INFECTION AND IMMUNITY, OCt. 1990, p. 3462-3464 0019-9567/90/103462-03$02.00/0 Copyright © 1990, American Society for Microbiology
Vol. 58, No. 10
Linkage of Sucrose-Metabolizing Genes in Streptococcus
mutans
DENNIS PERRY'* AND HOWARD K. KURAMITSU2 Department of Microbiology-Immunology, Northwestern University Medical and Dental Schools, Chicago, Illinois 60611,1 and Department of Pediatric Dentistry, University of Texas Health Science Center, San Antonio, Texas 78284-78882 Received 19 April 1990/Accepted 24 July 1990
Antibiotic resistance markers inserted adjacent to different cloned genes from Streptococcus mutans were used to determine the relative positions of these genes on the chromosome. The results showed that these genes, fru-i and gbp, are closely linked to the gifA-ftf-scrB cluster. However, gfD was linked neither to this cluster nor
to gtJB-gtfC.
The metabolism of dietary sucrose by Streptococcus mutans allows this organism to adhere tenaciously to tooth surfaces, and subsequent acid production results in caries (5). Apparently, the colonization of tooth surfaces by S. mutans is a highly complex process which is further compounded by the multitude of enzymes that appear to be involved in the metabolism of sucrose by these organisms. Although it has been conclusively shown that glucosyltransferase enzymes mediate the conversion of sucrose to insoluble glucans, the roles of other sucrose-metabolizing enzymes have not been clearly described. It seems likely, however, that a number of these enzymes act in concert to enhance the ability of the organisms to colonize tooth surfaces. In this regard, it has been generally demonstrated that genes with related functions are often clustered together on the chromosome in order to facilitate the coordinate regulation of their expression. Thus, it was of interest to examine the relative positions of sucrose-metabolizing genes on the S. mutans chromosome. Results have demonstrated tight linkage between the gtJB and gtfC genes (glucosyltransferases catalyzing the synthesis of primarily insoluble glucans [10]). More recently, we demonstrated that the S mutans genes coding for sucrose phosphorylase (gtfA [9]), fructosyltransferase (ftf), and sucrose-6-phosphate hydrolase (scrB) were clustered together on the S. mutans chromosome but unlinked to gtJR-gtfC (6). The relative positions of these genes were mapped by inserting different antibiotic resistance markers adjacent to or within the relevant genes on the chromosomes of these organisms. This approach has been continued in order to show that the genes fru-J (fructanase [1]) and gbp (glucanbinding protein [8]) are also linked to the gftA-ftf-scrB cluster. However, the recently isolated gtfD gene (glucosyltransferase catalyzing the synthesis of soluble glucan [3]) was neither linked to this cluster nor linked to gtfB-gtfC. The 4.5-kilobase (kb) EcoRI fragment of gbp derived from pMLG43 (8) and the 3.5-kb HindIll fragment offru-J derived from pFRU1 (1) were each inserted into the streptococcal mapping vector pVA891 containing a erythromycin resistance gene (erm; 7). Similarly, a chimeric plasmid containing the 1.7-kb EcoRI-HindIII fragment of gtJD derived from pNH4888 (3) also was constructed. These chimeric plasmids then were transformed into wild-type S. mutans GS-5 selecting for erythromycin resistance. Subsequently, a representative isolate of each of the Emr transformants (Gbp Emr, Frul Emr, and GtfD Em) was transformed with a chimeric *
plasmid containing a tetracycline resistance gene (tet) inserted within the ftf or scrB gene with a mini-Mu transposon (4, 6). DNA from the resulting Emr Tetr transformants was used to transform wild-type GS-5. Transformants were selected on Trypticase soy agar (BBL Microbiology Systems, Cockeysville, Md.) plus 0.2% yeast extract (TSAY) containing either erythromycin (10 ,ug/ml) or tetracycline (4 ,ug/ml) and replica plated onto media containing both antibiotics to determine the number of Emr Tetr transformants. The results showed that fru-J cotransferred with ftf and scrB at frequencies of 97.0 and 72.0% respectively, whereas gbp cotransferred with ftf and scrB at frequencies of 67.5 and 96.7%, respectively (Table 1). In contrast, the frequencies of cotransfer of gtJD withftf and scrB were both 0.5%, and thus gtJD was unlinked to these two genes. In addition, gtfD was not linked to the gtfB-gtfC cluster, since the frequency of cotransfer with gtJB was only 1.4%. An analysis of the cotransfer data to determine map distances would placefru-J close toftf and gbp close to scrB. Presumably, fru-l would also be tightly linked to gtfA, since previous results showed that the latter gene is linked to ftf (6). However, the map position offru-J relative to gtfA could be determined only indirectly, since both genes contained Emr markers. Therefore, a chimeric plasmid which contained a kanamycin resistance gene (kan) inserted adjacent tofru-J was constructed. Plasmid pFRU184 was constructed by inserting the 3.5-kb HindIII fru-l fragment into the HindIII site of pACYC184 (2). The 1.5-kb kan gene, which was isolated from p161 (D. LeBlanc, personal communication) after digestion with EcoRI, was inserted into the EcoRI site of pFRU184, resulting in pFRU184k. The kan gene in p161 is expressed in both Escherichia coli and S. mutans, TABLE 1. Cotransfer of GS-5 genes phenotypea
