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  • 1
    Online Resource
    Online Resource
    Red Fluorescent Proteins for Gene Expression and Protein Localization Studies in Streptococcus pneumoniae and Efficient Transformation with DNA Assembled via the Gibson Assembly Method
    American Society for Microbiology ; 2015
    In:  Applied and Environmental Microbiology Vol. 81, No. 20 ( 2015-10-15), p. 7244-7252
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 81, No. 20 ( 2015-10-15), p. 7244-7252
    Abstract: During the last decades, a wide range of fluorescent proteins (FPs) have been developed and improved. This has had a great impact on the possibilities in biological imaging and the investigation of cellular processes at the single-cell level. Recently, we have benchmarked a set of green fluorescent proteins (GFPs) and generated a codon-optimized superfolder GFP for efficient use in the important human pathogen Streptococcus pneumoniae and other low-GC Gram-positive bacteria. In the present work, we constructed and compared four red fluorescent proteins (RFPs) in S. pneumoniae . Two orange-red variants, mOrange2 and TagRFP, and two far-red FPs, mKate2 and mCherry, were codon optimized and examined by fluorescence microscopy and plate reader assays. Notably, protein fusions of the RFPs to FtsZ were constructed by direct transformation of linear Gibson assembly (isothermal assembly) products, a method that speeds up the strain construction process significantly. Our data show that mCherry is the fastest-maturing RFP in S. pneumoniae and is best suited for studying gene expression, while mKate2 and TagRFP are more stable and are the preferred choices for protein localization studies. The RFPs described here will be useful for cell biology studies that require multicolor labeling in S. pneumoniae and related organisms.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.02033-15
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2015
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    detail.hit.zdb_id: 1478346-0
    SSG: 12
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  • 2
    Online Resource
    Online Resource
    Chromosome segregation drives division site selection in Streptococcus pneumoniae
    Proceedings of the National Academy of Sciences ; 2017
    In:  Proceedings of the National Academy of Sciences Vol. 114, No. 29 ( 2017-07-18)
    Details
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 114, No. 29 ( 2017-07-18)
    Abstract: Accurate spatial and temporal positioning of the tubulin-like protein FtsZ is key for proper bacterial cell division. Streptococcus pneumoniae (pneumococcus) is an oval-shaped, symmetrically dividing opportunistic human pathogen lacking the canonical systems for division site control (nucleoid occlusion and the Min-system). Recently, the early division protein MapZ was identified and implicated in pneumococcal division site selection. We show that MapZ is important for proper division plane selection; thus, the question remains as to what drives pneumococcal division site selection. By mapping the cell cycle in detail, we show that directly after replication both chromosomal origin regions localize to the future cell division sites, before FtsZ. Interestingly, Z-ring formation occurs coincidently with initiation of DNA replication. Perturbing the longitudinal chromosomal organization by mutating the condensin SMC, by CRISPR/Cas9-mediated chromosome cutting, or by poisoning DNA decatenation resulted in mistiming of MapZ and FtsZ positioning and subsequent cell elongation. Together, we demonstrate an intimate relationship between DNA replication, chromosome segregation, and division site selection in the pneumococcus, providing a simple way to ensure equally sized daughter cells.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    URL: Article
    DOI: 10.1073/pnas.1620608114
    RVK:
    TA 1000
    RVK:
    WA 15000
    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2017
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    SSG: 11
    SSG: 12
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  • 3
    Online Resource
    Online Resource
    CcrZ is a pneumococcal spatiotemporal cell cycle regulator that interacts with FtsZ and controls DNA replication by modulating the activity of DnaA
    Springer Science and Business Media LLC ; 2021
    In:  Nature Microbiology Vol. 6, No. 9 ( 2021-08-09), p. 1175-1187
    Details
    In: Nature Microbiology, Springer Science and Business Media LLC, Vol. 6, No. 9 ( 2021-08-09), p. 1175-1187
    Abstract: Most bacteria replicate and segregate their DNA concomitantly while growing, before cell division takes place. How bacteria synchronize these different cell cycle events to ensure faithful chromosome inheritance by daughter cells is poorly understood. Here, we identify Cell Cycle Regulator protein interacting with FtsZ (CcrZ) as a conserved and essential protein in pneumococci and related Firmicutes such as Bacillus subtilis and Staphylococcus aureus . CcrZ couples cell division with DNA replication by controlling the activity of the master initiator of DNA replication, DnaA. The absence of CcrZ causes mis-timed and reduced initiation of DNA replication, which subsequently results in aberrant cell division. We show that CcrZ from Streptococcus pneumoniae interacts directly with the cytoskeleton protein FtsZ, which places CcrZ in the middle of the newborn cell where the DnaA-bound origin is positioned. This work uncovers a mechanism for control of the bacterial cell cycle in which CcrZ controls DnaA activity to ensure that the chromosome is replicated at the right time during the cell cycle.
    Type of Medium: Online Resource
    ISSN: 2058-5276
    URL: Article
    DOI: 10.1038/s41564-021-00949-1
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2021
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  • 4
    Online Resource
    Online Resource
    BactMAP: An R package for integrating, analyzing and visualizing bacterial microscopy data
    Wiley ; 2020
    In:  Molecular Microbiology Vol. 113, No. 1 ( 2020-01), p. 297-308
    Details
    In: Molecular Microbiology, Wiley, Vol. 113, No. 1 ( 2020-01), p. 297-308
    Abstract: High‐throughput analyses of single‐cell microscopy data are a critical tool within the field of bacterial cell biology. Several programs have been developed to specifically segment bacterial cells from phase‐contrast images. Together with spot and object detection algorithms, these programs offer powerful approaches to quantify observations from microscopy data, ranging from cell‐to‐cell genealogy to localization and movement of proteins. Most segmentation programs contain specific post‐processing and plotting options, but these options vary between programs and possibilities to optimize or alter the outputs are often limited. Therefore, we developed BactMAP (Bacterial toolbox for Microscopy Analysis & Plotting), a command‐line based R package that allows researchers to transform cell segmentation and spot detection data generated by different programs into various plots. Furthermore, BactMAP makes it possible to perform custom analyses and change the layout of the output. Because BactMAP works independently of segmentation and detection programs, inputs from different sources can be compared within the same analysis pipeline. BactMAP complies with standard practice in R which enables the use of advanced statistical analysis tools, and its graphic output is compatible with ggplot2, enabling adjustable plot graphics in every operating system. User feedback will be used to create a fully automated Graphical User Interface version of BactMAP in the future. Using BactMAP, we visualize key cell cycle parameters in Bacillus subtilis and Staphylococcus aureus , and demonstrate that the DNA replication forks in Streptococcus pneumoniae dissociate and associate before splitting of the cell, after the Z‐ring is formed at the new quarter positions. BactMAP is available from https://veeninglab.com/bactmap .
    Type of Medium: Online Resource
    ISSN: 0950-382X , 1365-2958
    URL: Issue
    URL: Article
    DOI: 10.1111/mmi.v113.1
    DOI: 10.1111/mmi.14417
    Language: English
    Publisher: Wiley
    Publication Date: 2020
    detail.hit.zdb_id: 1501537-3
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