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Home , Deinococcus deserti, Deinococcus radiodurans

J Biosci (2021)46:10 Ó Indian Academy of Sciences

DOI: 10.1007/s12038-020-00123-5 (0123456789().,-volV)(0123456789().,-volV)

New insights into the activation of Radiation Response regulon in radiodurans

1,2 1,2 ANAGANTI NARASIMHA and BHAKTI BASU * 1Molecular Biology Division, Bhabha Atomic Research Centre, Mumbai 400 085, India 2Homi Bhabha National Institute, Training School Complex, Anushakti Nagar, Mumbai 400 094, India

*Corresponding author (Email, [email protected])

MS received 3 September 2020; accepted 17 November 2020

The highly radiation-resistant bacterium responds to gamma radiation or desiccation through the coordinated expression of belonging to Radiation and Desiccation Resistance/Response (RDR) regulon. RDR regulon is operated through cis-acting sequence RDRM (Radiation Desiccation Response Motif), trans-acting repressor DdrO and protease IrrE (also called PprI). The present study evaluated whether RDR regulon controls the response of D. radiodurans to various other DNA damaging stressors, to which it is resistant, such as UV rays, mitomycin C (MMC), methyl methanesulfonate (MMS), ethidium bromide (EtBr), etc. Activation of 3 RDR regulon genes (ddrB, gyrB and DR1143) was studied by tagging their promoter sequences with a highly sensitive GFP reporter. Here we demonstrated that all the DNA damaging stressors elicited activation of RDR regulon of D. radiodurans in a dose-dependent and RDRM-/ IrrE-dependent manner. However, ROS-mediated indirect effects [induced by hydrogen peroxide (H2O2), methyl viologen (MV), heavy metal/metalloid (zinc or tellurite), etc.] did not activate RDR regulon. We also showed that level of activation was inversely proportional to cellular abundance of repressor DdrO. Our data strongly suggests that direct DNA damage activates RDR regulon in D. radiodurans.

Keywords. Deinococcus radiodurans; Radiation Desiccation Response regulon; Radiation Desiccation Response Motif; promoter; DdrO; dose-response dynamics

1. Introduction from severely damaged DNA, especially through the error-free repair of hundreds of lethal double-strand rapidly respond to environmental breaks (DSBs) (Blasius et al. 2008). This extraordinary stresses by coordinated expression of a number of emanates from an efficient repair of DNA genes, either arranged in operon(s) in prokaryotes or strand breaks, -protective functions, non-contiguous in , operated through a multiplicity and compact structure of genome, etc. (Daly common regulatory mechanism called regulon (Geisel et al. 2010; Krisko and Radman 2010). 2011). Transcriptional coordination of a regulon is D. radiodurans responds to gamma irradiation or mediated through trans-acting (s) that specifi- desiccation stresses by upregulating the expression of a cally binds to cis-acting regulatory sequence(s) in the number of genes during the post-stress recovery phase. promoter region of the operon(s)/genes it regulates Transcriptomic and proteomic analyses during recovery (Alkema et al. 2004; Zhang et al. 2012). An extremo- of D. radiodurans from gamma irradiation or desicca- phile Deinococcus radiodurans displays remarkable tion unraveled a coordinated induction of genes/ resistance to gamma radiation (Battista 1997). The that contribute to DNA recombination/repair, cellular organism is capable of reconstituting a functional detoxification and a few hypothetical Deinococcus- Electronic supplementary material: The online version of this article (https://doi.org/10.1007/s12038-020-00123-5) contains supplementary material, which is available to authorized users. http://www.ias.ac.in/jbiosci 10 Page 2 of 16 A Narasimha and B Basu specific proteins (Gutman et al. 1993;Liuet al. 2003; study. The dose-response relationship established for the Tanaka et al. 2004;Luet al. 2009; Bentchikou et al. RDR regulon genes revealed intrinsic differences 2010; Basu and Apte 2012). RDR (Radiation and Des- between different genes. Further, we probed the role of iccation Resistance/Response) regulon was predicted ROS mediated indirect effects in regulation by RDR through comparative genomics (Makarova et al. 2007). regulon. We found that RDR regulon was not induced A 17 bp palindromic cis-regulatory sequence called by oxidizing agents [hydrogen peroxide (H2O2), methyl RDRM (Radiation Desiccation Response Motif) was viologen (MV), heavy metal/metalloid (zinc or tellu- revealed from the comparison of genome sequences of rite)]. We demonstrate that the degree of RDR regulon D. radiodurans and D. geothermalis, and analyzing activation is inversely proportional to DdrO abundance. flanking sequences of genes that were strongly upregu- The data presented here expands the scope of RDR lated by gamma radiation in D. radiodurans,severalof regulon of D. radiodurans beyond radiation and desic- which displayed radiation-sensitive phenotype upon cation response. inactivation (Tanaka et al. 2004; Makarova et al. 2007). In D. radiodurans, RDR regulon comprises 29 ORFs, mostly arranged non-contiguously, except for cinA and 2. Materials and methods hut operons (Makarova et al. 2007). RDRM sequence, essential for basal repression as well as gamma radia- 2.1 The bacterial strains and growth conditions tion-induced expression of several RDR regulon genes in D. radiodurans (Ujaoney et al. 2010; Anaganti et al. All the bacterial strains used in this study are listed in 2016; Anaganti et al. 2017)isoperatedthroughtrans- table 1. D. radiodurans cells were grown in TGY acting repressor DdrO that suppresses expression of medium (1% tryptone, 0.1 glucose and 0.5% yeast RDR regulon genes under non-stress conditions (Devi- extract) at 32°C either in broth with constant shaking at gne et al. 2015; Anaganti et al. 2016). Activation of 150 rpm or on TGY agar plates (TGY with 1.5% bacto RDR regulon genes upon gamma irradiation is achieved agar). E. coli cells were grown in Luria Bertani (LB) through cleavage of DdrO protein by metalloprotease medium at 37°C, in broth with constant shaking at 150 IrrE (also called PprI) (Wang et al. 2015). The level of rpm or on agar plates at static conditions. Media were activation of different RDR regulon genes is governed supplemented with appropriate antibiotics (5 lg/ml of by sequence conservation and location of RDRM with Kanamycin for D. radiodurans and 50 lg/ml of respect to the core promoter, etc. (Anaganti et al. 2017). Kanamycin or 100 lg/ml of carbenicillin for E. coli) In addition to gamma rays and desiccation, D. whenever required. Growth was monitored either by radiodurans is resistant to very high doses of UV rays, measuring optical density at 600nm at appropriate time chemical mutagens, oxidizing agents etc. (Battista 1997; points or by counting the colony-forming units (CFU) Makarova et al. 2001;Blasiuset al. 2008). However, on agar plates following 48 h of incubation at 32°C. mapping of RDR regulon genes expression in response to these stressors is limited. Only a few genes such as ssb, recA, etc. are reported upregulated following 2.2 Stress conditions exposure to UV rays, MMC, etc. (Ujaoney et al. 2010; Selvam et al. 2013;Dulermoet al. 2015). Additionally, D. radiodurans cells were grown overnight to the early the expression of RDR regulon genes at sub-lethal stationary phase (OD600 = 2.5 ± 0.2). The cells were concentrations of all these stressors also remains com- resuspended in fresh TGY at a cell density of OD600 = pletely unexplored. In the present study, we systemati- 3.0. The cell suspension was exposed to various cally investigated the response of D. radiodurans to six genotoxic stressors as per details given below. Fol- DNA damaging stressors that include ionizing radiations lowing each stress, the cells were resuspended in fresh (gamma rays and UV rays), desiccation, and chemical TGY broth (at starting OD600 = 0.5) and allowed to mutagens [mitomycin C (MMC), methyl methanesul- recover under standard growth conditions for 6 h. fonate (MMS) and ethidium bromide (EtBr)]. We chose i) Gamma irradiation: The cells were exposed to 3 RDR regulon genes, viz. ddrB (DR0070), gyrB various doses of gamma radiation (0 kGy–6kGy) in a (DR0906) and DR1143 to evaluate their activation pat- gamma irradiator (GC-5000, BRIT, India) at a dose rate tern using promoter-reporter fusions. Activation of of 2 kGy/h, as per the protocol described earlier expression was recorded through highly sensitive green (Anaganti et al. 2017). Similarly treated cells, except fluorescent proteins (GFP) expression. Here we report for gamma radiation, acted as the control. Following that all the stressors activated RDR regulon genes under stress, cells were recovered. RDR regulon in D. radiodurans Page 3 of 16 10 Table 1. List of bacterial strains and used in this study

