Fructosyltransferase Genes Research Articles

Cariogenicity of Streptococcus mutans strains with defects in fructan metabolism assessed in a program-fed specific-pathogen-free rat model.

To define the role of dental plaque fructans and the enzymes involved in their metabolism in the initiation and progression of dental caries, we constructed otherwise-isogenic mutants of Streptococcus mutans defective in the ability to synthesize fructans, to degrade fructans, or to do both. The cariogenic potential of these organisms was evaluated in a specific-pathogen-free rat model in which the feeding patterns of the animals were controlled by means of a König-Hofer programed feeder. Specifically, rats were infected with wild-type S. mutans UA159 or derivatives of this strain which contained an insertionally-inactivated fructanase (fruA) gene, fructosyltransferase (ftf) gene, or which had both genes inactivated. The animals were fed 17 meals per day of Diet 2000 containing 56% sucrose at 70-minute intervals for five weeks, and caries experience was evaluated. Animals infected with S. mutans with a mutated fruA gene only had statistically significant decreases in sulcal caries severity. Such a decrease was not observed in previous studies with ad libitum-fed animals (Wexler et al., 1992). The manifestation of diminished virulence in the programmed feeding model, but not in ad libitum-fed animals, supports the concept that the primary contribution of FruA to virulence is through the utilization of fructans storage polysaccharides. Animals infected with strains carrying the ftf mutation or simultaneous mutations in ftf and fruA did not display decreased virulence, perhaps indicating that sucrose utilization pathways may compete for substrate in vivo, or that accumulation of fructans may affect the ecology or the physicochemical characteristics of dental plaque in such a way as to reduce its cariogenic potential. The results of this study also emphasize that the contribution of a particular virulence determinant to the caries process may be highly dependent on the experimental design, feeding regimen and diet, and the presence or absence of other enzymatic activities.

Identification of the Streptococcus mutans frp gene as a potential regulator of fructosyltransferase expression

Four putative open reading frames (ORFs) were previously identified in the regions flanking the Streptococcus mutans GS-5 fructosyltransferase (FTF) gene. One of these, ORF 3, appeared to code for a low-molecular-mass protein containing amino acid sequences sharing homology with several Gram-positive bacterial DNA-binding proteins and it was suggested that the ORF 3 gene product might be an FTF regulatory protein (FRP). In order to characterize this protein, we have purified the biotinylated tag-FRP fusion protein using the PinPoint protein purification system and this fusion protein was used in gel shift assays with DNA fragments containing the fif promoter region. FRP bound specifically to the upstream region of the ftf promoter containing the inverted repeat structure that is present upstream of the −35 sequence. In contrast, FRP did not bind to DNA fragments lacking the inverted repeat structure. The results of these experiments suggest that FRP interacts with the inverted repeat region upstream of the ftf promoter and such interactions may regulate FTF expression.

Identification of the Streptococcus mutans frp gene as a potential regulator of fructosyltransferase expression.

Four putative open reading frames (ORFs) were previously identified in the regions flanking the Streptococcus mutans GS-5 fructosyltransferase (FTF) gene. One of these, ORF 3, appeared to code for a low-molecular-mass protein containing amino acid sequences sharing homology with several Gram-positive bacterial DNA-binding proteins and it was suggested that the ORF 3 gene product might be an FTF regulatory protein (FRP). In order to characterize this protein, we have purified the biotinylated tag-FRP fusion protein using the PinPoint protein purification system and this fusion protein was used in gel shift assays with DNA fragments containing the ftf promoter region. FRP bound specifically to the upstream region of the ftf promoter containing the inverted repeat structure that is present upstream of the -35 sequence. In contrast, FRP did not bind to DNA fragments lacking the inverted repeat structure. The results of these experiments suggest that FRP interacts with the inverted repeat region upstream of the ftf promoter and such interactions may regulate FTF expression.

Microbial fructan production in transgenic potato plants and tubers

Fructans (fructose polymers) derived from plants usually have a very low degree of polymerisation (DP) and this limits the technical application of this versatile carbohydrate polymer. Previously we showed that the expression of bacterial fructosyltransferase genes in transgenic plants results in the accumulation of high molecular weight fructans with a DP of over 25,000. Here we report on our progress in accumulating such high DP fructans in potato plants and tubers. In these plants growth, tuber formation and carbohydrate partitioning were analyzed. Young plants showed no difference in growth between wildtype and fructan-producing (KP) plants. However, at a certain timepoint growth in KP plants is reduced and photosynthate starts to accumulate in source leaves. In these plants no diurnal turnover of starch and sucrose is observed. At harvest, the sink organs (roots, tubers) are reduced in weight. Depending on fructan content the KP tubers display a brown phenotype and yield was reduced. The starch content in the KP tubers is inversely correlated with the fructan level, whereas the sucrose, glucose, fructose and protein levels increase substantially, concomitant with fructan concentration. The cellular location of the fructans was investigated via immunofluorescence using monoclonal antibodies against levan and this showed the presence of fructan located along the cell rim instead of in the expected vacuolar location. It is concluded that fructan accumulation in these plants dramatically affects growth and carbohydrate partitioning and possible solutions for these problems are discussed.

Sucrose-derived exopolymers have site-dependent roles in Streptococcus mutans-promoted dental decay

We have constructed a panel of mutants of S. mutans V403 which are defective in one or more of the glucosyltransferase genes ( gtfB,C or D) or the fructosyltransferase gene ( ftf). These strains have been tested for virulence in a gnotobiotic rat caries model with reference to both buccal (smooth surface) and sulcal (pit and fissure) carious lesions. Our data suggest differing roles for degradable and non-degradable polymers at buccal and sulcal sites. Non-degradable polymers (made by products of the gtfB and C genes) contributed significantly to the severity of smooth surface lesions. However, our studies suggested their role in pit and fissure lesions was not as important as the role of degradable polymers. Specifically, a mutant deficient in the major insoluble glucan synthesizing activity (product of the gtfB gene) was 25% more cariogenic on sulcal surfaces than was the wild-type V403 strain. We propose that extracellular glucosyltransferases and fructosyltransferase compete for sucrose and that this competition influences pathogenicity at differing tooth sites.

