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

Abstract

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.

Changes in cspL , cspP , and cspC mRNA Abundance as a Function of Cold Shock and Growth Phase in Lactobacillus plantarum

“…For example, a family of five csp genes (cspA to cspE) is present in Lactococcus lactis. Their expression is strongly induced upon cold shock, except for CspE, which is already abundant at optimal temperature (46,47).…”

Sequence Analysis of the Lactococcal Plasmid pNP40: a Mobile Replicon for Coping with Environmental Hazards

“…Previous studies on the chromosomal CspC and CspD homologues have illustrated the physiological response mediated by these cold shock proteins; expression of CspD was enhanced by cold shock and resulted in increased survival at low temperatures, whereas only a modest increase in CspC expression accompanied cold shock, which was found to directly alter the levels of other cold shock proteins (71)(72)(73).…”

“…Freeze-thaw challenge and DNA damage assays. Freeze-thaw challenge experiments were performed essentially as described elsewhere (71,72). Briefly, cells were grown in GM17 medium at 30°C to an OD 600 of 0.5 and subjected to a cold shock by rapid temperature downshift from 30 to 10°C for 0, 2, or 4 h, after which 1 ml of these cultures was frozen at Ϫ20°C.…”

Dynamic Stresses of Lactic Acid Bacteria Associated to Fermentation Processes

“…The roles of these proteins are at the levels of membrane fluidity, DNA supercoiling and transcription and translation. Few papers have described cold shock proteins and mechanisms in LAB, in particular they have focused on Lactococcus lactis and L. plantarum [39][40][41][42]. Kim et al [39,40] tested different LAB to evaluate cold shock effects on cryotolerance.…”