Gene regulation for the extreme resistance to ionizing radiation of Deinococcus radiodurans

Journal Pre-proof Gene regulation for the extreme resistance to ionizing radiation of Deinococcus radiodurans

Wuzhou Wang, Yun Ma, Junyan He, Huizhou Qi, Fangzhu Xiao, Shuya He PII:

S0378-1119(19)30667-5

DOI:

https://doi.org/10.1016/j.gene.2019.144008

Reference:

GENE 144008

To appear in:

Gene

Received date:

14 May 2019

Revised date:

24 July 2019

Accepted date:

24 July 2019

Please cite this article as: W. Wang, Y. Ma, J. He, et al., Gene regulation for the extreme resistance to ionizing radiation of Deinococcus radiodurans, Gene (2018), https://doi.org/ 10.1016/j.gene.2019.144008

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Journal Pre-proof Title: Gene regulation for the extreme resistance to ionizing radiation of Deinococcus radiodurans Wuzhou Wang1, 3, Yun Ma1, Junyan He1, 3, Huizhou Qi2, 3, Fangzhu Xiao3, Shuya He1, 3,＊ 1. Institute of Biochemistry and Molecular Biology, Hengyang Medical College, University of South China, Hengyang, 421001, China. 2. Function laboratory center, Hengyang Medical College, University of South China, Hengyang, 421001, China.

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3. Hengyang Key Laboratory for Biological Effects of Nuclear Radiation, University of South China, Hengyang, 421001, China. Corresponding author: Shuya He

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＊

South China, Hengyang, 421001, China.

Abstract

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Tel: +86 7348281293

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Email: [email protected]

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Hengyang Key Laboratory for Biological Effects of Nuclear Radiation, University of

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Deinococcus radiodurans is a model microorganism used for studies on DNA repair and antioxidation due to its extraordinary tolerance to ionizing radiation and

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other DNA-damaging agents. Various transcriptome analyses have revealed that hundreds of genes are induced and that many other genes are repressed during recovery of D. radiodurans following irradiation, suggesting that gene regulation is of great importance for the extreme resistance of this microorganism to ionizing radiation. In this article, we focus on some reported strategies that are employed by D. radiodurans to regulate the genes implicated in its extreme tolerance to ionizing radiation for a comprehensive understanding of the reasons this bacterium can survive such extraordinary stress. We expect this review to shed light on potential radioprotective agents and applications for use in a range of fields. Keywords: Radioresistance, RDRM, Two-component systems, sRNA, Deinococcus radiodurans Introduction

Journal Pre-proof Over the last few decades, with the wide utilization of nuclear technology in a broad range of fields, there have been increasing situations in which humans are exposed to ionizing radiation. According to UNSCEAR, the largest source of ionizing radiation affecting humans is medical irradiation, such as that associated with radiotherapy for cancer patients, which produces side effects; computed tomography for diagnosis; and interventional medicine (Shannoun, 2015). Sometimes nuclear accidents suddenly happen, e.g., the nuclear power station explosion in Fukushima, and these produce such high levels of ionizing radiation that it is difficult for people

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in the area to escape its effects. In addition to the artificial ionizing radiation

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mentioned above, humans have incidental contact to natural ionizing radiation, e.g., cosmic rays from long journeys in the air and radon emanation in newly constructed

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houses (McLean et al., 2017).

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It is well known that humans have defense systems against very low doses of

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ionizing radiation but are unable to resist high doses. Ionizing radiation, which carries high energy and can ionize the molecules through which it passes, comprises two

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types: particulate (α and β particles) and electromagnetic (X and gamma rays) (Cox and Battista, 2005), both of which generate numerous ROS (reactive oxygen species)

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after cascading reactions. ROS can cause tissue or cell damage or death by (1) peroxidizing membrane lipids and further disrupting membrane composition, (2) oxidizing and/or cross-linking proteins, (3) breaking and inactivating sugar chains, and (4) damaging DNA (Ghosal et al., 2005). Since few radioprotectors are currently available for use in prevention and therapy, there is an urgent demand for exploring new and more effective protective agents that can be used to protect high-risk individuals (e.g., cancer patients treated by radiotherapy, medical professionals, astronauts and individuals irradiated during a nuclear disaster) from radiation damage (Munteanu et al., 2015). On Earth, some species have been discovered to have the capability to resist extreme ionizing radiation (Lim et al., 2019). Among these, Deinococcus radiodurans has been extensively studied as a good model organism due to the easy genetic manipulation (Minton, 1994) as well as the availability of the complete published genome sequence.

Journal Pre-proof Deinococcus radiodurans is a sphere-shaped, red-pigmented, nonmotile, nonsporulating, nonpathogenic, gram-positive, and aerobic bacterium existing in the form of diads or tetrads (Minton and Daly, 1995). The name of this prokaryote originates from the Greek deinos (strange) and coccus (a berry) (Slade and Radman, 2011). Since the first isolation from canned ground meat (Anderson et al., 1956), sterilized by irradiation but still capable of causing spoilage, D. radiodurans has attracted increasing attention because of its unprecedented resistance to ionizing radiation (Krisko and Radman, 2013). Surprisingly, in the exponential phase, D.

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radiodurans can survive a high dose of 5,000 Gy without evidence of mutation

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(Battista et al., 1999; Blasius et al., 2008). For comparison, exponentially growing D. radiodurans is approximately 30-fold more resistant to irradiation than Escherichia

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coli and 1,000-fold more resistant than humans (Slade and Radman, 2011). After the

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discovery of this extraordinarily radiation resistant organism, biologists have been

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trying to elucidate the mechanisms of the radiation stress tolerance of Deinococci. When faced with hazardous stimuli such as irradiation, organisms must regulate gene

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expression in response to ambient pressures. Various transcriptome analyses have revealed that hundreds of genes were induced and that many other genes were

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suppressed during the recovery of D. radiodurans after ionizing radiation (Liu et al., 2003; Tanaka et al., 2004; Luan et al., 2014), suggesting that gene regulation plays a significant role in the extreme resistance to ionizing radiation. In this article, we focus on some reported strategies that are employed by D. radiodurans to regulate genes implicated in the extreme tolerance to ionizing radiation for a comprehensive understanding of the reasons this bacterium can survive such an extraordinary stress. Characteristic feature of the D. radiodurans genome In 1999, the 3.28-megabase genome sequence of D. radiodurans, containing 3193 open reading frames (ORFs), was published, which revealed that it comprises two chromosomes (I: 2,648,615 bp and II: 412,340 bp) as well as two plasmids (177,466 bp and 45,702 bp) (White et al., 1999). The genome of D. radiodurans has a high GC content, reaching up to 66.6% (Slade and Radman, 2011). It is noteworthy that the genome analysis showed that, unlike other organisms, D. radiodurans

