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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 modulates the temporal induction of different DNA- and RNA-degrading colicins.Expression of the (A) cea2::lacZ and the (B) cea6::lacZ fusions in strain BW25113 (wt) and the ΔiscR and ΔasnC mutants. For panels A and B each value represents the mean ± SD of at least two independent measurements, the arrow indicates the time of the addition of a sub-lethal concentration of nalidixic acid (37 μM) and the dashed lines represent OD600. C) Growth curves of BW25113 (wt) and ΔasnC cells harbouring naturally occurring plasmids encoding either the DNA degrading colicin E2, the tRNA cleaving colicin E5 or the rRNA cleaving colicin E6. The arrow indicates the time of addition of nalidixic acid. Experiments were performed in duplicate and representative growth curves are shown. D) Assays of colicin production in BW25113 and ΔasnC 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 applied into wells in agar plates, overlaid with soft agar harbouring the colicin sensitive strain DH5α pBR322. The experiments were performed in duplicate. E) AsnC protein has little effect on the expression of the pore-forming colicin K. The activity of the cka promoter was measured in strain BW25113 (wt) and its ΔiscR or ΔasnC mutant derivatives. Each value represents the mean ± SD of four independent measurements, the arrow indicates the time of addition of a sub-lethal concentration of nalidixic acid (37 μM) and the dashed lines represent the OD600.
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pgen.1005354.g006: AsnC modulates the temporal induction of different DNA- and RNA-degrading colicins.Expression of the (A) cea2::lacZ and the (B) cea6::lacZ fusions in strain BW25113 (wt) and the ΔiscR and ΔasnC mutants. For panels A and B each value represents the mean ± SD of at least two independent measurements, the arrow indicates the time of the addition of a sub-lethal concentration of nalidixic acid (37 μM) and the dashed lines represent OD600. C) Growth curves of BW25113 (wt) and ΔasnC cells harbouring naturally occurring plasmids encoding either the DNA degrading colicin E2, the tRNA cleaving colicin E5 or the rRNA cleaving colicin E6. The arrow indicates the time of addition of nalidixic acid. Experiments were performed in duplicate and representative growth curves are shown. D) Assays of colicin production in BW25113 and ΔasnC 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 applied into wells in agar plates, overlaid with soft agar harbouring the colicin sensitive strain DH5α pBR322. The experiments were performed in duplicate. E) AsnC protein has little effect on the expression of the pore-forming colicin K. The activity of the cka promoter was measured in strain BW25113 (wt) and its ΔiscR or ΔasnC mutant derivatives. Each value represents the mean ± SD of four independent measurements, the arrow indicates the time of addition of a sub-lethal concentration of nalidixic acid (37 μM) and the dashed lines represent the OD600.

Mentions: To determine whether AsnC modulates the expression of other colicins, we assayed DNase colicin E2 (cea2) and rRNase colicin E6 (cea6) promoter activities following SOS induction with nalidixic acid using cea6::lacZ and cea2::lacZ promoter fusions in the wild-type, ΔiscR and ΔasnC strains. Results illustrated in Fig 6A and 6B show that the disruption of asnC resulted in elevated cea2 and cea6 promoter activity immediately after DNA damage induction, when compared to the wild-type and the ΔiscR strains. This indicates that AsnC, rather than IscR, is a key transcriptional repressor of the cea2 and cea6 promoters. In addition, we transferred the colicinogenic plasmids for these DNase and RNase colicins into the ΔasnC and wild-type strains. Following the induction of DNA damage, cell growth (Fig 6C) and colicin production (Fig 6D) was monitored in both strains. In the absence of asnC, cells failed to reach as high optical density as in the wild-type strain, suggesting that elevated colicin expression and cell lysis had taken place (Fig 6C). Cell extracts were prepared from cultures before and after DNA damage induction and colicin levels compared by a colicin production bioassay (Fig 6D). After SOS induction, nuclease colicin synthesis was induced earlier for colicins E2, E5 and E6 in the ΔasnC strain, in comparison to the wild-type strain, indicating that AsnC directly modulates the expression of a number of other colicin genes. Alignment of the cea8 promoter region sequence with corresponding sequences from colicin E2, E5 and E6 indicated that these promoters are very similar (S4 Fig) and, thus, similar co-ordinated regulation is perhaps to be expected.


