9 results on '"Siersma T"'
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2. A novel, high stringency selection system allows screening of few clones for high protein expression
- Author
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VANBLOKLAND, H, primary, KWAKS, T, additional, SEWALT, R, additional, VERHEES, J, additional, KLAREN, V, additional, SIERSMA, T, additional, KORSE, J, additional, TEUNISSEN, N, additional, BOTSCHUIJVER, S, additional, and VANMER, C, additional
- Published
- 2007
- Full Text
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3. The Multifaceted Antibacterial Mechanisms of the Pioneering Peptide Antibiotics Tyrocidine and Gramicidin S.
- Author
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Wenzel M, Rautenbach M, Vosloo JA, Siersma T, Aisenbrey CHM, Zaitseva E, Laubscher WE, van Rensburg W, Behrends JC, Bechinger B, and Hamoen LW
- Subjects
- Bacillus subtilis ultrastructure, Cell Membrane drug effects, Cell Wall drug effects, DNA Damage drug effects, DNA-Binding Proteins metabolism, Microbial Sensitivity Tests, Microscopy, Electron, Anti-Bacterial Agents pharmacology, Bacillus subtilis drug effects, Gramicidin pharmacology, Tyrocidine pharmacology
- Abstract
Cyclic β-sheet decapeptides from the tyrocidine group and the homologous gramicidin S were the first commercially used antibiotics, yet it remains unclear exactly how they kill bacteria. We investigated their mode of action using a bacterial cytological profiling approach. Tyrocidines form defined ion-conducting pores, induce lipid phase separation, and strongly reduce membrane fluidity, resulting in delocalization of a broad range of peripheral and integral membrane proteins. Interestingly, they also cause DNA damage and interfere with DNA-binding proteins. Despite sharing 50% sequence identity with tyrocidines, gramicidin S causes only mild lipid demixing with minor effects on membrane fluidity and permeability. Gramicidin S delocalizes peripheral membrane proteins involved in cell division and cell envelope synthesis but does not affect integral membrane proteins or DNA. Our results shed a new light on the multifaceted antibacterial mechanisms of these antibiotics and explain why resistance to them is virtually nonexistent. IMPORTANCE Cyclic β-sheet decapeptides, such as tyrocidines and gramicidin S, were among the first antibiotics in clinical application. Although they have been used for such a long time, there is virtually no resistance to them, which has led to a renewed interest in this peptide class. Both tyrocidines and gramicidin S are thought to disrupt the bacterial membrane. However, this knowledge is mainly derived from in vitro studies, and there is surprisingly little knowledge about how these long-established antibiotics kill bacteria. Our results shed new light on the antibacterial mechanism of β-sheet peptide antibiotics and explain why they are still so effective and why there is so little resistance to them., (Copyright © 2018 Wenzel et al.)
- Published
- 2018
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4. Daptomycin inhibits cell envelope synthesis by interfering with fluid membrane microdomains.
- Author
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Müller A, Wenzel M, Strahl H, Grein F, Saaki TNV, Kohl B, Siersma T, Bandow JE, Sahl HG, Schneider T, and Hamoen LW
- Abstract
Daptomycin is a highly efficient last-resort antibiotic that targets the bacterial cell membrane. Despite its clinical importance, the exact mechanism by which daptomycin kills bacteria is not fully understood. Different experiments have led to different models, including ( i ) blockage of cell wall synthesis, ( ii ) membrane pore formation, and ( iii ) the generation of altered membrane curvature leading to aberrant recruitment of proteins. To determine which model is correct, we carried out a comprehensive mode-of-action study using the model organism Bacillus subtilis and different assays, including proteomics, ionomics, and fluorescence light microscopy. We found that daptomycin causes a gradual decrease in membrane potential but does not form discrete membrane pores. Although we found no evidence for altered membrane curvature, we confirmed that daptomycin inhibits cell wall synthesis. Interestingly, using different fluorescent lipid probes, we showed that binding of daptomycin led to a drastic rearrangement of fluid lipid domains, affecting overall membrane fluidity. Importantly, these changes resulted in the rapid detachment of the membrane-associated lipid II synthase MurG and the phospholipid synthase PlsX. Both proteins preferentially colocalize with fluid membrane microdomains. Delocalization of these proteins presumably is a key reason why daptomycin blocks cell wall synthesis. Finally, clustering of fluid lipids by daptomycin likely causes hydrophobic mismatches between fluid and more rigid membrane areas. This mismatch can facilitate proton leakage and may explain the gradual membrane depolarization observed with daptomycin. Targeting of fluid lipid domains has not been described before for antibiotics and adds another dimension to our understanding of membrane-active antibiotics., Competing Interests: The authors declare no conflict of interest.
