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, 76 (9), 2747-53

Role of Antioxidant Enzymes in Bacterial Resistance to Organic Acids

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Role of Antioxidant Enzymes in Bacterial Resistance to Organic Acids

Jose M Bruno-Bárcena et al. Appl Environ Microbiol.

Abstract

Growth in aerobic environments has been shown to generate reactive oxygen species (ROS) and to cause oxidative stress in most organisms. Antioxidant enzymes (i.e., superoxide dismutases and hydroperoxidases) and DNA repair mechanisms provide protection against ROS. Acid stress has been shown to be associated with the induction of Mn superoxide dismutase (MnSOD) in Lactococcus lactis and Staphylococcus aureus. However, the relationship between acid stress and oxidative stress is not well understood. In the present study, we showed that mutations in the gene coding for MnSOD (sodA) increased the toxicity of lactic acid at pH 3.5 in Streptococcus thermophilus. The inclusion of the iron chelators 2,2'-dipyridyl (DIP), diethienetriamine-pentaacetic acid (DTPA), and O-phenanthroline (O-Phe) provided partial protection against 330 mM lactic acid at pH 3.5. The results suggested that acid stress triggers an iron-mediated oxidative stress that can be ameliorated by MnSOD and iron chelators. These findings were further validated in Escherichia coli strains lacking both MnSOD and iron SOD (FeSOD) but expressing a heterologous MnSOD from S. thermophilus. We also found that, in E. coli, FeSOD did not provide the same protection afforded by MnSOD and that hydroperoxidases are equally important in protecting the cells against acid stress. These findings may explain the ability of some microorganisms to survive better in acidified environments, as in acid foods, during fermentation and accumulation of lactic acid or during passage through the low pH of the stomach.

Figures

FIG. 1.
FIG. 1.
Response of S. thermophilus to lactic acid stress. Unadapted (open symbols) and adapted (closed symbols) cells of exponentially growing S. thermophilus parent strain A054 (□, ▪) and its isogenic ΔsodA mutant strain, KO2-4 (○, •), preexposed or not for 30 min to 33 mM dl-lactic acid (pH 5.5), were challenged in MRS medium containing 330 mM dl-lactic acid (pH 3.5). At specific time intervals, samples were diluted and plated on agar medium to monitor cell viability. The data are means of triplicate points.
FIG. 2.
FIG. 2.
Effect of iron chelators in protecting S. thermophilus KO2-4 against lactic acid toxicity. Unadapted exponentially growing cells of S. thermophilus KO2-4 (AO54 ΔsodA) were exposed at 37°C in MRS medium containing 330 mM dl-lactic acid (pH 3.5) and in the presence of increasing concentrations of chelators (2, 2′-dipyridyl, diethylenetriamine pentaacetic acid, and O-phenanthroline). Ten-microliter aliquots were removed at 0, 15, and 30 min from the different treatments, spotted on solid medium, and incubated at 37°C as described in Materials and Methods.
FIG. 3.
FIG. 3.
Effect of heterologous MnSOD from S. thermophilus AO54 on the survival and adaptative response of the sodA sodB mutant of E. coli (NC906) exposed to lactic acid stress. Exponentially growing cells of E. coli NC906 (▵, ▴) and NC906/pSODA (○, ) were preexposed to 33 mM dl-lactic acid (pH 5.5) (closed symbols) or not exposed (open symbols). After 30 min of treatment, cells (preexposed or not exposed) were resuspended in MRS medium containing 330 mM dl-lactic acid (pH 3.5). At specific time intervals, samples were diluted and plated on LB agar medium to monitor cell viability. The data are means of triplicate points.
FIG. 4.
FIG. 4.
Roles of MnSOD and FeSOD in the absence of hydroperoxidases (KatG and KatE) on the survival and adaptative response of exponentially growing E. coli cells exposed to lactic acid stress. (A) Cells were not preadapted. (B) Cells were preadapted by exposure to 33 mM dl-lactic acid (pH 5.5) for 30 min. The unadapted and adapted cells were resuspended in MRS medium containing 330 mM dl-lactic acid (pH 3.5). At specific time intervals, samples were diluted and plated on LB agar medium to monitor cell viability. ▪, parent Kat strain (UM2); •, SodA Kat (UM2A); ▴, SodB Kat (UM2B); and ▾, SodA SodB Kat (UM2AB). The data are means of triplicate points.
FIG. 5.
FIG. 5.
Schematic presentation showing how MnSOD, iron chelators, or hydroperoxidases could protect cells against oxidative stress mediated by lactic acid. Reaction 1 shows the oxidation of labile iron-sulfur clusters by O2; reaction 2 shows the regeneration of Fe(II) from Fe(III) by the O2 (the sum of reactions 2 and 3 is also known as the Haber-Weiss reaction); reaction 3 shows the generation of HO. by Fenton chemistry. Protective molecules and/or mechanisms are shown in boxes: MnSOD inhibits reactions 1, 2, and 3; hydroperoxidases also inhibit reaction 3; iron chelators inhibit reaction 3. Lactic acid provides protons and forms an iron-lactate complex that can enhance the generation of HO..

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