Journal Pre-proof exhibits many redundant in genes involved in DNA repair or ROS scavenging; for instance, it has 23 genes belonging to the Nudix family, 2 different 8-oxo-guanine glycosylases, 3 catalases and 4 superoxide dismutases (White et al., 1999; Makarova et al., 2001). The remarkable redundancy of genes seemingly contributes to the radioresistance of D. radiodurans, since one mutant of several relative genes does not seem to influence the corresponding function. The RDRM-dependent PprI/DdrO-mediated pathway contributes to the extreme radiation tolerance of D. radiodurans

palindromic

sequence

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17-bp

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RDRM, the abbreviation for radiation/desiccation response motif, is a conserved

[TT(A/C)(T/C)G(T/C)NN(T/A)N(A/G)(A/G)C(G/A)C(G/A)(T/G)AA]

existing

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upstream of many radiation-induced genes (e.g., recA, pprA, gyrA, gyrB, ssb and

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ddrO) in D. radiodurans (Ujaoney et al., 2010; Blanchard et al., 2017). Several key

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radiation-induced genes are briefly described below. Ionizing radiation can severely impair the genome of D. radiodurans and lead to hundreds of DNA double-strand breaks (DSB) (Mattimore and Battista, 1996). A recA mutant was remarkably

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sensitive to γ-irradiation (Moseley et al., 1972). RecA, which differs from homologs

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in other bacteria (Warfel and LiCata, 2015) and is likely required to form filaments on duplex DNA in D. radiodurans (Hsu et al., 2011; Pobegalov et al., 2015), has been shown to play a central role in each of two processes of genome reconstitution: extended

synthesis-dependent

strand

annealing

(ESDSA)

and

homologous

recombination (Zahradka et al., 2006; Rajpurohit et al., 2016; Sharma et al., 2019). PprA (pleiotropic protein promoting DNA repair A), first identified by Narumi et al., is another key pleiotropic protein conferring extreme radioresistance to D. radiodurans (Narumi et al., 2004; Adachi et al., 2019). After irradiation, PprA forms a multiprotein complex with many other proteins involved in DNA DSB repair (Kota and Misra, 2008). Irradiated cells devoid of PprA suffered from a severe disorder in DNA segregation and cell division (Devigne et al., 2013). The induction of the many important

genes

involved

in

radiation

damage

repair

suggests

that

the

RDRM-dependent pathway might be the predominant contributor to the unordinary

Journal Pre-proof radioresistance of D. radiodurans. This cis-regulatory element was originally delineated by Makarova et al., who used a bioinformatics approach (Makarova et al., 2007). Their investigation showed that the Deinococcus-specific RDRM sequence was located upstream of many DNA damage response-related genes, but at that time, the answer to the question of what protein targets the RDRM sequence was unknown. In radiosensitive bacteria, the LexA-based SOS radiation response is well characterized (Cheo et al., 1991; Courcelle et al., 2001). Under conditions without DNA damage stress, recA and other SOS-associated genes bound to LexA are

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repressed and thus are expressed only at the basal level. In contrast, when DNA is

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damaged, RecA is active and triggers the self-cleavage of the SOS repressor LexA, resulting in an elevated expression of the previously suppressed genes and the

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beginning of DNA damage recovery. As we know now, the unique bacterium D.

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radiodurans can survive excessive doses of ionizing radiation capable of cause severe

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DNA damage. Such an amazing phenotype implies that there are complicated regulatory networks responsible for the adaptation of the cellular processes to

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environmental changes in vivo. Scientists have even made a great effort to find a system equivalent to that of D. radiodurans but have failed. Although two lexA

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paralogs (dra0344 and dra0074) are present in this organism, neither are induced nor implicated in RecA induction after exposure to ionizing radiation (Narumi et al., 2001; Sheng et al., 2004; Satoh et al., 2006). Interestingly, transcriptomic and proteomics analyses have revealed that, regardless of the dose of ionizing radiation treatment (high or low), one predicted regulator was always upregulated in D. radiodurans and was designated DdrO (DR2574) (Liu et al., 2003; Tanaka et al., 2004). Furthermore, computational analysis revealed that this protein belongs to the Xre family and is specific for D. radiodurans. The encoding gene (dr2574) is preceded by an RDRM sequence. Hence, a hypothesis that DdrO is the global regulator of the RDR regulon in D. radiodurans was presented (Makarova et al., 2007). Nevertheless, devoid of experimental evidence, the regulatory mechanism remained unclear. Interestingly, the depletion of DdrO resulted in induced expression of PprA, GyrA and DdrB proteins, which demonstrated that, in experiments, DdrO performs as a regulator by repressing

Journal Pre-proof the expression of the RDR regulon genes (Devigne et al., 2015). PprI (DR0167), also termed IrrE, was identified at the beginning of the 21st century from natural mutants that were dramatically sensitive to ionizing radiation (Earl et al., 2002; Hua et al., 2003). The crystal structure showed that the PprI protein consists of three domains, one of which is a zinc peptidase-like domain (Vujicic-Zagar et al., 2009). Exogenous expression of pprI from D. radiodurans could enhance the resistance of E. coli to ionizing radiation through a significant induction of RecA protein (Gao et al., 2003). Due to its powerful function of switching on DNA

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damage response and cellular survival networks by inducing numerous crucial DNA

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repair-related genes (such as recA and pprA) following irradiation, this unique gene has attracted much attention (Lu et al., 2009). Expression of PprI improved the

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tolerance of Lactococcus lactis to various stresses (Dong et al., 2015). Moreover, PprI

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provided protection from radiation damage for eukaryotes (Dong et al., 2015; Wen et

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al., 2016). However, understanding the biochemical basis for the role of PprI in gene regulation was initially confounded by divergent findings. A previous study reported

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that DNA binding was necessary for PprI to perform important roles in the radiation damage recovery of D. radiodurans (Lu et al., 2012), while others showed that PprI

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did not bind DNA (Ohba et al., 2005; Vujicic-Zagar et al., 2009). The contradiction was not reconciled until the observation that cleavage of DdrO by PprI was associated with proteolysis activity (Ludanyi et al., 2014; Devigne et al., 2015; Wang et al., 2015). Concerning the regulatory role of PprI in organisms without DdrO, we speculate that active PprI might also cleave some other proteins that act as repressors of stress tolerance-related genes. Taking gyrB, one of the RDR regulon genes, as an example, the effects of RDRM on the regulation of RDR regulon were carefully studied. A series of RDRM variants of the gyrB gene promoter was constructed, and each variant was inserted into the promoter of the groESL gene, which is not preceded by the RDRM (Anaganti et al., 2017). Relying on a GFP reporter, the effect of several different kinds of variants on promoter activity, under normal or irradiation conditions, was assessed. Notably, the partial deletion of RDRM exhibited increased GFP expression upon

