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Molecular Biology and Genetic Tools to Investigate Functional Redundancy Among Fe-S Cluster Carriers in E. coli

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Fe-S Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2353))

Abstract

Iron-sulfur (Fe-S) clusters are among the oldest protein cofactors, and Fe-S cluster-based chemistry has shaped the cellular metabolism of all living organisms. Over the last 30 years, thanks to molecular biology and genetic approaches, numerous actors for Fe-S cluster assembly and delivery to apotargets have been uncovered. In prokaryotes, Escherichia coli is the best-studied for its convenience of growth and its genetic amenability. During evolution, redundant ways to secure the supply of Fe-S clusters to the client proteins have emerged in E. coli. Disrupting gene expression is essential for gene function exploration, but redundancy can blur the interpretations as it can mask the role of important biogenesis components. This chapter describes molecular biology and genetic strategies that have permitted to reveal the E. coli Fe-S cluster conveying component network, composition, organization, and plasticity. In this chapter, we will describe the following genetic methods to investigate the importance of E. coli Fe-S cluster carriers: one-step inactivation of chromosomal genes in E. coli using polymerase chain reaction (PCR) products, P1 transduction, arabinose-inducible expression system, mevalonate (MVA) genetic by-pass, sensitivity tests to oxidative stress and iron starvation, β-galactosidase assay, gentamicin survival test, and Hot Fusion cloning method.

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Acknowledgments

Thanks are due to all members of the Py group and the Barras group (past in Marseille, and present in Paris) who contributed to studies on Fe-S cluster biogenesis. We thank the Ezraty group (Marseille) for sharing material resources and fruitful discussions. Funding sources were from the Center National de la Recherche Scientifique, Aix-Marseille Université, Agence Nationale de la Recherche (JPAIMR «Combinatorial», Blanc «FRACOL»), ERACoBioTech « Iron Plug’n Play », and Association Française de l’Ataxie de Friedreich.

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  1. Laboratoire de Chimie Bactérienne, Institut de Microbiologie de la Méditerranée, CNRS UMR 7283, Aix-Marseille University, Marseille, France

    Yohann Duverger & Béatrice Py

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  1. Yohann Duverger
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  2. Béatrice Py
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Correspondence to Béatrice Py .

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  1. Department of Chemistry, Wake Forest University, Winston Salem, NC, USA

    Patricia C. Dos Santos

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Duverger, Y., Py, B. (2021). Molecular Biology and Genetic Tools to Investigate Functional Redundancy Among Fe-S Cluster Carriers in E. coli . In: Dos Santos, P.C. (eds) Fe-S Proteins. Methods in Molecular Biology, vol 2353. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1605-5_1

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  • DOI: https://doi.org/10.1007/978-1-0716-1605-5_1

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1604-8

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