Analysis of the role of 7 kDa cold-shock proteins of Lactococcus lactis MG1363 in cryoprotection

Low-temperature adaptation and cryoprotection were studied in the lactic ac id bacterium Lactococcus lactis MG1363. An approximately 100-fold increased survival after freezing was observed when cells were shocked to 10 degrees C for 4 h compared to mid-exponential-phase cells grown at 30 degrees C, i ndicating an active protection against freezing. Using two-dimensional gel electrophoresis a group of 7 kDa cold-induced proteins (CSPs) was identifie d that corresponds to a previously described family of csp genes of L. lact is MG1363 (Wouters et al., 1998, Microbiology 144, 2885-2893), The 7 kDa CS Ps appeared to be the most strongly induced proteins upon cold shock to 10 degrees C. Northern blotting and two-dimensional gel electrophoresis showed that the csp genes were maximally expressed at 10 degrees C, while inducti on was lower at 20 and 4 degrees C. However, preincubation at 20 and 4 degr ees C, as well as stationary-phase conditions, also induced cryoprotection (approx. 30-, 130- and 20-fold, respectively, compared to 30 degrees C mid- exponential phase). For all treatments leading to an increased freeze survi val (exposure to 4, 10 and 20 degrees C and stationary-phase conditions), i ncreased levels of three proteins (26, 43 and 45 kDa) were observed for whi ch a role in cryoprotection might be suggested. Increased freeze survival c oincides with increased CSP expression, except for stationary-phase conditi ons. However, the level of observed freeze protection does not directly cor relate with the csp gene expression levels. In addition, for the first time specific overproduction of a CSP in relation to freeze survival was studie d. This revealed that L, lactis cells overproducing CspD at 30 degrees C sh ow a 2-10-fold increased survival after freezing compared to control cells. This indicates that the 7 kDa cold-shock protein CspD may enhance the surv ival capacity after freezing but that other factors supply additional cryop rotection.