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Development of a highly sensitive luciferase-based reporter system to study two-step protein secretion in cyanobacteria

View ORCID ProfileDavid A. Russo, Julie A. Z. Zedler, Fabian D. Conradi, Nils Schuergers, Poul Erik Jensen, View ORCID ProfileConrad W. Mullineaux, View ORCID ProfileAnnegret Wilde, View ORCID ProfileGeorg Pohnert
doi: https://doi.org/10.1101/2021.10.08.463751
David A. Russo
1Bioorganic Analytics, Institute for Inorganic and Analytical Chemistry, Friedrich Schiller University Jena, Jena, Germany
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  • For correspondence: david.russo@uni-jena.de
Julie A. Z. Zedler
2Matthias Schleiden Institute for Genetics, Bioinformatics and Molecular Botany, Friedrich Schiller University Jena, Jena, Germany
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Fabian D. Conradi
3School of Biological and Behavioural Sciences, Queen Mary University of London, London, United Kingdom
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Nils Schuergers
4Molecular Genetics of Prokaryotes, Institute of Biology III, University of Freiburg, Freiburg, Germany
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Poul Erik Jensen
5Department of Food Science, University of Copenhagen, Frederiksberg C, Denmark
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Conrad W. Mullineaux
3School of Biological and Behavioural Sciences, Queen Mary University of London, London, United Kingdom
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  • ORCID record for Conrad W. Mullineaux
Annegret Wilde
4Molecular Genetics of Prokaryotes, Institute of Biology III, University of Freiburg, Freiburg, Germany
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Georg Pohnert
1Bioorganic Analytics, Institute for Inorganic and Analytical Chemistry, Friedrich Schiller University Jena, Jena, Germany
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Abstract

Cyanobacteria, ubiquitous oxygenic photosynthetic bacteria, interact with the environment and their surrounding microbiome through the secretion of a variety of small molecules and proteins. The release of these compounds is mediated by sophisticated multi-protein complexes, also known as secretion systems. Genomic analyses indicate that protein and metabolite secretion systems are widely found in cyanobacteria; however little is known regarding their function, regulation and secreted effectors. One such system, the type IVa pilus system (T4aPS), is responsible for the assembly of dynamic cell surface appendages, type IVa pili (T4aP), that mediate ecologically relevant processes such as phototactic motility, natural competence and adhesion. Several studies have suggested that the T4aPS can also act as a two-step protein secretion system in cyanobacteria akin to the homologous type II secretion system in heterotrophic bacteria. To determine whether the T4aP are involved in two-step secretion of non-pilin proteins, we developed a NanoLuc-based quantitative secretion reporter for the model cyanobacterium Synechocystis sp. PCC 6803. The NLuc reporter presented a wide dynamic range with at least one order of magnitude more sensitivity than traditional immunoblotting. Application of the reporter to a collection of Synechocystis T4aPS mutants demonstrated that two-step protein secretion in cyanobacteria is independent of T4aP. In addition, our data suggest that secretion differences typically observed in T4aPS mutants are likely due to a disruption of cell envelope homeostasis. This study opens the door to explore protein secretion in cyanobacteria further.

Importance Protein secretion allows bacteria to interact and communicate with the external environment. Secretion is also biotechnologically relevant, where it is often beneficial to target proteins to the extracellular space. Due to a shortage of quantitative assays, many aspects of protein secretion are not understood. Here we introduce a NanoLuc (NLuc)-based secretion reporter in cyanobacteria. NLuc is highly sensitive and can be assayed rapidly and in small volumes. The NLuc reporter allowed us to clarify the role of type IVa pili in protein secretion and identify mutations that increase secretion yield. This study expands our knowledge on cyanobacterial secretion and offers a valuable tool for future studies of protein secretion systems in cyanobacteria.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted October 09, 2021.
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Development of a highly sensitive luciferase-based reporter system to study two-step protein secretion in cyanobacteria
David A. Russo, Julie A. Z. Zedler, Fabian D. Conradi, Nils Schuergers, Poul Erik Jensen, Conrad W. Mullineaux, Annegret Wilde, Georg Pohnert
bioRxiv 2021.10.08.463751; doi: https://doi.org/10.1101/2021.10.08.463751
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Development of a highly sensitive luciferase-based reporter system to study two-step protein secretion in cyanobacteria
David A. Russo, Julie A. Z. Zedler, Fabian D. Conradi, Nils Schuergers, Poul Erik Jensen, Conrad W. Mullineaux, Annegret Wilde, Georg Pohnert
bioRxiv 2021.10.08.463751; doi: https://doi.org/10.1101/2021.10.08.463751

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