Prokaryotes

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    Protocols in Current Issue
    Rapid Genome Engineering of Pseudomonas Assisted by Fluorescent Markers and Tractable Curing of Plasmids
    Authors:  Daniel C. Volke, Nicolas T. Wirth and Pablo I. Nikel, date: 02/20/2021, view: 862, Q&A: 0

    Precise genome engineering has become a commonplace technique for metabolic engineering. Also, insertion, deletion and alteration of genes and other functional DNA sequences are essential for understanding and engineering cells. Several techniques

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    Rapid Genome Engineering of Pseudomonas Assisted by Fluorescent Markers and Tractable Curing of Plasmids
    Authors:  Daniel C. Volke, Nicolas T. Wirth and Pablo I. Nikel, date: 02/20/2021, view: 862, Q&A: 0
    [Abstract]

    Precise genome engineering has become a commonplace technique for metabolic engineering. Also, insertion, deletion and alteration of genes and other functional DNA sequences are essential for understanding and engineering cells. Several techniques have been developed to this end (e.g., CRISPR/Cas-assisted methods, homologous recombination, or λ

    ...
    Resolving Structural Changes of Photoreceptors in Living Escherichia coli via In-cell Infrared Difference Spectroscopy
    Authors:  Lukas Goett-Zink, Jessica L. Klocke and Tilman Kottke, date: 02/05/2021, view: 340, Q&A: 0
    [Abstract]

    Several in-cell spectroscopic techniques have been developed recently to investigate the structure and mechanism of proteins in their native environment. Conditions in vivo differ dramatically from those selected for in vitro experiments. Accordingly, the cellular environment can affect the protein mechanism for example by molecular crowding or

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    Determination of Antibacterial Activity of Film Coatings against Four Clinically Relevant Bacterial Strains
    [Abstract]

    Antibacterial coatings have currently gained great importance in biomedical technology investigations. Because of the spatial arrangement of the film coatings, evaluation of antibacterial activity presents a new challenge regarding traditional bacterial counting methods. In this protocol, four clinically relevant pathogens, Salmonella typhimurium,

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    Analysis of Pseudomonas aeruginosa c-di-GMP High and Low Subpopulations Using Flow-assisted Cell Sorting (FACS) and Quantitative Reverse Transcriptase PCR (qRT-PCR)
    Authors:  Catherine R. Armbruster and Matthew R. Parsek, date: 01/20/2021, view: 525, Q&A: 0
    [Abstract]

    Cyclic diguanylate monophosphate (c-di-GMP) is a second messenger signaling molecule that drives the transition from planktonic to the biofilm mode of growth in many bacterial species. Pseudomonas aeruginosa has at least two surface sensing systems that produce c-di-GMP in response to surface attachment, the Wsp and Pil-Chp systems. We recently

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    Assessment of Diadenylate Cyclase and c-di-AMP-phosphodiesterase Activities Using Thin-layer and Ion Exchange Chromatography
    Authors:  Andreas Latoscha, David Jan Drexler, Gregor Witte and Natalia Tschowri, date: 01/05/2021, view: 522, Q&A: 0
    [Abstract]

    All living cells use cyclic nucleotides as second messengers for signal sensing and transduction. Cyclic di-3′,5′-adenosine monophosphate (c-di-AMP) is primarily involved in the control of bacterial and euryarcheal osmoadaptation and is produced by diadenylate cyclases from two molecules of ATP. Specific phosphodiesterases hydrolyze

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    Preparation of Bacterial Outer Membrane Vesicles for Characterisation of Periplasmic Proteins in Their Native Environment
    Authors:  Johannes Thoma and Björn M. Burmann, date: 12/20/2020, view: 852, Q&A: 0
    [Abstract]

    Bacterial outer membrane vesicles (OMVs) are naturally formed by budding from the outer membrane of Gram-negative bacteria. OMVs consist of a lipid bilayer identical in composition to the original outer membrane and contain periplasmic content within their lumen. Enriched with specific envelope proteins, OMVs make for an excellent native-like

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    Pea Aphid Rearing, Bacterial Infection and Hemocyte Phagocytosis Assay
    Authors:  Li Ma, Lu Liu and Zhiqiang Lu, date: 12/20/2020, view: 538, Q&A: 0
    [Abstract]

    Insects rely on the simple but effective innate immune system to combat infection. Cellular and humoral responses are interconnected and synergistic in insects’ innate immune system. Phagocytosis is one major cellular response. It is difficult to collect clean hemolymph from the small insect like pea aphid. Here, we provide a practicable

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    Antimicrobial Sensitivity Assay for Bdellovibrio bacteriovorus
    Authors:  Emanuele Marine and Klaas M. Pos, date: 12/20/2020, view: 615, Q&A: 0
    [Abstract]

    Bdellovibrio bacteriovorus, an obligate predatory bacterium [i.e., bacteria that kill and feed on other bacteria (prey)], has the potential to be used as a probiotic for the disinfection of surfaces or for the treatment of bacterial infections. One option is to use this organism in combination with antimicrobials to potentiate the effectiveness of

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    Charging State Analysis of Transfer RNA from an α-proteobacterium
    Authors:  Liang Yin and Caroline S. Harwood, date: 12/05/2020, view: 499, Q&A: 0
    [Abstract] Transfer RNA (tRNA) is an essential link between the genetic code and proteins. During the process of translation, tRNA is charged with its cognate amino acid and delivers it to the ribosome, thus serving as a substrate of protein synthesis. To analyze the charging state of a particular tRNA, total RNA is purified and analyzed on an acid-urea gel. ...
    Ribosome Purification from an α-proteobacterium and rRNA Analysis by Northern Blot
    Authors:  Liang Yin and Caroline S. Harwood, date: 12/05/2020, view: 481, Q&A: 0
    [Abstract] Ribosomes are an integral part of cellular life. They are complex molecular machines consisting of multiple ribosomal proteins and RNAs. To study different aspects of ribosome composition, many methods have been developed over the decades. Here, we describe how to purify ribosomes from the α-proteobacterium Rhodopseudomonas palustris ...



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