Journal Pre-proof DdrO cleavage after irradiation. These observations indicated that the RDRM sequence played a dual role in the regulation of the RDR regulon in D. radiodurans. Briefly, in this manner, under normal conditions, DdrO as a repressor binds to the RDRM site of the RDR regulon genes in D. radiodurans, including ddrO itself. Once subjected to radiation stress, the protease activity of PprI is triggered to reduce the expression of the DdrO protein, thereby abolishing repression and orchestrating recovery pathways (see Fig. 1). However, when the stress disappears, the cleavage of DdrO needs to be switched off to reinstate the accumulation of DdrO protein and

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repression of the RDR regulon genes as in the unstressed condition. If the DdrO

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cleavage is not inhibited, cells will be unable to survive because of the disordered biochemical state in vivo. As this kind of radiation damage response is mostly

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conserved in radiation resistant Deinococcus bacteria, it appears to dominantly

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contribute to the radioresistance of D. radiodurans. The question of how PprI is

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activated remains controversial. A recent publication indicated that PprI-dependent DdrO cleavage was probably activated by increased availability of zinc ions for PprI,

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since stress from irradiation could cause the release of zinc ions from other zinc metalloproteases (Blanchard et al., 2017). Another study showed that the protease

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activity of PprI that cleaves DdrO was dependent on manganese ions (Wang et al., 2015). Further research is required to reach agreement on this complex issue. Involvement of G-quadruplexes in the radioresistance of D. radiodurans G-quadruplexes (also called G4s or G4 nucleotide structures) are G-rich DNA or RNA regions that form cyclic arrays of four guanines through Hoogsten hydrogen bonds with the aid of monovalent cations such as potassium or sodium ions (Rhodes and Lipps, 2015). With the advancement of research, it is now known that these types of structures are widely distributed in genomes of diverse organisms, ranging from prokaryotes to eukaryotes (Todd et al., 2005; Rawal et al., 2006; Johnson et al., 2008). The architecture of a G4 is topologically polymorphic and can exist in intramolecular or intermolecular folding domains (Burge et al., 2006; Patel et al., 2007). According to the direction in which the strands are folded, G-quadruplexes are either parallel or antiparallel (Lipps and Rhodes, 2009). With the aid of specific proteins,

Journal Pre-proof G-quadruplexes can be controlled to fold or unfold, and an increasing number of studies have shown that unfolding these structures is important to genome integrity (Bochman et al., 2012). Bioinformatics analyses have indicated that potential G-quadruplexes (pG4s) are not randomly distributed in the genomes of organisms but rather are prone to clustering in special regions such as promoters and telomeres (Huppert, 2008). An increasing number of studies have implicated G-quadruplexes in genomic stability and many cellular processes, including replication, recombination, transcription, translation and so on (Du et al., 2008; Cahoon and Seifert, 2009;

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Paeschke et al., 2011).

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The D. radiodurans genome has high GC content. Guanine-rich areas of the genome may form pG4 DNA structures in vivo. To further elucidate the mechanisms

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for the extraordinary radioresistance of D. radiodurans, some work concerning

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G-quadruplexes has been undertaken. Previously, N. Beaume et al. tried to determine

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the relationship between the existence of potential G4 (pG4) DNA structures in promoters and specific functions in various bacteria (Beaume et al., 2013). Cluster

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analysis of tens of thousands of promoters in 19 species of bacteria, including D. radiodurans, in functional classes indicated that the pG4 DNA structure might confer

unsupervised

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a specific function to microorganisms. For the study of radioresistance, clustering

utilizing

pG4-containing

promoters

of

twenty-one

radioresistance-related genes revealed that three radiation resistant bacteria, including D. radiodurans, were grouped together. Subsequent experiments showed that several important radiation damage repair genes harboring promoter-pG4 motifs (recA, recQ, recF, recO and recR) were downregulated in the presence of the G4-DNA-binding ligand, resulting in a decline in the radioresistance of D. radiodurans (Beaume et al., 2013). To ascertain the role of G4 DNA structures in this strange microorganism, in an extension of the previous study, Misra’s team synthesized guanine runs present upstream in some radioresistance-related genes (pprI, recQ, recF, mutL and radA) and observed the formation of G4 DNA driven by potassium ions in vitro (Kota et al., 2015). Mixed and parallel G4 DNA structures were formed by guanine runs present upstream of mutL and recQ, respectively. Under radiation stress conditions, in D.

Journal Pre-proof radiodurans, the activity of the mutL promoter was stimulated, whereas that of recQ was repressed. When the cells were exposed to a combination of G4-DNA-binding drugs and ionizing radiation, both recQ expression and radioresistance were reduced, suggesting a role for the G4 DNA structure in the radioresistance of this organism. RecQ, with a unique architecture in D. radiodurans, was capable of repairing DNA damage, and RecQ-deficient cells were sensitive to gamma ray irradiation (Killoran and Keck, 2008; Chen et al., 2009). This key protein should be upregulated following irradiation. G4 DNA structures are assumed to repress transcription by blocking

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polymerases (Bochman et al., 2012). Based on this assumption, we hypothesized that,

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under radiation stress, some unknown proteins in D. radiodurans could resolve the G-quadruplex structure in the promoter region of recQ to stimulate the transcription

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of RecQ. However, the binding of G4-targeted drugs stabilized the formation of

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G-quadruplexes, thereby blocking the polymerase needed to drive transcription.

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Folding and unfolding of G-quadruplexes to regulate the D. radiodurans genome has been preliminarily investigated. In one publication, a protein called Topoisomerase IB

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was found to relax intramolecular recF-G-quadruplexes and was therefore speculated to unwind G-quadruplexes under normal conditions in D. radiodurans (Kota and

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Misra, 2015). To date, little data are available to address some vital issues; for instance, what components does D. radiodurans exploit to form or resolve the G4 DNA structures, and how do the G4 DNA structures in D. radiodurans regulate expression of the crucial genes that correlate to radiation damage recovery? Therefore, in the future, more studies will be required to understand the mechanisms associated with the G4 DNA structures used by D. radiodurans to resist radiation stress. Transcriptional regulators involved in ionizing radiation tolerance After exposure to extreme ionizing radiation, D. radiodurans cells suffer a wide variety of devastation. It is necessary for D. radiodurans to orderly switch on a large number of processes for damage recovery, such as DNA repair, antioxidation and cell cleaning. Transcriptional regulators are indispensable for activating the genes participating in these processes. Some previously studied transcriptional regulators responsible for ionizing radiation tolerance are listed in Table 1. Among these proteins,

Journal Pre-proof PprI and DdrO were described in the previous section. In this section, we mainly discuss other regulatory proteins. DR0265, of the GntR family, was identified through use of transposon mutations (Dulermo et al., 2015). The C-terminal effector-binding domain of the HutC subfamily is part of this protein. HutC negatively regulates the hut operon associated with histidine metabolism (Dulermo et al., 2015). The hut operon in D. radiodurans appears to have an RDR function, since it contains the RDRM sequence (Makarova et al., 2007). However, the consensus sequence for the HutC subfamily has been

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identified in the form of 5’ GT-N(1)-TA-N(1)-AC 3’ (Bejerano-Sagie et al., 2006).