No. of Emr Tetr/ no. of Emrb
No. of Emr Tetr/ no. of Tetrb
Fru1 Ftf Frul ScrB Gbp Ftf Gbp ScrB GtfD Ftf GtfD ScrB GtfD GftB
74/74 52/100 54/100 103/103 0/100 1/100 4/209
57/61 93/100 81/100 99/106 1/100 0/100 1/142
DNA donor
Cotransferc
97.0 72.0 67.5 96.7
0.5 0.5 1.4
a The sucrose-metabolizing phenotypes of the DNA donors (e.g., Frul Ftf) were selected on the basis of Emr and Tetr, respectively. Strain GS-5 was transformed with 0.05 pg of each DNA per ml. b Transformants were selected on TSAY containing the individual antibiotics and then replica plated to TSAY containing both antibiotics. c Percent cotransfer = [no. of Emr Tetr/(no. of Emr + no of Tetr)] x 100.
Corresponding author. 3462
VOL. 58, 1990
NOTES
TABLE 2. Transformation of GS-5 with GtfA Frul Ftf DNA' No. of transformants/ 0.1 ml
Phenotype GtfA Em'. .................................. Frul KMr .............. ..................... Ftf Tetr ........... ........................ GtfA Em' Ftf Tetr ................................... GtfA Em' Frul Kmr ................................... Frul KMr Ftf Tetr ................................... GtfA Emr Frul KMr Ftf Tetr .........................
99 1,115 149
gtfA
frul ftf
2 1
a Cells were transformed with 0.05 p.g of DNA per ml, and different dilutions were spread onto TSAY containing the appropriate antibiotics.
although sensitive strains of the latter organism require 850 ,ug of kanamycin per ml to completely inhibit growth. pFRU184k was transformed into a GS-5 strain containing both gtfA (Emr) and ftf (Tetr), selecting for Emr Kmr Tetr transformants. DNA from one of these transformants was used to determine linkage among the three genes. When wild-type GS-5 cells were exposed to limiting concentrations of this DNA, transformation to resistance to all three individual antibiotics occurred, although the number of Kmr transformants was approximately 7- to 11-fold greater (Table 2). As expected, a significant number of Emr Tetr transformants also were observed, since it had been shown previously that gftA (Emr) andftf(Tet)r were linked (6). Virtually no Emr Kmr, KMr Tetr, or Em' KMr Tetr transformants were detected. However, when either the Emr or the Tetr transformants were replica plated to media containing either of the other two antibiotics, a significant number of double transformants, including KMr, were observed. In contrast, Kmr transformants, when replica plated, yielded less than 1% cotransfer with either Emr or Tetr. Thus the cotransfer of the Em', Tetr, and Kmir markers was asymmetrical in that the proportion of the Em' and Tetr transformants that also acquired Kmr was considerably higher than the proportion of the KMr transformants that also acquired Emr or Tetr. Similar observations were made by Perry et al. (7) during the transformation of Met' and gtfA (Emr), which are linked in S. mutans. These markers also transferred at unequal frequencies, with Met+ occurring some 50 times more frequently than Em'. Most likely, the transformation frequencies were influenced by the relative sizes of the homologous and inserted heterologous fragments of donor DNA (7). The results of these experiments indicated that gtfA,fru-1, and ftf are tightly linked (Table 3). An analysis of the cotransfer data strongly suggested thatfru-J (Kmr) is located closer toftf(Tetr) than to gtfA (Em') and most likely located between the last two genes (Fig. 1). This conclusion is based on the observation that approximately 98% of the Emr Tetr transformants were also KMr when tested by replica plating (data not shown), which would be expected if fru-l (Kmr) were located between the other two genes. In addition, the TABLE 3. Cotransfer of gtfA, fru-J and ft? Resistance phenotype
transformants/total
% Cotransfer
Emr Tetr/Emr + Tetr Emr Kmr/Em' KMr Tet'/Tetr
227/243 101/106 136/137
93.4 95.3 99.3
a GS-5 cells were transformed with 0.05 ,ug of GtfA Frul Ftf DNA per ml, and the GtfA, Frul, and Ftf phenotypes were selected on the basis of Emr, Kmr, and Tetr, respectively. The number of double transformants and the percent cotransfer were determined as described in the footnotes to Table 1.
