Control of Enzyme II^scr and Sucrose-6-Phosphate Hydrolase Activities in Streptococcus mutans by Transcriptional Repressor ScrR Binding to the cis-Active Determinants of the scr Regulon

Bacterial strains.

The S. mutans strains used in this study are listed in Table 1. Escherichia coli DH5α (supE44 hsdR17 recA1 endA1 gyrA96 thi-1 relA1) was utilized as a general cloning host. E. coli M15 (Nals Strs Rifs Thi− Lac− Ara+ Gal+ Mtl− F− RecA+ Uvr+ Lon+) was used for expression of fusion proteins. S. mutans was grown in Todd-Hewitt Broth (Invitrogen, Carlsbad, Calif. ) or defined medium (0. 2% l-glutamic acid, 0. 02% l-cysteine, 0. 09% l-leucine, 0. 1% NH4Cl, 0. 25% K2HPO4, 0. 25% KH2PO4, 0. 4% NaHCO3, 0. 12% MgSO4 · 7H2O, 0. 002% MnCl2 · 4H2O, 0. 002% FeSO4 · 7H2O, 0. 06% Na-pyruvate, 0. 0001% riboflavin, 0. 00005% thiamine-HCl, 0 VSports. 00001% biotin, 0. 0001% nicotinic acid, 0. 00001% p-aminobenzoic acid, 0. 00005% Ca-pantothenate, 0. 0001% pyridoxal-HCl, 0. 00001% folic acid) supplemented with 1% sugar (2). E. coli strains were grown in L broth (Invitrogen), and transformants were selected on L agar plates supplemented with the antibiotics indicated below.

DNA manipulations.

DNA isolation, restriction endonuclease digestion, PCR, sequencing, Southern blotting, ligation, transformation, and other DNA manipulations were carried out as described by Ausubel et al. (1). Restriction endonucleases and other DNA-modifying enzymes were obtained from Invitrogen, New England Biolabs Inc. (Beverly, Mass.), and Promega Corp. (Madison, Wis.) and used according to the specifications of the suppliers. DNA fragments were isolated from agarose gels by using a QIAEXII kit (QIAGEN, Valencia, Calif.). Double-stranded PCR template sequencing was performed by using a Sequenase 7-deaza-dGTP sequencing kit (U.S. Biochemicals, Cleveland, Ohio).

Overexpression and purification of the fusion protein ScrR-H.

A pair of primers complementary to the scrR gene without a stop codon at the 3′ terminus and an additional NcoI site (underlined) added to the 5′ terminus (dsF-1, 5′-AACCATGGTTGCAAAATTAACAG-3′; ds-3, 5′-CAACACTTTCTCCTGATAAAAGAC-3′) were designed to amplify the scrR gene. After digestion with NcoI and phosphorylation by T4 kinase, the 960-bp DNA fragment was cloned into the NcoI/BglII (blunted with the Klenow fragment)-digested vector pQE-60 (QIAGEN), which contains the six-His tag gene downstream of the multiple cloning site. The recombinant plasmid pQEScrR contained the entire scrR sequence (CATCACCATCACCATCAC) under the control of the T5 promoter and the lac operators. The DNA sequence of scrR in pQEScrR was verified by sequencing.

The transformant harboring plasmid pQEscrR was cultured in 1 liter of L broth containing 100 μg of ampicillin per ml and 25 μg of kanamycin per ml at 37°C to an optical density of 0.5 to 0.6 at 600 nm and then induced by 0.1 mM isopropyl-β-d-thiogalactopyranoside (IPTG). After induction at 37°C for 4 h, bacteria were harvested by centrifugation at 4,000 × g for 20 min and resuspended in 20 ml of lysis buffer C (50 mM NaH₂PO₄, 300 mM NaCl, 2 mM imidazole, pH 8.0). A soluble fraction obtained by sonication and centrifugation was applied to the Ni-nitrilotriacetic acid spin column (QIAGEN), and ScrR-H was eluted according to the directions of the supplier with slight modifications. Protein purity was evaluated by sodium dodecyl sulfate (SDS)-12.5% polyacrylamide gel electrophoresis and Western blotting with RGS · His antibody (QIAGEN).

