TY - JOUR
T1 - Use of a phosphorylation site mutant to identify distinct modes of gene repression by the control of virulence regulator (CovR) in Streptococcus pyogenes
AU - Horstmann, Nicola
AU - Sahasrabhojane, Pranoti
AU - Yao, Hui
AU - Su, Xiaoping
AU - Shelburne, Samuel A.
N1 - Publisher Copyright:
© 2017 American Society for Microbiology.
PY - 2017/9/1
Y1 - 2017/9/1
N2 - Control of the virulence regulator/sensor kinase (CovRS) two-component system (TCS) serves as a model for investigating the impact of signaling pathways on the pathogenesis of Gram-positive bacteria. However, the molecular mechanisms by which CovR, an OmpR/PhoB family response regulator, controls virulence gene expression are poorly defined, partly due to the labile nature of its aspartate phosphorylation site. To better understand the regulatory effect of phosphorylated CovR, we generated the phosphorylation site mutant strain 10870-CovR-D53E, which we predicted to have a constitutive CovR phosphorylation phenotype. Interestingly, this strain showed CovR activity only for a subset of the CovR regulon, which allowed for classification of CovR-influenced genes into D53E-regulated and D53E-nonregulated groups. Inspection of the promoter sequences of genes belonging to each group revealed distinct promoter architectures with respect to the location and number of putative CovR-binding sites. Electrophoretic mobility shift analysis demonstrated that recombinant CovR-D53E protein retains its ability to bind promoter DNA from both CovR-D53E-regulated and -nonregulated groups, implying that factors other than mere DNA binding are crucial for gene regulation. In fact, we found that CovR-D53E is incapable of dimerization, a process thought to be critical to OmpR/PhoB family regulator function. Thus, our global analysis of CovRD53E indicates dimerization-dependent and dimerization-independent modes of CovR-mediated repression, thereby establishing distinct mechanisms by which this critical regulator coordinates virulence gene expression.
AB - Control of the virulence regulator/sensor kinase (CovRS) two-component system (TCS) serves as a model for investigating the impact of signaling pathways on the pathogenesis of Gram-positive bacteria. However, the molecular mechanisms by which CovR, an OmpR/PhoB family response regulator, controls virulence gene expression are poorly defined, partly due to the labile nature of its aspartate phosphorylation site. To better understand the regulatory effect of phosphorylated CovR, we generated the phosphorylation site mutant strain 10870-CovR-D53E, which we predicted to have a constitutive CovR phosphorylation phenotype. Interestingly, this strain showed CovR activity only for a subset of the CovR regulon, which allowed for classification of CovR-influenced genes into D53E-regulated and D53E-nonregulated groups. Inspection of the promoter sequences of genes belonging to each group revealed distinct promoter architectures with respect to the location and number of putative CovR-binding sites. Electrophoretic mobility shift analysis demonstrated that recombinant CovR-D53E protein retains its ability to bind promoter DNA from both CovR-D53E-regulated and -nonregulated groups, implying that factors other than mere DNA binding are crucial for gene regulation. In fact, we found that CovR-D53E is incapable of dimerization, a process thought to be critical to OmpR/PhoB family regulator function. Thus, our global analysis of CovRD53E indicates dimerization-dependent and dimerization-independent modes of CovR-mediated repression, thereby establishing distinct mechanisms by which this critical regulator coordinates virulence gene expression.
KW - Dimerization
KW - Pathogenesis
KW - Regulation of gene expression
KW - Streptococcus pyogenes
KW - Transcriptional regulation
KW - Two-component regulatory systems
KW - Virulence regulation
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U2 - 10.1128/JB.00835-16
DO - 10.1128/JB.00835-16
M3 - Article
C2 - 28289082
AN - SCOPUS:85027973629
SN - 0021-9193
VL - 199
JO - Journal of bacteriology
JF - Journal of bacteriology
IS - 18
M1 - e00835-16
ER -