04 Fakultät Energie-, Verfahrens- und Biotechnik
Permanent URI for this collectionhttps://elib.uni-stuttgart.de/handle/11682/5
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Item Open Access Construction of a super-competent Bacillus subtilis 168 using the PmtlA-comKS inducible cassette(2015) Rahmer, Regine; Morabbi Heravi, Kambiz; Altenbuchner, JosefCompetence is a physiological state that enables Bacillus subtilis 168 to take up and internalize extracellular DNA. In practice, only a small subpopulation of B. subtilis 168 cells becomes competent when they enter stationary phase. In this study, we developed a new transformation method to improve the transformation efficiency of B. subtilis 168, specially in rich media. At first, different competence genes, namely comK, comS, and dprA, were alone or together integrated into the chromosome of B. subtilis 168 under control of mannitol-inducible PmtlA promoter. Overexpression of both comK and comS increased the transformation efficiency of B. subtilis REG19 with plasmid DNA by 6.7-fold compared to the wild type strain 168. This transformation efficiency reached its maximal level after 1.5 h of induction by mannitol. Besides, transformability of the REG19 cells was saturated in the presence of 100 ng dimeric plasmid or 3000 ng chromosomal DNA. Studying the influence of global regulators on the development of competence pointed out that important competence development factors, such as Spo0A, ComQXPA, and DegU, could be removed in REG19. On the other hand, efficient REG19 transformation remained highly dependent on the original copies of comK and comS regardless of the presence of PmtlA-comKS. Finally, novel plasmid-free strategies were used for transformation of REG19 based on Gibson assembly.Item Open Access The transcription factor TAL1 and miR-17-92 create a regulatory loop in hematopoiesis(2020) Meyer, Annekarin; Herkt, Stefanie; Kunze-Schumacher, Heike; Kohrs, Nicole; Ringleb, Julia; Schneider, Lucas; Kuvardina, Olga N.; Oellerich, Thomas; Häupl, Björn; Krueger, Andreas; Seifried, Erhard; Bonig, Halvard; Lausen, JoernA network of gene regulatory factors such as transcription factors and microRNAs establish and maintain gene expression patterns during hematopoiesis. In this network, transcription factors regulate each other and are involved in regulatory loops with microRNAs. The microRNA cluster miR-17-92 is located within the MIR17HG gene and encodes six mature microRNAs. It is important for hematopoietic differentiation and plays a central role in malignant disease. However, the transcription factors downstream of miR-17-92 are largely elusive and the transcriptional regulation of miR-17-92 is not fully understood. Here we show that miR-17-92 forms a regulatory loop with the transcription factor TAL1. The miR-17-92 cluster inhibits expression of TAL1 and indirectly leads to decreased stability of the TAL1 transcriptional complex. We found that TAL1 and its heterodimerization partner E47 regulate miR-17-92 transcriptionally. Furthermore, miR-17-92 negatively influences erythroid differentiation, a process that depends on gene activation by the TAL1 complex. Our data give example of how transcription factor activity is fine-tuned during normal hematopoiesis. We postulate that disturbance of the regulatory loop between TAL1 and the miR-17-92 cluster could be an important step in cancer development and progression.Item Open Access PRMT6 activates cyclin D1 expression in conjunction with the transcription factor LEF1(2021) Schneider, Lucas; Herkt, Stefanie; Wang, Lei; Feld, Christine; Wesely, Josephine; Kuvardina, Olga N.; Meyer, Annekarin; Oellerich, Thomas; Häupl, Björn; Seifried, Erhard; Bonig, Halvard; Lausen, JoernThe establishment of cell type specific gene expression by transcription factors and their epigenetic cofactors is central for cell fate decisions. Protein arginine methyltransferase 6 (PRMT6) is an epigenetic regulator of gene expression mainly through methylating arginines at histone H3. This way it influences cellular differentiation and proliferation. PRMT6 lacks DNA-binding capability but is recruited by transcription factors to regulate gene expression. However, currently only a limited number of transcription factors have been identified, which facilitate recruitment of PRMT6 to key cell cycle related target genes. Here, we show that LEF1 contributes to the recruitment of PRMT6 to the central cell cycle regulator CCND1 (Cyclin D1). We identified LEF1 as an interaction partner of PRMT6. Knockdown of LEF1 or PRMT6 reduces CCND1 expression. This is in line with our observation that knockdown of PRMT6 increases the number of cells in G1 phase of the cell cycle and decreases proliferation. These results improve the understanding of PRMT6 activity in cell cycle regulation. We expect that these insights will foster the rational development and usage of specific PRMT6 inhibitors for cancer therapy.Item Open Access Regulation of mtl operon promoter of Bacillus subtilis: requirements of its use in expression vectors(2011) Morabbi Heravi, Kambiz; Wenzel, Marian; Altenbuchner, JosefSeveral vector systems have been developed to express any gene desired to be studied in Bacillus subtilis. Among them, the transcriptionally regulated promoters involved in carbohydrate utilization are a research priority. Expression systems based on Bacillus promoters for xylose, maltose, and mannose utilization, as well as on the heterologous E. coli lactose promoter, have been successfully constructed. The promoter of the mtlAFD operon for utilization of mannitol is another promising candidate for its use in expression vectors. In this study, we investigated the regulation of the mtl genes in order to identify the elements needed to construct a strong mannitol inducible expression system in B. subtilis. Regulation of the promoters of Bacillus subtilis mtlAFD operon (PmtlA) and mtlR (PmtlR) encoding the activator were investigated by fusion to lacZ. Identification of the PmtlA and PmtlR transcription start sites revealed the sigma A like promoter structures. Also, the operator of PmtlA was determined by shortening, nucleotide exchange, and alignment of PmtlA and PmtlR operator regions. Deletion of the mannitol-specific PTS genes (mtlAF) resulted in PmtlA constitutive expression demonstrating the inhibitory effect of EIICBMtl and EIIAMtl on MtlR in the absence of mannitol. Disruption of mtlD made the cells sensitive to mannitol and glucitol. Both PmtlA and PmtlR were influenced by carbon catabolite repression (CCR). However, a CcpA deficient mutant showed only a slight reduction in PmtlR catabolite repression. Similarly, using PgroE as a constitutive promoter, putative cre sites of PmtlA and PmtlR slightly reduced the promoter activity in the presence of glucose. In contrast, glucose repression of PmtlA and PmtlR was completely abolished in a ptsG deletion mutant and significantly reduced in a MtlR (H342D) mutant.