, EIITre
exhibited minimal activity under any growth condition except when trehalose was present. Therefore, this PTS cannot be induced by sucrose (or any other sugar tested) in UA159. However, it is possible that this PTS exhibits some affinity for sucrose after it is induced by trehalose. Alternatively, the results presented here may be strain specific, since it has been known that there are some strain-specific variations in sugar transport (37
Similar to sucrose, maltose serves as a primer for the synthesis of water-soluble dextrans (10
). These molecules are cleaved by dextranase to isomaltosaccharides that can be taken up by S. mutans
. Therefore, the maltose/maltotriose ABC transporter (SMU.1568 to -1571) may also be responsible for transport of longer isomaltosaccharides. RT-PCR data revealed elevated transcription of one PTS (SMU.2047) in the presence of maltose and maltotriose (Fig. ). This transporter has been previously characterized as a glucose transporter (1
). Our transcriptional analysis also suggests its involvement in maltose and maltotriose uptake.
As mentioned earlier, in addition to EIILac
genes, all genes for the lactose-tagatose 6-phosphate pathway were also induced in the presence of lactose. This was an expected result because following lactose uptake, lactose-6-phosphate is cleaved by phospho-β-galactosidase (encoded by the last gene of the operon) to galactose-6-phosphate and glucose (15
). Galactose-6-phosphate is then metabolized by the tagatose-6-phosphate pathway. Interestingly, the galactose operon was also induced, suggesting that the galactose moiety of lactose is metabolized simultaneously through the Leloir and tagatose-6-phosphate pathways. The Leloir pathway seems to be the primary pathway for galactose catabolism when S. mutans
grows on galactose as a sole carbon source. The first enzyme of the Leloir pathway, galactokinase, catalyzes the phosphorylation of galactose to galactose-1-phosphate. Inactivation of the galactokinase gene completely abolished the growth of S. mutans
in galactose (3
). Therefore, the primary galactose uptake might not be mediated by a PTS. However, when galactokinase is intact, both the Leloir and the tagatose-6-phosphate pathways are active following growth in galactose (3
). Our microarray data confirmed this result (see Table S5 in the supplemental material). Therefore, S. mutans
might possess an enzyme that converts some intermediate of the Leloir pathway into one of the intermediates of the tagatose-6-phosphate pathway (and vice versa). Interestingly, transcription of the EIILac
genes was fully induced in galactose, suggesting that this PTS might also transport galactose. If this were true, galactose would be phosphorylated during PTS transport and utilized through the tagatose-6-phosphate pathway. However, strain UA159 cannot grow on galactose if galactokinase is inactivated. Therefore, the lactose PTS might not be a primary transporter for galactose. Although the microarray data (see Table S5 in the supplemental material) clearly showed induction of lactose, tagatose-6-phosphate, and galactose operons following growth in galactose, we could not with confidence detect a non-PTS transporter dedicated to galactose transport. This was most likely due to a different growth rate of UA159 in galactose compared to other sugars, in which a large number of genes exhibited differential transcription.
Russell and colleagues have shown that the MSM transporter was responsible for uptake and metabolism of melibiose, raffinose, and isomaltotriose and for the metabolism of sucrose (28
). Our results confirmed that the ABC transporter of the MSM operon is the major transporter for raffinose uptake. Induction of the fructose transporter (Fig. ) clearly shows the presence of a fructose moiety released from raffinose by extracellular enzymes, presumably fructan hydrolase (fructanase, exo-β-d
) and fructosyltransferase (13
). These enzymes are capable of cleaving the fructose moiety from raffinose, which is then efficiently taken up by the cell and utilized as an energy source. The lactose transporter might also be able to take up raffinose or the disaccharide known as melibiose that is left following the release of fructose. Melibiose is cleaved by intracellular alpha-galactosidase to galactose and glucose, and these monosaccharides are utilized as an energy source (28
). The microarray data clearly show that all three monosaccharides of raffinose and stachyose (galactose, glucose, and fructose) are utilized by S. mutans
during the mid-log phase of growth and that galactose is metabolized simultaneously through the Leloir and tagatose-6-phosphate pathways.
