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CovR-controlled global regulation of gene expression in Streptococcus mutans.

Dmitriev A, Mohapatra SS, Chong P, Neely M, Biswas S, Biswas I - PLoS ONE (2011)

Bottom Line: Genes encoded by the GI TnSmu2 were found to be dramatically reduced in IBS10, while genes encoded by the GI TnSmu1 were up regulated in the mutant.The microarray data were further confirmed by real-time RT-PCR analyses.Our results indicate that CovR truly plays a significant role in the regulation of several virulence related traits in this pathogenic streptococcus.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas, United States of America.

ABSTRACT
CovR/S is a two-component signal transduction system (TCS) that controls the expression of various virulence related genes in many streptococci. However, in the dental pathogen Streptococcus mutans, the response regulator CovR appears to be an orphan since the cognate sensor kinase CovS is absent. In this study, we explored the global transcriptional regulation by CovR in S. mutans. Comparison of the transcriptome profiles of the wild-type strain UA159 with its isogenic covR deleted strain IBS10 indicated that at least 128 genes (∼6.5% of the genome) were differentially regulated. Among these genes, 69 were down regulated, while 59 were up regulated in the IBS10 strain. The S. mutans CovR regulon included competence genes, virulence related genes, and genes encoded within two genomic islands (GI). Genes encoded by the GI TnSmu2 were found to be dramatically reduced in IBS10, while genes encoded by the GI TnSmu1 were up regulated in the mutant. The microarray data were further confirmed by real-time RT-PCR analyses. Furthermore, direct regulation of some of the differentially expressed genes was demonstrated by electrophoretic mobility shift assays using purified CovR protein. A proteomic study was also carried out that showed a general perturbation of protein expression in the mutant strain. Our results indicate that CovR truly plays a significant role in the regulation of several virulence related traits in this pathogenic streptococcus.

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Differential expression of fructosyltransferase in wild-type and ΔcovR strains of NG-8.(A). Colony morphology of IBS06 (covR mutant) and NG-8 (isogenic wild-type parent) on mitis-salivarius agar medium. Plates were incubated at 37°C under microaerophilic conditions for 48 hrs. (B) Semi-quantitative RT-PCR analysis of ftf and gyrA for the strains NG-8 and ΔcovR (IBS06). The gyrA gene was included as an internal control to ensure that equal amounts of RNA were used for each RT-PCR reaction. Experiments were repeated at least twice with two independent RNA isolations. (C) Analysis of extracellular proteins from the wild-type and the ΔcovR strains. Supernatant proteins from overnight cultures were precipitated by 20% TCA, washed with acetone, and resuspended in PBS. Equal amounts of protein were loaded in each lane, and samples were run on SDS-PAGE (4–20%) gels and stained with Coomassie blue. Bands marked with arrowheads were excised from the stained gel, and identified by mass spectrometry. Lanes: M, Fermentas prestained marker; 1, NG-8; 2, IBS06. Proteins identified by mass spectrometry are indicated at the right. (D) Western blot analysis of FTF (fructosyltransferase) expression. NG-8 (wild-type, lane 1) and IBS06 (covR, lane 2) were grown overnight in THY broth and whole-cell extracts were prepared. Equal amounts of cell extracts were separated on 4–20% SDS-PAGE gels and reacted with anti-FTF antibody (E). In vitro binding of CovR to the promoter of ftf (Pftf). EMSA was performed with His-tagged CovR as described in the text. An increasing concentration of CovR was added to 0.1 pmole of the putative promoters as follows: lane 1, 0 µM; lane 2, 0.5 µM; lane 3, 1.25 µM; lane 4, 2.5 µM. Lane 5 contains 1.25 µM CovR with 10 pmole of non-labelled Pftf DNA.
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pone-0020127-g006: Differential expression of fructosyltransferase in wild-type and ΔcovR strains of NG-8.(A). Colony morphology of IBS06 (covR mutant) and NG-8 (isogenic wild-type parent) on mitis-salivarius agar medium. Plates were incubated at 37°C under microaerophilic conditions for 48 hrs. (B) Semi-quantitative RT-PCR analysis of ftf and gyrA for the strains NG-8 and ΔcovR (IBS06). The gyrA gene was included as an internal control to ensure that equal amounts of RNA were used for each RT-PCR reaction. Experiments were repeated at least twice with two independent RNA isolations. (C) Analysis of extracellular proteins from the wild-type and the ΔcovR strains. Supernatant proteins from overnight cultures were precipitated by 20% TCA, washed with acetone, and resuspended in PBS. Equal amounts of protein were loaded in each lane, and samples were run on SDS-PAGE (4–20%) gels and stained with Coomassie blue. Bands marked with arrowheads were excised from the stained gel, and identified by mass spectrometry. Lanes: M, Fermentas prestained marker; 1, NG-8; 2, IBS06. Proteins identified by mass spectrometry are indicated at the right. (D) Western blot analysis of FTF (fructosyltransferase) expression. NG-8 (wild-type, lane 1) and IBS06 (covR, lane 2) were grown overnight in THY broth and whole-cell extracts were prepared. Equal amounts of cell extracts were separated on 4–20% SDS-PAGE gels and reacted with anti-FTF antibody (E). In vitro binding of CovR to the promoter of ftf (Pftf). EMSA was performed with His-tagged CovR as described in the text. An increasing concentration of CovR was added to 0.1 pmole of the putative promoters as follows: lane 1, 0 µM; lane 2, 0.5 µM; lane 3, 1.25 µM; lane 4, 2.5 µM. Lane 5 contains 1.25 µM CovR with 10 pmole of non-labelled Pftf DNA.

