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

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.

Figure 1

Chromosomal DNA from acid-challenged cells. The integrity of chromosomal DNA deteriorates with exposure of cells to acid challenge. Log-phase cells of parent strain (ATCC43895) were acid challenged at pH 2.0 for 15–240 min (time of exposure is indicated above each well). Genomic DNA was extracted, purified, and quantified before equal amounts were loaded into an agarose gel. (A) Visualization of ethidium bromide staining. (B) Relative intensity of each sample lane plotted against assigned migration distance.

Figure 2

Bal31 digestion of chromosomal DNA from acid-challeged cells. Bal31 nuclease digestion of chromosomal DNA recovered from acid-stressed cells revealed increased damage in dps, recA, and dps recA strains of E. coli O157:H7. Log-phase cells from the parent strain (ATCC43895), dps (FRIK47992), recA (FRIK4704-kcj05), and dps recA (FRIK4704-kcj06) were acid stressed at pH 2.0 for 2 h before genomic DNA was extracted and quantified. One micro-gram of DNA was digested with Bal31 nuclease and the entire digestion mixture was loaded into an agarose gel. (A) Visualization of ethidium bromide staining. (B) Relative intensity of each sample lane plotted against assigned migration distance.

Figure 3

Decreased acid tolerance in dps, recA, and dps recA strains. Acid challenges of parent (◆ ATCC43895), dps (▲ FRIK47992), recA (■ FRIK4704-kcj05), and dps recA (● FRIK4704-kcj06) strains were performed and the percent survival for each strain was monitored over time. Averages of at least three independent trials with standard error of the mean represented by error bars are presented.

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.

Figure 5

The Dps:DNA complex is more difficult to disrupt at low pH. Solutions containing a mixture of Dps and DNA (ratio 33:1, w/w) (300 ng total DNA) were incubated for 1 h, adjusted to the designated pH as indicated at the top of the wells, and incubated for 2 h at room temperature. DNA was extracted either with (A) with chloroform:isoamyl alcohol (24:1) in the presence of 2% SDS (+SDS) or with (B) phenol:chloroform (1:1) (-SDS), followed by precipitation with ethanol before being resolved in an agarose gel. Lane M, 1 kb DNA marker (Promega). Lane L, linear pUC18 DNA from HindIII digestion. The arrow points to the position of supercoiled plasmid DNA on each gel.

Figure 6

Low pH restores the DNA binding and protection activities of heat-denatured Dps. Heat-inactivated Dps (10 μg, 75°C, 15 min) was mixed with supercoiled pUC18 plasmid DNA (Dps:DNA (w/w) ratio of 33:1) and incubated for 1 h. The pH of the mixture was adjusted to the pH value indicated above each lane and incubated for 2 h at room temperature and analyzed by 1% agarose gel electrophoresis. Lane M, 1 kb DNA marker (Promega). Lane N, control experiment using native Dps protein, incubated at pH 7.0. Lane S, supercoiled pUC18 plasmid DNA. Lane L, HindIII linearized pUC18 plasmid. Agarose gels are visualized following staining with ethidium bromide. (A) No extraction step to purify DNA from protein. (B) DNA was extracted using chloroform:isoamyl alcohol (24:1) in the presence of 2% SDS.

Figure 7

CD spectra of Dps. CD spectra of Dps revealed different secondary conformations at different pH. The spectrum for Dps at pH 3.6 is typical of secondary structures consisting primarily of α-helices.