Strain/ Description References Bacterial strains D. radiodurans DirrE ATCC BAA-816, Wild type A gift from M. Daly (Daly et al. 2010) D. radiodurans The irrE gene knock out mutant of D. radiodurans Anaganti et al. (2016) E. coli DH5a F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoRnupG Lab collection - ? U80dlacZDM15 D(lacZYA-argF)U169, hsdR17(rKmK), k– - - - R E. coli BL21 F ompT gal dcmhsdSB(rB mB) k(DE3) pLysS(cm ) Lab collection Plasmids pKG Promoter probe shuttle vector 4745 bp, KanR Anaganti et al. (2016) pKG-PDR0070–1 pKG carrying ddrB (DR_0070) gene promoter of D. radiodurans Anaganti et al. (2016) (EcoRI/SpeI), KanR pKG-PDR0906–1 pKG with gyrB (DR_0906) gene promoter of D. radiodurans Anaganti et al. (2016) (EcoRI/SpeI), KanR pKG-PDR0906–2 Same as pKG- PDR0906–1 but with 17bp RDRM sequence deleted Anaganti et al. (2017) pKG-PDR1143 pKG with DR_1143 gene promoter of D. radiodurans (EcoRI/SpeI), Anaganti et al. (2016) KanR pKG-PDR0606–1 pKG with groESL (DR_0606) gene promoter of D. radiodurans Anaganti et al. (2016) (EcoRI/SpeI), KanR pET21-ddrO pET21a(?) vector with ddrO (DR2475) gene (NdeI/HindIII), AmpR Anaganti et al. (2016)

