Conservation and diversity of the IrrE/DdrO-controlled radiation response in radiation-resistant Deinococcus bacteria

Abstract

The extreme radiation resistance of Deinococcus bacteria requires the radiation-stimulated cleavage of protein DdrO by a specific metalloprotease called IrrE. DdrO is the repressor of a predicted radiation/desiccation response (RDR) regulon, composed of radiation-induced genes having a conserved DNA motif (RDRM) in their promoter regions. Here, we showed that addition of zinc ions to purified apo-IrrE, and short exposure of Deinococcus cells to zinc ions, resulted in cleavage of DdrO in vitro and in vivo, respectively. Binding of IrrE to RDRM-containing DNA or interaction of IrrE with DNA-bound DdrO was not observed. The data are in line with IrrE being a zinc peptidase, and indicate that increased zinc availability, caused by oxidative stress, triggers the in vivo cleavage of DdrO unbound to DNA. Transcriptomics and proteomics of Deinococcus deserti confirmed the IrrE-dependent regulation of predicted RDR regulon genes and also revealed additional members of this regulon. Comparative analysis showed that the RDR regulon is largely well conserved in Deinococcus species, but also showed diversity in the regulon composition. Notably, several RDR genes with an important role in radiation resistance in Deinococcus radiodurans, for example pprA, are not conserved in some other radiation-resistant Deinococcus species.

Keywords: gene regulation; proteomics; regulon; transcriptional repressor; transcriptomics; zinc peptidase.

Figure 1

Different metal ions restore peptidase activity of apo‐IrrE in vitro. Purified IrrE was incubated with EDTA to chelate divalent metal ions. After removing EDTA, different metal ions were tested for restoration of peptidase activity of IrrE (5 μmol/L), which was monitored by the cleavage of its substrate DdrO (20 μmol/L). (a) IrrE and metal ions in equimolar concentration. Lane M contains molecular weight marker proteins (masses, in kDa, are indicated). (b) Lower concentration of metal ions compared to IrrE. (c) Excess zinc inhibits IrrE‐mediated DdrO cleavage

Figure 2

Zinc shock induces IrrE‐dependent DdrO cleavage in vivo. Exponential phase cultures were exposed for 10 min to 250 μmol/L of the indicated metal ions, and analyzed by Western blotting using an antiserum raised against DdrO_C of D. deserti. Forty μg of cell extract protein was loaded in each lane, except for lane P where 20 ng of purified DdrO_C was loaded. (a) D. deserti wild‐type strain (WT). (b) D. radiodurans wild‐type (D. rad. WT) and D. deserti irrE mutant (ΔirrE) and wild‐type strains

Figure 3

Effect of IrrE on binding of DdrO to RDRM‐containing promoter regions. (a) DdrO (0.8 μmol/L) was incubated with DNA fragments containing the promoter of dnaK, ddrA or ddrD, or the intergenic region of the divergent ddr O_C and Deide_20580 genes that are both radiation‐induced (DNA fragments at 51, 64, 57 and 57 nmol/L, respectively). The sample in lane 5 also contained 1 μg of Poly(dI‐dC). (b) Electrophoretic mobility shift assays (EMSA) with DNA fragments containing ddr O_C‐Deide_20580 intergenic region or ddrA promoter region in presence of IrrE. When both IrrE (or IrrE‐E83Q) and DdrO were present (1.7 μmol/L each), either IrrE and DdrO were pre‐incubated at 37°C (lane 5) or room temperature (lanes 6, 8, 14 and 16) prior to addition of DNA (symbols + and ‐ in grey background), or DdrO and DNA were incubated prior to addition of and incubation with IrrE (lanes 7, 9, 15 and 17; white symbols in black background)