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 modulates the temporal induction of different DNA- and RNA-degrading colicins.Expression of the (A) cea2::lacZ and the (B) cea6::lacZ fusions in strain BW25113 (wt) and the ΔiscR and ΔasnC mutants. For panels A and B each value represents the mean ± SD of at least two independent measurements, the arrow indicates the time of the addition of a sub-lethal concentration of nalidixic acid (37 μM) and the dashed lines represent OD600. C) Growth curves of BW25113 (wt) and ΔasnC cells harbouring naturally occurring plasmids encoding either the DNA degrading colicin E2, the tRNA cleaving colicin E5 or the rRNA cleaving colicin E6. The arrow indicates the time of addition of nalidixic acid. Experiments were performed in duplicate and representative growth curves are shown. D) Assays of colicin production in BW25113 and ΔasnC 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 applied into wells in agar plates, overlaid with soft agar harbouring the colicin sensitive strain DH5α pBR322. The experiments were performed in duplicate. E) AsnC protein has little effect on the expression of the pore-forming colicin K. The activity of the cka promoter was measured in strain BW25113 (wt) and its ΔiscR or ΔasnC mutant derivatives. Each value represents the mean ± SD of four independent measurements, the arrow indicates the time of addition of a sub-lethal concentration of nalidixic acid (37 μM) and the dashed lines represent the OD600.
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Related In: Results  -  Collection

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pgen.1005354.g006: AsnC modulates the temporal induction of different DNA- and RNA-degrading colicins.Expression of the (A) cea2::lacZ and the (B) cea6::lacZ fusions in strain BW25113 (wt) and the ΔiscR and ΔasnC mutants. For panels A and B each value represents the mean ± SD of at least two independent measurements, the arrow indicates the time of the addition of a sub-lethal concentration of nalidixic acid (37 μM) and the dashed lines represent OD600. C) Growth curves of BW25113 (wt) and ΔasnC cells harbouring naturally occurring plasmids encoding either the DNA degrading colicin E2, the tRNA cleaving colicin E5 or the rRNA cleaving colicin E6. The arrow indicates the time of addition of nalidixic acid. Experiments were performed in duplicate and representative growth curves are shown. D) Assays of colicin production in BW25113 and ΔasnC 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 applied into wells in agar plates, overlaid with soft agar harbouring the colicin sensitive strain DH5α pBR322. The experiments were performed in duplicate. E) AsnC protein has little effect on the expression of the pore-forming colicin K. The activity of the cka promoter was measured in strain BW25113 (wt) and its ΔiscR or ΔasnC mutant derivatives. Each value represents the mean ± SD of four independent measurements, the arrow indicates the time of addition of a sub-lethal concentration of nalidixic acid (37 μM) and the dashed lines represent the OD600.
Mentions: To determine whether AsnC modulates the expression of other colicins, we assayed DNase colicin E2 (cea2) and rRNase colicin E6 (cea6) promoter activities following SOS induction with nalidixic acid using cea6::lacZ and cea2::lacZ promoter fusions in the wild-type, ΔiscR and ΔasnC strains. Results illustrated in Fig 6A and 6B show that the disruption of asnC resulted in elevated cea2 and cea6 promoter activity immediately after DNA damage induction, when compared to the wild-type and the ΔiscR strains. This indicates that AsnC, rather than IscR, is a key transcriptional repressor of the cea2 and cea6 promoters. In addition, we transferred the colicinogenic plasmids for these DNase and RNase colicins into the ΔasnC and wild-type strains. Following the induction of DNA damage, cell growth (Fig 6C) and colicin production (Fig 6D) was monitored in both strains. In the absence of asnC, cells failed to reach as high optical density as in the wild-type strain, suggesting that elevated colicin expression and cell lysis had taken place (Fig 6C). Cell extracts were prepared from cultures before and after DNA damage induction and colicin levels compared by a colicin production bioassay (Fig 6D). After SOS induction, nuclease colicin synthesis was induced earlier for colicins E2, E5 and E6 in the ΔasnC strain, in comparison to the wild-type strain, indicating that AsnC directly modulates the expression of a number of other colicin genes. Alignment of the cea8 promoter region sequence with corresponding sequences from colicin E2, E5 and E6 indicated that these promoters are very similar (S4 Fig) and, thus, similar co-ordinated regulation is perhaps to be expected.

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