Escherichia coli Periplasmic Thiol Peroxidase Acts as Lipid Hydroperoxide Peroxidase and the Principal Antioxidative Function during Anaerobic Growth

Abstract

To clarify the enzymatic property of Escherichia coli periplasmic thiol peroxidase (p20), the specific peroxidase activity toward peroxides was compared with other bacterial thiol peroxidases. p20 has the most substrate preference and peroxidase activity toward organic hydroperoxide. Furthermore, p20 exerted the most potent lipid peroxidase activity. Despite that the mutation of p20 caused the highest susceptibility toward organic hydroperoxide and heat stress, the cellular level of p20 did not respond to the exposure of oxidative stress. Expression level of p20 during anaerobic growth was sustained at the ϳ50% level compared with that of the aerobic growth. Viability of aerobic p20⌬ without glucose was reduced to the ϳ65% level of isogenic strains, whereas viability of aerobic p20⌬ with 0.5% glucose supplement was sustained. The deletion of p20 resulted in a gradual loss of the cell viability during anaerobic growth. At the stationary phase, the viability of p20⌬ was down to ϳ10% level of parent strains. An analysis of the protein carbonyl contents of p20⌬ as a marker for cellular oxidation indicates that severe reduction of viability of anaerobic p20⌬ was caused by cumulative oxidative stress. P20⌬ showed hypersensitivity toward membrane-soluble organic hydroperoxides. An analysis of protein carbonyl and lipid hydroperoxide contents in the membrane of the stress-imposed p20⌬ demonstrates that the severe reduction of viability was caused by cumulative oxidative stress on the membrane. Taken together, present data uncover in vivo function for p20 as a lipid hydroperoxide peroxidase and demonstrate that, as the result, p20 acts as the principal antioxidant in the anaerobic habitats.

Salmonella enterica Serovar Typhimurium Colonizing the Lumen of the Chicken Intestine Grows Slowly and Upregulates a Unique Set of Virulence and Metabolism Genes

“…Bacteria protect themselves from reactive oxygen species with a range of antioxidant defense enzymes, including thiol peroxidase. It was found that tpx acts as a lipid peroxidase to inhibit bacterial membrane oxidation and acts as a principle antioxidant for E. coli during anaerobic growth (14). It is possible that tpx may be functioning in a similar way here.…”

Pseudomonas aeruginosa Thiol Peroxidase Protects against Hydrogen Peroxide Toxicity and Displays Atypical Patterns of Gene Regulation

“…Previous observations have revealed contrasting tpx expression profiles. E. coli tpx is constitutively expressed and does not respond to treatments with peroxides, superoxide generators, or thiol-depleting agents (10). However, a proteomic analysis of Mycobacterium Tpx levels revealed increasing amounts of protein expression after exposure to thiol stress (19).…”

The Campylobacter jejuni Thiol Peroxidases Tpx and Bcp Both Contribute to Aerotolerance and Peroxide-Mediated Stress Resistance but Have Distinct Substrate Specificities

“…These are thiol peroxidase (Tpx) and the bacterioferritin comigratory protein (Bcp). Tpx and Bcp appear to be able to use a wide variety of peroxides as substrates in vitro, such as hydrogen peroxide, organic peroxides, and lipid peroxides (6,32,43). Tpx has been reported to be periplasmic in Escherichia coli (4) but has been shown to use the cytoplasmic thioredoxin system as an electron donor (46), so the location of the protein remains unclear.…”

“…Pure thioredoxin (Trx) and thioredoxin reductase (TrxR) from E. coli were obtained from Sigma-Aldrich (United Kingdom). The reaction mixture contained 50 mM HEPES-NaOH (pH 7.0), 20 M NADPH, 20 g Trx, 6.25 g TrxR, 1 M pure enzyme (Tpx or Bcp), and various concentrations of peroxides (hydrogen peroxide, tert-butyl-hydroperoxide, cumene hydroperoxide, or linoleic acid hydroperoxide). Reactions were carried out in a total volume of 1 ml at 37°C.…”