DNA mobility shift assays.

The DNA mobility shift assays were performed as described by Ausubel et al. (1) with the following modifications. Two to 20 ng of the DNA probe (10,000 to 40,000 cpm) was incubated with the amounts of ScrR-H protein indicated below in 15 μl of binding buffer (12% glycerol, 12 mM HEPES-NaOH [pH 7.9], 4 mM Tris-HCl [pH 7.9], 60 mM KCl, 1 mM EDTA, 1 mM dithiothreitol) at 30°C for 15 min. After incubation, the DNA-protein complexes and free DNA were resolved on nondenaturing 4% polyacrylamide gels with a running buffer containing 6.7 mM Tris · Cl (pH 7.9), 0.3 mM sodium acetate, and 1 mM EDTA. Gels were dried and subjected to autoradiography. The negative controls included a reaction of the DNA probe only without proteins and a reaction of the DNA probe with a nonspecific protein, maltose-binding protein (MBP; New England Biolabs Inc.).

For determination of the ability of ScrR-H to bind the intergenic region between the scrA and scrB genes, three DNA fragments were generated by PCR. Probe Pab from bp 19 to 234 (Fig. 1) contains both promoter regions of the scrA and scrB genes and was amplified by primer Pab-1 (5′-CTACTTTGCTATAATCCATTTGCA-3′) and primer Pab-2 (5′-CATCGTTTATCTACTCCTAATAA-3′). Probe Pa from bp 19 to 139 includes only the scrA promoter with a 20-bp imperfect inverted repeat and was amplified by primers Pab-1 and Pa-2 (5′-TATGTAAAACGATTGACATATTGG-3′). Probe Pb from nucleotides 140 to 234 contains a 37-bp imperfect direct-repeat sequence in the promoter region of scrB and was generated with primers Pb-1 (5′-AAATTCTTTTTTGCAACAAAAG-3′) and Pab-2. Pab was end labeled with [γ-³²P]ATP and T4 polynucleotide kinase as described previously (1).

DNase I protection assays.

The DNase I protection assays were carried out using the Core footprinting system (Promega Co.). The reaction mixture (50 μl) containing 10,000 cpm of ³²P-labeled DNA probe and the ScrR-H protein (0 to 18 μg) in binding buffer (25 mM Tris · Cl [pH 8.0], 50 mM KCl, 6.25 mM MgCl₂, 0.5 mM EDTA, 10% glycerol, 0.5 mM dithiothreitol) was incubated for 10 min on ice according to the directions of the supplier. DNase I was added at a final concentration of 5 U/ml, and the mixture was further incubated at room temperature for 1 min. The digestion was stopped by adding 90 μl of stop solution (200 mM NaCl, 30 mM EDTA, 1% SDS, 100 μg of yeast RNA per ml) and extracted once with phenol-CHCl₃ (1:1) and CHCl₃. After ethanol precipitation, the pellet was washed with 70% ethanol, dissolved in 10 μl of loading buffer (0.1 M NaOH-formamide [1:2], 0.1% xylene cyanol, 0.1% bromophenol blue), and analyzed on a 6% denatured polyacrylamide gel. For a size ladder, sequencing was performed with the PabE primer (5′-GGAATTCCTACTTTGCTATAATCCATTTGCA-3′) (the nucleotides added for the EcoRI site are underlined) for the sense strand and with the Pdp-3 primer (5′-CAAGGAGACAAAGCTAC-3′) for the antisense strand with a Sequenase kit (U.S. Biochemicals).

DNA templates from bp 19 to 326 (Fig. 1) were generated by PCR with primers PabE and Pdp-3. The DNA fragments were labeled at the 5′ ends with [γ-³²P]ATP and T4 polynucleotide kinase. For preparation of the sense strand for DNase I protection assays, the labeled probe was digested with EcoRV and selectively precipitated by ethanol. An antisense strand probe was prepared by digestion with EcoRI after being labeled with ³²P.

Construction of the scrR-defective mutants in the S. mutans scrA::lacZ strain.