Mentions: Our transcriptome data suggest that one of the virulence-related genes, ftf (SMU.2028), was moderately up-regulated in the covR mutant strain. FTF, which synthesizes fructan polymers from sucrose, can be found in either cell-associated or extracellular form. Expression of FTF varies greatly among various S. mutans isolates. Although FTF can be identified in UA159, it appears that FTF is one of the most abundant proteins in the culture supernatant of NG-8 [48]. Therefore, we chose to investigate the role of CovR in the production of FTF in this strain, and constructed a covR mutant strain, IBS06. IBS06 appeared to produce heavily mucoid colonies compared to NG-8 when streaked on mitis-salivarious agar plates (Fig. 6A). The extracellular protein profiles of the wild-type (NG-8) and the ΔcovR mutant (IBS06) were also determined by resolution via 4–20% gradient SDS-PAGE. Two distinct bands of approximately 170-kDa were elevated in the ΔcovR mutant IBS06 relative to the wild-type NG-8 strain. Mass-spectrometry analysis confirmed that the larger band corresponds to GtfB, while the smaller band corresponds to GtfC. As previously shown, these two bands were also up regulated in IBS10 supernatant fractions (Fig. 3A). In contrast, the supernatant fraction of NG-8 and its isogenic covR mutant strain contain a band that was absent in UA159 and its derivatives. This band, which is ∼87.0 kDa in size, was more abundant (∼3-fold) in the mutant strain (IBS06) compared to the wild-type strain (NG-8). Mass-spectrometry analysis confirmed the identity of the band as FTF protein. To verify our mass-spectrometry result, we employed western blot analysis (Fig. 6D). Mid-exponential phase culture supernatants from the wild-type (NG-8) and the covR mutant (IBS06) strains were separated by SDS-PAGE, and probed with anti-FTF antibody. As expected, IBS06 showed increased presence of FTF in the supernatant compared to NG-8. Furthermore, purified CovR protein was able to bind to the promoter region of the ftf gene during EMSA analysis (Fig. 6E). This binding was specific, since excess unlabelled competitor DNA was able to disrupt the complex formation. Thus, taken together our results strongly suggest that in addition to glucosyltransferases (GtfB/C), CovR also regulates fructosyltransferase production in S. mutans strain NG-8.


CovR-controlled global regulation of gene expression in Streptococcus mutans.