In the presence of trehalose, transcription of an operon encoding putative EIITre
(SMU.2038) and trehalose-6-phosphate hydrolase (SMU.2037) was increased. The microarray result confirmed that this operon indeed encodes a major trehalose-inducible EIITre
and accompanied hydrolase. Presumably, the PTS takes up and phosphorylates trehalose to trehalose-6-phosphate that is then hydrolyzed to glucose and glucose-6-phosphate. Although the presence of an inducible trehalose PTS was demonstrated previously (25
), this is the first study that characterizes the trehalose operon and its transcription.
is capable of utilizing β-glucosides such as cellobiose, esculin, arbutin, and salicin. Metabolic and transport genes for these sugars are organized in three loci. Cellobiose and salicin are utilized by proteins encoded in the cel
locus (SMU.1596 to -1601) (22
). The second locus, designated bgl
(SMU.977 to -985), consists of the genes for PTS transport (EII, SMU.980) and the metabolism of esculin (7
). Arbutin is hydrolyzed by its own phospho-β-glucosidase (encoded by the arb
gene, SMU.1102), but it is transported by the cel
). Although the microarray data showed that transcription of EIICel
(SMU.1596, SMU.1598, and SMU.1600) varied severalfold in different sugars, the signal was always very low, suggesting that this operon is substrate inducible and there was only a basal level of transcription under all previously tested conditions (Fig. ). To test this hypothesis, a microarray analysis was performed following growth in cellobiose. As expected, the genes for EIICel
, the accompanying phospho-β-glucosidase (SMU.1601), and the regulator (SMU.1599) were differentially transcribed, with an induction level much higher than the basal level of transcription (Fig. ; see Table S11 in the supplemental material). We conclude that cellobiose is transported by an inducible PTS and subsequently hydrolyzed by phospho-β-glucosidase, CelA.
Previous studies have demonstrated that S. mutans
possessed two independent inducible PTSs for transport of mannitol and sorbitol (20
). The accompanying catabolic enzymes, mannitol-1-phosphate and sorbitol-6-phosphate dehydrogenases that convert the respective phosphorylated sugars to fructose-6-phosphate, were also inducible (20
). However, our results showed that mannitol induced both mannitol and sorbitol operons, suggesting that both operons were utilized for transport and metabolism of mannitol in UA159. Consequently, sorbitol EII was renamed EIISorb/Mntl
Analysis of global transcription profiles did not detect significant induction of any sugar transporter in the presence of glucose compared to any other condition. However, genes for five EIIs were consistently highly transcribed, and two ABC transporters were moderately expressed, whereas the other transporters showed very low transcription in glucose (Fig. and ; see Tables S1 and S3 to S10 in the supplemental material). The presence of a glucose PTS in S. mutans
was reported decades ago (9
). In fact, S. mutans
harbors at least two glucose PTS transporters (21
). One of them has been characterized and demonstrated to take up mannose, glucose, and 2-deoxyglucose (2
). Another is responsible for glucose, 2-deoxyglucose, and α-methylglucoside transport (1
). Comparison of the two glucose PTSs to the genome database (4
) showed that they were designated SMU.1877 to -1879 (SMU.1877, mannose/glucose-specific EIIAB; SMU.1878, mannose/glucose-specific EIIC; SMU.1879, mannose/glucose-specific EIID) and SMU.2047 (glucose/maltose-specific EIIABC), respectively (Fig. ). RT-PCR data revealed that the second glucose PTS was also a maltose transporter. Both glucose transporters were consistently highly transcribed in UA159. Additionally, both ABC sugar transporters ABCRaf
showed differential transcription in glucose, suggesting that they might have some affinity for this carbohydrate.
The five PTS transporters that were highly transcribed in glucose showed a similar level of transcription in all other sugars used in this study (Fig. and ). It is not completely clear which carbohydrates are substrates for these transporters. As mentioned earlier, EIIMal/Glu
is specific for maltose and glucose, and EIIMan/Glu
is specific for mannose and glucose. Sato and colleagues have cloned and sequenced a sucrose-specific transporter of S. mutans
). Comparison of this sequence to the genome database showed that one of the five highly expressed EIIs is EIISuc
. In addition to the inducible fructose PTS, S. mutans
also possesses a constitutive fructose PTS (11
). Comparison of its sequence to the genome database revealed that EIIFru
was also one of the five highly transcribed transporters. The specificity of the fifth highly transcribed EII (SMU.270 to -272) remains unknown.
