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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

AsnC delays cea8 expression.A) A Coomassie blue stained SDS-PAGE protein gel, which shows the protein profile of the eluates obtained from a control (lane 1) and cea8 affinity chromatography (lane 2) experiment. Proteins 1–9, which are denoted by boxes, were trypsin digested and analysed by mass spectrometry. Candidate proteins identified by affinity chromatography are listed in S2 Table. B) The cea8 promoter region was cloned into the lac expression vector pRW50 and transformed into E. coli strain BW25113 (wt) and various mutant derivatives. The arrow indicates the time of addition of nalidixic acid (37 μM) and the dashed line represents optical density (OD600). β-galactosidase activity was determined at hourly intervals. Each value represents the mean ± SD of at least three independent measurements. C) Representative growth curves of BW25113 (wt) and ΔasnC cells, carrying plasmid pColE8, which naturally expresses the DNA degrading E8 colicin. The arrow indicates the time of addition of nalidixic acid. D) Assays of colicin production in BW25113 and ΔasnC cells, carrying the pColE8. Equal amounts of cells were collected at hourly time points after the time of addition of nalidixic acid (0 h), indicated by an arrow in panel C, and crude cell extracts were applied into wells in agar plates overlaid with soft agar, harbouring the colicin sensitive strain DH5α pBR322. All experiments were performed in duplicate.
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pgen.1005354.g002: AsnC delays cea8 expression.A) A Coomassie blue stained SDS-PAGE protein gel, which shows the protein profile of the eluates obtained from a control (lane 1) and cea8 affinity chromatography (lane 2) experiment. Proteins 1–9, which are denoted by boxes, were trypsin digested and analysed by mass spectrometry. Candidate proteins identified by affinity chromatography are listed in S2 Table. B) The cea8 promoter region was cloned into the lac expression vector pRW50 and transformed into E. coli strain BW25113 (wt) and various mutant derivatives. The arrow indicates the time of addition of nalidixic acid (37 μM) and the dashed line represents optical density (OD600). β-galactosidase activity was determined at hourly intervals. Each value represents the mean ± SD of at least three independent measurements. C) Representative growth curves of BW25113 (wt) and ΔasnC cells, carrying plasmid pColE8, which naturally expresses the DNA degrading E8 colicin. The arrow indicates the time of addition of nalidixic acid. D) Assays of colicin production in BW25113 and ΔasnC cells, carrying the pColE8. Equal amounts of cells were collected at hourly time points after the time of addition of nalidixic acid (0 h), indicated by an arrow in panel C, and crude cell extracts were applied into wells in agar plates overlaid with soft agar, harbouring the colicin sensitive strain DH5α pBR322. All experiments were performed in duplicate.

Mentions: To investigate the delay in cea8 induction in SOS-induced cells we used a pull-down assay [11], using a cleared cell extract from mid-logarithmic grown, SOS induced, E. coli cells, and a biotinylated 179 bp cea8 promoter fragment as a bait. Eluted proteins were separated by SDS-polyacrylamide gel electrophoresis and nine bands were analysed by mass spectroscopy (Fig 2A). We identified 30 transcription regulators and nucleoid associated factors that had associated with the bait (S1 Table). To screen for their ability to regulate cea8 expression after DNA damage induction with nalidixic acid, we measured cea8::lac activity in deletion mutants from the Keio collection [16] and we selected strains in which a 3-fold increase in cea8 promoter activity, in comparison to the wild-type strain, was observed (S2 Table). Thus, we focused on the AsnC, StpA, OmpR, YbjK, YihW, YegW and MngR proteins and measured cea8 promoter activity following SOS induction using pRW50 cea8::lac fusion in the corresponding deletion mutant strains throughout the bacterial growth curve. Results presented in Fig 2B show that disruption of asnC resulted in the biggest effects on cea8 promoter induction after DNA damage. An intermediate increase in promoter activity was observed in the strain deficient for stpA, whilst the other deletions had a minimal effect, with our data confirming that IscR does not regulate colicin E8 expression. The StpA protein, a paralogue of the nucleoid-associated protein H-NS, forms a rigid filament along DNA, and can cause DNA bridging [17]. Furthermore, StpA can act as an RNA chaperone [18] and a transcriptional repressor [19,20], thus, it may be involved in colicin gene expression. However, here we focused on AsnC, and assayed its binding to cea8 promoter region and its effect on colicin E8 synthesis. To do this, we introduced the ΔasnC allele into a strain that harbours a cea8-encoding plasmid. After treatment of cells with a subinhibitory concentration of nalidixic acid that induced DNA damage, cell growth and colicin production was compared in the wild-type and the ΔasnC mutant. Our results show that AsnC enhances viability of the strain harbouring the colicin E8-encoding plasmid (Fig 2C). Bioassays were also used to follow colicin levels in crude cell extracts prepared from cells before and after SOS induction. The results show that, in the ΔasnC strain, colicin E8 was produced an hour earlier in comparison to the delayed synthesis in the wild-type strain (Fig 2D). This suggests that AsnC directly represses cea8 promoter activity and, in concert with LexA, ensures regulated and delayed expression of the cea8 gene.


