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Escherichia coli YafP protein modulates DNA damaging property of the nitroaromatic compounds.

Gutierrez A, Elez M, Clermont O, Denamur E, Matic I - Nucleic Acids Res. (2011)

Bottom Line: Using a murine septicaemia model, we showed that YafP activity reduced the bacterial fitness in the absence of PolIV.The YafP antimutator activity was independent of the PolIV activity.Given that YafP was annotated as a putative acetyltransferase, it could be that YafP participates in the metabolic transformation of genotoxic compounds, hence modulating the balance between their mutagenicity and cytotoxicity.

View Article: PubMed Central - PubMed

Affiliation: Faculté de Médecine Paris Descartes, Inserm U1001, Université Paris Descartes, Paris, France.

ABSTRACT
Escherichia coli SOS functions constitute a multifaceted response to DNA damage. We undertook to study the role of yafP, a SOS gene with unknown function. yafP is part of an operon also containing the dinB gene coding for DNA Polymerase IV (PolIV). Our phylogenetic analysis showed that the gene content of this operon is variable but that the dinB and the yafP genes are conserved in the majority of E. coli natural isolates. Therefore, we studied if these proteins are functionally linked. Using a murine septicaemia model, we showed that YafP activity reduced the bacterial fitness in the absence of PolIV. Similarly, YafP increased cytotoxicity of two DNA damaging nitroaromatic compounds, 4-nitroquinoline-1-oxide (NQO) and nitrofurazone, in the absence of PolIV. The fact that PolIV counterbalances YafP-induced cytotoxicity could explain why these two genes are transcriptionally linked. We also studied the involvement of YafP in genotoxic-stress induced mutagenesis and found that PolIV and YafP reduced NQO-induced mutagenicity. The YafP antimutator activity was independent of the PolIV activity. Given that YafP was annotated as a putative acetyltransferase, it could be that YafP participates in the metabolic transformation of genotoxic compounds, hence modulating the balance between their mutagenicity and cytotoxicity.

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Synthesis of the YafP–YFP protein fusion. A translational fusion between yafP gene and the yellow fluorescent protein (YFP) was used to monitor the synthesis of the YafP–YFP protein fusion in cells. (A) Growth curve of untreated cells. a, b and c represent three different time points at which the cells were sampled for (B) measurement of fluorescence using FACS analysis. (C) Cells sampled at b time point were treated with 1 µg/ml of the SOS-inducing DNA damaging agent Mitomycine C (MMC) and fluorescence was measured 30 min and 45 min after. Experiments were repeated three times. Representative curves are shown.
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Figure 2: Synthesis of the YafP–YFP protein fusion. A translational fusion between yafP gene and the yellow fluorescent protein (YFP) was used to monitor the synthesis of the YafP–YFP protein fusion in cells. (A) Growth curve of untreated cells. a, b and c represent three different time points at which the cells were sampled for (B) measurement of fluorescence using FACS analysis. (C) Cells sampled at b time point were treated with 1 µg/ml of the SOS-inducing DNA damaging agent Mitomycine C (MMC) and fluorescence was measured 30 min and 45 min after. Experiments were repeated three times. Representative curves are shown.

Mentions: In order to study the conditions under which YafP is translated, we constructed a translational fusion between the yafP gene and the yellow fluorescent protein (YFP) and used fluorescence activated cell sorting (FACS) to monitor the fluorescence of cells. We followed cell fluorescence at different cell growth phases in the absence of SOS-inducing treatment (Figure 2A and B). Cell fluorescence was low during logarithmic growth phase, but it became much stronger upon entry to stationary phase. Exponentially growing cells treated with the SOS-inducing DNA damaging agent MMC produced a large amount of YafP–YFP protein fusion (Figure 2C). This is in accordance with the observation that the expression of E. coli K-12 dinB operon, including yafP, increases upon UV-induced SOS response (3,13). Therefore, it can be concluded that YafP protein is synthesized in CFT073 cells in spite of the fact that it requires translational frameshifting and that the regulation of CFT073 dinB operon appears to be similar to that one in K-12.Figure 2.