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Furthermore, it has been reported that the hut operon in Corynebacterium resistens, which has an organization similar to that of D. radiodurans, was induced by the IclR

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factor (Schroder et al., 2012). Accordingly, it seems impossible that HutC controls the

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hut operon in D. radiodurans. D. radiodurans devoid of DR0265 are 44-fold more

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sensitive to ionizing radiation at a dose of 20 kGy than the wild type. The mutant was also sensitive to H2O2. It has been speculated that DR0265 might regulate

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ROS-scavenging genes (Dulermo et al., 2015). Nevertheless, the target genes of this transcriptional regulator must be further investigated.

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DdrI (DNA damage response I, DR0997), a member of the CRP family, is a transcriptional activator for genes correlated with radiation damage recovery in D. radiodurans. CRP, a global transcriptional regulator that extensively exists in various bacteria, comprises two typical domains: a cNMP-binding domain and an HTH domain (Lawson et al., 2004; Gallagher et al., 2009). After conformational changes caused by the formation of the complex in the presence of cAMP, CRP is activated and interacts with RNAP, facilitating the binding of RNAP to the promoter and thus initiating the transcription (Savery et al., 1995; Fujimoto et al., 2002). It has been reported that the CRP protein plays significant roles in the adjustment to numerous cellular actions, including energy metabolism, cell division and tolerance to high temperatures and starvation (Britos et al., 2011; Shimada et al., 2011; Tsai et al., 2017). Transcriptome analyses showed that DdrI was highly induced post ionizing radiation, suggesting that this protein might contribute to the radioresistance of D. radiodurans

Journal Pre-proof (Liu et al., 2003; Tanaka et al., 2004). In addition, it has been demonstrated that DdrI acted as a dimer and required cAMP for DNA binding in vitro, corroborating the notion that DdrI is a CRP-like transcription factor (Meyer et al., 2018). Disruption of DdrI sensitized D. radiodurans cells to ionizing radiation (Yang et al., 2016; Meyer et al., 2018). According to the available data, DdrI may contribute to the radioresistance of D. radiodurans by regulating the transcription of genes involved in antioxidative response, DNA repair, chromosome segregation and other cellular processes. Heterologous expression of CRP in E. coli could rescue some of the defective

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phenotypes of DdrI-disrupted D. radiodurans cells, suggesting that E. coli CRP shares

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some targets with DdrI in D. radiodurans (Meyer et al., 2018). In addition to DR0997, there are three other members of the CRP family in D. radiodurans, DR2362,

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DR1646 and DR0834 (Yang et al., 2016), whose roles are not yet understood.

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PprM (DR0907) was initially identified as a modulator of the DNA damage

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response that depended on PprI in D. radiodurans through 2D gel analysis in which wild-type and pprI-disrupted cells were compared (Ohba et al., 2009). It has been

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considered a novel transcription regulator of great importance for conferring the extreme resistance to ionizing radiation onto D. radiodurans. Knockout of pprM

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resulted in significant sensitivity of D. radiodurans to gamma irradiation, indicating that this protein plays an important role in exceptional radioresistance. Under normal conditions, PprM is essential for the induction of KatE1, which is the central catalase in D. radiodurans (Jeong et al., 2016). E. coli with transferred pprM showed increased tolerance to oxidative stress (Park et al., 2016). Our team has previously observed that the absence of PprM in D. radiodurans led to a distinct decline in the production of deinoxanthin, a key compound that scavenges the ROS produced by ionizing radiation (Zeng et al., 2017). These available results implied that PprM was likely to contribute to prominent radiation stress tolerance via regulation of antioxidation-associated genes. Although PprM is a homolog of Csp (cold shock protein) that is well known for being induced under cold stress and is believed to destabilize RNA secondary structures that do not facilitate transcription or translation as a chaperone (Phadtare and Severinov, 2010; Li et al., 2017), it is not induced in

Journal Pre-proof response to cold stress and is abundantly expressed under normal conditions. In contrast, this protein was induced under heat stress (Airo et al., 2004). A recent study showed pleiotropic effects of PprM disruption on the proteome of D. radiodurans. Several important proteins that were involved in crucial cellular pathways post irradiation recovery, such as Alr, RfbB, SpeA, Lon, and RpsB (Anaganti et al., 2019), were repressed. There are at least two csp genes in the other seven radiation-resistant Deinococcus species (Lim et al., 2019). Csp proteins identified in all Deinococci genomes contain a C-terminal region of unknown function that appears to be different

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from that from other bacteria (Lim et al., 2019). In addition to the RNA-binding motif,

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the domain for DNA binding is also present in this protein. Hence, PprM might act not only as a chaperone but also as a transcription regulator that has an aggressive role

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in the radioresistance of D. radiodurans; however, DNA binding still needs to be

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experimentally substantiated.

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DR0865 is a homolog of Fur (ferric uptake regulator), which is correlated with the homeostasis of intracellular divalent manganese ions in D. radiodurans (Ul

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Hussain Shah et al., 2014). Computational analysis revealed that DR0865 displayed 26% and 24% similarity with the Fur protein from E. coli and Helicobacter pylori,

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respectively. This regulator has three metal-binding domains. According to the crystal structure of H. pylori Fur, domain II can bind to DNA by sensing changes in the concentration of metals, while domain III does not bind to DNA but affects DNA binding (Dian et al., 2011; Ul Hussain Shah et al., 2014). The DR0865 mutant showed an accumulation of intracellular manganese, a significant growth defect under unstressed conditions and sensitivity to gamma rays, suggesting that this regulatory protein is likely to be implicated in radioresistance. Although a suitable intracellular Mn(II) concentration has an antioxidative effect that protects D. radiodurans from oxidative stress (Daly et al., 2004), an excessive Mn(II) concentration could induce overproduction of toxic ROS (Ghosal et al., 2005) and inhibit protein and RNA synthesis (Faulkner and Helmann, 2011). Further transcriptional analysis under excess Mn(II)-induced stress showed that DR0865 could positively regulate expression of the manganese efflux pump gene mntE and negatively control expression of Mn ABC

Journal Pre-proof transporter genes (Ul Hussain Shah et al., 2014). Nevertheless, comparative transcriptional analyses between wild-type and DR0865-deficient cells under gamma irradiation might help us obtain a better understanding of the regulatory role of DR0865 in the ionizing radiation resistance of this miraculous bacterium. DR2539 is a DtxR-like regulator with approximately 230 amino acid residues (Chen et al., 2010). The domain containing residues 1-65 is predicted to bind DNA. Comparative analyses of amino acid sequences implied that DR2539 seemed to be an iron-dependent or Mn-dependent regulator of D. radiodurans. The DtxR family could

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regulate ion uptake to maintain Mn/Fe homeostasis (Andrews et al., 2003). It has also

