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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

IscR does not modulate the temporal induction of nuclease colicins.A) Measured β-galactosidase activities of strain BW25113, carrying the lac expression vector pRW50 containing various nuclease colicin promoter fragments. Each value represents the mean ± SD of at least three independent measurements, the arrow indicates the time of addition of a sub-lethal concentration of nalidixic acid (37 μM) and the dashed lines represent optical density (OD600). B) The binding of purified LexA protein to a P32 end-labelled cea8 fragment was investigated using DNase I footprint analysis. The location of the two LexA binding sites, LexA1 and LexA2 is indicated by orange boxes and the -10 and -35 promoter elements by blue boxes. The final concentration of LexA protein used in reactions was 12.5, 25, 50 and 100 nM (lanes 2–5). C) The binding of purified LexA and RNA polymerase (RNAP) to a P32 end-labelled cea8 fragment was investigated using EMSA analysis. DNA fragments were first incubated for 15 min with various concentrations of LexA (100, 200 or 400 nM) followed by the addition of 100 nM RNAP with further incubation for 15 min at 37°C. The location of free DNA, the LexA/DNA and RNAP/DNA complexes is indicated. D) The binding of purified IscR protein to P32 end-labelled colicin K (cka) or cea8 promoter fragments was assayed using EMSA analysis. The concentration of IscR used was 0.4, 0.8, 1.6 and 3.2 μM. The location of free DNA and the IscR/DNA complex is marked. E) Assays of colicin production in BW25113 and its ΔiscR derivative cells, carrying various colicin-encoding plasmids. Equal amounts of cells were collected at hourly time points after the addition of nalidixic acid (0 h) and crude cell extracts were placed into wells in agar plates overlaid with soft agar, harbouring the E. coli K-12 indicator strain DH5α pBR322. Experiments were performed in duplicate and representative growth curves are shown in S2 Fig.
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pgen.1005354.g001: IscR does not modulate the temporal induction of nuclease colicins.A) Measured β-galactosidase activities of strain BW25113, carrying the lac expression vector pRW50 containing various nuclease colicin promoter fragments. Each value represents the mean ± SD of at least three independent measurements, the arrow indicates the time of addition of a sub-lethal concentration of nalidixic acid (37 μM) and the dashed lines represent optical density (OD600). B) The binding of purified LexA protein to a P32 end-labelled cea8 fragment was investigated using DNase I footprint analysis. The location of the two LexA binding sites, LexA1 and LexA2 is indicated by orange boxes and the -10 and -35 promoter elements by blue boxes. The final concentration of LexA protein used in reactions was 12.5, 25, 50 and 100 nM (lanes 2–5). C) The binding of purified LexA and RNA polymerase (RNAP) to a P32 end-labelled cea8 fragment was investigated using EMSA analysis. DNA fragments were first incubated for 15 min with various concentrations of LexA (100, 200 or 400 nM) followed by the addition of 100 nM RNAP with further incubation for 15 min at 37°C. The location of free DNA, the LexA/DNA and RNAP/DNA complexes is indicated. D) The binding of purified IscR protein to P32 end-labelled colicin K (cka) or cea8 promoter fragments was assayed using EMSA analysis. The concentration of IscR used was 0.4, 0.8, 1.6 and 3.2 μM. The location of free DNA and the IscR/DNA complex is marked. E) Assays of colicin production in BW25113 and its ΔiscR derivative cells, carrying various colicin-encoding plasmids. Equal amounts of cells were collected at hourly time points after the addition of nalidixic acid (0 h) and crude cell extracts were placed into wells in agar plates overlaid with soft agar, harbouring the E. coli K-12 indicator strain DH5α pBR322. Experiments were performed in duplicate and representative growth curves are shown in S2 Fig.

Mentions: To study the induction of various nuclease colicins after DNA damage, we assayed the activity of colicin promoters in E. coli K-12 strain BW25113. In this experiment, different DNA fragments, carrying colicin promoters, were cloned into the lac expression vector, pRW50, to give colicin promoter::lac fusions. After inducing the DNA damage response with a sub-inhibitory concentration of nalidixic acid, we observed that the promoters of the DNA degrading colicins E2, E7 and E8, of colicins E5 and D, targeting tRNA, and of the rRNA cleaving colicin E6, are only induced after a prolonged delay (Fig 1A). Note that there is little expression from nuclease colicin promoters in the absence of DNA damage (S1 Fig).


