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Acid stress damage of DNA is prevented by Dps binding in Escherichia coli O157:H7.

Jeong KC, Hung KF, Baumler DJ, Byrd JJ, Kaspar CW - BMC Microbiol. (2008)

Bottom Line: Dps (DNA-binding protein in starved cells) mutants of E. coli have reduced acid tolerance when compared to the parent strain although the role of Dps in acid tolerance is unclear.The contribution of RecA to acid tolerance indicated that DNA repair was important even when Dps was present.Low pH appeared to strengthen the Dps-DNA association and the secondary structure of Dps retained or formed alpha-helices at low pH.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Bacteriology, University of Wisconsin, Madison, WI 53706, USA. kcjeong@borcim.wustl.edu

ABSTRACT

Background: Acid tolerance in Escherichia coli O157:H7 contributes to persistence in its bovine host and is thought to promote passage through the gastric barrier of humans. Dps (DNA-binding protein in starved cells) mutants of E. coli have reduced acid tolerance when compared to the parent strain although the role of Dps in acid tolerance is unclear. This study investigated the mechanism by which Dps contributes to acid tolerance in E. coli O157:H7.

Results: The results from this study showed that acid stress lead to damage of chromosomal DNA, which was accentuated in dps and recA mutants. The use of Bal31, which cleaves DNA at nicks and single-stranded regions, to analyze chromosomal DNA extracted from cells challenged at pH 2.0 provided in vivo evidence of acid damage to DNA. The DNA damage in a recA mutant further corroborated the hypothesis that acid stress leads to DNA strand breaks. Under in vitro assay conditions, Dps was shown to bind plasmid DNA directly and protect it from acid-induced strand breaks. Furthermore, the extraction of DNA from Dps-DNA complexes required a denaturing agent at low pH (2.2 and 3.6) but not at higher pH (>pH4.6). Low pH also restored the DNA-binding activity of heat-denatured Dps. Circular dichroism spectra revealed that at pH 3.6 and pH 2.2 Dps maintains or forms alpha-helices that are important for Dps-DNA complex formation.

Conclusion: Results from the present work showed that acid stress results in DNA damage that is more pronounced in dps and recA mutants. The contribution of RecA to acid tolerance indicated that DNA repair was important even when Dps was present. Dps protected DNA from acid damage by binding to DNA. Low pH appeared to strengthen the Dps-DNA association and the secondary structure of Dps retained or formed alpha-helices at low pH. Further investigation into the precise interplay between DNA protection and damage repair pathways during acid stress are underway to gain additional insight.

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Dps protects DNA from acid-induced damage in vitro. M, 1 kb DNA marker (Promega). L, HindIII linearized pUC18 plasmid. (A) Representative data set on determination of optimal amount of Dps required to bind supercoiled pUC18 plasmid (300 ng). Lane 1, untreated pUC18 plasmid. Lanes 2 to 4, pUC18 plasmid incubated with Dps:DNA ratio (w/w) of 3.3, 16.3, and 33.3, respectively. (B) Representative data set showing Dps protection of DNA from acid-induced damage in vitro. Supercoiled pUC18 DNA was either mixed with Dps (ratio 33:1, w/w, +Dps) or with control buffer (-Dps) for 1 h. The pH of the reactions was then adjusted as indicated above the wells and the samples were incubated for 2 h at room temperature. DNA was then extracted with chloroform:isoamyl alcohol (24:1) in the presence of 2% SDS, precipitated with ethanol, and resolved by agarose gel electrophoresis.
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Figure 4: Dps protects DNA from acid-induced damage in vitro. M, 1 kb DNA marker (Promega). L, HindIII linearized pUC18 plasmid. (A) Representative data set on determination of optimal amount of Dps required to bind supercoiled pUC18 plasmid (300 ng). Lane 1, untreated pUC18 plasmid. Lanes 2 to 4, pUC18 plasmid incubated with Dps:DNA ratio (w/w) of 3.3, 16.3, and 33.3, respectively. (B) Representative data set showing Dps protection of DNA from acid-induced damage in vitro. Supercoiled pUC18 DNA was either mixed with Dps (ratio 33:1, w/w, +Dps) or with control buffer (-Dps) for 1 h. The pH of the reactions was then adjusted as indicated above the wells and the samples were incubated for 2 h at room temperature. DNA was then extracted with chloroform:isoamyl alcohol (24:1) in the presence of 2% SDS, precipitated with ethanol, and resolved by agarose gel electrophoresis.

Mentions: In vitro experiments were conducted to demonstrate conclusively that the physical association of Dps with DNA provided DNA protection from acid damage. First, the binding of Dps to DNA with varying concentrations of Dps was examined to determine the Dps:DNA ratio that bound all plasmid DNA under the assay conditions employed. The results from gel mobility shift assays showed that Dps completely bound pUC18 DNA (300 ng) at a ratio of approximately 33:1 (w/w) (Fig. 4A). Using this ratio, purified Dps and plasmid DNA were incubated in solutions of varying pH. After incubation, protein was extracted using chloroform:isoamyl alcohol (24:1) in the presence of 2% SDS and the plasmid integrity was determined by the relative abundance of super-coiled, nicked, and linearized forms of the plasmid. Results showed that plasmid incubated with Dps remained in the supercoiled form at all challenge pH values, with little detectable nicking (Fig 4B). In contrast, when Dps was omitted from the assay, the plasmid was nicked and/or linearized at pH 3.6 and at pH 2.6. At pH 2.2, the plasmid DNA was degraded when Dps was absent.


