CE12 - Génétique, génomique et ARN

Recovery or cell death in stress-exposed bacteria – NOVOREP

To Live or to Die, that’s the Response

The project has the ambition to fully characterize, from signal to cellular responses, a novel bacterial stress response mechanism in which two proteins, a metalloprotease and a transcriptional regulator, control expression of known and yet unknown genes, leading to either cell survival or apoptotic- like death.

Recovery or cell death in stress-exposed bacteria

Bacteria are frequently exposed to harmful or unfavourable conditions, not only in their natural environment, but also in industrial applications or when exposed to the human defence system or antibiotics. Bacteria have evolved a number of defence, stress response and repair mechanisms, which are crucial for their survival, but which may cause human health problems in case of surviving pathogens. Deciphering the underlying molecular mechanisms of these diverse bacterial defence strategies is essential for developing new therapeutic and industrial applications. In Deinococcus, which are extremely resistant to radiation and oxidative stress, we have demonstrated that genes contributing to stress survival are finely regulated by a novel but only partially elucidated mechanism consisting of a metalloprotease that specifically cleaves the transcriptional repressor of the radiation response. This repressor is essential for cell viability and its prolonged depletion induces apoptotic-like cell death, a phenomenon poorly described in bacteria. Uncharacterized proteins similar to this protease/repressor pair are present in many other bacteria, including human pathogens, in which they may regulate a stress response. Combining genetics, functional genomics, molecular biology, biochemistry, microscopy and structural biology, our project aims at (1) identifying the stress-induced molecular signal that triggers repressor cleavage, (2) determining how the repressor interacts with the protease and with its target DNA, (3) identifying and characterizing the genes involved in the apoptotic-like death, and (4) characterizing similar protease/repressor pairs of other bacteria including pathogens. This research project has the potential for innovative and groundbreaking discoveries, with results that may pave the way to applications in the fields of biotechnology and drug design.

Various methods will be used to characterize the so-called radiation/desiccation response (RDR) controlled by metallopeptidase IrrE and transcriptional repressor DdrO in Deinococcus. In addition, several proteins pairs similar to IrrE and DdrO from other bacteria will be characterized. The methods used include genetics (construction of gene deletion mutants and genes encoding proteins with deleted domains or with amino acid substitutions), microbiology and microscopy (characterization of wild-type and mutant strains after exposure to several stress conditions and/or to various agents), biochemistry (to characterize interactions and activity of purified proteins), structural biology (including X-ray crystallography; of proteins and of macromolecule complexes), transcriptomics (RNA-Seq; to identify genes regulated directly or indirectly by DdrO at different time points after induction of the response), ChIP-Seq (to identify DNA target sites of DdrO in cells).

(June 2021)
We have shown that metallopeptidase IrrE is activated in Deinococcus by increased availability of zinc ions. Radiation and oxidative stress induce changes in redox homeostasis, and we propose that a part of the zinc pool coordinated with cysteine residues in proteins is released due to their oxidation and becoming more available to IrrE (Magerand et al, 2021).
DdrO has been characterized using various methods, including crystallography. We showed that DdrO is composed of a DNA-binding domain and a dimerization domain. The latter is required for DNA binding of entire DdrO. The site-specific cleavage by IrrE of the dimerization domain of DdrO abolishes dimerization and DNA binding of DdrO. The data also strongly suggest that IrrE cleaves DdrO monomers. The importance of several residues of DdrO, as revealed in the crystal structures of DdrO, was confirmed biochemically and in Deinococcus cells (de Groot et al, 2019).

(June 2021)
Experiments are under way to characterize other aspects of the response mechanism and its outcomes.

Magerand R, Rey P, Blanchard L, de Groot A. 2021. Redox signaling through zinc activates the radiation response in Deinococcus bacteria. Sci Rep. 11(1):4528. doi: 10.1038/s41598-021-84026-x.

de Groot A, Siponen MI, Magerand R, Eugénie N, Martin-Arevalillo R, Doloy J, Lemaire D, Brandelet G, Parcy F, Dumas R, Roche P, Servant P, Confalonieri F, Arnoux P, Pignol D, Blanchard L. 2019. Crystal structure of the transcriptional repressor DdrO: insight into the metalloprotease/repressor-controlled radiation response in Deinococcus. Nucleic Acids Res. 47(21):11403-11417. doi: 10.1093/nar/gkz883.

Blanchard L, de Groot A. 2021. Coexistence of SOS-Dependent and SOS-Independent Regulation of DNA Repair Genes in Radiation-Resistant Deinococcus Bacteria. Cells 10(4):924. doi: 10.3390/cells10040924.

Our project falls within the framework of the diverse bacterial defence and stress response mechanisms, for which discoveries are in full expansion. Bacteria are frequently exposed to harmful or unfavourable conditions, not only in their natural environment, but also in industrial applications or when exposed to the human defence system or antibiotics. Bacteria have evolved a number of defence, stress response and repair mechanisms, which are crucial for their survival, but which may cause human health problems in case of surviving pathogens. Deciphering the underlying molecular mechanisms of these diverse bacterial defence strategies is essential for developing new therapeutic and industrial applications. In Deinococcus, which are extremely resistant to radiation and oxidative stress, we have demonstrated that genes contributing to stress survival are finely regulated by a novel but only partially elucidated mechanism consisting of a metalloprotease that specifically cleaves the transcriptional repressor of the radiation response. This repressor is essential for cell viability and its prolonged depletion induces apoptotic-like cell death, a phenomenon poorly described in bacteria. Uncharacterized proteins similar to this protease/repressor pair are present in many other bacteria, including human pathogens, in which they may regulate a stress response. Combining genetics, functional genomics, molecular biology, biochemistry, microscopy and structural biology, our project aims at (1) identifying the stress-induced molecular signal that triggers repressor cleavage, (2) determining how the repressor interacts with the protease and with its target DNA, (3) identifying and characterizing the genes involved in the apoptotic-like death, and (4) characterizing similar protease/repressor pairs of other bacteria including pathogens. This research project has the potential for innovative and groundbreaking discoveries, with results that may pave the way to applications in the fields of biotechnology and drug design.

Project coordination

Arjan DE GROOT (Institut de biosciences et biotechnologies d'Aix-Marseille)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

BIAM Institut de biosciences et biotechnologies d'Aix-Marseille
I2BC Institut de Biologie Intégrative de la Cellule

Help of the ANR 382,192 euros
Beginning and duration of the scientific project: December 2019 - 48 Months

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