Events associated with DNA replication disruption are not observed in hydrogen peroxide-treated Escherichia coli.

UV irradiation induces pyrimidine dimers that block polymerases and disrupt the replisome. Restoring replication depends on the recF pathway proteins which process and maintain the replication fork DNA to allow the lesion to be repaired before replication resumes. Oxidative DNA lesions, such as those induced by hydrogen peroxide (H2O2), are often thought to require similar processing events, yet far less is known about how cells process oxidative damage during replication. Here we show that replication is not disrupted by H2O2-induced DNA damage in vivo. Following an initial inhibition, replication resumes in the absence of either lesion removal or RecF-processing. Restoring DNA synthesis depends on the presence of manganese in the medium, which we show is required for replication, but not repair to occur. The results demonstrate that replication is enzymatically inactivated, rather than physically disrupted by H2O2-induced DNA damage; indicate that inactivation is likely caused by oxidation of an iron-dependent replication or replication-associated protein that requires manganese to restore activity and synthesis; and address a long standing paradox as to why oxidative glycosylase mutants are defective in repair, yet not hypersensitive to H2O2. The oxygen-sensitive pausing may represent an adaptation that prevents replication from occurring under potentially lethal or mutagenic conditions.

Static Growth Promotes PrrF and 2-Alkyl-4(1H)-Quinolone Regulation of Type VI Secretion Protein Expression in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that is frequently associated with both acute and chronic infections. P. aeruginosa possesses a complex regulatory network that modulates nutrient acquisition and virulence, but our knowledge of these networks is largely based on studies with shaking cultures, which are not likely representative of conditions during infection. Here, we provide proteomic, metabolic, and genetic evidence that regulation by iron, a critical metallonutrient, is altered in static P. aeruginosa cultures. Specifically, we observed a loss of iron-induced expression of proteins for oxidative phosphorylation, tricarboxylic acid (TCA) cycle metabolism under static conditions. Moreover, we identified type VI secretion as a target of iron regulation in P. aeruginosa cells under static but not shaking conditions, and we present evidence that this regulation occurs via PrrF small regulatory RNA (sRNA)-dependent production of 2-alkyl-4(1H)-quinolone metabolites. These results yield new iron regulation paradigms in an important opportunistic pathogen and highlight the need to redefine iron homeostasis in static microbial communities.IMPORTANCE Host-mediated iron starvation is a broadly conserved signal for microbial pathogens to upregulate expression of virulence traits required for successful infection. Historically, global iron regulatory studies in microorganisms have been conducted in shaking cultures to ensure culture homogeneity, yet these conditions are likely not reflective of growth during infection. Pseudomonas aeruginosa is a well-studied opportunistic pathogen and model organism for iron regulatory studies. Iron homeostasis is maintained through the Fur protein and PrrF small regulatory sRNAs, the functions of which are highly conserved in many other bacterial species. In the current study, we examined how static growth affects the known iron and PrrF regulons of P. aeruginosa, leading to the discovery of novel PrrF-regulated virulence processes. This study demonstrates how the utilization of distinct growth models can enhance our understanding of basic physiological processes that may also affect pathogenesis.

Manganese Is Required for the Rapid Recovery of DNA Synthesis following Oxidative Challenge in Escherichia coli.

Divalent metals such as iron and manganese play an important role in the cellular response to oxidative challenges and are required as cofactors by many enzymes. However, how these metals affect replication after oxidative challenge is not known. Here, we show that replication in Escherichia coli is inhibited following a challenge with hydrogen peroxide and requires manganese for the rapid recovery of DNA synthesis. We show that the manganese-dependent recovery of DNA synthesis occurs independent of lesion repair, modestly improves cell survival, and is associated with elevated rates of mutagenesis. The Mn-dependent mutagenesis involves both replicative and translesion polymerases and requires prior disruption by H2O2 to occur. Taking these findings together, we propose that replication in E. coli is likely to utilize an iron-dependent enzyme(s) that becomes oxidized and inactivated during oxidative challenges. The data suggest that manganese remetallates these or alternative enzymes to allow genomic DNA replication to resume, although with reduced fidelity.IMPORTANCE Iron and manganese play important roles in how cell's cope with oxygen stress. However, how these metals affect the ability of cells to replicate after oxidative challenges is not known. Here, we show that replication in Escherichia coli is inhibited following a challenge with hydrogen peroxide and requires manganese for the rapid recovery of DNA synthesis. The manganese-dependent recovery of DNA synthesis occurs independently of lesion repair and modestly improves survival, but it also increases the mutation rate in cells. The results imply that replication in E. coli is likely to utilize an iron-dependent enzyme(s) that becomes oxidized and inactivated during oxidative challenges. We propose that manganese remetallates these or alternative enzymes to allow genomic DNA replication to resume, although with reduced fidelity.

Replication Rapidly Recovers and Continues in the Presence of Hydroxyurea in Escherichia coli.

In both prokaryotes and eukaryotes, hydroxyurea is suggested to inhibit DNA replication by inactivating ribonucleotide reductase and depleting deoxyribonucleoside triphosphate pools. In this study, we show that the inhibition of replication in Escherichia coli is transient even at concentrations of 0.1 M hydroxyurea and that replication rapidly recovers and continues in its presence. The recovery of replication does not require the alternative ribonucleotide reductases NrdEF and NrdDG or the translesion DNA polymerases II (Pol II), Pol IV, and Pol V. Ribonucleotides are incorporated at higher frequencies during replication in the presence of hydroxyurea. However, they do not contribute significantly to the observed synthesis or toxicity. Hydroxyurea toxicity was observed only under conditions where the stability of hydroxyurea was compromised and by-products known to damage DNA directly were allowed to accumulate. The results demonstrate that hydroxyurea is not a direct or specific inhibitor of DNA synthesis in vivo and that the transient inhibition observed is most likely due to a general depletion of iron cofactors from enzymes when 0.1 M hydroxyurea is initially applied. Finally, the results support previous studies suggesting that hydroxyurea toxicity is mediated primarily through direct DNA damage induced by the breakdown products of hydroxyurea, rather than by inhibition of replication or depletion of deoxyribonucleotide levels in the cell.IMPORTANCE Hydroxyurea is commonly suggested to function by inhibiting DNA replication through the inactivation of ribonucleotide reductase and depleting deoxyribonucleoside triphosphate pools. Here, we show that hydroxyurea only transiently inhibits replication in Escherichia coli before replication rapidly recovers and continues in the presence of the drug. The recovery of replication does not depend on alternative ribonucleotide reductases, translesion synthesis, or RecA. Further, we show that hydroxyurea toxicity is observed only in the presence of toxic intermediates that accumulate when hydroxyurea breaks down, damage DNA, and induce lethality. The results demonstrate that hydroxyurea toxicity is mediated indirectly by the formation of DNA damage, rather than by inhibition of replication or depletion of deoxyribonucleotide levels in the cell.