Microbiology

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    Protocols in Current Issue
    Split-Chloramphenicol Acetyl Transferase Assay to Study Protein-Protein Interactions and Ubiquitylation in Escherichia coli
    Authors:  Amir Florentin, Alina Kordonsky, Elon Yariv, Reut Avishid, Noa Efron, Edache Akogwu and Gali Prag, date: 09/05/2022, view: 234, Q&A: 0
    [Abstract]

    Protein-protein interactions and protein modifications play central roles in all living organisms. Of the more than 200 types of post-translational modifications, ubiquitylation is the most abundant, and it profoundly regulates the functionality of the eukaryotic proteome. Various in vitro and in vivo methodologies to study protein interactions

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    Tracking the Reversed Oxidative Tricarboxylic Acid Cycle in Bacteria
    [Abstract]

    Different pathways for autotrophic CO2 fixation can be recognized by the presence of genes for their specific key enzymes. On this basis, (meta)genomic, (meta)transcriptomic, or (meta)proteomic analysis enables the identification of the role of an organism or a distinct pathway in primary production. However, the recently discovered variant of the

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    Activity-based Crosslinking to Identify Substrates of Thioredoxin-domain Proteins in Malaria Parasites
    Authors:  David W. Cobb, Grace S. Woods and Vasant Muralidharan, date: 02/20/2022, view: 1390, Q&A: 0
    [Abstract] Malaria remains a major public health issue, infecting nearly 220 million people every year. The spread of drug-resistant strains of Plasmodium falciparum around the world threatens the progress made against this disease. Therefore, identifying druggable and essential pathways in P. falciparum parasites remains a major area of ...
    Isolation of Mitochondria from Ustilago maydis Protoplasts
    [Abstract]

    Ustilago maydis, a basidiomycete that infects Zea mays, is one of the top ten fungal models for studying DNA repair, signal transduction pathways, and dimorphic transitions, among other processes. From a metabolic point of view, U. maydis lacks fermentative capacity, pointing to mitochondria as a key player in central metabolism. Oxidative

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    An Assay to Determine NAD(P)H: Quinone Oxidoreductase Activity in Cell Extracts from Candida glabrata
    Authors:  Anamika Battu, Rajaram Purushotham and Rupinder Kaur, date: 11/05/2021, view: 1319, Q&A: 0
    [Abstract]

    Flavodoxin-like proteins (Fld-LPs) are an important constituent of the oxidative stress defense system in several organisms and highly conserved from bacteria to humans. These proteins possess NAD(P)H:quinone oxidoreductase activity and convert quinones to hydroquinones through two-electron reduction, using NAD(P)H and quinone as electron donor

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    Simple Methods for the Preparation of Colloidal Chitin, Cell Free Supernatant and Estimation of Laminarinase
    Author:  Ananthamurthy Koteshwara, date: 10/05/2021, view: 2136, Q&A: 0
    [Abstract]

    Colloidal chitin (CC) is a common substrate used in research work involving chitin-active enzymes (chitinases). Cell free supernatant (CFS) is prepared from fermented broth. Preparation of CC and CFS usually involve large amounts of liquid, which must be separated from the solids. This necessitates the use of a large volume centrifugation

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    Construction of a Highly Diverse mRNA Library for in vitro Selection of Monobodies
    [Abstract]

    Recently, we developed transcription/translation coupled with the association of puromycin linker (TRAP) display as a quick in vitro selection method to obtain antibody-like proteins. For the in vitro selection, it is important to prepare mRNA libraries among which the diversity is high. Here, we describe a method for the preparation of monobody

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    In vitro Nitrate Reductase Activity Assay of Mycolicibacterium smegmatis Crude Extract
    Authors:  Wei Tan, Zhi-Hui Shao and Guo-Ping Zhao, date: 07/20/2021, view: 1753, Q&A: 0
    [Abstract]

    Nitrate is one of the major inorganic nitrogen sources for microorganisms. Many bacterial and archaeal lineages can express cytoplasmic assimilatory nitrate reductase (NAS), which catalyzes the rate-limiting reduction of nitrate to nitrite in the nitrate assimilation pathway. Here, we present a detailed protocol for measuring in vitro nitrate

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    Assay for Assessing Mucin Binding to Bacteria and Bacterial Proteins
    [Abstract]

    Legionella pneumophila, a Gram-negative bacterium and the causative agent of Legionnaires’ disease, exports over 300 effector proteins/virulence factors, through its type II (T2SS) and type IV secretion systems (T4SS). One such T2SS virulence factor, ChiA, not only functions as a chitinase, but also as a novel mucinase, which we believe aids

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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: 2637, 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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