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been revealed that DR2539 could regulate the intracellular Mn/Fe ratio and repress the expression of katE (Chen et al., 2010). MntH (DR1709), a manganese transporter,

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is associated with a high concentration of intracellular Mn(II) in D. radiodurans (Sun

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et al., 2010). An electrophoretic gel mobility shift assay showed that DR2539 could

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bind specifically to the promoter of the mntH gene, and DR2539 was found to strictly repress the transcription of mntH (Sun et al., 2012). The issue of whether repression

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of the Mn-dependent transporter gene and katE by DR2539 can be abolished by an unknown protein in the wild-type bacterium under oxidative stress requires further

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investigation. During DR2539 repression reversal, the expression of the Mn-dependent transporter gene and katE will be induced, and thus, the ability to defend oxidation will be enhanced. This mechanism of action might explain the effect of DR2539 on the unusual oxidative stress resistance of D. radiodurans. DR0171 was considered to play a role in the adaptation to radiation stress as a transcriptional regulator (Makarova et al., 2001). DR0171 was highly induced after treatment with acute ionizing radiation (Liu et al., 2003). The D. radiodurans dr0171-null mutant exhibited distinct vulnerability to radiation stress (Lu et al., 2011). Under radiation stress, reassembly of genomic DNA was delayed in cells devoid of DR0171. Comparative transcriptional analyses between the dr0171 mutant and wild type showed that approximately 153 genes involved in cellular processing, signaling and metabolism were repressed under irradiation. Confirmation of the interaction of

Journal Pre-proof DR0171 with some key target genes will clarify the regulatory effects of this protein on the extraordinary radioresistance of D. radiodurans. OxyR, a member of the LysR family, is a transcription factor that senses elevated levels of H2O2 and induces the expression of several antioxidative defensive genes (Zheng and Storz, 2000). An alignment of OxyR homologs revealed that the Cys199 and Cys208 residues of E. coli OxyR are conserved. When exposed to H2O2, the Cys199 residue is initially oxidized to a reactive intermediate (Cys-SOH), and then, the intermediate reacts with the other conserved residue (Cys208) to generate an

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intramolecular disulfide bond that locks OxyR in an activated form (Zheng et al.,

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1998). Activated OxyR has the ability to bind to diverse sequences upstream of the promoters of the oxidative stress-responsive regulon and switch on transcription

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(Storz et al., 1990). There are two OxyR homologs, DR0615 and DRA0336, in D.

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radiodurans. The deletion of dr0615 resulted in hypersensitivity to oxidative stress (Chen et al., 2008). Compared with OxyR from E. coli, DROxyR (DR0615) has only

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one conserved cysteine (Cys210), which is oxidized under exposure to oxidative stress

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and is necessary for sensing H2O2. Transcriptional analyses indicated that DROxyR acts as a positive transcriptional regulator (e.g., thioredoxin gene and katE) as well as

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a negative transcriptional regulator (e.g., mntH) (Chen et al., 2008). mntH is a gene that regulates Mn/Fe ion homeostasis. Two transcriptional regulators, DR0865 and DR2539, also control Mn/Fe ion homeostasis. The question of whether these three regulators interact with each other to create an intricate network that contributes to oxidative stress resistance needs to be elucidated. Similarly, the absence of the other OxyR homolog (DRA0336) also led to an increased sensitivity to H2O2 (Yin et al., 2010). In this protein, the Cys228 residue was essential to its function. These available data suggest that DRA0336 and DR0615 function together as transcription factors to protect cells from oxidative damage. HucR (hypothetical uricase regulator, DR1159) has been characterized as a novel uric acid-responsive transcriptional regulator belonging to the Mar family in D. radiodurans (Wilkinson and Grove, 2004). This protein dimerizes at an overlapping promoter region with pseudopalindromic sequence between hucR and an adjacent

Journal Pre-proof gene that encodes uricase. There are at least two binding sites for the ligand of uric acid with different affinities (Wilkinson and Grove, 2005). In the presence of uric acid, the transcription of uricase was stimulated, and uricase activity was increased, suggesting that HucR acted as a repressor to regulate aromatic catabolism and thus maintain the levels of uric acid in D. radiodurans (Wilkinson and Grove, 2004). As a scavenger of ROS, uric acid has been shown to provide an antioxidant defense to organisms (Ames et al., 1981; Santos et al., 1999; Kean et al., 2000). Based on this fact, we suggest the following possible mechanism of action: After infliction of

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ionizing radiation, purines in the genome of D. radiodurans cells are damaged by

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high levels of ROS. The catabolism of these impaired bases leads to the accumulation of uric acid, which is harmful to cells. Binding of uric acid to HucR relieves the

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repression of the transcription of the uricase that catalyzes uric acid to allantoin.

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Using this strategy, HucR maintains a constant concentration of uric acid in D. radiodurans cells, probably contributing to the extreme resistance to oxidative stress

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caused by γ-irradiation. The deletion of hucR and the determination of oxidative stress

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tolerance should be undertaken in the future to substantiate this hypothesis. RecX (DR1310) is another central regulatory protein beneficial for the

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radioresistance of D. radiodurans. This protein negatively regulates the antioxidant activity in D. radiodurans, since two crucial ROS-scavenging enzymes (catalase and superoxide dismutase) activities are markedly enhanced in the recX-null mutant (Sheng et al., 2005a). Two-dimensional electrophoresis combined with the MALDI-TOF method revealed that, compared to the wild type, 12 proteins associated with DNA repair and stress response were upregulated in the recX mutant, while 23 proteins responsible for cellular metabolism were downregulated. This result indicated that RecX was of importance in the metabolic pathway under unstressed conditions (Sheng et al., 2009). It has been reported that this protein not only repressed the expression of RecA but also inhibited RecA activities, including LexA cleavage, ATPase activity and strand exchange (Sheng et al., 2005b). Furthermore, RecX regulation of recA induction is not dependent on RecA activity (Sheng et al., 2010). Under normal conditions, RecX was utilized by D. radiodurans to preserve the

Journal Pre-proof stability of the genome (Sheng et al., 2005b). It is well known that DdrO can repress RecA expression and that the cleavage of DdrO by PprI can abrogate this downregulation (Devigne et al., 2015). At present, a question is raised regarding the relationship of RecX and DdrO on the regulation of RecA expression. Under radiation stress, the mechanism by which RecX influences the reversed repression of RecA expression requires an in-depth investigation. Two-component systems in response to ionizing radiation stress Two-component systems constitute a prevalent strategy by which bacteria sense

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and respond to a wide range of signals and stressors (Capra and Laub, 2012;

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Groisman, 2016). This type of signal transduction is essential for optimizing fitness, ensuring metabolic resources, adapting to changing habitats and increasing chances

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for survival (Hibbing et al., 2010; Harapanahalli et al., 2015; Gomez-Mejia et al.,

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2018). The typical two-component system is composed of an HK (histidine kinase)

