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Silencing of DNase Colicin E8 Gene Expression by a Complex Nucleoprotein Assembly Ensures Timely Colicin Induction.

Kamenšek S, Browning DF, Podlesek Z, Busby SJ, Žgur-Bertok D, Butala M - PLoS Genet. (2015)

Bottom Line: We demonstrate that a large AsnC nucleosome-like structure, in conjunction with two LexA molecules, prevent cea8 transcription initiation and that AsnC binding activity is directly modulated by L asparagine.We show that L-asparagine is an environmental factor that has a marked impact on cea8 promoter regulation.Our results show that AsnC also modulates the expression of several other DNase and RNase colicin genes but does not substantially affect pore-forming colicin K gene expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia.

ABSTRACT
Colicins are plasmid-encoded narrow spectrum antibiotics that are synthesized by strains of Escherichia coli and govern intraspecies competition. In a previous report, we demonstrated that the global transcriptional factor IscR, co dependently with the master regulator of the DNA damage response, LexA, delays induction of the pore forming colicin genes after SOS induction. Here we show that IscR is not involved in the regulation of nuclease colicins, but that the AsnC protein is. We report that AsnC, in concert with LexA, is the key controller of the temporal induction of the DNA degrading colicin E8 gene (cea8), after DNA damage. We demonstrate that a large AsnC nucleosome-like structure, in conjunction with two LexA molecules, prevent cea8 transcription initiation and that AsnC binding activity is directly modulated by L asparagine. We show that L-asparagine is an environmental factor that has a marked impact on cea8 promoter regulation. Our results show that AsnC also modulates the expression of several other DNase and RNase colicin genes but does not substantially affect pore-forming colicin K gene expression. We propose that selection pressure has "chosen" highly conserved regulators to control colicin expression in E. coli strains, enabling similar colicin gene silencing among bacteria upon exchange of colicinogenic plasmids.

No MeSH data available.


Related in: MedlinePlus

Binding of AsnC protein to cea8 is altered by L-asparagine.A) Organisation of the colicin E8 promoter region. The figure shows the DNA sequence of the cea8 promoter from position -93 to +100. The -35 and -10 core promoter elements and the predicted start of transcription (+1) are shown in bold type and the start of translation (ATG) is underlined. The location of the two LexA binding sites (LexA1 and LexA2) is shown by orange boxes. The AsnC-induced hypersensitive sites observed by DNase I footprinting are starred. B) The binding of purified AsnC protein to a P32 end-labelled cea8 fragment in the presence and absence of L-asparagine (± L-Asn) was investigated using EMSA analysis. The concentration of AsnC in lanes 2–7 and 9–14 was 0.5, 1.05, 2.1, 4.2, 8.4 and 12.6 μM, respectively. The location of free DNA, the position of the wells and the various AsnC/DNA complexes is indicated. C) An in vitro DNase I footprint experiment analysing the binding of purified AsnC to the cea8 promoter. The concentration of AsnC in lanes 2–6 and 8–12 was 0.5, 1.0, 2.1, 4.2, and 6.3 μM, respectively. The AsnC-induced hypersensitive sites are starred (also see panel A) and the location of the different protection patterns observed due to L-asparagine is indicated by red boxes.
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pgen.1005354.g003: Binding of AsnC protein to cea8 is altered by L-asparagine.A) Organisation of the colicin E8 promoter region. The figure shows the DNA sequence of the cea8 promoter from position -93 to +100. The -35 and -10 core promoter elements and the predicted start of transcription (+1) are shown in bold type and the start of translation (ATG) is underlined. The location of the two LexA binding sites (LexA1 and LexA2) is shown by orange boxes. The AsnC-induced hypersensitive sites observed by DNase I footprinting are starred. B) The binding of purified AsnC protein to a P32 end-labelled cea8 fragment in the presence and absence of L-asparagine (± L-Asn) was investigated using EMSA analysis. The concentration of AsnC in lanes 2–7 and 9–14 was 0.5, 1.05, 2.1, 4.2, 8.4 and 12.6 μM, respectively. The location of free DNA, the position of the wells and the various AsnC/DNA complexes is indicated. C) An in vitro DNase I footprint experiment analysing the binding of purified AsnC to the cea8 promoter. The concentration of AsnC in lanes 2–6 and 8–12 was 0.5, 1.0, 2.1, 4.2, and 6.3 μM, respectively. The AsnC-induced hypersensitive sites are starred (also see panel A) and the location of the different protection patterns observed due to L-asparagine is indicated by red boxes.

Mentions: The AsnC protein is a member of the Lrp/AsnC family of regulators, which often assemble to form wheel-like octamers and whose DNA binding activity can be modified by small molecules, such as amino acids [13]. AsnC regulates the expression of its own gene, asnC, and the asnA gene, encoding for a synthetase that catalyses the ammonia-dependent conversion of aspartate to asparagine [14]. To investigate the binding of AsnC to the cea8 promoter (Fig 3A), we over-expressed and purified the AsnC protein and performed in vitro experiments in the presence or absence of the amino acid L-asparagine. EMSA experiments show that several AsnC molecules can interact with cea8 (Fig 3) and that in the presence of L-asparagine a number of distinct complexes can be observed (Fig 3B). In the absence of L-asparagine, at higher AsnC concentrations, DNA remained in the wells of the gel, indicating that high molecular weight nucleoprotein complexes had formed.


