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Rescue of Escherichia coli cells from UV-induced death and filamentation by caspase-3 inhibitor

  • Surbhi Wadhawan
  • Satyendra GautamEmail author
Original Article
  • 3 Downloads

Abstract

Escherichia coli cells have been observed earlier to display caspase-3-like protease activity (CLP) and undergo programmed cell death (PCD) when exposed to gamma rays. The presence of an irreversible caspase-3 inhibitor (Ac-DEVD-CMK) during irradiation was observed to increase cell survival. Since radiation is known to induce SOS response, the effect of a caspase-3 inhibitor on SOS response was studied in E. coli. UV, a well-known SOS inducer, was used in the current study. Cell filamentation in E. coli upon UV exposure was found to be inhibited by ninefold in the presence of a caspase-3 inhibitor. CLP activity was found to increase twofold in UV-exposed cells than in control (non-treated) cells. Further, bright fluorescing filaments were observed in UV-exposed E. coli cells treated with FITC-DEVD-FMK, a fluorescent dye tagged with an irreversible caspase-3 inhibitor (DEVD-FMK), indicating the presence of active CLP in these cells. Unlike caspase-3 inhibitor, a serine protease inhibitor, phenylmethanesulfonyl fluoride (PMSF), was not found to improve cell survival after UV treatment. Additionally, a SOS reporter system known as SIVET (selectable in vivo expression technology) assay was performed to reconfirm the inhibition of SOS induction in the presence of caspase-3 inhibitor. SIVET assay is used to quantify cells in which the SOS response has been induced leading to a scorable permanent selectable change in the cell. The SIVET induction frequency (calculated as the ratio of SIVET-induced cells to total viable cells) increased around tenfold in UV-exposed cultures. The induction frequency was found to decrease significantly to 51 from 80% in the cells pre-incubated with caspase-3 inhibitor. On the contrary, caspase-3 inhibitor failed to improve cell survival of E. coli ΔrecA and E. coli DM49 (SOS non-inducible) cells post UV treatment. Summing together, the results indicated a possible linkage of SOS response and the PCD process in E. coli. The findings also indicated that functional SOS pathway is required for CLP-like activity; however, the exact mechanism remains to be elucidated.

Keywords

Bacteria Escherichia coli Programmed cell death SOS repair UV radiation SIVET 

Notes

Acknowledgements

The authors acknowledge Keio collection, Japan, for E. coli wt and recA knockout strains provided to our institute (Bhabha Atomic Research Centre, Mumbai, India) and Dr. S. H. Mangoli (Molecular Biology Division, B.A.R.C, Mumbai) for sharing these strains with us. The authors thank Dr. Anubrata Das (Molecular Biology Division, B.A.R.C, Mumbai) for sharing E. coli DM49 strain with us. The authors also acknowledge Prof. M. Z. Humayun (Rutgers New Jersey Medical School, New Jersey, USA) for gifting us E. coli MG1655 and E. coli SG104 strains.

Supplementary material

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Figure S1

General scheme of SIVET assay (adapted from Livny and Friedman, 2004).Selectable in vivo expression technology (SIVET) system consists of two main components: (a) a gene encoding the TnpR resolvase inserted downstream of a defective H-19B prophage, and (b) a chloramphenicol transacetylation gene (cat) disrupted by tetracycline (tet) gene. This tet gene is flanked by a modified resolvase target sequence (resC). During SOS induction, the prophage promoter is de-repressed and the resulting activity of resolvase excises the tet gene and one resC site. Excision results in a DNA fragment with tet gene (it is eventually lost during segregation) and a sequence with functional cat gene converting the cell from TetR CmS phenotype to a TetS CmR phenotype. Therefore, the ratio of CmR cells to total viable cells is a measure of prophage induction, which is indicative of SOS response. (PNG 816 kb)

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High resolution image (TIF 26387 kb)
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Figure S2

Cell morphology ofE. coliΔrecAand DM49 cells after UV exposure (70 mJ m−2 s−1). Crystal violet stained (A)E. coli ΔrecA control cells (no UV treatment); (B)E. coli ΔrecA cells treated with UV for 6 s; (C)E. coli ΔrecA cells treated with UV for 12 s; (D)E. coli DM49 control cells (no UV treatment); (E)E. coli DM49 cells treated with UV for 12 s. Cells were examined under a microscope (Carl Zeiss, Germany) using oil immersion objective (100X). (PNG 4393 kb)

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High resolution image (TIF 26387 kb)

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Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.Food Technology DivisionBhabha Atomic Research CentreMumbaiIndia
  2. 2.Homi Bhabha National InstituteMumbaiIndia

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