Physiological and Regulatory Effects of Controlled Overproduction of Five Cold Shock Proteins of Lactococcus lactis MG1363

Abstract

The physiological and regulatory effects of overproduction of five cold shock proteins (CSPs) of Lactococcus lactis were studied. CspB, CspD, and CspE could be overproduced at high levels (up to 19% of the total protein), whereas for CspA and CspC limited overproduction (0.3 to 0.5% of the total protein) was obtained. Northern blot analysis revealed low abundance of the cspC transcript, indicating that the stability of cspC mRNA is low. The limited overproduction of CspA is likely to be caused by low stability of CspA since when there was an Arg-Pro mutation at position 58, the level of CspA production increased. Using two-dimensional gel electrophoresis, it was found that upon overproduction of the CSPs several proteins, including a number of coldinduced proteins of L. lactis, were induced. Strikingly, upon overproduction of CspC induction of CspB, putative CspF, and putative CspG was also observed. Overproduction of CspB and overproduction of CspE result in increased survival when L. lactis is frozen (maximum increases, 10-and 5-fold, respectively, after 4 freeze-thaw cycles). It is concluded that in L. lactis CSPs play a regulatory role in the cascade of events that are initiated by cold shock treatment and that they either have a direct protective effect during freezing (e.g., RNA stabilization) or induce other factors involved in the freeze-adaptive response or both.In a variety of bacteria, cold shock proteins (CSPs) are the major induced proteins upon exposure to cold shock. Different functions, e.g., as transcriptional activators, RNA chaperones, and anti-freeze proteins, have been attributed to CSPs (for reviews see references 11 and 36). CspA of Escherichia coli and CspB of Bacillus subtilis have been shown to bind singlestranded DNA, and E. coli CspA has been shown to act as a transcriptional activator for the hns and gyrA genes encoding proteins involved in DNA supercoiling (2,17,22). E. coli CspA and B. subtilis CspB B have very similar five-stranded ␤-barrel structures with several outward-facing residues important for single-stranded DNA binding. Furthermore, CSPs contain two highly conserved RNA-binding motifs, named RNP-1 and RNP-2, and indeed, mRNA-binding capacity has been demonstrated for E. coli CspA and B. subtilis CspB (13,15). It has been proposed that members of the CSP family bind to RNA in a cooperative manner and function as RNA chaperones, thereby facilitating the translation process (13). Disruption of B. subtilis cspB results in a freeze-sensitive phenotype (33) and also affects the level of induction of other cold-induced proteins (CIPs) upon temperature downshock in B. subtilis (12). Deletion of three CSPs in B. subtilis was shown to be lethal (13). Since not all members of the CSP family are cold induced, it has been suggested that CSPs play a role in multiple cellular processes, such as chromosomal condensation and/or cell division (36).The mesophilic lactic acid bacterium Lactococcus lactis is widely used to start industrial food fermentations. A variety of genes involved in t...

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