Silencing of DNase Colicin E8 Gene Expression by a Complex Nucleoprotein Assembly Ensures Timely Colicin Induction.

Kamenšek S, Browning DF, Podlesek Z, Busby SJ, Žgur-Bertok D, Butala M - PLoS Genet. (2015)

Binding of AsnC protein to cea8 is altered by L-asparagine.A) Organisation of the colicin E8 promoter region. The figure shows the DNA sequence of the cea8 promoter from position -93 to +100. The -35 and -10 core promoter elements and the predicted start of transcription (+1) are shown in bold type and the start of translation (ATG) is underlined. The location of the two LexA binding sites (LexA1 and LexA2) is shown by orange boxes. The AsnC-induced hypersensitive sites observed by DNase I footprinting are starred. B) The binding of purified AsnC protein to a P32 end-labelled cea8 fragment in the presence and absence of L-asparagine (± L-Asn) was investigated using EMSA analysis. The concentration of AsnC in lanes 2–7 and 9–14 was 0.5, 1.05, 2.1, 4.2, 8.4 and 12.6 μM, respectively. The location of free DNA, the position of the wells and the various AsnC/DNA complexes is indicated. C) An in vitro DNase I footprint experiment analysing the binding of purified AsnC to the cea8 promoter. The concentration of AsnC in lanes 2–6 and 8–12 was 0.5, 1.0, 2.1, 4.2, and 6.3 μM, respectively. The AsnC-induced hypersensitive sites are starred (also see panel A) and the location of the different protection patterns observed due to L-asparagine is indicated by red boxes.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482635&req=5

pgen.1005354.g003: Binding of AsnC protein to cea8 is altered by L-asparagine.A) Organisation of the colicin E8 promoter region. The figure shows the DNA sequence of the cea8 promoter from position -93 to +100. The -35 and -10 core promoter elements and the predicted start of transcription (+1) are shown in bold type and the start of translation (ATG) is underlined. The location of the two LexA binding sites (LexA1 and LexA2) is shown by orange boxes. The AsnC-induced hypersensitive sites observed by DNase I footprinting are starred. B) The binding of purified AsnC protein to a P32 end-labelled cea8 fragment in the presence and absence of L-asparagine (± L-Asn) was investigated using EMSA analysis. The concentration of AsnC in lanes 2–7 and 9–14 was 0.5, 1.05, 2.1, 4.2, 8.4 and 12.6 μM, respectively. The location of free DNA, the position of the wells and the various AsnC/DNA complexes is indicated. C) An in vitro DNase I footprint experiment analysing the binding of purified AsnC to the cea8 promoter. The concentration of AsnC in lanes 2–6 and 8–12 was 0.5, 1.0, 2.1, 4.2, and 6.3 μM, respectively. The AsnC-induced hypersensitive sites are starred (also see panel A) and the location of the different protection patterns observed due to L-asparagine is indicated by red boxes.
Mentions: The AsnC protein is a member of the Lrp/AsnC family of regulators, which often assemble to form wheel-like octamers and whose DNA binding activity can be modified by small molecules, such as amino acids [13]. AsnC regulates the expression of its own gene, asnC, and the asnA gene, encoding for a synthetase that catalyses the ammonia-dependent conversion of aspartate to asparagine [14]. To investigate the binding of AsnC to the cea8 promoter (Fig 3A), we over-expressed and purified the AsnC protein and performed in vitro experiments in the presence or absence of the amino acid L-asparagine. EMSA experiments show that several AsnC molecules can interact with cea8 (Fig 3) and that in the presence of L-asparagine a number of distinct complexes can be observed (Fig 3B). In the absence of L-asparagine, at higher AsnC concentrations, DNA remained in the wells of the gel, indicating that high molecular weight nucleoprotein complexes had formed.

Bottom Line: We demonstrate that a large AsnC nucleosome-like structure, in conjunction with two LexA molecules, prevent cea8 transcription initiation and that AsnC binding activity is directly modulated by L asparagine.We show that L-asparagine is an environmental factor that has a marked impact on cea8 promoter regulation.Our results show that AsnC also modulates the expression of several other DNase and RNase colicin genes but does not substantially affect pore-forming colicin K gene expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia.

ABSTRACT
Colicins are plasmid-encoded narrow spectrum antibiotics that are synthesized by strains of Escherichia coli and govern intraspecies competition. In a previous report, we demonstrated that the global transcriptional factor IscR, co dependently with the master regulator of the DNA damage response, LexA, delays induction of the pore forming colicin genes after SOS induction. Here we show that IscR is not involved in the regulation of nuclease colicins, but that the AsnC protein is. We report that AsnC, in concert with LexA, is the key controller of the temporal induction of the DNA degrading colicin E8 gene (cea8), after DNA damage. We demonstrate that a large AsnC nucleosome-like structure, in conjunction with two LexA molecules, prevent cea8 transcription initiation and that AsnC binding activity is directly modulated by L asparagine. We show that L-asparagine is an environmental factor that has a marked impact on cea8 promoter regulation. Our results show that AsnC also modulates the expression of several other DNase and RNase colicin genes but does not substantially affect pore-forming colicin K gene expression. We propose that selection pressure has "chosen" highly conserved regulators to control colicin expression in E. coli strains, enabling similar colicin gene silencing among bacteria upon exchange of colicinogenic plasmids.

No MeSH data available.


Related in: MedlinePlus