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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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CovR is required for competence.Fold-difference in transformation efficiency of the S. mutans UA159 (wild-type) compared to IBS10 (ΔcovR) with two circular plasmid DNAs (pDL276 and pOri23) and linear DNA (a PCR fragment from plasmid pIB75, see text). Mean values with standard error from two (pOri23 and pIB75) or three (pDL276) independent experiments are shown.
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pone-0020127-g002: CovR is required for competence.Fold-difference in transformation efficiency of the S. mutans UA159 (wild-type) compared to IBS10 (ΔcovR) with two circular plasmid DNAs (pDL276 and pOri23) and linear DNA (a PCR fragment from plasmid pIB75, see text). Mean values with standard error from two (pOri23 and pIB75) or three (pDL276) independent experiments are shown.

Mentions: Our transcriptome study indicated that several competence related genes, including coiA, dprA, comF/comFC, comEA/comEC, and the genes belonging to the comY operon (Table S2) were down regulated in the covR mutant strains. The extent of down regulation varied between 1.6 to 3.6-fold. Quantitative RT-PCR confirmed that at least dprA was also down regulated in the covR mutant to a similar extent as in the transcriptome study. Thus, tit appears that a functional CovR is required for optimal competence gene expression. To determine if activation of these genes by CovR correlates with competence of S. mutans, both the wild-type UA159 and and ΔcovR mutant IBS10 strains were used as recipient for the foreign DNA. Three different types of DNA molecules were used: pDL276 plasmid that replicates via rolling-circle mechanism; pOri23 plasmid that replicates via theta mechanism; and a linear DNA fragment generated by PCR that contains an erythromycin resistance marker flanked by ∼0.5-kb homology corresponding to the SMU.261 locus (a locus unlinked and unrelated to CovR). Both of the pDL276 and pOri23 plasmids were able to replicate in S. mutans, providing resistance to kanamycin and erythromycin, respectively, whereas the PCR product encoding erythromycin resistance (Table S1) could provide this phenotype only after integration into the chromosome. In the latter case, in addition to the defect in transformation, possible deficiencies in the recombination pathway could also be detected. As shown Fig. 2, in all cases, the transformation efficiency of the IBS10 strain was lower compared to the wild- type strain. A reduction of ∼2.7-fold was obtained with transformation of the linear DNA fragment, while a reduction of ∼5.0-fold was obtained for the replicating plasmid transformations. Thus, we conclude that CovR plays an important role in the competence of S. mutans.


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)

CovR is required for competence.Fold-difference in transformation efficiency of the S. mutans UA159 (wild-type) compared to IBS10 (ΔcovR) with two circular plasmid DNAs (pDL276 and pOri23) and linear DNA (a PCR fragment from plasmid pIB75, see text). Mean values with standard error from two (pOri23 and pIB75) or three (pDL276) independent experiments are shown.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105014&req=5

pone-0020127-g002: CovR is required for competence.Fold-difference in transformation efficiency of the S. mutans UA159 (wild-type) compared to IBS10 (ΔcovR) with two circular plasmid DNAs (pDL276 and pOri23) and linear DNA (a PCR fragment from plasmid pIB75, see text). Mean values with standard error from two (pOri23 and pIB75) or three (pDL276) independent experiments are shown.
Mentions: Our transcriptome study indicated that several competence related genes, including coiA, dprA, comF/comFC, comEA/comEC, and the genes belonging to the comY operon (Table S2) were down regulated in the covR mutant strains. The extent of down regulation varied between 1.6 to 3.6-fold. Quantitative RT-PCR confirmed that at least dprA was also down regulated in the covR mutant to a similar extent as in the transcriptome study. Thus, tit appears that a functional CovR is required for optimal competence gene expression. To determine if activation of these genes by CovR correlates with competence of S. mutans, both the wild-type UA159 and and ΔcovR mutant IBS10 strains were used as recipient for the foreign DNA. Three different types of DNA molecules were used: pDL276 plasmid that replicates via rolling-circle mechanism; pOri23 plasmid that replicates via theta mechanism; and a linear DNA fragment generated by PCR that contains an erythromycin resistance marker flanked by ∼0.5-kb homology corresponding to the SMU.261 locus (a locus unlinked and unrelated to CovR). Both of the pDL276 and pOri23 plasmids were able to replicate in S. mutans, providing resistance to kanamycin and erythromycin, respectively, whereas the PCR product encoding erythromycin resistance (Table S1) could provide this phenotype only after integration into the chromosome. In the latter case, in addition to the defect in transformation, possible deficiencies in the recombination pathway could also be detected. As shown Fig. 2, in all cases, the transformation efficiency of the IBS10 strain was lower compared to the wild- type strain. A reduction of ∼2.7-fold was obtained with transformation of the linear DNA fragment, while a reduction of ∼5.0-fold was obtained for the replicating plasmid transformations. Thus, we conclude that CovR plays an important role in the competence of S. mutans.

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