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and an RR (response regulator) (Zschiedrich et al., 2016). Generally, the HK comprises a signal recognition domain; a transmission domain; a histidine-containing

na

domain, DHp; and an ATP-binding catalytic domain. The partner component, RR, contains a receiver domain and an effector domain. When sensing environmental

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stimuli, a histidine residue in the Dhp domain of HK is autophosphorylated by the catalytic domain of the kinase, and the phosphoryl group is subsequently transferred to an aspartate residue in the receiver domain of RR. This so-called phosphoryl relay results in a conformational change in RR and subsequently triggers activation of a coupled domain that induces the transcription of genes responsive to environmental changes, enabling adaptation. The genome of D. radiodurans is predicted to encode 23 histidine kinase domains and 29 receiver domains (Makarova et al., 2001). Hence, it is envisioned that these unique proteins can build intricate signal networks, which might contribute to the surprising radioresistance of this microorganism. To date, some two-component systems involved in radioresistance have been characterized (see Fig. 2). A radRS operon comprising drb0090 that encodes a putative response regulator (RadR) and drb0091 that encodes a putative histidine kinase (RadS) have been shown to play a

Journal Pre-proof crucial role in the radiation damage response (Desai et al., 2011). A radR/radS double mutant and a radR mutant both displayed significant sensitivity to DNA-damaging agents and delayed DSB repair. Experiments in vitro revealed that recombinant RadS are autophosphorylated and transferred γ-phosphate from ATP to RadR. Despite this preliminary characterization, the genes induced by the RadR regulator need to be determined. Interestingly, a previous study reported that all 12 HK mutants, especially five (dr0860, dr1174, dr1556, dr2244 and dr2419), exhibited increased sensitivity to acute γ-irradiation compared with the wild type of D. radiodurans (Im et al., 2013).

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The absence of DR2415, which is a cognate response regulator of DR2416, resulted

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in a marked decline in radioresistance, suggesting that DrtR/S (DR2415/DR2416) might be another DNA damage response TCS. Another response regulator, DRB0081,

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has been demonstrated to regulate the expression of the kdp operon implicated in

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potassium homeostasis (Dani et al., 2017). After exposure to 5 kGy ionizing radiation,

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the survival fraction of the drb0081 mutant was reduced relative to the wild type. The role of potassium ions in the stability of the replication process and maintenance of

na

genome integrity accounted for the radiation-sensitive phenotype of this RR-null mutant (Hughes and Cidlowski, 1999; Durand et al., 2016). Heterogeneous expression

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of the response regulator DR1558 could reduce ROS levels in E. coli because of the increased KatE catalase activity (Appukuttan et al., 2016; Park et al., 2019). These data reflected the role of DR1558 in oxidative stress resistance. DrRRA (DR2418) was identified as a response regulator that plays a central role in the extraordinary radioresistance of D. radiodurans (Wang et al., 2008). The deletion of DrRRA led to many defects in D. radiodurans. The antioxidant activities of superoxide dismutases and catalases were decreased. The expression of two highly radioresistance-related proteins (RecA and PprA) was reduced. Additionally, the reassembly of the numerous genome fragments caused by radiation insult was also delayed. Since the deficiency of DR2419 resulted in considerable impairment to the radioresistance of D. radiodurans, it was assumed to be a putative partner of the HK of the response regulator DrRRA (Im et al., 2013). DqsR (Deinococcus quorum sensing Regulator, DR0987) regulates quorum sensing and gene expression by sensing extracellular

Journal Pre-proof signaling molecules termed AHLs (acylated homoserine lactones). Quorum sensing plays a crucial role in many cellular processes, including biofilm formation, luminescence, and virulence (Bassler et al., 1997; Ahmed et al., 2019; Khider et al., 2019). Quorum sensing and quorum quenching are coexisting action in bacteria. Under normal conditions, the amount of AHLs was restricted by quorum quenching enzymes; however, when D. radiodurans cells were exposed to ionizing radiation, the concentration of extracellular AHLs was increased by induction of AHL synthetic enzymes. The formed AHL-DqsR complex regulates a number of stress-related genes,

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including cell cleansing genes and Mn/Fe ion transport genes. Compared to

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unirradiated control cells, the dqsR mutant was sensitive to oxidative stress (Lin et al., 2016a). Reported data concerning quorum sensing are still very inadequate. We do not

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know how the AHL synthetic enzymes are activated. Another study revealed that a

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furanosyl borate diester, serving as a signaling molecule, was involved in the

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regulation of the stress-responsive genes of D. radiodurans (Lin et al., 2016b). Apart from these two chemical compounds, we do not know whether there are any other

na

signaling molecules in quorum sensing or not. Therefore, further investigations are essential to understand the mechanisms in detail. In addition to the two-component

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systems, D. radiodurans also encodes 16 putative GGDEF domain-containing cyclases that play a key role in diguanylate signaling (Makarova et al., 2001). The cyclic diguanylate signaling system is an important signal transduction system, but studies concerning it have been insufficient. Surprisingly, the signal transduction system of D. radiodurans has eukaryotic characteristics. This unique composition might, to some extent, contribute to the radioresistance of D. radiodurans. DR2518 was identified as a eukaryotic type Ser/Thr kinase by Rajpurohit et al. (Rajpurohit and Misra, 2010). Disruption of DR2518 induced by hypersensitivity to ionizing radiation and complete inhibition of DSB repair, suggesting that it acted as a DNA repair-related protein kinase to contribute to the radioresistance of D. radiodurans. Regulatory RNAs potentially regulate radioresistance in D. radiodurans Regulatory RNAs, also referred to as sRNAs because of their small sizes, are generally noncoding RNAs (ncRNAs) (Gottesman, 2004). sRNAs have been

Journal Pre-proof identified and reported to act major players in bacteria to handle a vast array of stresses, such as oxidative stress, temperature stress, iron deficiency stress, pH stress, and metabolite stress (Repoila and Gottesman, 2001; Opdyke et al., 2004; Jonas and Melefors, 2009; Holmqvist and Wagner, 2017). Hundreds of sRNAs have been predicted to be present in E. coli, and approximately one hundred have been experimentally validated (Raghavan et al., 2011). To the best of our knowledge that is based on available results, most sRNAs interact with mRNA targets by an antisense mechanism, resulting in inhibition of translation via the blocking of ribosome binding

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to the Shine-Dalgarno sequence or mRNA degradation (Desnoyers et al., 2013).

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Broad distributions of sRNAs in three kingdoms of life and a considerable number sRNA species existing in each well-studied organism imply their important status in

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various cellular processes (Raghavan et al., 2011; Hinton et al., 2014; Baldrich et al.,

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2019).

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In recent years, several studies have reported that some sRNAs are highly expressed in D. radiodurans when under stress from high doses of ionizing radiation.