Although the five PTSs were highly transcribed under every condition tested in this study, some showed transcription that was further elevated if the particular sugar was present. The gene for high-affinity EIISuc, previously characterized as being constitutive, showed a further increase in transcription in sucrose compared to glucose (Fig. ). Similarly, RT-PCR results revealed that the transcription of the genes for EIIMal/Glu and EIIFru was elevated severalfold in the presence of maltose and sucrose, respectively (Fig. ). These data confirm that the transcription of the “constitutive” EII genes can be further increased under the appropriate environmental conditions.
Evidence that S. mutans
constantly expresses five PTSs suggests that their substrates, glucose, fructose, maltose, and sucrose, might be preferable sugars for this organism. These are also the main dietary sugars, and therefore this bacterium is capable of their immediate utilization as they become available. It is obvious that these transporters are important for S. mutans
, since the cell expresses them continuously. In addition to their role in instant sugar uptake, it is also possible that these PTSs are involved in the regulation of sugar transport and in catabolite repression. Experiments conducted with EIIMan/Glu
mutants indicated that preferential utilization of glucose over lactose depended on the presence of this EII (36
Transcription of the genes for PTS EI and Hpr was consistently high following growth in different carbohydrates, suggesting that these genes were not regulated by a particular sugar used in this study. EIIβ-Glu
(SMU.980) was not induced by any sugar used in this study, but it has been previously suggested that it transported a β-glucoside (7
). Two EIIs (SMU.114 and -115 and SMU.100 to -103) were repressed under every condition tested in this study, and therefore their sugar specificity remains unknown.
Analysis of the global transcription patterns following growth on 13 different carbohydrates revealed two types of sugar transporters in S. mutans. Members of the PTS family, the primary carbohydrate transporters of gram-positive bacteria, transported most of the sugars. In fact, monosaccharides, disaccharides, β-glucosides, and sugar alcohols were all transported by PTSs. In contrast, the ABC transporters appear to be specialized for transport of oligosaccharides in S. mutans.
Differential expression profiles not only identified the sugar transporters but also confirmed the presence of the previously studied genes involved in the catabolism of sugars and allowed identification of new genes and operons. The microarray results, in conjunction with genome sequencing and annotation, provided information about the exact locations and compositions of the operons involved in sugar transport and metabolism. In nine operons, transporter genes for EII are an integral part of operons consisting of genes encoding enzymes necessary for the hydrolysis and catabolism of the transported sugar. Colocalization of the genes for sugar transport and catabolism indicates their coregulation. Transcription of the majority of the sugar transporters and accompanied catabolic genes was induced by their specific substrate. The common feature of all of these operons was the presence of a regulatory gene that was presumably responsible for their transcriptional regulation.
Although most of the sugars tested in this study specifically induced genes for their own transport and catabolism, five of the PTSs were consistently highly expressed regardless of the sugar source. These transcription profiles were consistent with the published functional studies for glucose/mannose and fructose PTSs. Microarray data provided evidence that the maltose/glucose PTS and the sucrose PTS were also consistently highly expressed, suggesting that glucose, fructose, maltose, and sucrose might be the preferred sugars for S. mutans
. Additionally, these transporters might be involved in the regulation of sugar transport and metabolism, as shown for EIIMan/Glu
All inducible PTSs showed very low transcription in the absence of the specific sugar substrate, suggesting their high specificity for particular sugars. The ABC transporters showed low to moderate transcription in different sugars, suggesting that they might transport multiple substrates. Interestingly, the transcription of these ABC transporters was very low in sucrose, suggesting their minor role in transport of this carbohydrate.
Our results demonstrate that S. mutans possesses inducible transporters for specific sugars and five PTS transporters that are consistently highly transcribed and presumably available for immediate uptake of the common dietary sugars. The capacity of S. mutans to rapidly transport and metabolize a wide range of sugars, whenever they become available, may be directly related to its survival in dental plaque and its cariogenic potential in humans.