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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 smt operon in the CovR deficient strain.(A) Protein profile of the wild-type UA159, the ΔcovR mutant IBS10, and the complemented IBS10 strain. Whole cell lysates from cultures grown to the mid exponential phase were resolved on a 4–20% gradient gel, and stained with PageBlue (Fermentas). M: Prestained molecular weight marker; lane 1: UA159 (wild-type); lane 2: IBS10 (covR mutant); and lane 3: IBS10/pIB30 (complemented covR mutant). The 313-kDa protein band was excised from the gel and analyzed by mass spectrometry to verify its identity. (B) RT-PCR analysis of the genes in the smt locus. RNA was harvested from cultures at the mid-exponential phase of growth and subjected to real time RT-PCR analysis using primer pairs specific for bacA, bacC, gyrA, psaA, and SMU.1349 genes, as described in the text. Strains used include: UA159 (wild-type), IBS10 (covR mutant), and IBS10/pIB30 (complemented covR mutant). Real time RT-PCR reactions were performed in triplicate and the mean values with standard deviations are shown.
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pone-0020127-g003: Differential expression of smt operon in the CovR deficient strain.(A) Protein profile of the wild-type UA159, the ΔcovR mutant IBS10, and the complemented IBS10 strain. Whole cell lysates from cultures grown to the mid exponential phase were resolved on a 4–20% gradient gel, and stained with PageBlue (Fermentas). M: Prestained molecular weight marker; lane 1: UA159 (wild-type); lane 2: IBS10 (covR mutant); and lane 3: IBS10/pIB30 (complemented covR mutant). The 313-kDa protein band was excised from the gel and analyzed by mass spectrometry to verify its identity. (B) RT-PCR analysis of the genes in the smt locus. RNA was harvested from cultures at the mid-exponential phase of growth and subjected to real time RT-PCR analysis using primer pairs specific for bacA, bacC, gyrA, psaA, and SMU.1349 genes, as described in the text. Strains used include: UA159 (wild-type), IBS10 (covR mutant), and IBS10/pIB30 (complemented covR mutant). Real time RT-PCR reactions were performed in triplicate and the mean values with standard deviations are shown.

Mentions: In an earlier study we also observed that a high molecular weight protein, which migrated to a point above the 175-kDa marker, was specifically expressed in the wild-type UA159, but was absent in the isogenic ΔcovR mutant IBS10 (see Fig. 1 of reference [18]. To confirm our previous observation, protein profiles of the wild-type (UA159) and the covR mutant IBS10 were resolved using a 4–20% SDS-PAGE. As shown in Fig. 3A, in the crude cell lysates prepared from the exponentially growing cultures, a band above the largest marker (250-kDa) appears to be expressed in the wild-type strain, but is absent in the crude lysates of IBS10. This band was also visible in the stationary phase culture lysates from UA159, but not in the lysates of IBS10 (data not shown). To identify this band, the band was excised from the gel, and the protein was analyzed by mass-spectrometry. Twenty peptide fragments were identified by mass-spectrometry analysis, and a BLAST search revealed that the peptides corresponded to BacA (SMU.1342), which is encoded by smt locus within the TnSmu2 GI. To verify that the expression of the BacA protein was indeed up-regulated by CovR, total cellular protein was also extracted from IBS10 carrying pIB30, a plasmid containing full-length wild-type covR [18], and its protein profile was compared with the wild-type UA159 and ΔcovR mutant strain IBS10 (Fig. 3A). The profile of the complemented covR mutant strain displayed the same band as the wild-type strain, suggesting that the loss of the BacA protein in crude cell extract of IBS10 was due to the inactivation of covR.


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 smt operon in the CovR deficient strain.(A) Protein profile of the wild-type UA159, the ΔcovR mutant IBS10, and the complemented IBS10 strain. Whole cell lysates from cultures grown to the mid exponential phase were resolved on a 4–20% gradient gel, and stained with PageBlue (Fermentas). M: Prestained molecular weight marker; lane 1: UA159 (wild-type); lane 2: IBS10 (covR mutant); and lane 3: IBS10/pIB30 (complemented covR mutant). The 313-kDa protein band was excised from the gel and analyzed by mass spectrometry to verify its identity. (B) RT-PCR analysis of the genes in the smt locus. RNA was harvested from cultures at the mid-exponential phase of growth and subjected to real time RT-PCR analysis using primer pairs specific for bacA, bacC, gyrA, psaA, and SMU.1349 genes, as described in the text. Strains used include: UA159 (wild-type), IBS10 (covR mutant), and IBS10/pIB30 (complemented covR mutant). Real time RT-PCR reactions were performed in triplicate and the mean values with standard deviations are shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105014&req=5

pone-0020127-g003: Differential expression of smt operon in the CovR deficient strain.(A) Protein profile of the wild-type UA159, the ΔcovR mutant IBS10, and the complemented IBS10 strain. Whole cell lysates from cultures grown to the mid exponential phase were resolved on a 4–20% gradient gel, and stained with PageBlue (Fermentas). M: Prestained molecular weight marker; lane 1: UA159 (wild-type); lane 2: IBS10 (covR mutant); and lane 3: IBS10/pIB30 (complemented covR mutant). The 313-kDa protein band was excised from the gel and analyzed by mass spectrometry to verify its identity. (B) RT-PCR analysis of the genes in the smt locus. RNA was harvested from cultures at the mid-exponential phase of growth and subjected to real time RT-PCR analysis using primer pairs specific for bacA, bacC, gyrA, psaA, and SMU.1349 genes, as described in the text. Strains used include: UA159 (wild-type), IBS10 (covR mutant), and IBS10/pIB30 (complemented covR mutant). Real time RT-PCR reactions were performed in triplicate and the mean values with standard deviations are shown.
Mentions: In an earlier study we also observed that a high molecular weight protein, which migrated to a point above the 175-kDa marker, was specifically expressed in the wild-type UA159, but was absent in the isogenic ΔcovR mutant IBS10 (see Fig. 1 of reference [18]. To confirm our previous observation, protein profiles of the wild-type (UA159) and the covR mutant IBS10 were resolved using a 4–20% SDS-PAGE. As shown in Fig. 3A, in the crude cell lysates prepared from the exponentially growing cultures, a band above the largest marker (250-kDa) appears to be expressed in the wild-type strain, but is absent in the crude lysates of IBS10. This band was also visible in the stationary phase culture lysates from UA159, but not in the lysates of IBS10 (data not shown). To identify this band, the band was excised from the gel, and the protein was analyzed by mass-spectrometry. Twenty peptide fragments were identified by mass-spectrometry analysis, and a BLAST search revealed that the peptides corresponded to BacA (SMU.1342), which is encoded by smt locus within the TnSmu2 GI. To verify that the expression of the BacA protein was indeed up-regulated by CovR, total cellular protein was also extracted from IBS10 carrying pIB30, a plasmid containing full-length wild-type covR [18], and its protein profile was compared with the wild-type UA159 and ΔcovR mutant strain IBS10 (Fig. 3A). The profile of the complemented covR mutant strain displayed the same band as the wild-type strain, suggesting that the loss of the BacA protein in crude cell extract of IBS10 was due to the inactivation of covR.

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