Dmitriev A, Mohapatra SS, Chong P, Neely M, Biswas S, Biswas I - PLoS ONE (2011)

Differential expression of fructosyltransferase in wild-type and ΔcovR strains of NG-8.(A). Colony morphology of IBS06 (covR mutant) and NG-8 (isogenic wild-type parent) on mitis-salivarius agar medium. Plates were incubated at 37°C under microaerophilic conditions for 48 hrs. (B) Semi-quantitative RT-PCR analysis of ftf and gyrA for the strains NG-8 and ΔcovR (IBS06). The gyrA gene was included as an internal control to ensure that equal amounts of RNA were used for each RT-PCR reaction. Experiments were repeated at least twice with two independent RNA isolations. (C) Analysis of extracellular proteins from the wild-type and the ΔcovR strains. Supernatant proteins from overnight cultures were precipitated by 20% TCA, washed with acetone, and resuspended in PBS. Equal amounts of protein were loaded in each lane, and samples were run on SDS-PAGE (4–20%) gels and stained with Coomassie blue. Bands marked with arrowheads were excised from the stained gel, and identified by mass spectrometry. Lanes: M, Fermentas prestained marker; 1, NG-8; 2, IBS06. Proteins identified by mass spectrometry are indicated at the right. (D) Western blot analysis of FTF (fructosyltransferase) expression. NG-8 (wild-type, lane 1) and IBS06 (covR, lane 2) were grown overnight in THY broth and whole-cell extracts were prepared. Equal amounts of cell extracts were separated on 4–20% SDS-PAGE gels and reacted with anti-FTF antibody (E). In vitro binding of CovR to the promoter of ftf (Pftf). EMSA was performed with His-tagged CovR as described in the text. An increasing concentration of CovR was added to 0.1 pmole of the putative promoters as follows: lane 1, 0 µM; lane 2, 0.5 µM; lane 3, 1.25 µM; lane 4, 2.5 µM. Lane 5 contains 1.25 µM CovR with 10 pmole of non-labelled Pftf DNA.
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Related In: Results  -  Collection