ii) UV irradiation: The cells (OD600 = 5.0) were lM)for1hat32°Cwithshakingat150rpm.Following deposited onto a filter membrane (0.45 micron, Milli- stress, cells were recovered. pore, 47 mm diameter) to form a thin cell layer on the membrane. The cells were then exposed to different doses of UV rays (0 kJ/m2–2 kJ/m2) at the dose rate of 2.3 Promoter activity assay 4 J/m2/s. Similarly treated cells, except for UV irradi- ation, acted as the control. Following stress, cells were During recovery, the promoter activity was monitored recovered. as detailed earlier (Anaganti et al. 2016). In brief, 500 iii) Desiccation: The cells were deposited onto the ll of the sample was withdrawn at appropriate time filter membrane as explained above and kept in a sterile intervals, the cells were washed with phosphate buffer Petri dish. Desiccation was carried out for 0–12 weeks as saline (PBS) and resuspended in PBS. The cell density per the protocol described earlier (Ujaoney et al. 2017). (OD600) and fluorescence (kex 489nm, kem 520nm) Similarly treated cells, except for desiccation, acted as were measured using a plate reader (Synergy H1, the control. Following stress, cells were recovered. BioTek, UK). Fluorescence was normalized against the iv) Chemical mutagens: The cells were exposed to cell density (Florescence/OD600nm). All graphs were various doses of DNA alkylating agents such as methyl plotted using origin-pro 8.0 software. methanesulfonate (MMS, 0–50 mM), mitomycin-C (MMC, 0–50 lg/ml) or DNA intercalating agent ethidium bromide (EtBr, 0–50 lg/ml) for 1 h at 32°C 2.4 Purification of DdrO protein and generation with shaking at 150 rpm. Similarly treated cells, except of anti-DdrO polyclonal antibodies for chemical mutagen, acted as the control. Following stress, cells were recovered. Cloning of response regulator ddrO (DR2574) gene v) Oxidizing agents: The cells were exposed to hydro- in pET21a vector and identification of correct clones gen peroxide (H2O2, 0–50 mM) or methyl viologen (MV, by DNA sequencing was reported earlier (Anaganti 0–20 or 250 lM) for 1 h at 32°C with shaking at 150 rpm. et al. 2016; Anaganti et al. 2017). E. coli BL21 cells Similarly treated cells, except for oxidizing agents, acted carrying pET21-ddrO plasmid were induced with as the control. Following stress, cells were recovered.vi) 1mM IPTG for over-expression of DdrO protein. The Metal : The cells were exposed to ZnCl2 (0–1000 cells were lysed by sonication and the histidin-tag- lM), MnCl2 (0–20 or 250 lM) or K2TeO3 (0–10 or 250 ged DdrO protein was purified using affinity 10 Page 4 of 16 A Narasimha and B Basu chromatography matrix (Ni-NTA Agarose, Qiagen) pKG-PddrB, pKG-PgyrB or pKG-PDR1143 were exposed as per manufacturer’s protocol. The purified protein to various DNA damaging agents such as gamma rays was resolved on SDS-PAGE gel and the DdrO pro- (6 kGy), UV rays (2 kJ/m2), desiccation (12 weeks), tein band corresponding to 14 kDa was excised from MMS (50 mM), MMC (5 lg/ml), EtBr (15 lg/ml), the gel, the protein was eluted and lyophilized. Anti- unless stated otherwise. Response to gamma irradiation DdrO polyclonal antibodies were prepared in rabbit, stress served as a positive control. at the commercial facility (Genei Laboratories Pvt. We demonstrate that similar to gamma rays, expo- Ltd., India). sure to UV rays, desiccation, MMS, MMC, EtBr also activated GFP expression from all the 3 promoters of RDR regulon genes (figure 1A–C). For all the three 2.5 Western blotting promoters, the highest activation was elicited by exposure to gamma rays, while the response to other The cellular proteins were extracted from D. radiodu- stressors varied with each promoter. For PddrB, rans cells by resuspending cells in Laemmli buffer decreasing order of activation was observed following followed by boiling for 5 min. The cellular proteins (30 exposure to gamma rays[desiccation[EtBr[MMS lg/ lane) were resolved by 15% SDS-PAGE. The [ MMC [ UV rays (figure 1A). For PgyrB, activation resolved proteins were electro-blotted onto nitrocellu- by gamma rays was equivalent to activation by EtBr, lose membrane, probed with anti-DdrO polyclonal followed by desiccation [ MMS [ MMC [ UV rays antibodies raised in rabbit (Dilution 1:1000) followed (figure 1B). For PDR1143, order of activation was by alkaline phosphotase enzyme-linked anti-rabbit IgG gamma rays [ MMS [ EtBr [ desiccation [ UV rays secondary antibodies (1:10,000 dilution, Sigma). The [ MMC (figure 1C). Overall, all three promoters blots were developed using NBT-BCIP substrate showed higher activation by gamma rays, desiccation, (Roche). The intensity of the bands was analyzed by MMS or EtBr while activation by MMC or UV rays GelQuant software. was comparatively lower. It has been reported earlier that following gamma irradiation, RDR regulon genes are regulated 3. Results through cis-acting element RDRM and trans-acting proteins DdrO and metalloprotease IrrE (Devigne 3.1 RDR regulon of D. radiodurans is activated et al. 2015;Wanget al. 2015). We evaluated whe- by a wide spectrum of DNA damaging stressors ther the same regulatory circuit is used for responding to other DNA damaging stressors as Tolerance of D. radiodurans to extremely high doses of well. For this purpose, we used PgyrB since its gamma rays, UV rays, prolonged desiccation as well as RDRM motif does not overlap with -10/-35-like unusually high concentrations of various chemical sequences, thereby allowing us to measure promoter mutagens and oxidizing agents is well established activity even after deletion of RDRM. PddrB and (Battista 1997; Tanaka et al. 2004; Daly et al. 2010; PDR1143 were excluded from this experiment since Slade and Radman 2011; Basu and Apte 2012). deletion of RDRM itself drastically reduced their However, except for gamma rays, the response of RDR promoter activity (Anaganti et al. 2016; Anaganti regulon and its regulation following exposure to vari- et al. 2017). We observed that similar to gamma ous other stressors remains unexplored. Promoter probe rays, activation of PgyrB was abolished following shuttle vector pKG, developed in our lab earlier, is a exposure to UV rays, desiccation, MMS, MMC or versatile tool to monitor the real-time expression and EtBr, when RDRM sequence was deleted from the response of deinococcal promoters to various stressors promoter or when promoter activity was scored (Anaganti et al. 2016). Here, we assessed the response under irrE- background (in D. radiodurans DirrE of 3 RDR regulon genes (ddrB, gyrB and DR1143) of knockout mutant) (figure 1D). Based on these D. radiodurans, to various stressors, by monitoring results, we conclude that the RDR regulon of D. activation of their respective promoters (PddrB,PgyrB radiodurans responds to a wide spectrum of DNA and PDR1143) as a function of GFP reporter expression. damaging stressors through a conserved RDRM- Recombinant D. radiodurans cells harboring either DdrO-IrrE regulatory circuit. RDR regulon in D. radiodurans Page 5 of 16 10