gbp
i~~~~~~~~---u-
-1-1.1
.01
86
1
scrB
3463
* .05-* I(-.20)
@
.-
-
.33
-
FIG. 1. Linkage map of GS-5 sucrose-metabolizing genes. Map distances were calculated from the values in Tables 1 and 3 by the following formula: distance = 1 - (% cotransfer/100). Values in parentheses were determined in a previous study (6).
relative map distances between scrB and fru-J and between scrB andftf (Fig. 1) also suggest thatfru-J lies between gtfA and ftf. However, the extreme closeness of ftf and fru-J suggests that physical mapping may be necessary to conclusively determine the relative positions of these two genes. In this regard, the cotransfer values between fru-J and ftf yielded map distances of 0.03 and 0.01 when fru-J was selected on the basis of Emr (Table 1) and KMr (Table 3), respectively. The map distance for these two genes (Fig. 1) was calculated from the cotransfer value in Table 3. The observed map distances betweenfru-J and scrB and between ftf and gbp (0.28 and 0.33, respectively) were much greater than expected for these genes (Fig. 1). As previously shown (6), chromosomal breakage during DNA isolation could account for these observations. It is not surprising that the five genes within the cluster are linked, although their exact positions on the chromosome relative to their specific functions are not readily apparent. All are involved in the metabolism of sucrose or a metabolic product of this disaccharide. While it is not unusual for genes with related functions to be unlinked, it was still somewhat surprising that gtfD was not closely linked to gtfB-gtfC. Therefore, gtfD and the gtfB-gtfC gene cluster may be
independently regulated.
We thank D. LeBlanc, R. Burne, and R. R. B. Russell for providing the plasmids p161, pFRU1, and pMLG43, respectively. This study was supported by Public Health Service grant DE-
06082 from the National Institute of Dental Research. LITERATURE CITED 1. Burne, R. A., K. Schilling, W. H. Bowen, and R. E. Yasbin. 1987. Expression, purification, and characterization of an exoP-D-fructosidase of Streptococcus mutans. J. Bacteriol. 169: 4507-4517. 2. Chang, A. C. Y., and S. N. Cohen. 1978. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic mini-plasmid. J. Bacteriol. 134: 1141-1156. 3. Hanada, N., and H. K. Kuramitsu. 1989. Isolation and characterization of the Streptococcus mutans gtfD gene, coding for primer-dependent soluble glucan synthesis. Infect. Immun. 57: 2079-2085. 4. Kuramitsu, H. K. 1987. Utilization of a mini-mu transposon to construct defined mutants in Streptococcus mutans. Mol. Mi-
3464
NOTES
crobiol. 1:229-231. 5. Loesche, W. J. 1986. Role of Streptococcus mutans in human dental decay. Microbiol. Rev. 50:353-380. 6. Perry, D., and H. K. Kuramitsu. 1989. Genetic linkage among cloned genes of Streptococcus mutans. Infect. Immun. 57:805809. 7. Perry, D., L. N. Nilsen, and H. K. Kuramitsu. 1985. Mapping of a cloned glucosyltransferase gene in Streptococcus mutans. Infect. Immun. 50:130-135.
INFECT. IMMUN. 8. Russell, R. R. B., D. Coleman, and G. Dougan. 1985. Expression of a gene for glucan-binding protein from Streptococcus mutans in Escherichia coli. J. Gen. Microbiol. 131:295-299. 9. Russell, R. R. B., H. Mukasa, A. Shimamura, and J. J. Ferretti. 1988. Streptococcus mutans gtfA gene specifies sucrose phosphorylase. Infect. Immun. 56:2763-2765. 10. Ueda, S., T. Shiroza, and H. K. Kuramitsu. 1988. Sequence analysis of the gtfC gene from Streptococcus mutans GS-5. Gene 69:101-109.
