The DNA-Binding Protein from Starved Cells (Dps) Utilizes Dual Functions To Defend Cells against Multiple Stresses

Abstract

Bacteria deficient in the DNA-binding protein from starved cells (Dps) are viable under controlled conditions but show dramatically increased mortality rates when exposed to any of a wide range of stresses, including starvation, oxidative stress, metal toxicity, or thermal stress. It remains unclear whether the protective action of Dps against specific stresses derives from its DNAbinding activity, which may exclude destructive agents from the chromosomal region, or its ferroxidase activity, which neutralizes and sequesters potentially damaging chemical species. To resolve this question, we have identified the critical residues of Escherichia coli Dps that bind to DNA and modulate iron oxidation. We uncoupled the biochemical activities of Dps, creating Dps variants and mutant E. coli strains that are defective in either DNA-binding or ferroxidase activity. Quantification of the contribution of each activity to the protection of DNA integrity and cellular viability revealed that both activities of Dps are required in order to counteract many differing stresses. These findings demonstrate that Dps plays a multipurpose role in stress protection via its dual activities, explaining how Dps can be of vital importance to bacterial viability over a wide range of stresses. IMPORTANCEThe DNA-binding protein from starved cells (Dps) protects bacterial cells against many different types of stressors. We find that DNA binding and iron oxidation by Dps are performed completely independently of each other. Both biochemical activities are required to protect E. coli against stressors, as well as to protect DNA from oxidative damage in vitro. These results suggest that many stressors may cause both oxidative stress and direct DNA damage.T he ability to adapt to changes in the environment is one of the key determinants of the fitness of a species. Bacteria have evolved a multitude of ways to survive and prosper under stressful conditions, ranging from extreme measures such as sporulation to the expression of specialized stress mediation proteins (1-3). One such protein vital in stress survival is the DNA-binding protein from starved cells (Dps) (4), which is conserved to a remarkable degree in more than 300 bacterial species (5). In Escherichia coli, Dps acts as a component of several stress response pathways; it can be independently upregulated as a member of the OxyR regulon in exponentially growing cells or via S in stationary-phase cells (6). The presence of Dps enhances bacterial survival of many different stresses, including starvation, heat shock, oxidative stress, and overexposure to iron (7,8). These protective effects of Dps expression are presumably due to one or both of its dual biochemical functions, DNA binding and ferroxidase activity (9), but the molecular mechanisms and physiological consequences of these activities are not yet fully elucidated.E. coli Dps binds to DNA in vitro with no apparent sequence specificity, forming a highly stable complex (7, 10). Dps is a minor component of the E. coli nucleoid ...

Hysteresis in DNA compaction by Dps is described by an Ising model

“…It has been proposed that fluctuations in divalent cation concentrations act as a trigger for biocrystal assembly in vivo (9, 16). Dps dodecamers have an overall negative surface charge that electrostatically repels the DNA backbone, whereas positively charged lysine residues located in the disordered N termini play an important role in DNA binding (10,13,15). Interestingly, in images of biocrystals, Dps dodecamers are also tightly packed, implying existence of extensive Dps-Dps interactions (14).…”

Single-Cell Analysis of the Dps Response to Oxidative Stress

“…Cells lacking the dps gene are more sensitive to oxidative stress, showing dramatically reduced viability and elevated DNA damage (16,18,23). Because the Dps protein is a key protector in stress survival, especially during the initial stage of the exposure, the nonprobabilistic initiation of Dps production allows all affected cells to respond to the oxidative damage.…”

“…The following primers were used (the sequences of the homologous regions are underlined): forward primer MDM9, 5=-TACTTAATCTCGTTAATTACTGGGACATAACATCAAG AGGATATGAAATTATGAGTACCGCTAAATTAG-3=; and reverse primer MDM10, 5=-AGGAAGCCGCTTTTATCGGGTACTAAAGTTCT GCACCATCAGCGATGGATTTACTTGTACAGCTCGTCCA-3=. The fragment was DpnI digested, purified, and then introduced with electroporation into a W3110 dps::cat-sacB strain (23). Homologous recombination was allowed to occur for 3 h in LB medium at 37°C while shaking at 250 rpm, and cells were plated on NaCl-free LB 10% sucrose agar (counterselective for sacB).…”

“…The E. coli dps-mCherry strain was created from the E. coli K-12 strain W3110 (CGSC 4474) by replacement of the genomic dps gene by a counterselectable cat-sacB cassette (23) and subsequent replacement with a dps-mCherry cassette.…”

“…Dps oligomers are composed of 12 identical monomers, each one folded into a compact four-helix bundle (21), surrounding a central cavity that can store up to 500 iron atoms (22). The DNA-binding and ferroxidase activities of Dps are biochemically separable, but they both contribute to the maintenance of DNA integrity and cellular viability (23).…”