Figure 1. Activation of RDR regulon genes by various DNA damaging stressors. Activation of (A)PddrB or (B)PgyrB promoters of D. radiodurans following exposure to gamma radiation (6 kGy), desiccation (12 weeks), EtBr (15 lg/ml), 2 MMS (50 mM), MMC (5 lg/ml) or UV rays (2 kJ/m ). The highest fold change in GFP expression for PddrB was recorded at 4 h of recovery following exposure to all the stressors, except for UV rays where it was recorded at 3 h for PddrB or PgyrB, respectively. (C) Activation of PDR1143 promoter of D. radiodurans following exposure to gamma radiation (6 kGy), desiccation (12 weeks), EtBr (15 lg/ml), MMS (10 mM), MMC (5 lg/ml) or UV rays (1.5 kJ/m2). The highest fold change in GFP expression for PddrB was recorded at 2 h of recovery following exposure to gamma rays, MMS or UV rays and at 4 h of recovery following exposure to desiccation, EtBr or MMC. (D) Effect of absence of IrrE or RDRM on activation of PgyrB promoter of D. radiodurans exposed to all the DNA damaging stressors (as mentioned in B). WT represents a condition in which both IrrE proteins as well as RDRM sequence were present, DRDRM represents a condition in which PgyrB promoter with RDRM sequence deletion was used and DirrE represents a condition in which GFP expression was scored in D. radiodurans DirrE mutant. Error bars indicate mean ± standard deviation derived from 3 independent biological replicates.

3.2 Dose-response dynamics of RDR regulon studied the expression dynamics of RDR regulon genes genes following exposure to different DNA following exposure to 6 kGy of gamma rays (Anaganti damaging agents et al. 2016). However, dose-response dynamics of RDR regulon at sub-lethal concentrations of gamma Expression dynamics of RDR regulon genes has been rays remains unknown. As for other stressors, there is a documented in the range of 3–15 kGy dose for gamma paucity of data, with only single time point expression rays, using different experimental approaches such as details available, if any. We systematically investigated transcriptomics, proteomics, etc. (Liu et al. 2003; the dose-response dynamics of PddrB,PgyrB and PDR1143 Tanaka et al. 2004; Basu and Apte 2012). Our lab during the recovery phase (0–6 h) following exposure 10 Page 6 of 16 A Narasimha and B Basu to a wide range of gamma rays (0–6 kGy), desiccation translation (Snodgrass et al. 2010;Dolz-Edoet al. (0–12 weeks), UV rays (0–2 kJ/m2), methyl methane- 2013), which may interfere with GFP expression. sulfonate (MMS, 0–50 mM), mitomycin-C (MMC, The lowest activation levels for PddrB were observed 0–50 lg/ml) or ethidium bromide (EtBr, 0–50 lg/ml). during recovery from UV irradiation. Here, a wide Among the 3 RDR regulon genes under investigation, range of UV irradiation (0.25–2 kJ/m2) activated best activation (in terms of fold change in GFP PddrB promoter with peak activation observed at 3 h expression) was observed for PddrB, followed by of recovery for all the doses tested (figure 2F). PDR1143, since the expression of both these promoters is The activation pattern of the gyrB gene promoter barely detectable under non-stress conditions (Anaganti (PgyrB) was found to be similar to that of PddrB in terms et al. 2016). PgyrB promoter is expressed under non- of doses of various stressors that elicited activation stress conditions (Anaganti et al. 2016) and thus, although the time point at which maximum activation showed lower activation when measured in terms of was achieved at each dose varied. For gamma rays, fold change in GFP expression. increased activation was observed with increasing dose The promoter of ddrB gene (PddrB) was activated (figure 3A). Similar to PddrB, increasing the dose of with as low as 500 Gy dose of gamma rays and EtBr increased activation of PgyrB till 15 lg/ml but GFP increasing doses were associated with increased expression decreased at higher doses, although PgyrB activation (figure 2A). Maximum activation of the was activated by the lowest concentration, 5 lg/ml of promoter was observed at 1 h of post-irradiation EtBr (figure 3B). Dose-response curves for desiccation recovery (PIR) in the lower dose range (up to 1 (figure 3C), MMS (figure 3D), MMC (figure 3E) and kGy), at 2 h of PIR for medium dose range (2–4 UV (figure 3F) showed 2–3 fold activation of PgyrB. kGy) or at 4 h of PIR for 6 kGy dose which is a Except for MMC, response to which saturated at 5 lg/ D37 dose for D. radiodurans (Harris et al. 2004) ml concentration, all other stressors increased promoter (figure 2A). Similarly, we report increasing activa- activity with increasing doses. Peak activation was tion of PddrB with increasing duration of desiccation observed at 1 h of recovery following exposure to 0.25 (figure 2B). Activation following 3 weeks of des- kJ/m2 orat2hofrecovery following 0.5–2 kJ/m2 iccation peaked at 2 h of post-desiccation recovery doses of UV rays (figure 3F). (PDR) while that after 6–12 weeks of desiccation Similar to the other 2 gene promoters, PDR1143 was peaked at 4 h of PDR (figure 2B). During recovery best activated by gamma rays, although peak activation of D. radiodurans cells from exposure to EtBr, the at each dose was achieved at 2 h of PIR (figure 4A). lowest dose tested (5 lg/ml) did not elicit any Following exposure to MMS, dose-response was linear activation, while increased activation was observed with maximum activation (between 2–3 folds) withincreasingdosesupto15lg/ml (figure 2C). achieved at 2 h for doses ranging from 5–30 mM, while When the dose of EtBr was increased further (20 for 50 mM MMS, peak activation was observed at 4 h lg/ml), the activation level actually decreased (fig- (figure 4B). PDR1143 responded to 5 lg/ml EtBr with ure 2C). Further increase in the dose (up to 50 lg/ peak activation at 1 h of recovery and higher doses ml) also returned decreased activation levels (Data peak activation was observed at 4 h of recovery not shown). The observed inhibitory effects of EtBr although the response was saturated beyond 15 lg/ml on GFP expression could be due to inhibition of EtBr (figure 4C). Desiccation, UV rays and MMC DNA/ RNA synthesis and transcription in presence caused only subtle activation of PDR1143 at all the doses of high concentrations of EtBr (Grant 1969; Haya- tested. PDR1143 responded to only 12 weeks of desic- kawa et al. 1998). Exposure to MMS resulted in cation (figure 4D) while the response to UV rays was 2 increased activation of PddrB with doses increasing observed only beyond 0.5 kJ/m (figure 4E). Like other from 10 mM to 50 mM (figure 2D). Maximum promoters, response to MMC saturated at 5 lg/ml activation was observed at 1 h with doses up to 20 concentration (figure 4F). mM or at 4 h with doses from 25–50 mM MMS The data presented here establish dose-response (figure 2D). Next, we studied the response of PddrB relationships of 3 RDR genes under study, in response to MMC. Here, we observed activation of the pro- to six DNA damaging agents. Although the response to moter at 5 lg/ml concentration of MMC with various types of DNA damage is regulated through a maximumactivationachievedat4hduringrecov- conserved regulatory circuit in general, each individual ery (figure 2E), however, with further increase in RDRM-promoter displayed unique response features dose, the activation level actually decreased. MMC which could be attributable to differences in their is known to exert an inhibitory effect on protein promoter structures, RDRM sequence and location, etc. RDR regulon in D. radiodurans Page 7 of 16 10