Vol. 58, No. 10
Linkage of Sucrose-Metabolizing Genes in Streptococcus
mutans
DENNIS PERRY'* AND HOWARD K. KURAMITSU2 Department of Microbiology-Immunology, Northwestern University Medical and Dental Schools, Chicago, Illinois 60611,1 and Department of Pediatric Dentistry, University of Texas Health Science Center, San Antonio, Texas 78284-78882 Received 19 April 1990/Accepted 24 July 1990
Antibiotic resistance markers inserted adjacent to different cloned genes from Streptococcus mutans were used to determine the relative positions of these genes on the chromosome. The results showed that these genes, fru-i and gbp, are closely linked to the gifA-ftf-scrB cluster. However, gfD was linked neither to this cluster nor
to gtJB-gtfC.
The metabolism of dietary sucrose by Streptococcus mutans allows this organism to adhere tenaciously to tooth surfaces, and subsequent acid production results in caries (5). Apparently, the colonization of tooth surfaces by S. mutans is a highly complex process which is further compounded by the multitude of enzymes that appear to be involved in the metabolism of sucrose by these organisms. Although it has been conclusively shown that glucosyltransferase enzymes mediate the conversion of sucrose to insoluble glucans, the roles of other sucrose-metabolizing enzymes have not been clearly described. It seems likely, however, that a number of these enzymes act in concert to enhance the ability of the organisms to colonize tooth surfaces. In this regard, it has been generally demonstrated that genes with related functions are often clustered together on the chromosome in order to facilitate the coordinate regulation of their expression. Thus, it was of interest to examine the relative positions of sucrose-metabolizing genes on the S. mutans chromosome. Results have demonstrated tight linkage between the gtJB and gtfC genes (glucosyltransferases catalyzing the synthesis of primarily insoluble glucans [10]). More recently, we demonstrated that the S mutans genes coding for sucrose phosphorylase (gtfA [9]), fructosyltransferase (ftf), and sucrose-6-phosphate hydrolase (scrB) were clustered together on the S. mutans chromosome but unlinked to gtJR-gtfC (6). The relative positions of these genes were mapped by inserting different antibiotic resistance markers adjacent to or within the relevant genes on the chromosomes of these organisms. This approach has been continued in order to show that the genes fru-J (fructanase [1]) and gbp (glucanbinding protein [8]) are also linked to the gftA-ftf-scrB cluster. However, the recently isolated gtfD gene (glucosyltransferase catalyzing the synthesis of soluble glucan [3]) was neither linked to this cluster nor linked to gtfB-gtfC. The 4.5-kilobase (kb) EcoRI fragment of gbp derived from pMLG43 (8) and the 3.5-kb HindIll fragment offru-J derived from pFRU1 (1) were each inserted into the streptococcal mapping vector pVA891 containing a erythromycin resistance gene (erm; 7). Similarly, a chimeric plasmid containing the 1.7-kb EcoRI-HindIII fragment of gtJD derived from pNH4888 (3) also was constructed. These chimeric plasmids then were transformed into wild-type S. mutans GS-5 selecting for erythromycin resistance. Subsequently, a representative isolate of each of the Emr transformants (Gbp Emr, Frul Emr, and GtfD Em) was transformed with a chimeric *
plasmid containing a tetracycline resistance gene (tet) inserted within the ftf or scrB gene with a mini-Mu transposon (4, 6). DNA from the resulting Emr Tetr transformants was used to transform wild-type GS-5. Transformants were selected on Trypticase soy agar (BBL Microbiology Systems, Cockeysville, Md.) plus 0.2% yeast extract (TSAY) containing either erythromycin (10 ,ug/ml) or tetracycline (4 ,ug/ml) and replica plated onto media containing both antibiotics to determine the number of Emr Tetr transformants. The results showed that fru-J cotransferred with ftf and scrB at frequencies of 97.0 and 72.0% respectively, whereas gbp cotransferred with ftf and scrB at frequencies of 67.5 and 96.7%, respectively (Table 1). In contrast, the frequencies of cotransfer of gtJD withftf and scrB were both 0.5%, and thus gtJD was unlinked to these two genes. In addition, gtfD was not linked to the gtfB-gtfC cluster, since the frequency of cotransfer with gtJB was only 1.4%. An analysis of the cotransfer data to determine map distances would placefru-J close toftf and gbp close to scrB. Presumably, fru-l would also be tightly linked to gtfA, since previous results showed that the latter gene is linked to ftf (6). However, the map position offru-J relative to gtfA could be determined only indirectly, since both genes contained Emr markers. Therefore, a chimeric plasmid which contained a kanamycin resistance gene (kan) inserted adjacent tofru-J was constructed. Plasmid pFRU184 was constructed by inserting the 3.5-kb HindIII fru-l fragment into the HindIII site of pACYC184 (2). The 1.5-kb kan gene, which was isolated from p161 (D. LeBlanc, personal communication) after digestion with EcoRI, was inserted into the EcoRI site of pFRU184, resulting in pFRU184k. The kan gene in p161 is expressed in both Escherichia coli and S. mutans, TABLE 1. Cotransfer of GS-5 genes phenotypea