Fructan as a New Carbohydrate Sink in Transgenic Potato Plants.

Fructans are polyfructose molecules that function as nonstructural storage carbohydrates in several plant species that are important crops. We have been studying plants for their ability to synthesize and degrade fructans to determine if this ability is advantageous. We have also been analyzing the ability to synthesize fructan in relation to other nonstructural carbohydrate storage forms like starch. To study this, we induced fructan accumulation in normally non-fructan-storing plants and analyzed the metabolic and physiological properties of such plants. The normally non-fructan-storing potato plant was modified by introducing the microbial fructosyltransferase genes so that it could accumulate fructans. Constructs were created so that the fructosyltransferase genes of either Bacillus subtilis (sacB) or Streptococcus mutans (ftf) were fused to the vacuolar targeting sequence of the yeast carboxypeptidase Y (cpy) gene. These constructs were placed under the control of the constitutive cauliflower mosaic virus 35S promoter and introduced into potato tissue. The regenerated potato plants accumulated high molecular mass (>5 [times] 106 D) fructan molecules in which the degree of polymerization of fructose units exceeded 25,000. Fructan accumulation was detected in every plant tissue tested. The fructan content in the transgenic potato plants tested varied between 1 and 30% of dry weight in leaves and 1 and 7% of dry weight in microtubers. Total nonstructural neutral carbohydrate content in leaves of soil-grown plants increased dramatically from 7% in the wild type to 35% in transgenic plants. Our results demonstrated that potato plants can be manipulated to store a foreign carbohydrate by introducing bacterial fructosyltransferase genes. This modification affected photosynthate partitioning in microtubers and leaves and increased nonstructural carbohydrate content in leaves.

Cariogenicity of Streptococcus mutans V403 glucosyltransferase and fructosyltransferase mutants constructed by allelic exchange

Streptococcus mutans produces several enzymes which metabolize sucrose. Three glucosyltransferase genes (gtfB, gtfC, and gtfD) and a single fructosyltransferase gene (ftf) encode enzymes which are important in formation of exopolysaccharides. Mutants of S. mutans V403 carrying single and multiple mutations of the gtfB, gtfC, gtfD, and ftf genes recently have been constructed by allelic exchange in our laboratory. Using selected strains from this panel of mutants, we examined the importance of water-insoluble glucan, water-soluble glucan, and fructan production in cariogenicity while controlling for the effects of strain and species variability. Genetic and biochemical characterization of mutants and assays of glucosyltransferase and fructosyltransferase activities were performed to ensure that the phenotypes of strains coincided with deficiencies predicted by genotype. The young gnotobiotic rat model of cariogenicity was used to assess virulence of the wild-type strain and isogenic mutants. Mutant strains were less virulent than the wild type in almost every location examined for caries on tooth surfaces and level of involvement of lesions (depth and severity). Inactivation of either gtfB and gtfC or ftf dramatically reduced virulence; the subsequent inactivation of gtfD did not enhance the effect of reduced virulence.

Isolation of DNA encoding sucrase genes from Streptococcus salivarius and partial characterization of the enzymes expressed in Escherichia coli.

Restriction enzyme fragments containing two sucrase genes have been isolated from a cosmid library of Streptococcus salivarius DNA. The genes were expressed in Escherichia coli cells, and the properties of both enzymes were studied in partially purified protein extracts from E. coli. One gene encoding an invertase-type sucrase was subcloned on a 2.4-kilobase-pair fragment. The sucrase enzyme had a Km for sucrose of 48 mM and a pH optimum of 6.5. The S. salivarius sucrase clone showed no detectable hybridization to a yeast invertase clone. Two overlapping subclones which had 1 kilobase pair of DNA in common were used to localize a fructosyltransferase gene. The fructosyltransferase had a Km of 93 mM and a pH optimum of 7.0. The product of the fructosyltransferase was a levan. A fructosyltransferase clone from Bacillus subtilis did not hybridize to S. salivarius DNA. The properties of the enzymes were compared with those of previously characterized sucrases.

Isolation and characterization of a fructosyltransferase gene from Streptococcus mutans GS-5.

A fructosyltransferase (FTF) gene from Streptococcus mutans GS-5 has been isolated from a lambda L47.1 clone bank. The gene was contained on an 11.7-kilobase GS-5 DNA fragment and was initially subcloned into plasmid pACYC184 as a 5.4-kilobase HindIII fragment. However, further analysis revealed that transcription of the FTF gene was initiated at the P1 promoter contained on the vector. It was possible to subclone the FTF gene with its presumed promoter as a 3.4-kilobase EcoRI fragment to produce the chimeric plasmid pSS22 expressing FTF activity. The cloned enzyme was purified to apparent homogeneity after ammonium sulfate precipitation, gel filtration, and DEAE-Bio-Gel-A chromatography followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme displayed a lower molecular weight (63,000) compared with the multiple activities detected in the culture fluids of strain GS-5. In addition, storage of the purified enzyme resulted in the formation of even lower-molecular-weight enzymatically active species. These results suggested that proteolytic degradation of the FTF occurs both in S. mutans and in Escherichia coli. In addition, a comparison of the properties of the cloned enzyme with those previously characterized from another serotype c S. mutans strain suggests that multiple FTF genes may be present in these organisms.