Figure 2. Dose-response dynamics of PddrB exposed to various DNA damaging stressors. D. radiodurans cells harboring pKG-PddrB were exposed to (A) gamma rays (0–6 kGy), (B) desiccation (0–12 weeks), (C) EtBr (0–20 lg/ml), (D) MMS (0–50 mM), (E) MMC (0–10 lg/ml) or (F) UV rays (0–2 kJ/m2). Fold change in GFP expression was recorded till 6 h of recovery. Error bars indicate mean ± standard deviation derived from 3 independent biological replicates.

3.3 ROS mediated indirect effects to evaluate the contribution of indirect effects in the do not contribute to activation of RDR regulon activation of RDR regulon. genes Gamma radiation, which caused the highest acti- vation of RDR regulon genes, produce highly reac- The hazardous effects of are caused tive and thus, extremely toxic hydroxyl radicals (HO˙ ) either by the direct action of radiation wherein a variety through radiolysis of water which either cause of bio-molecules directly absorb the photon energy and localized damage to DNA, proteins, lipids, etc., or in undergo localized damage [direct effect] or by indirect the presence of O2 transform into superoxide ions .– action of radiation wherein the absorbed energy is (O2 ) and hydrogen peroxide (H2O2) (Halliwell and transferred to other molecules resulting in diffused Gutteridge 2015). Here we studied dose-response damage [indirect effect] (Alizadeh et al. 2013). It has dynamics following exposure to H2O2 or methyl .– been estimated that, in general, direct interactions of viologen that primarily generates O2 , to study the gamma-ray photons with DNA result in *20% of contribution of indirect effects towards the activation damage while *80% of the DNA damage is caused by of RDR regulon. In addition, we also studied the indirect effects through radiation-induced ROS (Halli- dose-response dynamics of zinc mediated oxidative well and Gutteridge 1999). Apart from ionizing radia- stress. Recently, 250 lM zinc was shown to activate tion, several environmental stressors such as IrrE protein in D. deserti, leading to the speculation desiccation, MMC, salinity, , etc., enhance that -induced zinc release could be the the generation of reactive (ROS) (Shi activating signal for induction of RDR regulon et al. 2013; Xie et al. 2019). Therefore, it was pertinent (Blanchard et al. 2017). 10 Page 8 of 16 A Narasimha and B Basu

Figure 3. Dose-response dynamics of PgyrB exposed to various DNA damaging stressors. D. radiodurans cells harboring pKG-PgyrB were exposed to (A) gamma rays (0–6 kGy), (B) EtBr (0–20 lg/ml), (C) desiccation (0–12 weeks), (D) MMS (0–50 mM), (E) MMC (0–10 lg/ml) or (F) UV rays (0–2 kJ/m2). Other details were the same as described in legends to figure 2.