No. of Emr Tetr/ no. of Emrb
No. of Emr Tetr/ no. of Tetrb
Fru1 Ftf Frul ScrB Gbp Ftf Gbp ScrB GtfD Ftf GtfD ScrB GtfD GftB
74/74 52/100 54/100 103/103 0/100 1/100 4/209
57/61 93/100 81/100 99/106 1/100 0/100 1/142
DNA donor
Cotransferc
97.0 72.0 67.5 96.7
0.5 0.5 1.4
a The sucrose-metabolizing phenotypes of the DNA donors (e.g., Frul Ftf) were selected on the basis of Emr and Tetr, respectively. Strain GS-5 was transformed with 0.05 pg of each DNA per ml. b Transformants were selected on TSAY containing the individual antibiotics and then replica plated to TSAY containing both antibiotics. c Percent cotransfer = [no. of Emr Tetr/(no. of Emr + no of Tetr)] x 100.
Corresponding author. 3462
VOL. 58, 1990
NOTES
TABLE 2. Transformation of GS-5 with GtfA Frul Ftf DNA' No. of transformants/ 0.1 ml
Phenotype GtfA Em'. .................................. Frul KMr .............. ..................... Ftf Tetr ........... ........................ GtfA Em' Ftf Tetr ................................... GtfA Em' Frul Kmr ................................... Frul KMr Ftf Tetr ................................... GtfA Emr Frul KMr Ftf Tetr .........................
99 1,115 149
gtfA
frul ftf
2 1
a Cells were transformed with 0.05 p.g of DNA per ml, and different dilutions were spread onto TSAY containing the appropriate antibiotics.
although sensitive strains of the latter organism require 850 ,ug of kanamycin per ml to completely inhibit growth. pFRU184k was transformed into a GS-5 strain containing both gtfA (Emr) and ftf (Tetr), selecting for Emr Kmr Tetr transformants. DNA from one of these transformants was used to determine linkage among the three genes. When wild-type GS-5 cells were exposed to limiting concentrations of this DNA, transformation to resistance to all three individual antibiotics occurred, although the number of Kmr transformants was approximately 7- to 11-fold greater (Table 2). As expected, a significant number of Emr Tetr transformants also were observed, since it had been shown previously that gftA (Emr) andftf(Tet)r were linked (6). Virtually no Emr Kmr, KMr Tetr, or Em' KMr Tetr transformants were detected. However, when either the Emr or the Tetr transformants were replica plated to media containing either of the other two antibiotics, a significant number of double transformants, including KMr, were observed. In contrast, Kmr transformants, when replica plated, yielded less than 1% cotransfer with either Emr or Tetr. Thus the cotransfer of the Em', Tetr, and Kmir markers was asymmetrical in that the proportion of the Em' and Tetr transformants that also acquired Kmr was considerably higher than the proportion of the KMr transformants that also acquired Emr or Tetr. Similar observations were made by Perry et al. (7) during the transformation of Met' and gtfA (Emr), which are linked in S. mutans. These markers also transferred at unequal frequencies, with Met+ occurring some 50 times more frequently than Em'. Most likely, the transformation frequencies were influenced by the relative sizes of the homologous and inserted heterologous fragments of donor DNA (7). The results of these experiments indicated that gtfA,fru-1, and ftf are tightly linked (Table 3). An analysis of the cotransfer data strongly suggested thatfru-J (Kmr) is located closer toftf(Tetr) than to gtfA (Em') and most likely located between the last two genes (Fig. 1). This conclusion is based on the observation that approximately 98% of the Emr Tetr transformants were also KMr when tested by replica plating (data not shown), which would be expected if fru-l (Kmr) were located between the other two genes. In addition, the TABLE 3. Cotransfer of gtfA, fru-J and ft? Resistance phenotype
transformants/total
% Cotransfer
Emr Tetr/Emr + Tetr Emr Kmr/Em' KMr Tet'/Tetr
227/243 101/106 136/137
93.4 95.3 99.3
a GS-5 cells were transformed with 0.05 ,ug of GtfA Frul Ftf DNA per ml, and the GtfA, Frul, and Ftf phenotypes were selected on the basis of Emr, Kmr, and Tetr, respectively. The number of double transformants and the percent cotransfer were determined as described in the footnotes to Table 1.
