Temperature-dependent freeze-thaw tolerance and genetic characterization of stress response mechanisms of Listeria

Abstract

Listeria monocytogenes is a gram-positive foodborne pathogen that can cause severe illness (listeriosis) with high fatality rate. L. monocytogenes is ubiquitous in the environment and encounters a number of different stress conditions, including freeze-thaw stress. The growth temperature of the bacteria plays an important role in expression of several key virulence factors. However, our understanding of the impact of growth temperature on Listeria’s stress responses remains limited. In this study, we investigated the impact of growth temperature on the freeze-thaw tolerance of Listeria spp. Listeria cells grown at 37oC showed significantly higher tolerance against repeated freezing and thawing then cells grown at 4oC or 25oC (p<0.05). In order to address if this phenomenon is seen in other psychrotrophic foodborne pathogens, we investigated the impact of growth temperature on the freeze-thaw tolerance of Yersinia enterocolitica. However, impact of growth temperature on the cryotolerance of Y. enterocolitica was opposite of that observed with Listeria spp: Yersinia cells grown at 4oC were markedly more tolerant to the damaging effects of repeated freezing and thawing than 37oC-grown bacteria.

To identify genes responsible for the observed temperature-dependent cryotolerance of L. monocytogenes we constructed mutant libraries of two L. monocytogenes strains, F2365 (serotype 4b) and 10403S (serotype 1/2a). The mutant libraries were constructed with a mariner-based transposition system and high efficiency of transposon insertion was achieved. However, screening directly for impaired cryotolerance following repeated freezing and thawing in a 96-well plate format did not allow consistent differentiation between 37oC-grown and 4oC-grown cultures. Therefore, our subsequent studies focused on screening for the loss of tolerance to the stresses expected to take place during repeated freeze-thaw treatments. The stresses included oxidative stresses, cold stress, and osmotic stress. One mutant in a putative oxidative stress gene was identified based on its susceptibility to paraquat and was found to have markedly impaired freeze-thaw tolerance. Thermoregulated control of cryotolerance resembles that of key virulence genes but is exhibited by both pathogenic and non-pathogenic species of Listeria. Growth temperature-dependent cryotolerance may represent an adaptation of ancestral Listeria lineages that preceded the emergence of non-pathogenic clades, and may involve thermoregulated attributes at the transcriptional or proteomic levels. The impact of growth temperature on cryotolerance of Listeria was unexpected, and underlying mechanisms remain to be identified.