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)

IscR does not modulate the temporal induction of nuclease colicins.A) Measured β-galactosidase activities of strain BW25113, carrying the lac expression vector pRW50 containing various nuclease colicin promoter fragments. Each value represents the mean ± SD of at least three independent measurements, the arrow indicates the time of addition of a sub-lethal concentration of nalidixic acid (37 μM) and the dashed lines represent optical density (OD600). B) The binding of purified LexA protein to a P32 end-labelled cea8 fragment was investigated using DNase I footprint analysis. The location of the two LexA binding sites, LexA1 and LexA2 is indicated by orange boxes and the -10 and -35 promoter elements by blue boxes. The final concentration of LexA protein used in reactions was 12.5, 25, 50 and 100 nM (lanes 2–5). C) The binding of purified LexA and RNA polymerase (RNAP) to a P32 end-labelled cea8 fragment was investigated using EMSA analysis. DNA fragments were first incubated for 15 min with various concentrations of LexA (100, 200 or 400 nM) followed by the addition of 100 nM RNAP with further incubation for 15 min at 37°C. The location of free DNA, the LexA/DNA and RNAP/DNA complexes is indicated. D) The binding of purified IscR protein to P32 end-labelled colicin K (cka) or cea8 promoter fragments was assayed using EMSA analysis. The concentration of IscR used was 0.4, 0.8, 1.6 and 3.2 μM. The location of free DNA and the IscR/DNA complex is marked. E) Assays of colicin production in BW25113 and its ΔiscR derivative cells, carrying various colicin-encoding plasmids. Equal amounts of cells were collected at hourly time points after the addition of nalidixic acid (0 h) and crude cell extracts were placed into wells in agar plates overlaid with soft agar, harbouring the E. coli K-12 indicator strain DH5α pBR322. Experiments were performed in duplicate and representative growth curves are shown in S2 Fig.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005354.g001: IscR does not modulate the temporal induction of nuclease colicins.A) Measured β-galactosidase activities of strain BW25113, carrying the lac expression vector pRW50 containing various nuclease colicin promoter fragments. Each value represents the mean ± SD of at least three independent measurements, the arrow indicates the time of addition of a sub-lethal concentration of nalidixic acid (37 μM) and the dashed lines represent optical density (OD600). B) The binding of purified LexA protein to a P32 end-labelled cea8 fragment was investigated using DNase I footprint analysis. The location of the two LexA binding sites, LexA1 and LexA2 is indicated by orange boxes and the -10 and -35 promoter elements by blue boxes. The final concentration of LexA protein used in reactions was 12.5, 25, 50 and 100 nM (lanes 2–5). C) The binding of purified LexA and RNA polymerase (RNAP) to a P32 end-labelled cea8 fragment was investigated using EMSA analysis. DNA fragments were first incubated for 15 min with various concentrations of LexA (100, 200 or 400 nM) followed by the addition of 100 nM RNAP with further incubation for 15 min at 37°C. The location of free DNA, the LexA/DNA and RNAP/DNA complexes is indicated. D) The binding of purified IscR protein to P32 end-labelled colicin K (cka) or cea8 promoter fragments was assayed using EMSA analysis. The concentration of IscR used was 0.4, 0.8, 1.6 and 3.2 μM. The location of free DNA and the IscR/DNA complex is marked. E) Assays of colicin production in BW25113 and its ΔiscR derivative cells, carrying various colicin-encoding plasmids. Equal amounts of cells were collected at hourly time points after the addition of nalidixic acid (0 h) and crude cell extracts were placed into wells in agar plates overlaid with soft agar, harbouring the E. coli K-12 indicator strain DH5α pBR322. Experiments were performed in duplicate and representative growth curves are shown in S2 Fig.
Mentions: To study the induction of various nuclease colicins after DNA damage, we assayed the activity of colicin promoters in E. coli K-12 strain BW25113. In this experiment, different DNA fragments, carrying colicin promoters, were cloned into the lac expression vector, pRW50, to give colicin promoter::lac fusions. After inducing the DNA damage response with a sub-inhibitory concentration of nalidixic acid, we observed that the promoters of the DNA degrading colicins E2, E7 and E8, of colicins E5 and D, targeting tRNA, and of the rRNA cleaving colicin E6, are only induced after a prolonged delay (Fig 1A). Note that there is little expression from nuclease colicin promoters in the absence of DNA damage (S1 Fig).

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