Temperature-dependent requirement for catalase in aerobic growth of Listeria monocytogenes F2365

Abstract

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.

FIG. 1.

Comparison of the kat genomic regions between L. monocytogenes F2365 and L. monocytogenes EGD-e. Transposon insertion in ROA3 is shown (▾). The putative rho-independent terminators are shown by a lollipop symbol.

FIG. 2.

Impact of temperature on colony size of kat mutant ROA3. (A) Growth of F2365, ROA3, and ROA3C on TSAYE medium following 48 h of incubation at 37°C. (B) Growth of F2365, ROA3, ROA3C, and ROA3E on TSAYE medium following 7 days of incubation at 10°C.

FIG. 3.

Impact of temperature on growth of kat mutant ROA3 in liquid medium. L. monocytogenes F2365 (⧫) and ROA3 (▪) were grown at 37°C (A) and 4°C (B) statically; F2365 (⋄) and ROA3 (□) were grown at 4°C with agitation (150 rpm) in BHI broth, and growth was monitored as described in Materials and Methods.

FIG. 4.

Freeze-thaw tolerance of kat mutant ROA3. L. monocytogenes F2365 (triangles) and ROA3 (squares) were grown at 37°C (filled symbols) or at 25°C (open symbols) and exposed to 18 repeated freeze-thaw cycles. Survival was assessed as described in Materials and Methods.

FIG. 5.

Growth of kat mutant ROA3 in milk. Raw milk (filled symbols) and pasteurized milk (open symbols) were inoculated with L. monocytogenes F2365 (triangles) and ROA3 (squares) and incubated at 4°C over 21 days. Growth was monitored as described in Materials and Methods.