- Published
- 2016
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5. The conserved DNA-binding protein WhiA is involved in cell division in Bacillus subtilis.
- Author
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Surdova K, Gamba P, Claessen D, Siersma T, Jonker MJ, Errington J, and Hamoen LW
- Subjects
- Bacillus subtilis cytology, Bacillus subtilis growth & development, Bacterial Proteins genetics, Cell Cycle Proteins genetics, DNA Transposable Elements, DNA-Binding Proteins genetics, Gene Deletion, Mutagenesis, Insertional, Sequence Homology, Amino Acid, Bacillus subtilis physiology, Bacterial Proteins metabolism, Cell Cycle Proteins metabolism, Cell Division, DNA-Binding Proteins metabolism
- Abstract
Bacterial cell division is a highly coordinated process that begins with the polymerization of the tubulin-like protein FtsZ at midcell. FtsZ polymerization is regulated by a set of conserved cell division proteins, including ZapA. However, a zapA mutation does not result in a clear phenotype in Bacillus subtilis. In this study, we used a synthetic-lethal screen to find genes that become essential when ZapA is mutated. Three transposon insertions were found in yvcL. The deletion of yvcL in a wild-type background had only a mild effect on growth, but a yvcL zapA double mutant is very filamentous and sick. This filamentation is caused by a strong reduction in FtsZ-ring assembly, suggesting that YvcL is involved in an early stage of cell division. YvcL is 25% identical and 50% similar to the Streptomyces coelicolor transcription factor WhiA, which induces ftsZ and is required for septation of aerial hyphae during sporulation. Using green fluorescent protein fusions, we show that YvcL localizes at the nucleoid. Surprisingly, transcriptome analyses in combination with a ChIP-on-chip assay gave no indication that YvcL functions as a transcription factor. To gain more insight into the function of YvcL, we searched for suppressors of the filamentous phenotype of a yvcL zapA double mutant. Transposon insertions in gtaB and pgcA restored normal cell division of the double mutant. The corresponding proteins have been implicated in the metabolic sensing of cell division. We conclude that YvcL (WhiA) is involved in cell division in B. subtilis through an as-yet-unknown mechanism.
- Published
- 2013
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6. The use of a stringent selection system allows the identification of DNA elements that augment gene expression.
- Author
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Hoeksema F, van Blokland R, Siep M, Hamer K, Siersma T, den Blaauwen J, Verhees J, and Otte AP
- Subjects
- Animals, CHO Cells, Cricetinae, Cricetulus, Enzyme-Linked Immunosorbent Assay, Humans, Transfection, Retinoblastoma Protein genetics
- Abstract
The use of high stringency selection systems often results in the induction of very few recombinant mammalian cell lines, which limits the ability to isolate a cell line with favorable characteristics. The employment of for instance STAR elements in DNA constructs elevates the induced number of colonies and also the protein expression levels in these colonies. Here, we describe a method to systematically identify genomic DNA elements that are able to induce many stably transfected mammalian cell lines. We isolated genomic DNA fragments upstream from the human Rb1 and p73 gene loci and cloned them around an expression cassette that contains a very stringent selection marker. Due to the stringency of the selection marker, hardly any colony survives without flanking DNA elements. We tested fourteen ~3500 bp DNA stretches from the Rb1 and p73 loci. Only two ~3500 bp long DNA fragments, called Rb1E and Rb1F, induced many colonies in the context of the stringent selection system and these colonies displayed high protein expression levels. Functional analysis showed that the Rb1 DNA fragments contained no enhancer, promoter, or STAR activity. Our data show the potential of a methodology to identify novel gene expression augmenting DNA elements in an unbiased manner.
- Published
- 2011
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7. A novel, high stringency selection system allows screening of few clones for high protein expression.