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pone-0020127-g006: Differential expression of fructosyltransferase in wild-type and ΔcovR strains of NG-8.(A). Colony morphology of IBS06 (covR mutant) and NG-8 (isogenic wild-type parent) on mitis-salivarius agar medium. Plates were incubated at 37°C under microaerophilic conditions for 48 hrs. (B) Semi-quantitative RT-PCR analysis of ftf and gyrA for the strains NG-8 and ΔcovR (IBS06). The gyrA gene was included as an internal control to ensure that equal amounts of RNA were used for each RT-PCR reaction. Experiments were repeated at least twice with two independent RNA isolations. (C) Analysis of extracellular proteins from the wild-type and the ΔcovR strains. Supernatant proteins from overnight cultures were precipitated by 20% TCA, washed with acetone, and resuspended in PBS. Equal amounts of protein were loaded in each lane, and samples were run on SDS-PAGE (4–20%) gels and stained with Coomassie blue. Bands marked with arrowheads were excised from the stained gel, and identified by mass spectrometry. Lanes: M, Fermentas prestained marker; 1, NG-8; 2, IBS06. Proteins identified by mass spectrometry are indicated at the right. (D) Western blot analysis of FTF (fructosyltransferase) expression. NG-8 (wild-type, lane 1) and IBS06 (covR, lane 2) were grown overnight in THY broth and whole-cell extracts were prepared. Equal amounts of cell extracts were separated on 4–20% SDS-PAGE gels and reacted with anti-FTF antibody (E). In vitro binding of CovR to the promoter of ftf (Pftf). EMSA was performed with His-tagged CovR as described in the text. An increasing concentration of CovR was added to 0.1 pmole of the putative promoters as follows: lane 1, 0 µM; lane 2, 0.5 µM; lane 3, 1.25 µM; lane 4, 2.5 µM. Lane 5 contains 1.25 µM CovR with 10 pmole of non-labelled Pftf DNA.
Mentions: Our transcriptome data suggest that one of the virulence-related genes, ftf (SMU.2028), was moderately up-regulated in the covR mutant strain. FTF, which synthesizes fructan polymers from sucrose, can be found in either cell-associated or extracellular form. Expression of FTF varies greatly among various S. mutans isolates. Although FTF can be identified in UA159, it appears that FTF is one of the most abundant proteins in the culture supernatant of NG-8 [48]. Therefore, we chose to investigate the role of CovR in the production of FTF in this strain, and constructed a covR mutant strain, IBS06. IBS06 appeared to produce heavily mucoid colonies compared to NG-8 when streaked on mitis-salivarious agar plates (Fig. 6A). The extracellular protein profiles of the wild-type (NG-8) and the ΔcovR mutant (IBS06) were also determined by resolution via 4–20% gradient SDS-PAGE. Two distinct bands of approximately 170-kDa were elevated in the ΔcovR mutant IBS06 relative to the wild-type NG-8 strain. Mass-spectrometry analysis confirmed that the larger band corresponds to GtfB, while the smaller band corresponds to GtfC. As previously shown, these two bands were also up regulated in IBS10 supernatant fractions (Fig. 3A). In contrast, the supernatant fraction of NG-8 and its isogenic covR mutant strain contain a band that was absent in UA159 and its derivatives. This band, which is ∼87.0 kDa in size, was more abundant (∼3-fold) in the mutant strain (IBS06) compared to the wild-type strain (NG-8). Mass-spectrometry analysis confirmed the identity of the band as FTF protein. To verify our mass-spectrometry result, we employed western blot analysis (Fig. 6D). Mid-exponential phase culture supernatants from the wild-type (NG-8) and the covR mutant (IBS06) strains were separated by SDS-PAGE, and probed with anti-FTF antibody. As expected, IBS06 showed increased presence of FTF in the supernatant compared to NG-8. Furthermore, purified CovR protein was able to bind to the promoter region of the ftf gene during EMSA analysis (Fig. 6E). This binding was specific, since excess unlabelled competitor DNA was able to disrupt the complex formation. Thus, taken together our results strongly suggest that in addition to glucosyltransferases (GtfB/C), CovR also regulates fructosyltransferase production in S. mutans strain NG-8.

Bottom Line: Genes encoded by the GI TnSmu2 were found to be dramatically reduced in IBS10, while genes encoded by the GI TnSmu1 were up regulated in the mutant.The microarray data were further confirmed by real-time RT-PCR analyses.Our results indicate that CovR truly plays a significant role in the regulation of several virulence related traits in this pathogenic streptococcus.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas, United States of America.

ABSTRACT
CovR/S is a two-component signal transduction system (TCS) that controls the expression of various virulence related genes in many streptococci. However, in the dental pathogen Streptococcus mutans, the response regulator CovR appears to be an orphan since the cognate sensor kinase CovS is absent. In this study, we explored the global transcriptional regulation by CovR in S. mutans. Comparison of the transcriptome profiles of the wild-type strain UA159 with its isogenic covR deleted strain IBS10 indicated that at least 128 genes (∼6.5% of the genome) were differentially regulated. Among these genes, 69 were down regulated, while 59 were up regulated in the IBS10 strain. The S. mutans CovR regulon included competence genes, virulence related genes, and genes encoded within two genomic islands (GI). Genes encoded by the GI TnSmu2 were found to be dramatically reduced in IBS10, while genes encoded by the GI TnSmu1 were up regulated in the mutant. The microarray data were further confirmed by real-time RT-PCR analyses. Furthermore, direct regulation of some of the differentially expressed genes was demonstrated by electrophoretic mobility shift assays using purified CovR protein. A proteomic study was also carried out that showed a general perturbation of protein expression in the mutant strain. Our results indicate that CovR truly plays a significant role in the regulation of several virulence related traits in this pathogenic streptococcus.

Show MeSH
Related in: MedlinePlus