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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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Related in: MedlinePlus

Two-dimensional gel electrophoresis analysis of S. mutans.Whole cell lysates of the wild-type UA159 (A) and ΔcovR mutant IBS10 (B) are electrophoresed in 10% SDS-PAGE. Red and black circles in Fig. 2A indicate protein spots exclusively present in wild-type strain, or at least 3-fold abundant in the wild-type strain compared to the covR mutant, respectively. Red and black circles in Fig. 2B indicate protein spots exclusively present in the covR mutant strain, or at least 3-fold abundant in covR mutant strain compared to wild-type strain, respectively. Numbers correspond to the orf according to NCBI designation (SMU#). Molecular weight standards (kD) are shown.
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pone-0020127-g005: Two-dimensional gel electrophoresis analysis of S. mutans.Whole cell lysates of the wild-type UA159 (A) and ΔcovR mutant IBS10 (B) are electrophoresed in 10% SDS-PAGE. Red and black circles in Fig. 2A indicate protein spots exclusively present in wild-type strain, or at least 3-fold abundant in the wild-type strain compared to the covR mutant, respectively. Red and black circles in Fig. 2B indicate protein spots exclusively present in the covR mutant strain, or at least 3-fold abundant in covR mutant strain compared to wild-type strain, respectively. Numbers correspond to the orf according to NCBI designation (SMU#). Molecular weight standards (kD) are shown.

Mentions: Our one-dimensional SDS-PAGE analysis showed that at least three differentially expressed proteins (GtfB, GtfC, and BacA) could be easily detected, distinguishing the wild-type UA159 from the covR mutant IBS10 (Fig. 3A, [18]). To evaluate a possible relationship between transcriptional and translational regulations that is modulated by CovR, a two-dimensional gel electrophoresis approach was used to identify the protein spots that are differentially expressed in UA159 and IBS10. Crude cellular lysates from UA159 and IBS10 were prepared from mid-exponential-phase and subjected to proteomic analysis. A total of approximately 480 polypeptide spots could be detected in the pI range of 5.2 to 8.2 by silver staining (Fig. 5, data not shown). Three-fold difference in spot densities was used as cut-off to identify differentially expressed proteins/protein isoforms in the UA159 and IBS10 strains. Comparison of the proteomes from UA159 and IBS10 revealed that at least 41 protein spots had levels of expression altered by a change of three-fold or greater, with a P- value of ≤0.05. Among the differentially expressed proteins, 26 were down regulated while 15 spots were up regulated in IBS10 strain relative to those in the wild-type strain grown under the same condition. Fourteen spots of interest were excised from the gels, and the polypeptides were identified by mass-spectrometry. Corresponding protein identities are indicated in the Fig. 5 by their SMU# according to the GenBank S. mutans genome annotation [15]. For example, the spot identified as SMU.235 (hypothetical protein) was exclusively present in the UA159 strain, while the spot identified as SMU.155 (polynucleotide phosphorylase) was exclusively present in the IBS10 strain. On the other hand, the spot identified as SMU.1496 (galactose-6-phosphate isomerase) was present in both the strains, but was 3-fold more abundant in the ΔcovR mutant. In total, 18 peptides present in the 14 spots were unambiguously identified by mass-spectrometry. Overall, apart from GtfB and GtfC, protein expression pattern did not correlate with the microarray results.


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)

Two-dimensional gel electrophoresis analysis of S. mutans.Whole cell lysates of the wild-type UA159 (A) and ΔcovR mutant IBS10 (B) are electrophoresed in 10% SDS-PAGE. Red and black circles in Fig. 2A indicate protein spots exclusively present in wild-type strain, or at least 3-fold abundant in the wild-type strain compared to the covR mutant, respectively. Red and black circles in Fig. 2B indicate protein spots exclusively present in the covR mutant strain, or at least 3-fold abundant in covR mutant strain compared to wild-type strain, respectively. Numbers correspond to the orf according to NCBI designation (SMU#). Molecular weight standards (kD) are shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105014&req=5

pone-0020127-g005: Two-dimensional gel electrophoresis analysis of S. mutans.Whole cell lysates of the wild-type UA159 (A) and ΔcovR mutant IBS10 (B) are electrophoresed in 10% SDS-PAGE. Red and black circles in Fig. 2A indicate protein spots exclusively present in wild-type strain, or at least 3-fold abundant in the wild-type strain compared to the covR mutant, respectively. Red and black circles in Fig. 2B indicate protein spots exclusively present in the covR mutant strain, or at least 3-fold abundant in covR mutant strain compared to wild-type strain, respectively. Numbers correspond to the orf according to NCBI designation (SMU#). Molecular weight standards (kD) are shown.
Mentions: Our one-dimensional SDS-PAGE analysis showed that at least three differentially expressed proteins (GtfB, GtfC, and BacA) could be easily detected, distinguishing the wild-type UA159 from the covR mutant IBS10 (Fig. 3A, [18]). To evaluate a possible relationship between transcriptional and translational regulations that is modulated by CovR, a two-dimensional gel electrophoresis approach was used to identify the protein spots that are differentially expressed in UA159 and IBS10. Crude cellular lysates from UA159 and IBS10 were prepared from mid-exponential-phase and subjected to proteomic analysis. A total of approximately 480 polypeptide spots could be detected in the pI range of 5.2 to 8.2 by silver staining (Fig. 5, data not shown). Three-fold difference in spot densities was used as cut-off to identify differentially expressed proteins/protein isoforms in the UA159 and IBS10 strains. Comparison of the proteomes from UA159 and IBS10 revealed that at least 41 protein spots had levels of expression altered by a change of three-fold or greater, with a P- value of ≤0.05. Among the differentially expressed proteins, 26 were down regulated while 15 spots were up regulated in IBS10 strain relative to those in the wild-type strain grown under the same condition. Fourteen spots of interest were excised from the gels, and the polypeptides were identified by mass-spectrometry. Corresponding protein identities are indicated in the Fig. 5 by their SMU# according to the GenBank S. mutans genome annotation [15]. For example, the spot identified as SMU.235 (hypothetical protein) was exclusively present in the UA159 strain, while the spot identified as SMU.155 (polynucleotide phosphorylase) was exclusively present in the IBS10 strain. On the other hand, the spot identified as SMU.1496 (galactose-6-phosphate isomerase) was present in both the strains, but was 3-fold more abundant in the ΔcovR mutant. In total, 18 peptides present in the 14 spots were unambiguously identified by mass-spectrometry. Overall, apart from GtfB and GtfC, protein expression pattern did not correlate with the microarray results.

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