- Author
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van Blokland HJ, Kwaks TH, Sewalt RG, Verhees JA, Klaren VN, Siersma TK, Korse JW, Teunissen NC, Botschuijver S, van Mer C, Man SY, and Otte AP
- Subjects
- Animals, CHO Cells metabolism, Cricetinae, Cricetulus, Cell Line, Cloning, Molecular methods, Gene Dosage genetics, Gene Expression Regulation genetics, Transfection methods
- Abstract
To obtain highly productive mammalian cell lines, often large numbers of clones need to be screened. This is largely due to low selection stringencies, creating many, but low protein producing clones. To remedy this problem, a novel, very stringent selection system was designed, to create few, but high protein producing clones. In essence, a selection marker with a startcodon that confers attenuated translation initiation frequency was placed upstream of the gene of interest with a startcodon that confers optimal translation initiation. From the transcribed bicistronic mRNA, the selection marker is translated at a low frequency, and the protein of interest at a high frequency. This selection system is so stringent that clones form only rarely. However, application of anti-repressor elements, which increase promoter activity, did induce the formation of clones that expressed proteins at high levels. When combined with anti-repressor elements, this novel selection system can be a valuable tool to rapidly create few, but highly productive mammalian cell lines.
- Published
- 2007
- Full Text
- View/download PDF
8. Targeting of a histone acetyltransferase domain to a promoter enhances protein expression levels in mammalian cells.
- Author
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Kwaks TH, Sewalt RG, van Blokland R, Siersma TJ, Kasiem M, Kelder A, and Otte AP
- Subjects
- Animals, CHO Cells, Cell Cycle Proteins genetics, Cell Cycle Proteins metabolism, Cricetinae, Cricetulus, Genetic Enhancement methods, Histone Acetyltransferases, Promoter Regions, Genetic genetics, Protein Structure, Tertiary, Recombinant Fusion Proteins biosynthesis, Transcription Factors genetics, Transcription Factors metabolism, p300-CBP Transcription Factors, Acetyltransferases genetics, Acetyltransferases metabolism, Bacterial Proteins genetics, Bacterial Proteins metabolism, Gene Targeting methods, Protein Engineering methods, Serine Endopeptidases genetics, Serine Endopeptidases metabolism, Transfection methods
- Abstract
Silencing of transfected genes in mammalian cells is a fundamental problem that probably involves the (in)accessibility status of chromatin. A potential solution to this problem is to provide a cell with protein factors that make the chromatin of a promoter more open or accessible for transcription. We tested this by targeting such proteins to different promoters. We found that targeting the p300 histone acetyltransferase (HAT) domain to strong viral or cellular promoters is sufficient to result in higher expression levels of a reporter protein. In contrast, targeting the chromatin-remodeling factor Brahma does not result in stable, higher protein expression levels. The long-term effects of the targeted p300HAT domain on protein expression levels are positively reinforced, when also anti-repressor elements are applied to flank the reporter construct. These elements were previously shown to be potent blockers of chromatin-associated repressors. The simultaneous application of the targeted p300HAT domain and anti-repressor elements conveys long-term stability to protein expression. Whereas no copy number dependency is achieved by targeting of the p300HAT domain alone, copy number dependency is improved when anti-repressor elements are included. We conclude that targeting of protein domains such as HAT domains helps to facilitate expression of transfected genes in mammalian cells. However, the simultaneous application of other genomic elements such as the anti-repressor elements prevents silencing more efficiently.
- Published
- 2005
- Full Text
- View/download PDF
9. Identification of anti-repressor elements that confer high and stable protein production in mammalian cells.
- Author
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Kwaks TH, Barnett P, Hemrika W, Siersma T, Sewalt RG, Satijn DP, Brons JF, van Blokland R, Kwakman P, Kruckeberg AL, Kelder A, and Otte AP
- Subjects
- Animals, CHO Cells, Chromatin metabolism, Cricetinae, Cricetulus, Humans, Mammals, Molecular Sequence Data, Promoter Regions, Genetic, Protein Biosynthesis, Protein Engineering methods, Proteins genetics, Repressor Proteins metabolism, Chromatin genetics, Gene Expression Profiling methods, Gene Expression Regulation genetics, Recombinant Proteins biosynthesis, Recombinant Proteins genetics, Repressor Proteins genetics, Transgenes genetics
- Abstract
The expression of transgenic proteins is often low and unstable over time, a problem that may be due to integration of the transgene in repressed chromatin. We developed a screening technology to identify genetic elements that efficiently counteract chromatin-associated repression. When these elements were used to flank a transgene, we observed a substantial increase in the number of mammalian cell colonies that expressed the transgenic protein. Expression of the shielded transgene was, in a copy number-dependent fashion, substantially higher than the expression of unprotected transgenes. Also, protein production remained stable over an extended time period. The DNA elements are small, not exceeding 2,100 base pairs (bp), and they are highly conserved between human and mouse, at both the functional and sequence levels. Our results demonstrate the existence of a class of genetic elements that can readily be applied to more efficient transgenic protein production in mammalian cells.
- Published
- 2003
- Full Text
- View/download PDF
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