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)

AsnC delays cea8 expression.A) A Coomassie blue stained SDS-PAGE protein gel, which shows the protein profile of the eluates obtained from a control (lane 1) and cea8 affinity chromatography (lane 2) experiment. Proteins 1–9, which are denoted by boxes, were trypsin digested and analysed by mass spectrometry. Candidate proteins identified by affinity chromatography are listed in S2 Table. B) The cea8 promoter region was cloned into the lac expression vector pRW50 and transformed into E. coli strain BW25113 (wt) and various mutant derivatives. The arrow indicates the time of addition of nalidixic acid (37 μM) and the dashed line represents optical density (OD600). β-galactosidase activity was determined at hourly intervals. Each value represents the mean ± SD of at least three independent measurements. C) Representative growth curves of BW25113 (wt) and ΔasnC cells, carrying plasmid pColE8, which naturally expresses the DNA degrading E8 colicin. The arrow indicates the time of addition of nalidixic acid. D) Assays of colicin production in BW25113 and ΔasnC cells, carrying the pColE8. Equal amounts of cells were collected at hourly time points after the time of addition of nalidixic acid (0 h), indicated by an arrow in panel C, and crude cell extracts were applied into wells in agar plates overlaid with soft agar, harbouring the colicin sensitive strain DH5α pBR322. All experiments were performed in duplicate.
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Related In: Results  -  Collection

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pgen.1005354.g002: AsnC delays cea8 expression.A) A Coomassie blue stained SDS-PAGE protein gel, which shows the protein profile of the eluates obtained from a control (lane 1) and cea8 affinity chromatography (lane 2) experiment. Proteins 1–9, which are denoted by boxes, were trypsin digested and analysed by mass spectrometry. Candidate proteins identified by affinity chromatography are listed in S2 Table. B) The cea8 promoter region was cloned into the lac expression vector pRW50 and transformed into E. coli strain BW25113 (wt) and various mutant derivatives. The arrow indicates the time of addition of nalidixic acid (37 μM) and the dashed line represents optical density (OD600). β-galactosidase activity was determined at hourly intervals. Each value represents the mean ± SD of at least three independent measurements. C) Representative growth curves of BW25113 (wt) and ΔasnC cells, carrying plasmid pColE8, which naturally expresses the DNA degrading E8 colicin. The arrow indicates the time of addition of nalidixic acid. D) Assays of colicin production in BW25113 and ΔasnC cells, carrying the pColE8. Equal amounts of cells were collected at hourly time points after the time of addition of nalidixic acid (0 h), indicated by an arrow in panel C, and crude cell extracts were applied into wells in agar plates overlaid with soft agar, harbouring the colicin sensitive strain DH5α pBR322. All experiments were performed in duplicate.
Mentions: To investigate the delay in cea8 induction in SOS-induced cells we used a pull-down assay [11], using a cleared cell extract from mid-logarithmic grown, SOS induced, E. coli cells, and a biotinylated 179 bp cea8 promoter fragment as a bait. Eluted proteins were separated by SDS-polyacrylamide gel electrophoresis and nine bands were analysed by mass spectroscopy (Fig 2A). We identified 30 transcription regulators and nucleoid associated factors that had associated with the bait (S1 Table). To screen for their ability to regulate cea8 expression after DNA damage induction with nalidixic acid, we measured cea8::lac activity in deletion mutants from the Keio collection [16] and we selected strains in which a 3-fold increase in cea8 promoter activity, in comparison to the wild-type strain, was observed (S2 Table). Thus, we focused on the AsnC, StpA, OmpR, YbjK, YihW, YegW and MngR proteins and measured cea8 promoter activity following SOS induction using pRW50 cea8::lac fusion in the corresponding deletion mutant strains throughout the bacterial growth curve. Results presented in Fig 2B show that disruption of asnC resulted in the biggest effects on cea8 promoter induction after DNA damage. An intermediate increase in promoter activity was observed in the strain deficient for stpA, whilst the other deletions had a minimal effect, with our data confirming that IscR does not regulate colicin E8 expression. The StpA protein, a paralogue of the nucleoid-associated protein H-NS, forms a rigid filament along DNA, and can cause DNA bridging [17]. Furthermore, StpA can act as an RNA chaperone [18] and a transcriptional repressor [19,20], thus, it may be involved in colicin gene expression. However, here we focused on AsnC, and assayed its binding to cea8 promoter region and its effect on colicin E8 synthesis. To do this, we introduced the ΔasnC allele into a strain that harbours a cea8-encoding plasmid. After treatment of cells with a subinhibitory concentration of nalidixic acid that induced DNA damage, cell growth and colicin production was compared in the wild-type and the ΔasnC mutant. Our results show that AsnC enhances viability of the strain harbouring the colicin E8-encoding plasmid (Fig 2C). Bioassays were also used to follow colicin levels in crude cell extracts prepared from cells before and after SOS induction. The results show that, in the ΔasnC strain, colicin E8 was produced an hour earlier in comparison to the delayed synthesis in the wild-type strain (Fig 2D). This suggests that AsnC directly represses cea8 promoter activity and, in concert with LexA, ensures regulated and delayed expression of the cea8 gene.

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