We exposed recombinant D. radiodurans cells indirect effects per se do not activate RDR regulon harboring pKG-PgyrB to three different oxidizing genes in D. radiodurans. agents, viz. H2O2 (0–50 mM), methyl viologen (0–20 or 250 lM) or ZnCl2 (0–1000 lM), whereas expo- sure to MnCl2 (0–20 or 250 lM) served as a neg- 3.4 Activation of RDR genes is inversely ative control as it acts as an antioxidant (Daly et al. proportional to DdrO abundance 2004). We observed that none of the oxidizing agents activated PgyrB (figure 5A-C) as the GFP expression Next, we asked how calibrated activation of RDR levels remained similar to control (figure 5D) regulon is achieved during the response to different throughout the recovery phase. We also checked DNA damaging stressors. It has been shown that cel- RDR regulon activation following exposure to lular concentration of DdrO substantially decreases in K2TeO3 (0–10 or 250 lM), however, GFP expression the early phase of recovery following exposure of D. levels did not increase (supplementary figure 1). We radiodurans to 3.8 kGy gamma irradiation (Devigne have earlier reported ROS generation and depletion et al. 2015) which coincides with an increase in of reduction potential in D. radiodurans following expression of RDR regulon genes. Hence, we moni- exposure to potassium tellurite, however, induction of tored the abundance of DdrO protein in D. radiodurans DdrB protein or increase in the abundance of GyrB (WT or DirrE mutant) cells (harboring pKG-PgyrB) protein was not observed (Anaganti et al. 2015). exposed to different DNA damaging stressors and Thus, current findings are consistent with our earlier checked if any correlation exists between DdrO abun- findings. From this data, we infer that ROS mediated dance and RDR gene activation. Compared to RDR regulon in D. radiodurans Page 9 of 16 10

Figure 4. Dose-response dynamics of PDR1143 exposed to various DNA damaging stressors. D. radiodurans cells harboring pKG-PDR1143 were exposed to (A) gamma rays (0–6 kGy), (B) MMS (0–50 mM), (C) EtBr (0–20 lg/ml), (D) desiccation (0–12 weeks), (E) UV rays (0–2 kJ/m2)or(F) MMC (0–10 lg/ml). Other details were the same as described in legends to figure 2. unstressed cells, cells exposed to 6 kGy gamma irra- possibility of EtBr enhancing GFP fluorescence or diation showed about 20 folds decrease in DdrO acting through an additional regulatory mechanism abundance (figure 6A, C) which correlated well with could not be ruled out. The cells exposed to oxidizing the highest induction in GFP expression from RDRM- agents did not show activation of RDR regulon genes promoters (figure 6D). As predicted, D. radiodurans in our experiments (figure 5). Corroborating these DirrE cells exposed to 6 kGy gamma irradiation results, we also found that DdrO was not degraded in showed DdrO abundance similar to unstressed cells cells exposed to oxidizing agents or metals (figure 6B- and no change in GFP expression (figure 6A). Cells C) and no change in RDR regulon genes expression exposed to desiccation, MMS or EtBr showed a mod- following exposure to oxidizing agents (figure 6D). erate decrease in cellular levels of DdrO (figure 6A, C) From these results, we infer that the cellular abundance which translated into a moderate increase in expression of DdrO repressor controls the degree of activation of of RDR regulon genes except for EtBr (figure 6D). RDR regulon. DdrO was least degraded in cells exposed to MMC or UV rays (figure 6A, C) and it correlated well with the lowest induction in GFP expression observed in cells 4. Discussion exposed to MMC or UV rays (figure 6C, D). PgyrB activation by EtBr was found to be higher than by belonging to the genus Deinococcus are pro- MMS or desiccation and therefore, inconsistent with ficient in DNA repair, as a result of which they display the cellular abundance of DdrO. Although the exact extreme resistance to high doses of several DNA reason for this observation is not immediately clear, the damaging agents such as gamma rays, desiccation, UV 10 Page 10 of 16 A Narasimha and B Basu

Figure 5. Dose-response dynamics of PgyrB exposed to various oxidizing agents. D. radiodurans cells harboring pKG-PgyrB were exposed to (A)H2O2 (0–50 mM), (B) methyl viologen (0–20 lM), (C) ZnCl2 (0–1000 lM) or MnCl2 (0–20 lM). Other details were the same as described in legends to figure 2. rays, chemical mutagens, etc. (Battista 1997). Infor- Often, closely related species utilize orthologous mation gathered from the sequenced species of Dei- regulatory circuits to respond to a particular signal nococci indicate very high conservation of a regulatory (Perez and Groisman 2009). Similarly, an organism circuit called RDR regulon, comprising of cis-acting may employ a common regulatory circuit to sequence RDRM, trans-acting transcription factor orchestrate a response to different stressors with DdrO and its cognate metallo-peptidase IrrE (Blan- similar activation signal(s). So far, gene expression chard et al. 2017). Although, RDR regulon displays a mediated by RDR regulon is well studied with minor species-specific variation with respect to the respect to gamma irradiation and desiccation repertoire of RDRM-promoter genes, at least 16 RDR stresses, in several Deinococcal species (Ludanyi regulon genes were common to D. radiodurans, et al. 2014;Devigneet al. 2015;Wanget al. 2015; D. deserti, and D. geothermalis (Blanchard et al. Anaganti et al. 2017). Here we demonstrated that 2017). Similarly, a few deinococcal strains encode apart from gamma radiation or desiccation, UV paralogs of RDR regulon genes, for example, D. rays, MMS, MMC as well as EtBr could activate deserti encodes two functionally redundant DdrO RDR regulon genes in D. radiodurans in RDRM genes, encoded DdrOC and plasmid-en- and IrrE dependent manner, while ROS mediated coded DdrOP3 (Ludanyi et al. 2014). indirect effects did not contribute to activation. RDR regulon in D. radiodurans Page 11 of 16 10

