Enhanced levels of cold shock proteins in Listeria monocytogenes LO28 upon exposure to low temperature and high hydrostatic pressure

Abstract

Listeria monocytogenes is a psychrotrophic food-borne pathogen that is problematic for the food industry because of its ubiquitous distribution in nature and its ability to grow at low temperatures and in the presence of high salt concentrations. Here we demonstrate that the process of adaptation to low temperature after cold shock includes elevated levels of cold shock proteins (CSPs) and that the levels of CSPs are also elevated after treatment with high hydrostatic pressure (HHP). Two-dimensional gel electrophoresis combined with Western blotting performed with anti-CspB of Bacillus subtilis was used to identify four 7-kDa proteins, designated Csp1, Csp2, Csp3, and Csp4. In addition, Southern blotting revealed four chromosomal DNA fragments that reacted with a csp probe, which also indicated that a CSP family is present in L. monocytogenes LO28. After a cold shock in which the temperature was decreased from 37 degrees C to 10 degrees C the levels of Csp1 and Csp3 increased 10- and 3.5-fold, respectively, but the levels of Csp2 and Csp4 were not elevated. Pressurization of L. monocytogenes LO28 cells resulted in 3.5- and 2-fold increases in the levels of Csp1 and Csp2, respectively. Strikingly, the level of survival after pressurization of cold-shocked cells was 100-fold higher than that of cells growing exponentially at 37 degrees C. These findings imply that cold-shocked cells are protected from HHP treatment, which may affect the efficiency of combined preservation techniques.

FIG. 1.

Growth of L. monocytogenes LO28 at 37°C (▪) and after cold shock at 10°C (○).

FIG. 2.

Analysis of L. monocytogenes LO28 proteins. (A) Upper panel, one-dimensional gel electrophoresis of mid-exponential-phase cells (exp) (37°C), cells cold shocked at 10°C for 1, 2, 3, 4, and 20 h, and cells in the stationary phase (stat) at 37°C; lower panel, Western blot of an identical one-dimensional gel with anti-CspB from B. subtilis. (B) 2D-E of cell extracts of L. monocytogenes LO28 with pI values ranging from 3 to 10. Proteins induced at least threefold are enclosed in boxes, and the CSPs are circled. Left panel, exponential-phase cells at 37°C; right panel, cells 4 h after cold shock at 10°C.

FIG. 3.

Identification of CSPs in L. monocytogenes LO28. (A) Separation of cell extracts from cells in the exponential growth phase at 37°C (upper panel), from cells 4 h after cold shock at 10°C (middle panel), and from cells 20 h after cold shock at 10°C (lower panel). (B) Western blot, obtained with anti-CspB from B. subtilis, of cell extract from L. monocytogenes LO28 cells 20 h after cold shock at 10°C.

FIG. 4.

csp genes in L. monocytogenes LO28: Southern blot of L. monocytogenes LO28 PstI fragments in which the csp PCR product was used as the probe. The sizes of markers (in base pairs) are indicated on the left, and the sizes of the hybridizing fragments (in base pairs) are indicated on the right.

FIG. 5.

Response of L. monocytogenes LO28 to HHP treatment. (A) Growth (OD₆₂₀) at 37°C (⧫) and growth after 10 min of exposure to 50 MPa (□), 100 MPa (○), and 200 MPa (▵). (B) 2D-E with a pI range of 4 to 5 for cell extracts from untreated cells (upper panel) and cell extracts from cells treated for 10 min at 200 MPa (lower panel).

FIG. 6.

Reduction in the number of viable L. monocytogenes cells after exposure for 20 min to 200, 250, 300, and 350 MPa for cells growing exponentially at 37°C (⧫) and cells exposed to 10°C for 4 h (□).