gbp
i~~~~~~~~---u-
-1-1.1
.01
86
1
scrB
3463
* .05-* I(-.20)
@
.-
-
.33
-
FIG. 1. Linkage map of GS-5 sucrose-metabolizing genes. Map distances were calculated from the values in Tables 1 and 3 by the following formula: distance = 1 - (% cotransfer/100). Values in parentheses were determined in a previous study (6).
relative map distances between scrB and fru-J and between scrB andftf (Fig. 1) also suggest thatfru-J lies between gtfA and ftf. However, the extreme closeness of ftf and fru-J suggests that physical mapping may be necessary to conclusively determine the relative positions of these two genes. In this regard, the cotransfer values between fru-J and ftf yielded map distances of 0.03 and 0.01 when fru-J was selected on the basis of Emr (Table 1) and KMr (Table 3), respectively. The map distance for these two genes (Fig. 1) was calculated from the cotransfer value in Table 3. The observed map distances betweenfru-J and scrB and between ftf and gbp (0.28 and 0.33, respectively) were much greater than expected for these genes (Fig. 1). As previously shown (6), chromosomal breakage during DNA isolation could account for these observations. It is not surprising that the five genes within the cluster are linked, although their exact positions on the chromosome relative to their specific functions are not readily apparent. All are involved in the metabolism of sucrose or a metabolic product of this disaccharide. While it is not unusual for genes with related functions to be unlinked, it was still somewhat surprising that gtfD was not closely linked to gtfB-gtfC. Therefore, gtfD and the gtfB-gtfC gene cluster may be
independently regulated.
We thank D. LeBlanc, R. Burne, and R. R. B. Russell for providing the plasmids p161, pFRU1, and pMLG43, respectively. This study was supported by Public Health Service grant DE-
06082 from the National Institute of Dental Research. LITERATURE CITED 1. Burne, R. A., K. Schilling, W. H. Bowen, and R. E. Yasbin. 1987. Expression, purification, and characterization of an exoP-D-fructosidase of Streptococcus mutans. J. Bacteriol. 169: 4507-4517. 2. Chang, A. C. Y., and S. N. Cohen. 1978. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic mini-plasmid. J. Bacteriol. 134: 1141-1156. 3. Hanada, N., and H. K. Kuramitsu. 1989. Isolation and characterization of the Streptococcus mutans gtfD gene, coding for primer-dependent soluble glucan synthesis. Infect. Immun. 57: 2079-2085. 4. Kuramitsu, H. K. 1987. Utilization of a mini-mu transposon to construct defined mutants in Streptococcus mutans. Mol. Mi-
3464
NOTES
crobiol. 1:229-231. 5. Loesche, W. J. 1986. Role of Streptococcus mutans in human dental decay. Microbiol. Rev. 50:353-380. 6. Perry, D., and H. K. Kuramitsu. 1989. Genetic linkage among cloned genes of Streptococcus mutans. Infect. Immun. 57:805809. 7. Perry, D., L. N. Nilsen, and H. K. Kuramitsu. 1985. Mapping of a cloned glucosyltransferase gene in Streptococcus mutans. Infect. Immun. 50:130-135.
INFECT. IMMUN. 8. Russell, R. R. B., D. Coleman, and G. Dougan. 1985. Expression of a gene for glucan-binding protein from Streptococcus mutans in Escherichia coli. J. Gen. Microbiol. 131:295-299. 9. Russell, R. R. B., H. Mukasa, A. Shimamura, and J. J. Ferretti. 1988. Streptococcus mutans gtfA gene specifies sucrose phosphorylase. Infect. Immun. 56:2763-2765. 10. Ueda, S., T. Shiroza, and H. K. Kuramitsu. 1988. Sequence analysis of the gtfC gene from Streptococcus mutans GS-5. Gene 69:101-109.
