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Identification of Rhodococcus erythropolis Promoters Controlled by Alternative Sigma Factors Using In Vivo and In Vitro Systems and Heterologous RNA Polymerase

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Abstract

Rhodococcus erythropolis CCM2595 is a bacterial strain, which has been studied for its capability to degrade phenol and other toxic aromatic compounds. Its cell wall contains mycolic acids, which are also an attribute of other bacteria of the Mycolata group, such as Corynebacterium and Mycobacterium species. We suppose that many genes upregulated by phenol stress in R. erythropolis are controlled by the alternative sigma factors of RNA polymerase, which are active in response to the cell envelope or oxidative stress. We developed in vitro and in vivo assays to examine the connection between the stress sigma factors and genes activated by various extreme conditions, e.g., heat, cell surface, and oxidative stress. These assays are based on the procedures of such tests carried out in the related species, Corynebacterium glutamicum. We showed that the R. erythropolis CCM2595 genes frmB1 and frmB2, which encode S-formylglutathione hydrolases (named corynomycolyl transferases in C. glutamicum), are controlled by SigD, just like the homologous genes cmt1 and cmt2 in C. glutamicum. The new protocol of the in vivo and in vitro assays will enable us to classify R. erythropolis promoters according to their connection to sigma factors and to assign the genes to the corresponding sigma regulons. The complex stress responses, such as that induced by phenol, could, thus, be analyzed with respect to the gene regulation by sigma factors.

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Acknowledgements

We wish to thank Jan Nešvera and Jiří Matějů (Institute of Microbiology, Prague) for critical reading of the manuscript and helpful comments.

Funding

This work was supported by Grant 18-13254S from the Czech Science Foundation, Grants from Charles University (Projects GAUK 452120 and 338321), and Institutional Research Project RVO61388971 from the Institute of Microbiology of the CAS.

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Authors and Affiliations

  1. Institute of Microbiology of the CAS, v.v.i., Prague, Czech Republic

    Jan Blumenstein, Robert Rädisch, Václav Štěpánek, Michal Grulich, Hana Dostálová & Miroslav Pátek

  2. Department of Genetics and Microbiology, Faculty of Science, Charles University, Prague, Czech Republic

    Jan Blumenstein & Robert Rädisch

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  1. Jan Blumenstein
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Contributions

HD and MP conceived the study and wrote the initial draft. HD performed in vitro transcription, JB performed cloning and in vivo assays, RR performed the CRISPR-Cas9 constructions, VŠ carried out mutagenesis and database searches, and MG isolated and analyzed the proteins. All the authors read, edited, and approved the final manuscript.

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Correspondence to Miroslav Pátek.

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Blumenstein, J., Rädisch, R., Štěpánek, V. et al. Identification of Rhodococcus erythropolis Promoters Controlled by Alternative Sigma Factors Using In Vivo and In Vitro Systems and Heterologous RNA Polymerase. Curr Microbiol 79, 55 (2022). https://doi.org/10.1007/s00284-021-02747-8

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