Functions of the Cold Shock Proteins in Bacillus Subtilis

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.2021-07-1

The Bacillus subtilis Minimal Genome Compendium

To better understand cellular life, it is essential to decipher the contribution of individual components and their interactions. Minimal genomes are an important tool to investigate these interactions. Here, we provide a database of 105 fully annotated genomes of a series of strains with sequential deletion steps of the industrially relevant model bacterium Bacillus subtilis starting with the laboratory wild type strain B. subtilis 168 and ending with B. subtilis PG38, which lacks approximately 40% of the original genome. The annotation is supported by sequencing of key intermediate strains as well as integration of literature knowledge for the annotation of the deletion scars and their potential effects. The strain compendium presented here represents a comprehensive genome library of the entire MiniBacillus project. This resource will facilitate the more effective application of the different strains in basic science as well as in biotechnology

Functional Redundancy and Specialization of the Conserved Cold Shock Proteins in Bacillus subtilis

Faßhauer P, Busche T, Kalinowski J, et al. Functional Redundancy and Specialization of the Conserved Cold Shock Proteins in Bacillus subtilis. Microorganisms. 2021;9(7): 1434.Many bacteria encode so-called cold shock proteins. These proteins are characterized by a conserved protein domain. Often, the bacteria have multiple cold shock proteins that are expressed either constitutively or at low temperatures. In the Gram-positive model bacterium Bacillussubtilis, two of three cold shock proteins, CspB and CspD, belong to the most abundant proteins suggesting a very important function. To get insights into the role of these highly abundant proteins, we analyzed the phenotypes of single and double mutants, tested the expression of the csp genes and the impact of CspB and CspD on global gene expression in B. subtilis. We demonstrate that the simultaneous loss of both CspB and CspD results in a severe growth defect, in the loss of genetic competence, and the appearance of suppressor mutations. Overexpression of the third cold shock protein CspC could compensate for the loss of CspB and CspD. The transcriptome analysis revealed that the lack of CspB and CspD affects the expression of about 20% of all genes. In several cases, the lack of the cold shock proteins results in an increased read-through at transcription terminators suggesting that CspB and CspD might be involved in the control of transcription termination