Figure 6. Detection of DdrO repressor abundance and corresponding activation of PgyrB promoter, following exposure to various DNA damaging stressors. D. radiodurans (WT or DirrE) cells, were subjected to the indicated doses of (A) various genotoxic stresses or (B) various oxidizing agents. Cellular proteins (30lg), extracted immediately after stress, were resolved by 14% SDS-PAGE and electro-blotted onto the nitrocellulose membrane. DdrO protein was detected by immunostaining. Molecular weight markers are indicated on the left-hand side. Numbers below each band indicate fold change in abundance as adjudged by densitometry. Standard deviation was calculated from 3 independent biological replicates. (C) Graphical representation of fold change in abundance of DdrO protein following exposure to various stresses. Error bars indicate mean ± standard deviation derived from 3 independent biological replicates. (D) Fold change in promoter activity of PgyrB following exposure to various stresses at 4 h of recovery. Disagreement between DdrO abundance and PgyrB activation by EtBr is indicated by the # mark. Error bars indicate mean ± standard deviation derived from 3 independent biological replicates.

From these results, it is evident that the factors Literature reveals that direct interactions of these common to all the stressors used in this study must stressors with DNA induce a variety of DNA damages act as an activation signal. that bring about structural distortions in DNA that 10 Page 12 of 16 A Narasimha and B Basu further affect DNA-dependent vital cellular processes the proteome of D. radiodurans is preferentially pro- such as DNA replication, transcription, etc. Both tected from ROS-induced oxidative damage leaving its gamma rays and desiccation cause various types of genome vulnerable to damage (0.002–0.006 DSBs/Gy/ strand breaks in DNA (Potts 1994). UV radiation Mbp of the genome) similar to any other species (Slade induces DNA lesions such as cyclobutane-pyrimidine and Radman 2011). It is, therefore, interesting to dimers (CPDs) and 6–4 photoproducts (6–4PPs) and understand how the organism manages the DNA their Dewar valence Isomers (Sinha and Hader 2002). damage and oxidative stress signals following exposure Methyl methanesulfonate (MMS) alkylates both gua- to DNA damaging agents. nine and adenine at 7th and 3rd position to form While DNA repair needs the inducible expression of 7-methylguanine and 3-methyladenine, respectively, various DNA repair proteins, protection from ROS resulting in base mis-pairing, replication blocks, etc. needs a homeostatic balance of highly abundant (Lundin et al. 2005). Mitomycin-C (MMC) is known to in D. radiodurans. Several knockout be a potent DNA cross-linker. MMC is specifically mutants of D. radiodurans are differentially sensitive selective for 50-CpG-300 sequence and forms a mono- to either DNA damaging agents or oxidants. The adduct with G in the target strand while bis-adduct with mutants wherein the proteins related to DNA damage G opposite to C gives rise to inter-strand crosslinks repair are knocked out (recG, recN, pprA, uvrC, uvrB, (Tomasz 1995). Ethidium bromide preferentially binds drRRA, DR_0354, DR_1374 or DR_2606) display to GC-rich regions in DNA through reversible non- sensitivity to DNA damaging agents (gamma rays, covalent DNA intercalation. Although reversible, DNA desiccation, MMS, MMC or UV), but not to H2O2 intercalating agents are known to induce distortion of (Ujaoney et al. 2010; Selvam et al. 2013; Dulermo the double helix and may interfere with DNA replica- et al. 2015;Heet al. 2020). Conversely, deletion tion, transcription, etc. (Lambert et al. 1990; Reha et al. mutants of ROS sensing transcription factors (oxyR, 2002). Although some of the stressors do not directly DR_0615; oxyR2, DR_A0336 or mur, DR_0865) are produce DNA strand breaks, structural distortions by selectively sensitive to H2O2 without affecting the them are generally sufficient to pose obstacles to the radiation resistance of the organism (Chen et al. 2008; progression of the replication fork (Yeeles et al. 2013) Yin et al. 2010; Ul Hussain Shah et al. 2014). OxyR or repair of the distortions may generate broken-strand protein acts as an activator of catalase (KatA, intermediates. Indeed, reports indicate that the DNA DR_1998) and repressor of DNA binding ferritin-like fragments are generated to rescue the stalled replication protein (Dps1, DR_2263) and Mn?2 transporter fork to avoid lethality (Khan and Kuzminov 2012). (MntH, DR_1709) (Chen et al. 2008). OxyR2 protein Thus, in addition to direct DNA strand breaks, struc- acts as an activator of catalase (KatE, DR_A0259) (Yin tural distortions or stalled replication forks may act as a et al. 2010). Thus, both the oxyR mutants display signal for activation of RDR regulon. increased ROS levels due to decrease in antioxidant All known DNA-damaging agents concomitantly capacity. Similarly, deletion of Mur regulator induce oxidative stress (Slade and Radman 2011). The (DR_0865) imparted sensitivity to H2O2, gamma irra- antioxidant system of D. radiodurans is known to diation or UV rays due to dysregulation of Mn?2 effectively act against primary ROS such as hydroxyl homeostasis, but did not affect RDR regulon genes (Ul Á- radicals (OH˙ ), superoxide radicals (O2 ) and hydrogen Hussain Shah et al. 2014). Mur acts as an activator of ?2 peroxide (H2O2) (Slade and Radman 2011). D. radio- Mn efflux pump MntE (DR1236) and a repressor of durans possesses an immensely redundant antioxidant Mn?2 ABC transporter (Mnt, DR_2284, DR_2285 and capacity due to presence of a battery of highly DR2523). PerR-like protein (PerR, NCBI Ref. No. expressed enzymatic antioxidants (4 SODs, 3 catalases, KJ817356) acts as a repressor of KatA and Dps1 (Liu 2 peroxidases, 4 peroxiredoxins, 2 Dps proteins, etc.) et al. 2014). These mutant phenotypes suggest that as well as strong non-enzymatic antioxidants (Mn?2 defects in antioxidant homeostasis selectively affect the complexes, PQQ, carotenoids, deinoxanthine, etc.) and resistance of D. radiodurans to oxidizing agents couples it with (a) metabolic configuration tuned to without affecting the radiation resistance or induction minimize metabolically generated ROS, (b) signifi- of DNA repair genes. Our observation that oxidative cantly low prevalence of oxidizable amino lysine, stress does not activate RDR regulon is consistent with cysteine and methionine in its proteins and (c) intrinsic this data. We summarize our results and information properties of proteins that suppress vulnerability to available in the literature in a model for activation of oxidative stress (Ghosal et al. 2005; Chang et al. DNA Damage Response (DDR) regulon in D. radio- 2020). However, it is noteworthy to mention that only durans (figure 7). The model clearly demonstrates the RDR regulon in D. radiodurans Page 13 of 16 10