The chromosomal DNA of SP2ΔscrR (6) was used to transform (12) the S. mutans scrA::lacZ strain IS3AZ2. The resultant mutant, IS3AZ2 ΔscrR, carries a tetracycline resistance gene inserted within the scrR gene.

Mutation of the operators O_B and O_C in the S. mutans scrA::lacZ strain.

A spontaneous O_B deletion mutation plasmid of pSBL20, pAZ4-1 (6) (see below), with a spectinomycin resistance gene (8) instead of the lacZ gene inserted in the scrB gene, was transformed into the S. mutans scrA::lacZ strain IS3AZ2 (19), yielding ΔO_B strain AZ24-2. The O_B and O_C mutation plasmid pSCSP-1 was constructed by replacing the lacZ gene of pSBL60-1 with the same spectinomycin cassette. Transformation of pSCSP-1 into IS3AZ2 yielded the O_B and O_C mutant AZ23-4.

Mutation of the operators VSports app下载 - O_B and O_C in the S. mutans scrB::lacZ strain.

A spontaneous O_B deletion in plasmid pSBL20 (6) was obtained when recovery of the E. coli strain harboring the plasmid from stock culture yielded pSBL20B. Sequence analysis indicated that a 21-bp fragment from bp 200 to 220 including the Shine-Dalgarno site was deleted (Fig. 1) from pSBL20B. This O_B deletion plasmid with a lacZ gene translational insertion in the scrB gene was transformed into the S. mutans SP2 strain, yielding ΔO_B strain SP2C5-2. An O_C mutation was introduced into pSBL20B by replacing the O_C sequence with a 20-bp random sequence (5′-AAGCTTGTGTTACGTACACA-3′) by using an ExSite PCR-based site-directed mutagenesis kit (Stratagene, La Jolla, Calif.). The resultant plasmid, pSBL60-1, contained mutations in both the O_B and O_C sequences. Transformation of pSBL60-1 into the SP2 strain yielded the O_B and O_C mutant SP2C6-6.

"V体育官网入口" Northern blots.

The preparation of total RNA from S. mutans was as described previously (6). A DNA fragment of the lacZ gene was generated by PCR with a pair of primers (Lac-1, 5′-CAACGTCGTGACTGGGAAAAC-3′; Lac-2, 5′-CATTACCAGTTGGTCTGGTGTC-3′). The digoxigenin (DIG)-labeled DNA probe was generated with a DIG-High Prime kit (Boehringer Mannheim Corp., Indianapolis, Ind.). The RNA fragments were separated by electrophoresis of the denatured RNA sample (20 μg/lane) in 1% agarose gels containing 3% formaldehyde. The gel was washed three times with 10× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) for 10 min to remove formaldehyde, and blotting was carried out with 20× SSC buffer. The blotted membrane was washed with 6× SSC once for 1 min and fixed by UV cross-linking. Hybridization was then carried out with DIG Easy Hyb (Boehringer Mannheim Corp.) with a DIG-labeled probe (0.3 mg/ml) at 50°C according to the directions of the supplier.

Determination of β-galactosidase activity.

The S. mutans ΔO_B strain (AZ24-2) and O_B-O_C mutant (AZ23-4) were grown in defined medium supplemented with 1% concentrations of the sugars indicated below to mid-log phase. Cultures were then centrifuged, and the cells were assayed for β-galactosidase activity as described previously (6).

Production and purification of ScrR-H.

ScrR has homology with the proteins in the GalR-LacI family. The N terminus of this family is important to the specificity of the DNA binding function (23). Therefore, we constructed a protein with a C-terminal fusion. We placed scrR in pQE60 to fuse a six-histidine tag to the COOH-terminal end of the entire scrR sequence. The recombinant plasmid pQEScrR directed the synthesis of ScrR-H. Since the ScrR proteins in several gram-positive bacteria were shown to be autoaggregated, we examined culture and purification conditions to produce the soluble ScrR-H protein. Under the conditions we established, a considerable amount of ScrR-H was produced in the soluble fraction (Fig. 2).

DNA binding properties of ScrR.

