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 Desiccation Response regulon in Deinococcus 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 Deinococcus radiodurans responds to gamma radiation or desiccation through the coordinated expression of genes 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 genome from severely damaged DNA, especially through the error-free repair of hundreds of lethal double-strand Microorganisms rapidly respond to environmental breaks (DSBs) (Blasius et al. 2008). This extraordinary stresses by coordinated expression of a number of radioresistance emanates from an efficient repair of DNA genes, either arranged in operon(s) in prokaryotes or strand breaks, proteome-protective antioxidant functions, non-contiguous in eukaryotes, 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 protein(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/ proteins 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 gene 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 plasmids used in this study Strain/Plasmid Description References Bacterial strains D. radiodurans DirrE ATCC BAA-816, Wild type strain 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.