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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

LexA and AsnC can bind to cea8 promoter region simultaneously.A) The panel shows an EMSA analysing the binding of purified LexA and AsnC protein to a P32 end-labelled cea8 fragment in the presence of L-asparagine (+ L-Asn). 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. LexA protein was present in reactions at concentration of 400 nM. The location of free DNA, the position of the wells and the various protein/DNA complexes is marked. B) The binding of purified LexA and AsnC proteins to the cea8 promoter fragment was studied by DNase I footprinting in the presence and absence of L-asparagine (± L-Asn). AsnC was included at concentrations of 1.0, 2.1 and 4.2 μM and LexA at a concentration of 400 nM. The prominent hypersensitive bands, corresponding to positions +1 and -16, that are produced by the concurrent binding of AsnC and LexA, are starred. Red boxes indicate the position of AsnC interactions, which are affected by L-Asn. 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. C) The panel shows an extended run of the footprint analysis from panel B, focusing on positions -80 to -60 upstream of the transcription start site. Labels are identical to those described above. Panels D-F) show models of the different nucleoprotein complexes formed at the cea8 promoter by the binding of LexA and AsnC.
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pgen.1005354.g004: LexA and AsnC can bind to cea8 promoter region simultaneously.A) The panel shows an EMSA analysing the binding of purified LexA and AsnC protein to a P32 end-labelled cea8 fragment in the presence of L-asparagine (+ L-Asn). 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. LexA protein was present in reactions at concentration of 400 nM. The location of free DNA, the position of the wells and the various protein/DNA complexes is marked. B) The binding of purified LexA and AsnC proteins to the cea8 promoter fragment was studied by DNase I footprinting in the presence and absence of L-asparagine (± L-Asn). AsnC was included at concentrations of 1.0, 2.1 and 4.2 μM and LexA at a concentration of 400 nM. The prominent hypersensitive bands, corresponding to positions +1 and -16, that are produced by the concurrent binding of AsnC and LexA, are starred. Red boxes indicate the position of AsnC interactions, which are affected by L-Asn. 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. C) The panel shows an extended run of the footprint analysis from panel B, focusing on positions -80 to -60 upstream of the transcription start site. Labels are identical to those described above. Panels D-F) show models of the different nucleoprotein complexes formed at the cea8 promoter by the binding of LexA and AsnC.

Mentions: Since our data show that AsnC binds at multiple locations and alters the architecture of colicin E8 regulatory region, as well as binding within LexA target sites (Fig 3), we tested if AsnC and LexA can simultaneously bind to the cea8 promoter region. To investigate this, we performed EMSA analysis on the cea8 promoter fragment. Using purified LexA and AsnC, in the presence of L-asparagine, we observed a large nucleoprotein complex composed of at least two AsnC functional oligomers, presumably octamers [13], and two LexA dimers interacting at cea8 (Fig 4A). Note, that LexA was used at a concentration of 400 nM at which LexA repressor occupies both LexA binding sites within cea8 (Fig 1B). To determine whether occupancy of the DNA by AsnC affects the binding of LexA at the cea8 promoter region, we performed DNase I footprint analysis and compared signatures of LexA and AsnC in the presence or absence of L-asparagine. In both conditions, LexA repressors bound to tandem targets just downstream of the -10 promoter element (Fig 4B). As observed for AsnC binding in Fig 3C, the addition of L-asparagine also modulated the binding of AsnC in the LexA-AsnC nucleoprotein complex (Fig 4B and 4C). In the AsnC-LexA-cea8 complex, specific hypersensitive sites were apparent (determined by stars in Fig 4B), suggesting that the binding of both proteins subtly alters the structure or trajectory of the DNA around -10 element (Fig 4D–4F). Thus, we conclude that concurrent binding of LexA and AsnC to the cea8 regulatory region ensures delayed induction of the DNase E8 synthesis after DNA damage.


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)

LexA and AsnC can bind to cea8 promoter region simultaneously.A) The panel shows an EMSA analysing the binding of purified LexA and AsnC protein to a P32 end-labelled cea8 fragment in the presence of L-asparagine (+ L-Asn). 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. LexA protein was present in reactions at concentration of 400 nM. The location of free DNA, the position of the wells and the various protein/DNA complexes is marked. B) The binding of purified LexA and AsnC proteins to the cea8 promoter fragment was studied by DNase I footprinting in the presence and absence of L-asparagine (± L-Asn). AsnC was included at concentrations of 1.0, 2.1 and 4.2 μM and LexA at a concentration of 400 nM. The prominent hypersensitive bands, corresponding to positions +1 and -16, that are produced by the concurrent binding of AsnC and LexA, are starred. Red boxes indicate the position of AsnC interactions, which are affected by L-Asn. 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. C) The panel shows an extended run of the footprint analysis from panel B, focusing on positions -80 to -60 upstream of the transcription start site. Labels are identical to those described above. Panels D-F) show models of the different nucleoprotein complexes formed at the cea8 promoter by the binding of LexA and AsnC.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005354.g004: LexA and AsnC can bind to cea8 promoter region simultaneously.A) The panel shows an EMSA analysing the binding of purified LexA and AsnC protein to a P32 end-labelled cea8 fragment in the presence of L-asparagine (+ L-Asn). 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. LexA protein was present in reactions at concentration of 400 nM. The location of free DNA, the position of the wells and the various protein/DNA complexes is marked. B) The binding of purified LexA and AsnC proteins to the cea8 promoter fragment was studied by DNase I footprinting in the presence and absence of L-asparagine (± L-Asn). AsnC was included at concentrations of 1.0, 2.1 and 4.2 μM and LexA at a concentration of 400 nM. The prominent hypersensitive bands, corresponding to positions +1 and -16, that are produced by the concurrent binding of AsnC and LexA, are starred. Red boxes indicate the position of AsnC interactions, which are affected by L-Asn. 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. C) The panel shows an extended run of the footprint analysis from panel B, focusing on positions -80 to -60 upstream of the transcription start site. Labels are identical to those described above. Panels D-F) show models of the different nucleoprotein complexes formed at the cea8 promoter by the binding of LexA and AsnC.
Mentions: Since our data show that AsnC binds at multiple locations and alters the architecture of colicin E8 regulatory region, as well as binding within LexA target sites (Fig 3), we tested if AsnC and LexA can simultaneously bind to the cea8 promoter region. To investigate this, we performed EMSA analysis on the cea8 promoter fragment. Using purified LexA and AsnC, in the presence of L-asparagine, we observed a large nucleoprotein complex composed of at least two AsnC functional oligomers, presumably octamers [13], and two LexA dimers interacting at cea8 (Fig 4A). Note, that LexA was used at a concentration of 400 nM at which LexA repressor occupies both LexA binding sites within cea8 (Fig 1B). To determine whether occupancy of the DNA by AsnC affects the binding of LexA at the cea8 promoter region, we performed DNase I footprint analysis and compared signatures of LexA and AsnC in the presence or absence of L-asparagine. In both conditions, LexA repressors bound to tandem targets just downstream of the -10 promoter element (Fig 4B). As observed for AsnC binding in Fig 3C, the addition of L-asparagine also modulated the binding of AsnC in the LexA-AsnC nucleoprotein complex (Fig 4B and 4C). In the AsnC-LexA-cea8 complex, specific hypersensitive sites were apparent (determined by stars in Fig 4B), suggesting that the binding of both proteins subtly alters the structure or trajectory of the DNA around -10 element (Fig 4D–4F). Thus, we conclude that concurrent binding of LexA and AsnC to the cea8 regulatory region ensures delayed induction of the DNase E8 synthesis after DNA damage.

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