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 acid 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, indicating an active protection against freezing. Using two-dimensional gel electrophoresis a group of 7 kDa cold-induced proteins (CSPs) was identified that corresponds to a previously described family of csp genes of L. lactis MG1363 (Wouters et al., 1998, Microbiology 144, 2885-2893). The 7 kDa CSPs 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 induction was lower at 20 and 4 degrees C. However, pre-incubation at 20 and 4 degrees 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 survival (exposure to 4, 10 and 20 degrees C and stationary-phase conditions), increased levels of three proteins (26, 43 and 45 kDa) were observed for which a role in cryoprotection might be suggested. Increased freeze survival coincides with increased CSP expression, except for stationary-phase conditions. However, the level of observed freeze protection does not directly correlate with the csp gene expression levels. In addition, for the first time specific overproduction of a CSP in relation to freeze survival was studied. This revealed that L. lactis cells overproducing CspD at 30 degrees C show 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 survival capacity after freezing but that other factors supply additional cryoprotection.
Wouters
Jeroen A
JA
Microbial Ingredients Section, NIZO food research, Ede, The Netherlands2.|Laboratory for Food Microbiology, Division of Food Science, Wageningen University and Research Centre, Bomenweg 2, 6703 HD Wageningen, The Netherlands1.
Jeynov
Boyan
B
Laboratory for Food Microbiology, Division of Food Science, Wageningen University and Research Centre, Bomenweg 2, 6703 HD Wageningen, The Netherlands1.
Rombouts
Frank M
FM
Laboratory for Food Microbiology, Division of Food Science, Wageningen University and Research Centre, Bomenweg 2, 6703 HD Wageningen, The Netherlands1.
de Vos
Willem M
WM
Microbial Ingredients Section, NIZO food research, Ede, The Netherlands2.
Kuipers
Oscar P
OP
Microbial Ingredients Section, NIZO food research, Ede, The Netherlands2.
Abee
Tjakko
T
Laboratory for Food Microbiology, Division of Food Science, Wageningen University and Research Centre, Bomenweg 2, 6703 HD Wageningen, The Netherlands1.
D000222:Adaptation, Physiological; D001426:Bacterial Proteins / Q000502:physiology*; D015152:Blotting, Northern; D003080:Cold Temperature; D015180:Electrophoresis, Gel, Two-Dimensional; D015964:Gene Expression Regulation, Bacterial; D013294:Lactococcus lactis / Q000737:chemistry / Q000502:physiology*; D012329:RNA, Bacterial / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D013997:Time Factors
D001426:Bacterial Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; C077836:cold shock like protein 7kD, Bacteria