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After treatment with gamma irradiation at a dose of 3,000 Gy, 144 annotated noncoding RNAs were induced, of which 95 were putative novel antisense RNAs

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supposedly exerting important roles in radiation protection, e.g., in the glycolysis pathway (DRC0002 and DRC0003), homologous recombination (DR0198), and ABC transporter (DRB0121, DRB0122, DRB0123, DRB0124, DRB0125 and DR2145) action (Luan et al., 2014). Dra0234, only 171 bp in length, was highly induced in D. radiodurans after exposure to heavy γ-irradiation and might encode uncharacterized sRNAs in response to ionizing radiation stress (Liu et al., 2003). It has been reported that one sRNA could resemble a Y RNA, which has the ability to bind Rsr, an ortholog of a Ro protein, and contributes to radioresistance (Chen et al., 2000). To identify novel potential sRNAs in D. radiodurans and elucidate the radioresistance of this microbe from a new perspective, Tsai et al. conducted a study using computational prediction and a deep sequencing technique (Tsai et al., 2015). One hundred ninety-nine potential sRNA candidates were ultimately identified, and the expression of 41 was confirmed. Of interest, 8 confirmed sRNAs exhibited

Journal Pre-proof differential expression following a dose of 15 kGy ionizing radiation. Surprisingly, 7 orthologs were present in another radioresistance species of the family Deinococcaceae, D. geothermalis. Some potential sRNAs were predicted to bind mRNA targets that encode proteins involved in recovery from radiation damage, such as RecA, RadA and RuvA. Under oxidative stress from ionizing radiation, small sRNA genes (less than 400 bp) are more likely to sustain structural integrity compared to protein-encoding genes that are 1000-2000 bp in length. Hence, it is plausible to speculate that functional sRNAs could be transcribed from DSBs of the

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fragmented genome and could mediate the regulation of DNA repair genes for

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survival. Owing to difficulties in identifying their targets, the functions of most sRNAs remain unclear (Ivain et al., 2017). There are urgent demands for developing

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new and advanced technologies to accurately identify sRNA targets. This

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development can, to some extent, be expected to shed light on the issue of how

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sRNAs regulate gene expression for adaptation to extraordinary ionizing radiation. Other regulatory mechanisms

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In addition to the depictions offered above, D. radiodurans also employs other mechanisms to regulate genes that contribute radioresistance. Nitric oxide (NO)

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production has been found to be an endogenous modulator of deinoxanthin biosynthesis (Hansler et al., 2016). Knockout of the NO synthase gene resulted in a reduction in deinoxanthin production in D. radiodurans and a decrease in growth recovery post irradiation. Unexpectedly, the transcription of deinoxanthin biosynthesis genes remained unchanged compared to the wild-type strain. NO, as a bioactive signaling molecule, has been regarded as an influencer of protein activity through induction of posttranslational modifications. Deinoxanthin, the major carotenoid identified in D. radiodurans, has already been shown to be central in the maintenance of antioxidant activity and the resistance to ionizing radiation in this microorganism (Tian et al., 2007; Ji, 2010). Therefore, NO was thought to act as a posttranslational modulator of a putative hydroxylase and to be required for the extreme radioresistance of D. radiodurans. The FMN (flavin mononucleotide) riboswitch, which regulates the expression of genes involved in the biosynthesis and

Journal Pre-proof transport of riboflavin, is a highly conserved domain located in the 5’-UTR of mRNAs of prokaryotes (Yang et al., 2014). The FMN riboswitch of D. radiodurans is situated in the 5’-UTR of dr0153. The FMN riboswitch mutant exhibited obvious growth defects and increased susceptibility to oxidative stress, implicating it in the resistance to oxidative stress of D. radiodurans. Conclusions D. radiodurans is a prokaryote well known for its ability to resist extreme ionizing radiation at a dose of >10,000 Gy. In this review, we have discussed the

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mechanisms by which D. radiodurans rapidly regulates gene expression to recover

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from fierce radiation insult. The PprI/DdrO pair-based DNA damage response, also present in 11 other Deinococcus species, such as D. deserti, D. geothermalis, and D.

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proteolyticus, differs from the LexA-RecA response of many other radiosensitive

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bacteria, which might largely contribute to the extraordinary radioresistance of D.

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radiodurans. Nevertheless, some work will be required to clarify how PprI is activated to induce the cleavage of DdrO under radiation stress. Although

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approximately one hundred genes have been predicted to encode transcriptional regulators in the genome of D. radiodurans, only a few have been carefully

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investigated. Hence, the functions of most must be elucidated in the future. The small size of sRNA genes in D. radiodurans readily sustain integrity when surrounded by abominable ionizing radiation compared to genes that encode functional proteins, suggesting that sRNAs might serve a central role in the gene regulation of the adaptation to a hostile radiation environment. However, few data concerning the regulatory roles of sRNA are available until now owing to limited technology. It is necessary to develop more advanced technologies to identify sRNA targets, which would advance these studies. Transcriptome and proteome analyses of D. radiodurans are abundant, while those on metabolite profiling are nearly nonexistent. Transcriptome and proteome analyses reveal what will probably happen, whereas metabolomics studies show us what has indeed happened. Additional metabolomics analyses might help us find some small metabolites that act as signaling molecules to regulate gene expression in D. radiodurans for recovery from radiation damage.

Journal Pre-proof Moreover, the intricate regulatory networks for gene expression after exposure to ionizing radiation should be delineated, which might better explain the radioresistance of D. radiodurans. Most regulatory pathways also exist in other normal bacteria. We argue that the unique crosstalk of regulatory networks may be particularly efficient in D. radiodurans, enabling it to withstand excess irradiation. The continued studies of D. radiodurans as a model organism for radioresistance will open numerous possibilities for the application of this microorganism. The two-component system-based biosensor coupled with reporters can be used to monitor the dose of

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radiation environment. Considering that the exogenous expression of D. radiodurans

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pprI enhanced the radioresistance of eukaryotes (Wen et al., 2016), some key players involved in recovery from DNA damage in D. radiodurans might be genetically

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applied in the future to protect eukaryotes against high doses of ionizing radiation.

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Conflict of interest

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None. Acknowledgments

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This work was supported by the National Natural Science Foundation of China (Grant Nos. 81741143 and 11705085), the Natural Science Foundation of Hunan Province,

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China (Grant No. 2018JJ3458) and the Education Department of Hunan Province, China (Grant No. 17A186). References

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Journal Pre-proof White O., Eisen J.A., Heidelberg J.F., Hickey E.K., Peterson J.D., Dodson R.J., Haft D.H., Gwinn M.L., Nelson W.C., Richardson D.L., Moffat K.S., Qin H., Jiang L., Pamphile W., Crosby M., Shen M., Vamathevan J.J., Lam P., McDonald L., Utterback T., Zalewski C., Makarova K.S., Aravind L., Daly M.J., Minton K.W., Fleischmann R.D., Ketchum K.A., Nelson K.E., Salzberg S., Smith H.O., Venter J.C., Fraser C.M., 1999. Genome sequence of the radioresistant bacterium Deinococcus radiodurans R1. Science. 286(5444), 1571-1577. Wilkinson S.P., Grove A., 2004. HucR, a novel uric acid-responsive member of the MarR family of transcriptional regulators from Deinococcus radiodurans. J Biol Chem. 279(49), 51442-51450.

