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. 2012;7(4):e34791.
doi: 10.1371/journal.pone.0034791. Epub 2012 Apr 16.

The Escherichia coli SOS gene dinF protects against oxidative stress and bile salts

Jerónimo Rodríguez-Beltrán  1 , Alexandro Rodríguez-Rojas, Javier R Guelfo, Alejandro Couce, Jesús Blázquez
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"VSports在线直播" The Escherichia coli SOS gene dinF protects against oxidative stress and bile salts

Jerónimo Rodríguez-Beltrán et al. PLoS One. 2012.

Abstract

DNA is constantly damaged by physical and chemical factors, including reactive oxygen species (ROS), such as superoxide radical (O(2)(-)), hydrogen peroxide (H(2)O(2)) and hydroxyl radical (•OH). Specific mechanisms to protect and repair DNA lesions produced by ROS have been developed in living beings. In Escherichia coli the SOS system, an inducible response activated to rescue cells from severe DNA damage, is a network that regulates the expression of more than 40 genes in response to this damage, many of them playing important roles in DNA damage tolerance mechanisms. Although the function of most of these genes has been elucidated, the activity of some others, such as dinF, remains unknown VSports手机版. The DinF deduced polypeptide sequence shows a high homology with membrane proteins of the multidrug and toxic compound extrusion (MATE) family. We describe here that expression of dinF protects against bile salts, probably by decreasing the effects of ROS, which is consistent with the observed decrease in H(2)O(2)-killing and protein carbonylation. These results, together with its ability to decrease the level of intracellular ROS, suggests that DinF can detoxify, either direct or indirectly, oxidizing molecules that can damage DNA and proteins from both the bacterial metabolism and the environment. Although the exact mechanism of DinF activity remains to be identified, we describe for the first time a role for dinF. .

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Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Sequence alignment of DinF and NorM.
Aligment (ClustalW) of sequences from E. coli K12 was done according to Uniprot tools (http://www.uniprot.org) . Predicted transmembrane domains are highlighted in grey.
Figure 2
Figure 2. Viability after H2O2 treatment.
The data represent survival percentages after 30 min of 50 mM H2O2 treatment. Shown are the strains NR10831 (WT) and its mutant derivatives ΔdinF and ΔmutT harboring the empty vector pCA24N (black) or the plasmid expressing dinF, pDinF (gray). The error bars indicate the standard deviation of four independent replicates.
Figure 3
Figure 3. ROS levels in the E. coli wild type, dinF and mutT derivatives.
Strains harboring either the empty vector pCA24N (black) or the dinF-containing plasmid pDinF (gray) treated with DHR. Data represent the mean values of three independent measurements of the spontaneous fluorescence of 15,000 cells as measured by flow cytometry. The error bars indicate the standard deviation of three independent replicates.
Figure 4
Figure 4. Protein carbonylation.
Bar graph quantitating the protein carbonylation (femtomoles of DNP) in cells harboring the empty vector pCA24N (black) or the dinF-containing plasmid pDinF (gray) in the wild type, ΔdinF and ΔmutT derivative strains, following treatment with 10 mM H2O2 for 15 min. The data are the mean values from four separate experiments and error bars represent the standard deviation.
Figure 5
Figure 5. Effect of dinF expression on spontaneous mutation rate.
The upper graph shows the mutation rate (expressed as mutations/cell/generation) of the Lac− to Lac+ reversion for the wild type NR10831 (F′CC101) and its mutant derivatives, ΔdinF and ΔmutT harboring either the empty vector pCA24N (black) or the plasmid expressing dinF, pDinF (gray). The lower plot shows the mutation rate to rifampicin resistance of the wild type BW25113 and its mutant derivatives ΔdinF and ΔmutT harboring the same plasmids. Values were calculated by the MMS-ML method. Error bars represent 95% confidence intervals.
Figure 6
Figure 6. Fitness under bile salts challenge.
Black bars represent the mean results of competition experiments between the NR10831 ΔdinF ara+ (pCA24N) and its parental wild type strain NR10831 ara− (pCA24N), and viceversa. Grey bars represent the mean results of competition experiment of strain NR10831ΔdinF (pDinF) vs NR10831 (pCA24N) in order to see the effect of the complemented mutant. Error bars represent 95% confidence intervals around the mean.
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References

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