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Campylobacter jejuni Dps protein binds DNA in the presence of iron or hydrogen peroxide.

ABSTRACT: Iron is an essential cofactor for many enzymes; however, this metal can lead to the formation of reactive oxygen species. Ferritin proteins bind and oxidize Fe(2+) to Fe(3+), storing this metal in a nonreactive form. In some organisms, a particular subfamily of ferritins, namely, Dps proteins, have the ability to bind DNA. Here we show that the Campylobacter jejuni Dps has DNA binding activity that is uniquely activated by Fe(2+) or H2O2 at below neutral pH. The Dps-DNA binding activity correlated with the ability of Dps to self-aggregate. The Dps-DNA interaction was inhibited by NaCl and Mg(2+), suggesting the formation of ionic interactions between Dps and DNA. Alkylation of cysteines affected DNA binding in the presence of H2O2 but not in the presence of Fe(2+). Replacement of all cysteines in C. jejuni Dps with serines did not affect DNA binding, excluding the participation of cysteine in H2O2 sensing. Dps was able to protect DNA in vitro from enzymatic cleavage and damage by hydroxyl radicals. A C. jejuni dps mutant was less resistant to H2O2 in vivo. The concerted activation of Dps-DNA binding in response to low pH, H2O2, and Fe(2+) may protect C. jejuni DNA during host colonization.

SUBMITTER: Huergo LF

PROVIDER: S-EPMC3624597 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Campylobacter jejuni Dps protein binds DNA in the presence of iron or hydrogen peroxide.

Huergo Luciano F LF Rahman Hossinur H Ibrahimovic Adis A Day Christopher J CJ Korolik Victoria V

Journal of bacteriology 20130222 9

Iron is an essential cofactor for many enzymes; however, this metal can lead to the formation of reactive oxygen species. Ferritin proteins bind and oxidize Fe(2+) to Fe(3+), storing this metal in a nonreactive form. In some organisms, a particular subfamily of ferritins, namely, Dps proteins, have the ability to bind DNA. Here we show that the Campylobacter jejuni Dps has DNA binding activity that is uniquely activated by Fe(2+) or H2O2 at below neutral pH. The Dps-DNA binding activity correlat ...[more]

PMID: 23435977

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Project description:Dps is a multifunctional homododecameric protein that oxidizes Fe2+ ions accumulating them in the form of Fe2O3 within its protein cavity, interacts with DNA tightly condensing bacterial nucleoid upon starvation and performs some other functions. During the last two decades from discovery of this protein, its ferroxidase activity became rather well studied, but the mechanism of Dps interaction with DNA still remains enigmatic. The crucial role of lysine residues in the unstructured N-terminal tails led to the conventional point of view that Dps binds DNA without sequence or structural specificity. However, deletion of dps changed the profile of proteins in starved cells, SELEX screen revealed genomic regions preferentially bound in vitro and certain affinity of Dps for artificial branched molecules was detected by atomic force microscopy. Here we report a non-random distribution of Dps binding sites across the bacterial chromosome in exponentially growing cells and show their enrichment with inverted repeats prone to form secondary structures. We found that the Dps-bound regions overlap with sites occupied by other nucleoid proteins, and contain overrepresented motifs typical for their consensus sequences. Of the two types of genomic domains with extensive protein occupancy, which can be highly expressed or transcriptionally silent only those that are enriched with RNA polymerase molecules were preferentially occupied by Dps. In the dps-null mutant we, therefore, observed a differentially altered expression of several targeted genes and found suppressed transcription from the dps promoter. In most cases this can be explained by the relieved interference with Dps for nucleoid proteins exploiting sequence-specific modes of DNA binding. Thus, protecting bacterial cells from different stresses during exponential growth, Dps can modulate transcriptional integrity of the bacterial chromosome hampering RNA biosynthesis from some genes via competition with RNA polymerase or, vice versa, competing with inhibitors to activate transcription.

Project description:The expression of iron-regulated systems in gram-negative bacteria is generally controlled by the Fur protein, which represses the transcription of iron-regulated promoters by using Fe2+ as a cofactor. Mutational analysis of the Campylobacter jejuni fur gene was carried out by generation of a set of mutant copies of fur which had a kanamycin or chloramphenicol resistance gene introduced into the regions encoding the N and C termini of the Fur protein. The mutated genes were recombined into the C. jejuni NCTC 11168 chromosome, and putative mutants were confirmed by Southern hybridization. C. jejuni mutants were obtained only when the resistance genes were transcribed in the same orientation as the fur gene. The C. jejuni fur mutant grew slower than the parental strain. Comparison of protein profiles of fractionated C. jejuni cells grown in low- or high-iron medium indicated derepressed expression of three iron-regulated outer membrane proteins with molecular masses of 70, 75, and 80 kDa. Characterization by N-terminal amino acid sequencing showed the 75-kDa protein to be identical to CfrA, a Campylobacter coli siderophore receptor homologue, whereas the 70-kDa protein was identified as a new siderophore receptor homologue. Periplasmic fractions contained four derepressed proteins with molecular masses of 19, 29, 32, and 36 kDa. The 19-kDa protein has been previously identified, but its function is unknown. The cytoplasmic fraction contained two iron-repressed and two iron-induced proteins with molecular masses of 26, 55, 31, and 40 kDa, respectively. The two iron-repressed proteins have been previously identified as the oxidative stress defense proteins catalase (KatA) and alkyl hydroperoxide reductase (AhpC). AhpC and KatA were still iron regulated in the fur mutant, suggesting the presence of Fur-independent iron regulation. Further analysis of the C. jejuni iron and Fur regulons by using two-dimensional gel electrophoresis demonstrated the total number of iron- and Fur-regulated proteins to be lower than for other bacterial pathogens.

Dataset Information

Campylobacter jejuni Dps protein binds DNA in the presence of iron or hydrogen peroxide.

Publications

Campylobacter jejuni Dps protein binds DNA in the presence of iron or hydrogen peroxide.

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