Escherichia coli YafP protein modulates DNA damaging property of the nitroaromatic compounds.

Gutierrez A, Elez M, Clermont O, Denamur E, Matic I - Nucleic Acids Res. (2011)

Synthesis of the YafP–YFP protein fusion. A translational fusion between yafP gene and the yellow fluorescent protein (YFP) was used to monitor the synthesis of the YafP–YFP protein fusion in cells. (A) Growth curve of untreated cells. a, b and c represent three different time points at which the cells were sampled for (B) measurement of fluorescence using FACS analysis. (C) Cells sampled at b time point were treated with 1 µg/ml of the SOS-inducing DNA damaging agent Mitomycine C (MMC) and fluorescence was measured 30 min and 45 min after. Experiments were repeated three times. Representative curves are shown.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105422&req=5

Figure 2: Synthesis of the YafP–YFP protein fusion. A translational fusion between yafP gene and the yellow fluorescent protein (YFP) was used to monitor the synthesis of the YafP–YFP protein fusion in cells. (A) Growth curve of untreated cells. a, b and c represent three different time points at which the cells were sampled for (B) measurement of fluorescence using FACS analysis. (C) Cells sampled at b time point were treated with 1 µg/ml of the SOS-inducing DNA damaging agent Mitomycine C (MMC) and fluorescence was measured 30 min and 45 min after. Experiments were repeated three times. Representative curves are shown.
Mentions: In order to study the conditions under which YafP is translated, we constructed a translational fusion between the yafP gene and the yellow fluorescent protein (YFP) and used fluorescence activated cell sorting (FACS) to monitor the fluorescence of cells. We followed cell fluorescence at different cell growth phases in the absence of SOS-inducing treatment (Figure 2A and B). Cell fluorescence was low during logarithmic growth phase, but it became much stronger upon entry to stationary phase. Exponentially growing cells treated with the SOS-inducing DNA damaging agent MMC produced a large amount of YafP–YFP protein fusion (Figure 2C). This is in accordance with the observation that the expression of E. coli K-12 dinB operon, including yafP, increases upon UV-induced SOS response (3,13). Therefore, it can be concluded that YafP protein is synthesized in CFT073 cells in spite of the fact that it requires translational frameshifting and that the regulation of CFT073 dinB operon appears to be similar to that one in K-12.Figure 2.

Bottom Line: Using a murine septicaemia model, we showed that YafP activity reduced the bacterial fitness in the absence of PolIV.The YafP antimutator activity was independent of the PolIV activity.Given that YafP was annotated as a putative acetyltransferase, it could be that YafP participates in the metabolic transformation of genotoxic compounds, hence modulating the balance between their mutagenicity and cytotoxicity.

View Article: PubMed Central - PubMed

Affiliation: Faculté de Médecine Paris Descartes, Inserm U1001, Université Paris Descartes, Paris, France.

ABSTRACT
Escherichia coli SOS functions constitute a multifaceted response to DNA damage. We undertook to study the role of yafP, a SOS gene with unknown function. yafP is part of an operon also containing the dinB gene coding for DNA Polymerase IV (PolIV). Our phylogenetic analysis showed that the gene content of this operon is variable but that the dinB and the yafP genes are conserved in the majority of E. coli natural isolates. Therefore, we studied if these proteins are functionally linked. Using a murine septicaemia model, we showed that YafP activity reduced the bacterial fitness in the absence of PolIV. Similarly, YafP increased cytotoxicity of two DNA damaging nitroaromatic compounds, 4-nitroquinoline-1-oxide (NQO) and nitrofurazone, in the absence of PolIV. The fact that PolIV counterbalances YafP-induced cytotoxicity could explain why these two genes are transcriptionally linked. We also studied the involvement of YafP in genotoxic-stress induced mutagenesis and found that PolIV and YafP reduced NQO-induced mutagenicity. The YafP antimutator activity was independent of the PolIV activity. Given that YafP was annotated as a putative acetyltransferase, it could be that YafP participates in the metabolic transformation of genotoxic compounds, hence modulating the balance between their mutagenicity and cytotoxicity.

Show MeSH
Related in: MedlinePlus