Figure 7. A model for activation of RDR regulon in D. radiodurans. Information obtained from literature on various forms of DNA damage (direct effects) caused by gamma rays, desiccation, MMS, EtBr, MMC or UV rays or their repair intermediates is depicted. Indirect effects of gamma rays, desiccation UV rays or MMC through the generation of ROS or ROS generated by oxidizing agents such as H2O2, methyl viologen or metal ions, and their impact on DNA are shown at the left-hand bottom. Reactions carried out by enzymatic or non-enzymatic antioxidants are shown at the appropriate steps of ROS interconversion/ detoxification along with the activating (?) or repressing (a) action of antioxidant regulatory proteins. Mn?2 transporters are shown at the bottom and the regulatory effects of the regulatory proteins are depicted. Events that lead to activation or repression of RDR regulon are shown on the right-hand side. The extent of DdrO degradation by each DNA damaging stressor is indicated through the thickness of the arrow: the thicker the arrow, the greater degradation or lower DdrO abundance, and vice versa. Cross marks indicate the inability of oxidizing agents to signal DdrO degradation or the inability of IrrE to degrade DdrO bound to DNA. The activation signal for IrrE, which is still unknown, is shown by a question mark. Appropriate references from the bibliography are cited in parenthesis. 10 Page 14 of 16 A Narasimha and B Basu independent regulation of the DNA damage and DNA repair and oxidative stress alleviation. Mol. Cell. oxidative stress signals in D. radiodurans. It under- Proteomics 11 M111 011734 scores an important role of structural alterations in Battista JR 1997 Against all odds: The survival strategies of DNA in the activation of RDR regulon and dissects its Deinococcus radiodurans. Annu. Rev. Microbiol. 51 independence from ROS-mediated oxidative stress. 203–224 The data presented here strongly suggest that direct Bentchikou E, Servant P, Coste G and Sommer S 2010 A major role of the recfor pathway in DNA double-strand- DNA damage by various clastogenic agents activate break repair through esdsa in Deinococcus radiodurans. RDR regulon in D. radiodurans and thereby, expand- PLoS Genet. 6 e1000774. ing the regulatory network of RDR regulon beyond Blanchard L, Guerin P, Roche D, Cruveiller S, Pignol D, radiation and desiccation resistance/response. In view Vallenet D, Armengaud J and de Groot A 2017 Conser- of our results, renaming the regulatory circuit as DDR vation and diversity of the IrrE/DdrO-controlled radiation (DNA Damage Resistance/Response) regulon may response in radiation-resistant Deinococcus bacteria. better represent its regulatory scope. The response of Microbiologyopen 6 e00477 RDR regulon genes of D. radiodurans to a wide Blasius M, Sommer S and Hubscher U 2008 Deinococcus spectrum of DNA damaging stressors over a broad radiodurans: What belongs to the survival kit? Crit. Rev. dose range and on a single platform is documented for Biochem. Mol. Biol. 43 221–238 the first time, which would be immensely useful for Chang R, Stanley J, Robinson M, Sher J, Li Z, Chan Y, comparisons across different stresses. Omdahl A, Wattiez R et al. 2020 Protein structure, amino composition and sequence determine proteome vulnerability to oxidation-induced damage. EMBO J. e104523 Chen H, Xu G, Zhao Y, Tian B, Lu H, Yu X, Xu Z, Ying N Acknowledgements et al. 2008 A novel oxyr sensor and regulator of hydrogen peroxide stress with one cysteine residue in Deinococcus The research work reported in this manuscript is fun- radiodurans. 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Corresponding editor: BJ RAO