Temperature-Dependent Requirement for Catalase in Aerobic Growth of Listeria monocytogenes F2365

Listeria monocytogenes is a Gram-positive, psychrotrophic, facultative intracellular food-borne pathogen responsible for severe illness (listeriosis). The bacteria can grow in a wide range of temperatures (1 to 45°C), and low-temperature growth contributes to the food safety hazards associated with contamination of ready-to-eat foods with this pathogen. To assess the impact of oxidative stress responses on the ability of L. monocytogenes to grow at low temperatures and to tolerate repeated freeze-thaw stress (cryotolerance), we generated and characterized a catalase-deficient mutant of L. monocytogenes F2365 harboring a mariner-based transposon insertion in the catalase gene (kat). When grown aerobically on blood-free solid medium, the kat mutant exhibited impaired growth, with the extent of impairment increasing with decreasing temperature, and no growth was detected at 4°C. Aerobic growth in liquid was impaired at 4°C, especially under aeration, but not at higher temperatures (10, 25, or 37°C). Genetic complementation of the mutant with the intact kat restored normal growth, confirming that inactivation of this gene was responsible for the growth impairment. In spite of the expected impact of oxidative stress responses on cryotolerance, cryotolerance of the kat mutant was not affected.Listeria monocytogenes is a Gram-positive, facultative intracellular food-borne pathogen that has the ability to cause a severe disease (listeriosis) in humans and animals (13, 28, 30). L. monocytogenes is ubiquitously distributed in the environment and has the ability to grow over a wide range of temperatures (between 1 and 45°C) (13). Growth at low temperature has important implications for environmental persistence of the organism and for contamination of cold-stored, ready-to-eat foods, thus contributing to the food safety hazards associated with L. monocytogenes (19).L. monocytogenes is subjected to oxidative stress during both extracellular and intracellular growth and has evolved several responses to minimize the impact of reactive oxygen species (ROS). Catalase and superoxide dismutase (SOD) work synergistically in detoxification of ROS: superoxide anions are converted to H₂O₂ by SOD, with subsequent conversion of H₂O₂ into water and oxygen by catalase (22). Exposure to ROS may be especially acute during intracellular infection as well as under certain environmental conditions, such as those involved in repeated freezing and thawing (15, 16, 23, 29, 33).Previous studies revealed that the ability of L. monocytogenes to survive repeated freezing and thawing (cryotolerance) was markedly dependent on growth temperature, with bacteria grown at 37°C having significantly higher cryotolerance than those grown at either 4 or 25°C (1). However, mechanisms underlying Listeria''s cryotolerance have not been identified. Since oxidative damage is considered to take place during freezing and thawing, determinants such as catalase may be involved in cryotolerance.The catalase of L. monocytogenes has been investigated primarily in terms of its potential role in pathogenesis, with somewhat conflicting results. The isolation of catalase-negative strains from human listeriosis patients has led to the speculation that catalase is not required for human virulence (4, 8, 12, 31). On the other hand, under certain conditions (e.g., reduced serum levels), catalase-negative strains were impaired in their ability to survive in activated macrophages in comparison to catalase-positive strains (32). Furthermore, the catalase gene kat was among those for which expression was induced in infected cell cultures and in the spleens of mice infected with L. monocytogenes EGD-e, suggesting possible contributions to pathogenesis (5, 9).The potential role of catalase in environmental adaptations of L. monocytogenes such as growth at low temperature and cryotolerance was not addressed in these earlier investigations. In this study, we have characterized an isogenic mutant of L. monocytogenes F2365 to determine the involvement of catalase in growth at different temperatures, survival in selected foods, and cryotolerance of L. monocytogenes.