Acid stress damage of DNA is prevented by Dps binding in Escherichia coli O157:H7.

Jeong KC, Hung KF, Baumler DJ, Byrd JJ, Kaspar CW - BMC Microbiol. (2008)

Dps protects DNA from acid-induced damage in vitro. M, 1 kb DNA marker (Promega). L, HindIII linearized pUC18 plasmid. (A) Representative data set on determination of optimal amount of Dps required to bind supercoiled pUC18 plasmid (300 ng). Lane 1, untreated pUC18 plasmid. Lanes 2 to 4, pUC18 plasmid incubated with Dps:DNA ratio (w/w) of 3.3, 16.3, and 33.3, respectively. (B) Representative data set showing Dps protection of DNA from acid-induced damage in vitro. Supercoiled pUC18 DNA was either mixed with Dps (ratio 33:1, w/w, +Dps) or with control buffer (-Dps) for 1 h. The pH of the reactions was then adjusted as indicated above the wells and the samples were incubated for 2 h at room temperature. DNA was then extracted with chloroform:isoamyl alcohol (24:1) in the presence of 2% SDS, precipitated with ethanol, and resolved by agarose gel electrophoresis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2588596&req=5

Figure 4: Dps protects DNA from acid-induced damage in vitro. M, 1 kb DNA marker (Promega). L, HindIII linearized pUC18 plasmid. (A) Representative data set on determination of optimal amount of Dps required to bind supercoiled pUC18 plasmid (300 ng). Lane 1, untreated pUC18 plasmid. Lanes 2 to 4, pUC18 plasmid incubated with Dps:DNA ratio (w/w) of 3.3, 16.3, and 33.3, respectively. (B) Representative data set showing Dps protection of DNA from acid-induced damage in vitro. Supercoiled pUC18 DNA was either mixed with Dps (ratio 33:1, w/w, +Dps) or with control buffer (-Dps) for 1 h. The pH of the reactions was then adjusted as indicated above the wells and the samples were incubated for 2 h at room temperature. DNA was then extracted with chloroform:isoamyl alcohol (24:1) in the presence of 2% SDS, precipitated with ethanol, and resolved by agarose gel electrophoresis.
Mentions: In vitro experiments were conducted to demonstrate conclusively that the physical association of Dps with DNA provided DNA protection from acid damage. First, the binding of Dps to DNA with varying concentrations of Dps was examined to determine the Dps:DNA ratio that bound all plasmid DNA under the assay conditions employed. The results from gel mobility shift assays showed that Dps completely bound pUC18 DNA (300 ng) at a ratio of approximately 33:1 (w/w) (Fig. 4A). Using this ratio, purified Dps and plasmid DNA were incubated in solutions of varying pH. After incubation, protein was extracted using chloroform:isoamyl alcohol (24:1) in the presence of 2% SDS and the plasmid integrity was determined by the relative abundance of super-coiled, nicked, and linearized forms of the plasmid. Results showed that plasmid incubated with Dps remained in the supercoiled form at all challenge pH values, with little detectable nicking (Fig 4B). In contrast, when Dps was omitted from the assay, the plasmid was nicked and/or linearized at pH 3.6 and at pH 2.6. At pH 2.2, the plasmid DNA was degraded when Dps was absent.

Bottom Line: Dps (DNA-binding protein in starved cells) mutants of E. coli have reduced acid tolerance when compared to the parent strain although the role of Dps in acid tolerance is unclear.The contribution of RecA to acid tolerance indicated that DNA repair was important even when Dps was present.Low pH appeared to strengthen the Dps-DNA association and the secondary structure of Dps retained or formed alpha-helices at low pH.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Bacteriology, University of Wisconsin, Madison, WI 53706, USA. kcjeong@borcim.wustl.edu

ABSTRACT

Background: Acid tolerance in Escherichia coli O157:H7 contributes to persistence in its bovine host and is thought to promote passage through the gastric barrier of humans. Dps (DNA-binding protein in starved cells) mutants of E. coli have reduced acid tolerance when compared to the parent strain although the role of Dps in acid tolerance is unclear. This study investigated the mechanism by which Dps contributes to acid tolerance in E. coli O157:H7.

Results: The results from this study showed that acid stress lead to damage of chromosomal DNA, which was accentuated in dps and recA mutants. The use of Bal31, which cleaves DNA at nicks and single-stranded regions, to analyze chromosomal DNA extracted from cells challenged at pH 2.0 provided in vivo evidence of acid damage to DNA. The DNA damage in a recA mutant further corroborated the hypothesis that acid stress leads to DNA strand breaks. Under in vitro assay conditions, Dps was shown to bind plasmid DNA directly and protect it from acid-induced strand breaks. Furthermore, the extraction of DNA from Dps-DNA complexes required a denaturing agent at low pH (2.2 and 3.6) but not at higher pH (>pH4.6). Low pH also restored the DNA-binding activity of heat-denatured Dps. Circular dichroism spectra revealed that at pH 3.6 and pH 2.2 Dps maintains or forms alpha-helices that are important for Dps-DNA complex formation.

Conclusion: Results from the present work showed that acid stress results in DNA damage that is more pronounced in dps and recA mutants. The contribution of RecA to acid tolerance indicated that DNA repair was important even when Dps was present. Dps protected DNA from acid damage by binding to DNA. Low pH appeared to strengthen the Dps-DNA association and the secondary structure of Dps retained or formed alpha-helices at low pH. Further investigation into the precise interplay between DNA protection and damage repair pathways during acid stress are underway to gain additional insight.

Show MeSH
Related in: MedlinePlus