Molecular characterization of a family of cold-shock proteins of Lactococcus lactis

Lactic acid bacteria (LAB) are widely used as starter cultures in fermentation processes. The stress response of LAB during different industrial processes, and during low-temperature conditions in particular, requires a better understanding. For that reason a research project on the cold adaptation of Lactococcus lactis MG1363, a model LAB strain, was initiated. Research focused on the identification and characterization of a family of five csp genes, named cspA, cspB, cspC, cspD and cspE , encoding highly similar cold-shock proteins (CSPs; 65-85% identity). On the L. lactis MG1363 chromosome two tandem groups of csp genes ( cspA/cspB and cspC/cspD ) were identified, whereas cspE was found as a single gene.Transcription analysis showed that cspE is the only non-cold-induced csp gene, whereas the other csp genes are induced 10- to 40-fold at different times after cold shock. The 7-kDa CSPs, corresponding to the csp genes of L. lactis MG1363, were the highest induced proteins upon cold shock to 10°C as was shown by two-dimensional gel electrophoresis. Using the nisin-inducible expression system CspB, CspD and CspE could be overproduced to high levels. For CspA and CspC limited overproduction was obtained, that could be explained by low stability of cspC mRNA and by low stability of CspA. For L. lactis NZ9000ΔAB (deleted in cspAB ) and NZ9000ΔABE (deleted in cspABE ) no differences in growth at normal and at low temperature were observed, compared to that of the wild-type strain L. lactis NZ9000. The deletion of csp genes was compensated by increased expression of the remaining csp genes. These data indicate that the expression of csp genes in L. lactis is regulated by a tightly controlled transcription network.When L. lactis cells were shocked to 10°C for 4 h the survival to freezing increased approximately 100-fold compared to mid-exponential phase cells grown at 30°C. L. lactis cells overproducing CspB, CspD or CspE at 30°C show a 2-10 fold increased survival after freezing compared to control cells. The adaptive response to freezing conditions by prior exposure to 10°C was significantly delayed in strain NZ9000ΔABE compared to strains NZ9000 and NZ9000ΔAB.In combination, these data indicate that 7-kDa CSPs of L. lactis enhance the survival capacity after freezing. CSPs either have a direct protective effect during freezing, e.g. by RNA stabilization, and/or induce other factors involved in the freeze-adaptive response. A group of strongly cold-induced 7-kDa proteins was also identified for Streptococcus thermophilus and, indeed, enhanced production of these proteins also coincided with increased survival to freezing of this bacterium.Using two-dimensional gel electrophoresis, induction of several (non-7 kDa) cold-induced proteins (CIPs) of L. lactis was observed upon overproduction of CSPs. Furthermore, several CIPs were no longer cold induced in the csp -deleted strains, which indicates that CSPs might activate the expression of certain CIPs. A selection of CIPs of L. lactis was identified and appears to be implicated in a variety of cellular processes, e.g. transcriptional and translational control, sugar metabolism and signal sensing. Furthermore, it was shown that the maximal glycolytic activity measured at 30°C increases (approximately 2.5-fold) upon incubation at 10°C for two to four h, a process for which protein synthesis is required. Based on their cold induction and involvement in cold adaptation of glycolysis, it is proposed that the CcpA/HPr control circuit regulates a (unidentified) factor involved in the increased glycolytic activity.The research described in this thesis contributes to the understanding of the response of lactic acid bacteria to low temperatures and might yield applications for dairy industry, especially with respect to fermentation performance and the survival of starter bacteria during freezing.