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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

Binding of CovR to the putative promoter regions of the genes indicated below the respective panels.Increasing concentrations 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 specific DNA.
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pone-0020127-g004: Binding of CovR to the putative promoter regions of the genes indicated below the respective panels.Increasing concentrations 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 specific DNA.

Mentions: Our earlier studies demonstrated direct binding of CovR to promoters of four repressed genes and one activated gene. To further investigate the interaction of CovR with the regulated promoters, a total of 15 promoters were subjected to electrophoretic mobility shift assays (EMSA) with purified His-CovR protein. Among the promoters, CovR repressed four of them while the rest were activated by CovR (promoters used for EMSA are indicated in Table S2). For all 15 promoters except one, shifts in the mobility were observed when CovR was added, indicating direct binding of CovR to the DNA sequences upstream of both CovR-activated and -repressed genes (Fig. 4, data not shown). We did not observe any shift after incubation of CovR to the SMU.136 promoter, suggesting that regulation of this gene by CovR is indirect. We also did not observe any shift when CovR was added to a DNA fragment corresponding to the nlmAB operon, a result consistent with the absence of CovR regulation of this locus and allowing for this DNA fragment to be used as a negative control for the specificity of CovR binding to regulated promoters. Specificity of the binding interaction for each of the regulated promoters was also supported by competition with excess unlabeled probe (Fig. 4, lanes 5). Therefore, CovR specifically binds to the target promoters to activate or repress transcription of these genes, and supports the results of our microarray analysis.


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)

Binding of CovR to the putative promoter regions of the genes indicated below the respective panels.Increasing concentrations 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 specific DNA.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105014&req=5

pone-0020127-g004: Binding of CovR to the putative promoter regions of the genes indicated below the respective panels.Increasing concentrations 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 specific DNA.
Mentions: Our earlier studies demonstrated direct binding of CovR to promoters of four repressed genes and one activated gene. To further investigate the interaction of CovR with the regulated promoters, a total of 15 promoters were subjected to electrophoretic mobility shift assays (EMSA) with purified His-CovR protein. Among the promoters, CovR repressed four of them while the rest were activated by CovR (promoters used for EMSA are indicated in Table S2). For all 15 promoters except one, shifts in the mobility were observed when CovR was added, indicating direct binding of CovR to the DNA sequences upstream of both CovR-activated and -repressed genes (Fig. 4, data not shown). We did not observe any shift after incubation of CovR to the SMU.136 promoter, suggesting that regulation of this gene by CovR is indirect. We also did not observe any shift when CovR was added to a DNA fragment corresponding to the nlmAB operon, a result consistent with the absence of CovR regulation of this locus and allowing for this DNA fragment to be used as a negative control for the specificity of CovR binding to regulated promoters. Specificity of the binding interaction for each of the regulated promoters was also supported by competition with excess unlabeled probe (Fig. 4, lanes 5). Therefore, CovR specifically binds to the target promoters to activate or repress transcription of these genes, and supports the results of our microarray analysis.

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