In order to assess ScrR binding to the promoter regions of both the scrA and scrB genes, the ScrR fusion protein was utilized since ScrR could not be isolated from either N- or C-terminal-fusion proteins with ScrR due to autoaggregation (unpublished data). Various amounts of purified ScrR-H fusion protein were incubated with the ³²P-labeled probe Pab, and shifted bands were detected (Fig. 3). The free probe band could not be detected when ScrR-H was added to the reaction mixture (Fig. 3A, lanes 2 to 4), unlike in the control without ScrR-H (Fig. 3A, lane 1) or with the nonspecific protein MBP (Fig. 3A, lane 6). Instead, the DNA-ScrR complex migrated more slowly and finally was retarded in the sample well as the concentration of ScrR-H increased from 0.3125 to 2.5 μg (Fig. 3A). When unlabeled Pab, Pa, or Pb was added to the reaction mixture, the relative strengths of competition were in the order Pab > Pa > Pb. This finding indicated that there were ScrR binding sites in the Pa region as well as in the Pb region. The affinity of the binding site in Pa was higher than that in Pb (Fig. 3B and C). To further determine binding specificities, excess amounts of unlabeled probes and a series of heterologous DNA fragments, including the scrK promoter region downstream from the scrA gene, which encodes fructokinase (18), the S. mutans dgk structural gene, which encodes diacylglycerol kinase (24), and prtP, a structural gene from Treponema denticola (7), were added to a series of binding reaction mixtures. The results showed that, compared to the other heterologous competitors, the unlabeled Pab DNA fragment at a 10-fold excess competed effectively with ³²P-labeled Pab (Fig. 3B).

Identification of putative ScrR operator regions in the promoter sequences of scrA and scrB.

Results of the DNA mobility shift assays suggested the possibility of ScrR binding sites in the promoter regions of scrA and scrB. In order to identify these potential operator sequences, DNase I protection assays were carried out by using a 5′-end-labeled ³²P-PabE probe amplified with the primer pair PabE and Pdp-3 (Fig. 1). The probe was incubated with serial dilutions of ScrR-H fusion proteins and then subjected to DNase I digestion. The electrophoresis patterns of different amounts of ScrR (Fig. 4, lanes 2 to 4) were compared with a DNase I ladder (Fig. 4, lane 1) and the results of a competition assay in which unlabeled PabE was added to the reaction mixture of ScrR-H and ³²P-PabE (Fig. 4, lane 5). The experiment was reproducible for both the sense strand (Fig. 4A) and antisense strand (Fig. 4B) of PabE. The result showed that two regions with high affinity to ScrR were present in the promoter sequences. O_B contains a 35-bp imperfect direct repeat (from bp 187 to 221) located in the promoter region of scrB gene (Fig. 1). O_C (from bp 120 to 139) containing a 20-bp imperfect inverted-repeat sequence (5′-TATGTCAATCGTTTTACATA-3′) is located between the two promoters (Fig. 1). When ³²P-PabE was incubated with 1.2 pmol of ScrR-H, a protected region in the sequence of intergenic region of the scrA and scrB genes, named O_C, was observed (Fig. 4A). When the probe was incubated with 6 pmol of ScrR-H, the second protected region in the scrB promoter region, named O_B, was observed. These findings are consistent with the results of DNA mobility shift assays, which indicated that the affinity of Pa for ScrR is higher than that of Pb. The result that unlabeled PabE added to the reaction mixture competed with labeled PabE for ScrR binding and protection from DNase I digestion suggested that O_B and O_C are specific binding sites of ScrR.

Role of ScrR in the regulation of scrA expression. (V体育官网)

In order to determine whether ScrR plays a role as a regulatory protein in affecting scrA expression as it does for scrB (6), an scrR mutation was introduced into an scrA::lacZ strain by double-crossover recombination (see Materials and Methods). The mutated scrR gene in the resultant mutant (IS3AZ2 ΔscrR) was confirmed by Southern blotting with a DIG-labeled scrR probe (data not shown). The wild-type strain and the scrR mutant were grown in the presence of a defined broth containing 1% sucrose. The β-galactosidase activity in the mutant (6.1 ± 0.39 U) was about twofold higher than that in the wild type (3.2 ± 0.23 U) in repeated experiments. Similar results were also observed when these strains were grown with other sugars, such as fructose (3.1 ± 0.4 U in the scrR mutant versus 1.37 ± 0.25 U in the wild-type strain) and glucose (5.88 ± 0.09 U in the scrR mutant versus 3.7 ± 0.5 U in the wild-type strain). These results indicated that ScrR plays a role in the regulation of scrA expression similar to the role it plays with scrB.