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doi:10.1074/jbc.M405586200

HucR

from

Deinococcus

radiodurans.

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Mol

Biol.

350(4),

617-630.

-p

regulator

ro

Wilkinson S.P., Grove A., 2005. Negative cooperativity of uric acid binding to the transcriptional

doi:10.1016/j.jmb.2005.05.027

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Yang P., Chen Z., Shan Z., Ding X., Liu L., Guo J., 2014. Effects of FMN riboswitch on antioxidant

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activity in Deinococcus radiodurans under H2O2 stress. Microbiol Res. 169(5-6), 411-416. doi:10.1016/j.micres.2013.09.005

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Yang S., Xu H., Wang J., Liu C., Lu H., Liu M., Zhao Y., Tian B., Wang L., Hua Y., 2016. Cyclic AMP Receptor Protein Acts as a Transcription Regulator in Response to Stresses in Deinococcus

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radiodurans. PLoS One. 11(5), e0155010. doi:10.1371/journal.pone.0155010 Yin L., Wang L., Lu H., Xu G., Chen H., Zhan H., Tian B., Hua Y., 2010. DRA0336, another OxyR homolog, involved in the antioxidation mechanisms in Deinococcus radiodurans. J Microbiol. 48(4), 473-479. doi:10.1007/s12275-010-0043-8 Zahradka K., Slade D., Bailone A., Sommer S., Averbeck D., Petranovic M., Lindner A.B., Radman M., 2006. Reassembly of shattered chromosomes in Deinococcus radiodurans. Nature. 443(7111), 569-573. doi:10.1038/nature05160 Zeng Y., Ma Y., Xiao F., Wang W., He S., 2017. Knockout of pprM Decreases Resistance to Desiccation and Oxidation in Deinococcus radiodurans. Indian J Microbiol. 57(3), 316-321. doi:10.1007/s12088-017-0653-5 Zheng M., Aslund F., Storz G., 1998. Activation of the OxyR transcription factor by reversible disulfide bond formation. Science. 279(5357), 1718-1721. Zheng M., Storz G., 2000. Redox sensing by prokaryotic transcription factors. Biochem Pharmacol.

Journal Pre-proof 59(1), 1-6. Zschiedrich C.P., Keidel V., Szurmant H., 2016. Molecular Mechanisms of Two-Component Signal

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Transduction. J Mol Biol. 428(19), 3752-3775. doi:10.1016/j.jmb.2016.08.003

Journal Pre-proof Fig. 1. A schematic representation of RDRM-mediated regulation of RDR regulon genes. Under normal conditions, PprI, as a metalloprotease, is inactivated, and the RDR regulon genes (such as recA, PprA, gyrB, and ddrO) are repressed through binding of the DdrO protein to the RDRM located upstream of their respective promoter. However, when exposed to ionizing radiation, PprI is activated, and expression of the RDR regulon genes is triggered via cleavage of DdrO by activated PprI. Repression by DdrO is continuous when the radiation stress is abolished. RDRM:

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radiation/desiccation response motif; RDR: radiation/desiccation response.

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Fig. 2. Two-component systems in response to ionizing radiation stress in D. radiodurans. Upon ionizing radiation, the sensor domain of HK senses the irradiation

-p

stimulus, and a histidine residue in the DHp domain is autophosphorylated by the

re

catalytic domain of the kinase. The phosphoryl group is immediately transferred to an

lP

aspartate residue in the receiver domain of RR, resulting in activation of the coupled effector domain and induction of genes in response to ionizing radiation stress. HK:

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regulator.

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histidine kinase; DHp: dimerization and histidine phosphotransfer; RR: response

Journal Pre-proof Table 1 Transcriptional regulators involved in ionizing radiation tolerance Locus tag

Features and roles

References

DdrI

DR0997

A cyclic AMP receptor; regulates expression of genes involved in antioxidative responses, DNA repair, and various cellular pathways

Yang et al., 2016; Meyer et al., 2018

GntR

DR0265

Belongs to the HutC subfamily; involved in ROS scavenging

Dulermo et al., 2015

DdrO

DR2574

Regulates the expression of the RDR regulon genes preceded by RDRM

Devigne et al., 2015

PprM

DR0907

Contains a cold shock domain; a modulator of DNA damage response that depends on PprI and is involved in ROS scavenging

Ohba et al., 2009; Jeong et al., 2016; Zeng et al., 2017

Mur

DR0865

Involved in the regulation of manganese ion homeostasis

Ul Hussain Shah et al., 2014

IrrI

DR0171

Contains HTH DNA-binding domain; regulates metabolism and cellular processing and signaling

Lu et al., 2011

DtxR

DR2539

Regulates Mn/Fe ion homeostasis

Chen et al., 2010

Rex

DR1310

A transcriptional regulator of central carbon and energy metabolism as well as ROS scavenging

Sheng et al., 2005a; Sheng et al., 2009

A transcriptional activator (e.g., katE) as well as a transcriptional repressor (e.g., dps, mntH) Involved in antioxidation

Chen et al., 2008

re

lP

na

Jo ur

OxyR

-p

ro

of

Protein name

DR0615

DRA0336

Yin et al., 2010

HucR

DR1159

A member of the MarR family; responsible for oxidative stress and DNA damage

Wilkinson and Grove, 2004

PprI

DR0167

A zinc-dependent metalloprotease; a general switch responsible for DNA damage repair and cellular survival networks

Earl et al., 2002; Hua et al., 2003 Lu et al., 2009

Two-component systems

See next section in detail

Journal Pre-proof Highlights



We summarized that the RDRM-dependent PprI/DdrO-mediated pathway is different from the LexA-based SOS radiation response and might mainly contribute to the extreme radiation-tolerance of D. radiodurans.



We reviewed the roles of all reported transcriptional regulators which involved in ionizing radiation tolerance in D. radiodurans. We reviewed some unique strategies that were used by D. radiodurans to regulate

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genes associated with extreme tolerance to ionizing radiation.

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

Journal Pre-proof Abbreviations CRP: cAMP receptor protein DdrI: DNA damage response I DtxR: diphtheria toxin repressor Fur: ferric uptake regulator HK: histidine kinase HucR: hypothetical uricase regulator MntH: manganese acquisition transporter H

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PprI: inducer of pleiotropic proteins promoting DNA repair

ro

pG4s: potential G-quadruplexes

ROS: reactive oxygen species

Jo ur

na

RR: response regulator

lP

RDR: radiation/desiccation response

re

RDRM: radiation/desiccation response motif

-p

PprM: a modulator of the PprI-dependent DNA damage response

Figure 1

Figure 2