An Antioxidant Defense System in Radiation-Resistant Bacterium Deinococcus geothermalis against Oxidative Stress

Abstract

A radiation-resistant bacterium, Deinococcus geothermalis has various stress response mechanisms, including antioxidation. Features that maintain vitality at high radiation doses include the following: enzymatic scavengers of ROS such as catalase, SOD, and peroxidase; strain-specific DNA repair systems such as Deinococcal unique proteins; non-enzymatic responses such as manganese complexes, carotenoids, and DNA-binding proteins. This chapter summarizes the primary response mechanism by redox balance centered on the cystine transporter. It also reviews action characteristics of DNA-binding protein Dps and a putative LysR family protein, and effects on loss of function of the carotenoid biosynthesis genes by transposition of insertion sequences. Environmental adaptation and molecular evolution of radiation-resistant bacterium are also considered to explain the potentials of molecular behavior induced by oxidative stress.

1. Introduction

The radiation-resistant bacterium of genus Deinococcus is an essential resource for research to understand responses to oxidative stress and mechanisms for recovering direct double-strand break damage to DNA caused by gamma-radiation [1, 2, 3]. High gamma-ray resistance is caused by the unique DNA-damage repair proteins and various protective mechanisms in these radiation-resistant Deinococcus bacteria [4, 5, 6]. Many researchers have studied the properties of their unique proteins. These studies have expanded our scientific understanding [7, 8, 9]. Technological advances have recently been made to understand life phenomena through genomics, metabolomics, and proteomics studies [10, 11, 12]. Despite the remarkable progress in recent omics studies, it is still difficult to fully understand these cell recovery characteristics from various cellular stress damages. The accumulation of various creative research results will eventually lead to a complete understanding of such characteristics.

Only 20% of DNA damage is directly caused by radiation. In comparison, the remaining 80% is indirectly caused by reactive oxygen species (ROS) such as superoxide and hydroxyl radicals which are chemically reactive molecules that can damage cell structures such as cell membrane, proteins, and nucleic acids (DNA and RNA) [1, 10]. Bacteria have a natural ROS scavenging system composed of enzymatic antioxidants (e.g. catalase, peroxidase, superoxide reductase, and superoxide dismutase (SOD)), and non-enzymatic antioxidants (e.g. intracellular manganese, pyrroloquinoline quinone, carotenoids), small antioxidant thiols (e.g. cystine, bacillithiol, or mycothiol), and DNA-protecting proteins [2, 13, 14, 15].

Specific regulators tightly control many stress response defense systems. Enzymatic ROS scavengers are regulated by the global transcriptional regulator OxyR, a LysR family regulator [16, 17, 18, 19, 20]. OxyR of Deinococcus radiodurans is a 1-Cys-type that can activate the transcription of genes encoding catalase (katE), ferrous iron transporter (feoB), and iron(III) dicitrate transporter (drb0125). It is also a repressor of dps and mntH transcription to control antioxidant functions and Mn/Fe ion homeostasis [21].

These gene regulation systems are also susceptible to intracellular redox balance through specific ABC transporters and chemical modification of low-molecular-weight (LMW) thiol compounds using unique enzyme reactions. The cystine importer is one of the redox controlling ABC transporters [22, 23, 24, 25]. It could sense the redox balance and affect gene regulation for enzymatic defense through the OxyR activation [15, 20]. There are also some exceptional OxyR regulons in bacteria [26, 27, 28].

This redox balance affects various enzymatic and chemical modification processes through a progressive transformation. For example, acetylation is a conserved modification used to regulate various cellular pathways such as gene expression, protein synthesis, detoxification, and virulence. Acetyltransferase enzymes can transfer an acetyl moiety, usually from acetyl coenzyme A (AcCoA), onto a target substrate, thereby modulating the activity or stability [29]. Gcn5-related N-acetyltransferase (GNAT) members can acetylate the amino group of an extensive range of substrates. They are classified into three groups: (1) small molecule acetyltransferases such as aminoglycosides and mycothiol; (2) peptide acetyltrandferases such as the peptidoglycan that is part of the cell wall; and (3) protein acetyltransferases such as the histone family [30]. In Gram-positive Actinomycetes and Firmicutes, alternative LMW thiols such as mycothiol (MSH) and bacillithiol (BSH) play related as glutathione surrogates of Gram-negative bacteria, respectively [31].

