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Construction of in vitro transcription system for Corynebacterium glutamicum and its use in the recognition of promoters of different classes

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Abstract

To facilitate transcription studies in Corynebacterium glutamicum, we have developed an in vitro transcription system for this bacterium used as an industrial producer of amino acids and a model organism for actinobacteria. This system consists of C. glutamicum RNA polymerase (RNAP) core (α2, β, β′), a sigma factor and a promoter-carrying DNA template, that is specifically recognized by the RNAP holoenzyme formed. The RNAP core was purified from the C. glutamicum strain with the modified rpoC gene, which produced His-tagged β′ subunit. The C. glutamicum sigA and sigH genes were cloned and overexpressed using the Escherichia coli plasmid vector, and the sigma subunits σA and σH were purified by affinity chromatography. Using the reconstituted C. glutamicum holo-RNAPs, recognition of the σA- and σH-dependent promoters and synthesis of the specific transcripts was demonstrated. The developed in vitro transcription system is a novel tool that can be used to directly prove the specific recognition of a promoter by the particular σ factor(s) and to analyze transcriptional control by various regulatory proteins in C. glutamicum.

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Acknowledgments

This work was supported by Grants 204/09/J015 and P302/12/P633 from the Czech Science Foundation and by Institutional Research Project RVO61388971. We thank Jiří Janata for critical reading of the manuscript.

Conflict of interests

The authors declare that they have no conflict of interests.

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Authors and Affiliations

  1. Institute of Microbiology, AS CR, v.v.i., Vídeňská 1083, 14220, Prague 4, Czech Republic

    Jiří Holátko, Radoslav Šilar, Alžbeta Rabatinová, Hana Šanderová, Petr Halada, Jan Nešvera, Libor Krásný & Miroslav Pátek

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  1. Jiří Holátko
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  2. Radoslav Šilar
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  3. Alžbeta Rabatinová
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  4. Hana Šanderová
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  5. Petr Halada
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  6. Jan Nešvera
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  7. Libor Krásný
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  8. Miroslav Pátek
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Correspondence to Miroslav Pátek.

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Holátko, J., Šilar, R., Rabatinová, A. et al. Construction of in vitro transcription system for Corynebacterium glutamicum and its use in the recognition of promoters of different classes. Appl Microbiol Biotechnol 96, 521–529 (2012). https://doi.org/10.1007/s00253-012-4336-1

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  • DOI: https://doi.org/10.1007/s00253-012-4336-1

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