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. 2003 Oct;185(19):5791-9.
doi: 10.1128/JB.185.19.5791-5799.2003.

Control of enzyme IIscr and sucrose-6-phosphate hydrolase activities in Streptococcus mutans by transcriptional repressor ScrR binding to the cis-active determinants of the scr regulon

Bing Wang  1 , Howard K Kuramitsu
Affiliations

Control of enzyme IIscr and sucrose-6-phosphate hydrolase activities in Streptococcus mutans by transcriptional repressor ScrR binding to the cis-active determinants of the scr regulon (V体育安卓版)

Bing Wang et al. J Bacteriol. 2003 Oct.

Abstract (VSports)

In Streptococcus mutans, enzyme II(scr) and sucrose-6-phosphate hydrolase are two important enzymes in the transport and metabolism of dietary sucrose. The scr regulon of S. mutans is composed of three genes, scrA and scrB, which code for enzyme II(scr) and sucrose-6-phosphate hydrolase, respectively, and scrR, which codes for a GalR-LacI-type transcription regulator. It was previously shown that expression of both scrA and scrB is similarly induced by sucrose. Mutation in the scrR gene resulted in increased expression of scrB relative to that in the wild-type strain. In this study, we employed DNA mobility shift and DNase I protection assays with a purified ScrR-histidine tag fusion protein to examine the DNA binding properties of ScrR to the promoter regions of the scrA and scrB genes. The results showed that ScrR bound specifically to the promoter regions of both scrA and scrB. Two regions with high affinity for ScrR in the promoter sequences of the scrA and scrB genes were identified by DNase I protection assays. One, O(C), which includes a 20-bp imperfect inverted-repeat sequence, is located between the two promoters, and the other, O(B), is located within the scrB promoter region containing a 37-bp imperfect direct-repeat sequence. Mutations of O(B) and O(C) resulted in constitutive transcription and expression of both the scrA and scrB genes VSports手机版. Our results indicated that S. mutans coordinates the activities of enzyme II(scr) and sucrose-6-phosphate hydrolase by transcriptional repressor ScrR binding to the promoter regions of the scr regulon. .

