Role of the dpr Product in Oxygen Tolerance in Streptococcus mutans

Abstract

We have previously identified and characterized the alkyl hydroperoxide reductase of Streptococcus mutans, which consists of two components, Nox-1 and AhpC. Deletion of both nox-1 and ahpC had no effect on the sensitivity of S. mutans to cumene hydroperoxide or H 2 O 2 , implying that the existence of another antioxidant system(s) independent of the Nox-1-AhpC system compensates for the deficiency. Here, a new antioxidant gene (dpr for Dps-like peroxide resistance gene) was isolated from the S. mutans chromosome by its ability to complement an ahpCF deletion mutant of Escherichia coli with a tert-butyl hydroperoxide-hypersensitive phenotype. The dpr gene complemented the defect in peroxidase activity caused by the deletion of nox-1 and ahpC in S. mutans. Under aerobic conditions, the dpr disruption mutant carrying a spectinomycin resistance gene (dpr::Spc r mutant) grew as well as wild-type S. mutans in liquid medium. However, the dpr::Spc r mutant could not form colonies on an agar plate under air. In addition, neither the dpr::Spc r ahpC::Em r ::nox-1 triple mutant nor the dpr::Spc r sod::Em r double mutant was able to grow aerobically in liquid medium. The 20-kDa dpr gene product Dpr is an iron-binding protein. Synthesis of Dpr was induced by exposure of S. mutans cells to air. We propose a mechanism by which Dpr confers aerotolerance on S. mutans.Bacteria contain certain enzymes capable of reacting with oxygen, and they cannot avoid confronting harmful reactive oxygen species, including superoxide anion (O 2 Ϫ ), hydrogen peroxide (H 2 O 2 ), and organic hydroperoxide, if they are exposed to air. To live in the presence of oxygen, they have to convert these reactive oxygen species to nontoxic molecules. Therefore, enzymes such as superoxide dismutases (SOD), catalases, and peroxidases are ubiquitously distributed in aerotolerant bacteria.Lactic acid bacteria, including Streptococcus mutans, lack cytochromes and other heme-containing proteins. Most lactic acid bacteria, except several lactobacilli which acquire catalase activity if a source of heme is added to their growth medium (42), also lack catalase. However, they can grow in the presence of air. In view of the defense against oxygen toxicity, the lack of catalase in lactic acid bacteria is inconsistent with their aerotolerance. Mechanisms by which lactic acid bacteria cope with peroxide-mediated stress are therefore an area under active investigation. We previously identified H 2 O 2 -forming NADH oxidase (Nox-1) in S. mutans and found that Nox-1 is homologous with a flavoprotein component, AhpF, of Salmonella typhimurium alkyl hydroperoxide reductase (AhpR), consisting of AhpF and AhpC (16,17,30). We also identified ahpC, which is homologous with ahpC of S. typhimurium, upstream of nox-1 in S. mutans (30). Analyses of purified AhpC together with Nox-1 have verified that these proteins act as a bicomponent peroxidase system in S. mutans, catalyzing the NADH-dependent reduction of organic hydroperoxides or H 2 O 2 to their respective alcohols and/or H 2 ...

Substantial DNA damage from submicromolar intracellular hydrogen peroxide detected in Hpx - mutants of Escherichia coli

“…The digested PCR products were cloned into pCKR101 vector to generate the pDpr plasmid. Overproduction of active Dpr protein was confirmed by native PAGE stained with Ferene S (26 To avoid interactions between iron and conventional buffering compounds, none were used. Instead, solutions were adjusted with dilute NaOH to the desired pH, and the pH of the reaction solution was measured at the conclusion of the reaction to verify that it had not changed significantly (Ͻ0.2 pH units) (14).…”

Molecular Basis of H2O2 Resistance Mediated by Streptococcal Dpr

“…Dps family members share a conserved amino acid motif, similar to mammalian ferritin L-subunit iron nucleation center, in the N-terminal halves of the proteins (14,(22)(23)(24). Because several members, including the streptococcal Dpr (11,25), are known to bind iron (17,22,26,27), the conserved motif is believed to serve a functional role. It has been suggested that the motif catalyzes iron oxidation by its putative ferroxidase activity and also directs the formation of an iron core into the inner cavity of the oligomer (17,22,28,29).…”

“…Gram-positive bacteria, including the important human pathogens Streptococcus pyogenes and Streptococcus pneumoniae (11), relatively little is known of the molecular mechanisms of its action. The primary amino acid sequence shares similarity with Escherichia coli Dps (DNA-binding protein from starved cells) (12,13), a prototype for a large group of similar oligomeric proteins (14), which is abundantly expressed in starved cells and involved in DNA protection by DNA-Dps biocrystal formation (15,16).…”

Reassessment of Protein Stability, DNA Binding, and Protection of Mycobacterium smegmatis Dps

“…In this study, the subunit dissociation, DNA binding, and protection properties of native, untagged M. smegmatis Dps (DpsMs) 2 were assessed in parallel with those of the tagged protein (DpsMs-His) to establish possible differences between the two proteins ascribable to the C-terminal tag. Untagged and tagged dodecamers dissociated into dimers in a similar fashion.…”

“…The proteins of the Dps (DNA-binding proteins from starved cells) family are expressed by most bacteria under a variety of stress conditions to protect DNA against oxidative damage and other detrimental factors (1)(2)(3). DNA protection is achieved by a dual action.…”