"VSports手机版" Increased transcription of the scr regulon by mutation of the potential operators O_C and O_B.

Based on the sequences of the promoter regions of scrA and scrB (Fig. 1), the ScrR binding sequences appear to be very diverse, which suggested that the functions of these binding sites might be different. O_B and O_C contain an imperfect direct repeat and an imperfect inverted repeat, respectively, which have been shown to act as operators in GalR-LacI regulation (23). Since ScrR belongs to this group of proteins, we made mutations in the O_B and O_C regions to demonstrate the function of ScrR-operator interactions in the transcriptional repression of the target genes.

Initially, an O_B deletion plasmid (pAZ4-1) and an O_B-O_C mutation plasmid (pSCSP-1) were constructed and introduced into the S. mutans scrA::lacZ strain IS3AZ2 (10). Mutant AZ24-2 contains a deletion mutation in O_B. Besides the O_B deletion mutation, mutant AZ23-4 contains an additional O_C mutation resulting from the replacement of the O_C sequence with a 20-bp random sequence (5′-AAGCTTGTGTTACGTACACA-3′) (Fig. 5A). β-Galactosidase activities were determined in the wild-type strain IS3AZ2, the O_B mutant AZ24-2, and the O_B-O_C mutant AZ23-4. The expression of scrA::lacZ was elevated in both mutants compared to that in the wild-type strain in media containing 1% sucrose as well as in glucose and fructose media (Fig. 5B). The expression of scrA::lacZ was at least twofold higher in the O_B mutant (AZ24-2) than in the wild-type strain (IS3AZ2) in all the sugar-containing media. The O_B-O_C mutant (AZ23-4) showed about four- to fivefold-greater expression of scrA::lacZ than the wild-type strain. These results suggested that the ScrR interaction with O_B and O_C repressed scrA expression.

The O_B and O_C wild-type plasmid (pSBL20), O_B deletion plasmid (pSBL20B), and the O_B-O_C mutation plasmid (pSBL60-1) were also separately introduced into the S. mutans wild-type strain SP2. SP2C4 is an scrB::lacZ reporter strain with the wild-type promoter region. Mutant SP2C5-2 contains a deletion mutation in O_B. Mutant SP2C6-6 contains an additional O_C mutation resulting from the replacement of the O_C sequence with a 20-bp random sequence (5′-AAGCTTGTGTTACGTACACA-3′), besides the O_B deletion mutation (Fig. 6A). Since O_B overlaps the ribosome binding site of the scrB gene, deletion of O_B in the S. mutans scrB::lacZ fusion strains SP2C5-2 and SP2C6-6 resulted in negligible β-galactosidase activities compared to that of the wild-type scrB::lacZ strain (SP2C4) (data not shown). However, transcription of scrB::lacZ was increased in both mutants relative to that in the wild-type strain in the presence of 1% sucrose in Northern blots in which a DIG-labeled lacZ probe was used (Fig. 6B). The 6.7-kb band corresponds to the polycistronic transcript of the scrB::lacZ and erythromycin resistance genes in the wild-type strain SP2C4. The 5.5-kb band represents the polycistronic transcript of the scrB::lacZ and scrR gene. Transcription in the O_B-O_C mutant, SP2C6-6, was also enhanced relative to that in the O_B mutant, SP2C5-2. The same results were also observed when cells were grown in the presence of glucose and fructose (data not shown). These results were consistent with those for the expression of the scrA::lacZ fusion and suggested that the operators O_B and O_C not only control the transcription of the scrB gene but also regulate the expression of the scrA gene.