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Figures

FIG. 1.
FIG. 1.
DNA sequences of the promoter regions of the scrA and scrB genes. The −10 and −35 regions of the promoter sequence elements and translation start codons of scrA and scrB were determined previously (15). SD indicates the putative Shine-Dalgarno sequence. Primers for the PCR amplification of the probes used in the DNA mobility shift assay and DNase I protection assay (Fig. 4) are marked with an arrow over their nucleotide sequences. ScrR binding sites determined by the DNase I protection assay are boxed and named OB and OC. Solid-line arrows under these sequences denote inverted-repeat structures between the two promoters. Dashed-line arrows denoted imperfect direct-repeat structures in the promoter region of the scrB gene.
FIG. 2.
FIG. 2.
Purification of ScrR-H fusion proteins. SDS-polyacrylamide gel electrophoresis results are shown. Molecular mass standards are indicated at the left (from top to bottom: 200, 116, 97.4, 66, 45, and 31 kDa). Lane 1, soluble cell extract before IPTG induction; lane 2, soluble cell extract after IPTG induction; lane 3, purified ScrR-H.
FIG. 3.
FIG. 3.
Binding of ScrR-H to the promoter regions of the scrA and scrB genes by DNA mobility shift assays. Lane 1 was a control in which no ScrR-H was present. The positions of ScrR-H-bound (filled arrowhead) and free (open arrowhead) probes are shown. The position of the sample well is also indicated by an arrow. (A) Different amounts of ScrR-H were mixed with 0.02 pmol of 32P-Pab, namely, 0.3125 μg (lane 2), 0.625 μg (lane 3), 1.25 μg (lane 4), and 2.5 μg (lane 5), and 0.02 pmol of 32P-Pab was mixed with 5 μg of MBP (lane 6). (B) Competition experiment in the DNA mobility shift assays in which 32P-Pab was incubated with ScrR-H and different unlabeled DNA fragments. The concentrations of each unlabeled DNA fragment shown above the lanes were 0.4 pmol (lanes 3, 5, 7, 9, 11, and 13) and 0.8 pmol (lanes 4, 6, 8, 10, 12, and 14). Lane 2 was a control in which ScrR-H (15.6 ng) was mixed with 32P-Pab (0.04 pmol). (C) DNA fragments in the promoter regions of the scrA and scrB genes used in DNA mobility shift assays. Probe Pab from bp 19 to 234 contained both promoter regions of the scrA and scrB genes (dashed arrow). Probe Pa from bp 19 to 139 includes only the scrA promoter with a 20-bp imperfect inverted repeat (solid line). Probe Pb from nucleotides 140 to 234 contained a 37-bp imperfect direct-repeat sequence overlapping the Shine-Dalgarno (SD) (open box) region of scrB (dotted line). ScrR binding sites, OB and OC, are marked as hatched boxes. Translation start codons are indicated by bent arrows.
FIG. 4.
FIG. 4.
DNase I protection assay. The sense strand (A) and antisense strand (B) of PabE are shown. (A) In lanes 1 to 4, 32P-PabE (0.12 pmol) was incubated with 0, 0.12, 1.2, and 6 pmol of ScrR-H protein; in lane 5, 10 pmol of unlabeled PabE was added to the reaction mixture used in lane 4. A DNA sequence ladder using the PabE primer as a molecular ruler is shown in lanes 6 to 8. (B) In lanes 1 to 4, 32P-PabE (0.12 pmol) was incubated with 0, 0, 4, 8, and 16 pmol of ScrR-H protein; in lane 5, 10 pmol of unlabeled PabE was added to the reaction mixture used in lane 3. A DNA sequence ladder using the Pdp-3 primer as a molecular ruler is shown in lanes 6 to 8.
FIG. 5.
FIG. 5.
Mutation of OB and OC in the S. mutans scrB::lacZ strain. (A) Genetic and transcriptional organizations of the sucrose gene cluster of the S. mutans scrA::lacZ strain IS3AZ2, the OB mutant AZ24-2, and the OB-OC double mutant AZ23-4. An OB deletion mutation with a spectinomycin resistance gene, instead of the lacZ gene, was inserted into the scrB gene in the chromosome of ΔOB strain AZ24-2. The OC mutation was constructed by replacing the OC sequence with a 20-bp random sequence in the chromosome of the OB and OC mutant AZ23-4. The arrows indicate repeat sequences. (B) Comparison of the levels of expression of the S. mutans scrA::lacZ fusion in the wild type (IS3AZ2), OB mutant (AZ24-2), and OB plus OC mutant (AZ23-4). Overnight cultures of the S. mutans strains were inoculated into defined media supplemented with 1% glucose, sucrose, or fructose. The cultures were incubated at 37°C until an optical density at 600 nm of 0.8 to 0.9 was reached. β-Galactosidase activities were determined with ο-nitrophenyl-β-galactoside (ONPG) as the substrate. The experiment was carried out in triplicate, and the mean values and standard errors of the β-galactosidase activities are shown.
FIG. 6.
FIG. 6.
Mutation of OB and OC in the S. mutans scrB::lacZ strain. (A) Genetic and transcriptional organizations of the sucrose gene cluster of S. mutans SP2C4 (scrB::lacZ), SP2C5-2 (scrB::lacZ ΔOB), and SP2C6-6 (scrB::lacZ ΔOB ΔOC). The genes (open boxes and open arrows) are shown with the mapped promoters as well as the sizes of the transcripts after they were probed with a DIG-labeled lacZ probe in Northern blots. (B) Northern blot analysis of the RNA samples from S. mutans. Lanes: 1, SP2C4 (scrB::lacZ); 2, SP2C5-2 (scrB::lacZ ΔOB); 3, SP2C6-6(scrB::lacZ ΔOB ΔOC). The cells were grown in defined medium containing 1% sucrose. Sizes of the mRNA are shown.
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VSports最新版本 - References

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