Curation Information

Publication: CcpA and LacD.1 affect temporal regulation of Streptococcus pyogenes virulence genes.;Kietzman CC, Caparon MG;Infection and immunity 2010 Jan; 78(1):241-52 [19841076]
TF: CcpA [A0A0C6FYN7, view regulon]
Reported TF sp.: Streptococcus pyogenes HSC5
Reported site sp.: Streptococcus pyogenes HSC5
Created by: Matthew Coveyou
Curation notes: -

Experimental Process

DNA affinity purification using the exoA and lctO intergenic region was used to pull down a binding protein, identified by MALDI-TOF mass spectrometry as ccpA. A consensus search for catabolite responsive elements (CRE’s) identified two putative binding sites on the lctO probe (narrowed to one by further probe affinity analysis) and one each on the cfa and speB promoters. qRT-PCR was performed on growth phase- and ccpA-variable cultures; lctO and cfa shared similar expression patterns, repressed in exponential phase and derepressed in stationary phase in the wild type, while ccpA mutants demonstrated loss of growth-regulated repression. SpeB transcription was upregulated in stationary phase for both mutant and wild type, though mutant strains showed decreased transcription in both phases, suggesting that – though ccpA is a speB activator – it is not a growth phase regulator for that gene. DNA affinity confirmed specific binding of ccpA to the speB promoter. qRT-PCR of mouse lesions from ccpA-variable strains confirmed earlier expression data.

Transcription Factor Binding Sites

Sites reported in the paper
Sites matched in the genome

TGTAAGCGCTAACA
TGATAACGTTATAA
TGAGAACGGTCCCA

Gene Regulation

Regulated genes for each binding site are displayed below. Gene regulation diagrams show binding sites, positively-regulated genes, negatively-regulated genes, both positively and negatively regulated genes, genes with unspecified type of regulation. For each indvidual site, experimental techniques used to determine the site are also given.

Site sequence	Regulated genes	Experimental techniques	TF function	TF type
TGTAAGCGCTAACA	L897_01860,	Experimental technique details Consensus search (ECO:0005624) Consensus search - ECO:0005624 This is a weak form of in-silico search, in which the consensus sequence for the motif is compared to genomic positions and the number of mismatches (between candidate site and consensus) is used as a measure of site-quality. - Experimental technique details DNA affinity purification (ECO:0005629) DNA affinity purification - ECO:0005629 In DNA affinity purification, DNA from promoter regions thought to be bound by the protein is labelled (e.g. biotinylated) and bound to beads (or some type of matrix). The protein is then left to interact with DNA and eventually eluted. Bound protein will remain attached to beads. This can be detected through gel electophoresis. The protein can then be sequenced by mass-spectrometry. The techinque can be used to demonstrate binding of a purified protein, or to purify the binding protein from crude extract or a mix of proteins. - Experimental technique details MALDI-TOF Mass Spectrometry (ECO:0005527) MALDI-TOF Mass Spectrometry - ECO:0005527 MALDI-TOF mass spectrometry is used to determine the mass of larger compounds such as proteins. It is a frequently used method of establishing protein identity when constructing a proteome. - Experimental technique details qRT-PCR [RNA] (ECO:0001808) qRT-PCR [RNA] - ECO:0001808 Quantitative Reverse-Transcription PCR is a modification of PCR in which RNA is first reverse transcribed into cDNA and this is amplified measuring the product (qPCR) in real time. It therefore allows one to analyze transcription by directly measuring the product (RNA) of a gene's transcription. If the gene is transcribed more, the starting product for PCR is larger and the corresponding volume of amplification is also larger. -	repressor	dimer
TGATAACGTTATAA	L897_04845	Experimental technique details Consensus search (ECO:0005624) Consensus search - ECO:0005624 This is a weak form of in-silico search, in which the consensus sequence for the motif is compared to genomic positions and the number of mismatches (between candidate site and consensus) is used as a measure of site-quality. - Experimental technique details qRT-PCR [RNA] (ECO:0001808) qRT-PCR [RNA] - ECO:0001808 Quantitative Reverse-Transcription PCR is a modification of PCR in which RNA is first reverse transcribed into cDNA and this is amplified measuring the product (qPCR) in real time. It therefore allows one to analyze transcription by directly measuring the product (RNA) of a gene's transcription. If the gene is transcribed more, the starting product for PCR is larger and the corresponding volume of amplification is also larger. -	repressor	dimer
TGAGAACGGTCCCA	L897_08645,	Experimental technique details Consensus search (ECO:0005624) Consensus search - ECO:0005624 This is a weak form of in-silico search, in which the consensus sequence for the motif is compared to genomic positions and the number of mismatches (between candidate site and consensus) is used as a measure of site-quality. - Experimental technique details DNA affinity purification (ECO:0005629) DNA affinity purification - ECO:0005629 In DNA affinity purification, DNA from promoter regions thought to be bound by the protein is labelled (e.g. biotinylated) and bound to beads (or some type of matrix). The protein is then left to interact with DNA and eventually eluted. Bound protein will remain attached to beads. This can be detected through gel electophoresis. The protein can then be sequenced by mass-spectrometry. The techinque can be used to demonstrate binding of a purified protein, or to purify the binding protein from crude extract or a mix of proteins. - Experimental technique details qRT-PCR [RNA] (ECO:0001808) qRT-PCR [RNA] - ECO:0001808 Quantitative Reverse-Transcription PCR is a modification of PCR in which RNA is first reverse transcribed into cDNA and this is amplified measuring the product (qPCR) in real time. It therefore allows one to analyze transcription by directly measuring the product (RNA) of a gene's transcription. If the gene is transcribed more, the starting product for PCR is larger and the corresponding volume of amplification is also larger. -	activator	dimer