There has been growing interest in the survival mechanisms of psychoactive bacteria at repeated freeze-thaw cycles largely because successive freezing and thawing are common processes in nature. In addition, there is a considerable interest in the cryotolerance mechanisms of both bacteria related to food-spoilage and food-borne pathogens. It appears that overexpression of CSPs significantly improves cryotolerance, and helps to retard freezing or lessen the damage incurred upon freezing and thawing of the bacteria, yeasts, and plants (Kim et al. 1998a; Thomashow 1998; Broadbent and Lin 1999; Wouters et al. 1999; Thammavongs et al. 2000; Wouters et al. 2001; Minami et al. 2005).
In order to characterize freeze-thaw resistance, the single-cell isolates of the genus Exiguobacterium were subjected to repetitive freeze-thaw cycles (Vishnivetskaya et al. 2007). This study showed that bacteria grown in complex, structured (agar) medium had improved tolerance to the freeze-thaw challenge compared to bacteria grown in mass-action (liquid) medium, regardless of growth temperature. However, growth temperature was a determining factor of a cryotolerance in mass-action (liquid) habitat. Bacteria grown at 4°C in liquid medium tolerate freezing/thawing much better than when grown at 25°C. A subsequent study compared proteomic profiles of E. sibiricum 255-15 grown in liquid broth or an agar surface at both 4°C and 25°C to determine proteins important for cryotoler-ance (Qiu et al., unpublished). The bacteria with improved cryotolerance have revealed a general down-regulation of enzymes involved in major metabolic processes (glycolysis, anaerobic respiration, ATP synthesis, fermentation, electron transport, and sugar metabolism) as well as in the metabolism of lipids, amino acids, nucleotides and nucleic acids, while eight proteins (2'-5' RNA ligase, hypox-anthine phosphoribosyl transferase, FeS assembly ATPase SufC, thioredoxin reductase and four hypothetical proteins) were up-regulated (Qiu et al., unpublished). It has been shown that the repression of RNA species and over-expression of enzymes involved in amino acid biosynthesis during nutritional deprivation led to improved bacterial survivability (Jain et al. 2006). The overproduction of the CSPs in the mesophilic bacterium Lactobacillus plantarum transiently alleviated the reduction in growth rate, and led to an enhanced capacity to survive freezing (Derzelle et al. 2003). In E. sibiricum 255-15, only 15% of the total cellular proteins were overexpressed more than two-fold under different growth conditions. The induction of these proteins might have a potential role in freeze-thaw resistance.
The suppression of some enzymes in the cells grown on agar or at low temperatures indicated the reduction of biochemical reaction rates at these conditions. Therefore, it is reasonable to assume that bacterial cells with slowed metabolism and an enhanced system of replication, recombination, and repair easily tolerate severe environmental factors, e.g., repetitive freeze-thaw cycles.
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