| dc.description.abstracteng | RNA binding proteins are fundamental to the proper functioning of all cells. They are structural components in larger complexes such as ribosomes or regulate cellular processes that involve RNA such as transcription, translation, or the modification, processing, and decay of RNA. Some RNA binding proteins contain the cold shock domain which is highly conserved from bacteria to mammals. Bacterial cold shock proteins consist of a single cold shock domain that binds RNA and single stranded DNA. They have been extensively studied in various species and some act as RNA chaperones that destabilize secondary RNA structures to regulate transcriptional termination, RNA stability and processing, as well as translation. In the Gram-positive model organism Bacillus subtilis, the function(s) and targets of cold shock proteins have not been elucidated so far. This work identified the regulon of the cold shock proteins in B. subtilis and uncovered their involvement in many biological processes. The B. subtilis genome encodes the three cold shock protein paralogs CspB, CspC, and CspD. While csp single-mutants did not exhibit any obvious phenotype and a triple knockout was not possible, the cspB cspD double-knockout led to the loss of genetic competence, impairment of biofilm formation, aberrant gene expression, and a strong impairment of growth. This suggests CspC cannot fully replace the function of CspB and CspD. The cspB cspD double mutant formed suppressor mutants, which often harbored a point mutation that leads to upregulation of CspC. The overexpression of CspC in these suppressor mutants improved growth and genetic stability but did not restore genetic competence. This suggests CspC is functionally different from CspB and CspD. CspC was the only paralog that was induced at 15°C further highlighting the functional specialization. Comparison of the amino acid residue at position 58 which is important for functional specificity in Staphylococcus aureus, revealed that CspC harbors an alanine residue while CspB and CspD carry a proline residue at this position. Therefore, a CspC(A58P) variant was expressed in the cspB cspD double mutant background which improved genetic stability, growth, and also restored genetic competence. Hence, a single amino acid is responsible for the functional specificity of the cold shock proteins. Analysis of the cspB cspD double mutant transcriptome uncovered up- or downregulation for as many as 21% of genes suggesting numerous potential targets of CspB and CspD. One of these targets is the cspC 5’-UTR at which CspB and CspD but not CspC negatively regulated expression. Other targets were identified by analysis of read-through transcription at intergenic regions in the cspB cspD double mutant. An increased transcriptional read-through was found at the manR and liaH terminators. Conversely, transcriptional read-through was decreased at the terminator/ antiterminator switches between the pyrR-pyrP and pyrP-pyrB genes. These results demonstrate that the B. subtilis cold shock proteins have different biological functions and influence gene expression globally at least by regulation of transcription. This study may serve as a starting point for future research on cold shock protein function in B. subtilis. It presents methods and interesting targets to further explore the function of cold shock proteins. | de |