As antioxidant substances, carotenoid compounds also act as scavengers of ROS. Deinococcales species generally have a reddish color phenotype due to carotenoid biosynthesis. The metabolic pathway in Deinococcus is well conserved and industrially applicable [32].

As one of bacterial nucleoid proteins in gene expression specificity of growth phase-dependent manner, Dps (DNA-binding protein from starved cells) is initially suppressed at the exponential cell growth phase. It is then expressed in large quantities in the stationary growth phase to become the major protein [33, 34]. These sequential nucleoid protein transitions and overexpression of a particular protein demonstrate the function of a defense mechanism that can protect against cell damage during stress due to increased ROS and reduced nutrients that cells can consume. Dps proteins are found almost ubiquitously in bacterial genomes. Each bacterial genome contains species-specific Dps genes. Dps has multifaceted roles such as DNA binding, iron sequestration, and ferroxidase activity in various stress responses [35, 36, 37]. Dps was described initially in Escherichia coli as a protein that could protect the bacteria in a malnourished environment by DNA-binding [38]. Dps has a shell-like structure with a spherical hollow cavity in the center. This hollow cavity of Dps acts as an iron storage compartment and iron sequestration that is important in iron detoxification and homeostasis. Dps is a small DNA-binding protein having approximately 150 to 300 amino acid residues with a positive charge along the entire chain. While Dps is a significant protein when it is over-expressed, Dps plays another essential role in condensing and protecting the stationary-phase nucleoid from severe environmental stress such as oxidative and radioactive damages [34, 39, 40]. Therefore, many bacteria express more than one Dps depending on environmental factors around them. How the condensed DNA is untangled when proteins are expressed remains unclear. It merits further studies.

Bacterial insertion sequence (IS) elements consist of a gene encoding transposase (one or two), terminal inverted repeats (TIR), and direct repeats (DR). Their lengths are less than 3 kb [41, 42]. According to the database of IS finding platform such as ISfinder, IS types vary [43]. IS densities are significantly less in chromosomes than in plasmids in bacteria. Perhaps plasmids are the primary IS carrier [44]. These IS elements can be transferred by high temperature, γ-irradiation, oxidative stress, and substances that could damage DNA and result in gene breakdown [45, 46, 47]. Regulation of IS transposition is also affected by various factors such as transcriptional repressors and inhibitors, ribosome frameshifting, methylation, mRNA stability, and target sequences [48, 49, 50, 51]. Gene breakdown by transposition of IS elements does not just have deleterious aspects. IS-mediated gene inhibition offers various advantages such as virulence, antibiotic/xenobiotic resistance, metabolism, and small promoter obtained by IS migration in the genome of a strain [52].

A comprehensive paper on D. radiodurans’s antioxidant mechanism and control systems of specific regulator proteins in response to oxidation stress has been published during the preparation of this chapter [53]. Here, we will focus on antioxidation mechanisms in Deinococcus geothermalis, a radiation-resistant bacterium used within the research destination’s scope [54]. We characterized the cystine importer and used transcriptomics analysis to detect the critical players in intracellular antioxidant responses. The putative Dps protein and LysR family regulator’s functional role was then determined and IS transposition events were selected on carotenoid biosynthesis path defect (Figure 1). Understanding the sophisticated antioxidant system of living organisms is a problematic and challenging study like a puzzle game.

Figure 1.

WordCloud analysis of our owns four published papers [25, 55, 61, 62] by the WordCloud generator 40 quantity of MonkeyLearn.

2. An antioxidant defense system in a radiation-resistant bacterium

2.1 A cystine importer, redox balance and control of gene expression

D. geothermalis contains a cystine importer as a substrate-binding protein and a membrane permease Dgeo_1986-87 which is highly expressed at the late exponential growth phase [25, 55]. Its intracellular total thiol level is affected by the expression level of this cystine importer. Deinococcus has specific genes that can repair when DNA is damaged. Using dgeo_1986-87 a cystine importer disrupted mutant strain, we have detected the expression levels of unique DNA repair proteins, pprA, ddrA, and ddrB. These DNA repair proteins were highly up-regulated under oxidative stress conditions induced by 50 mM H₂O₂ [55]. However, when cystine importer expression is enhanced in a mutant Dgeo_1985R strain, DNA repair proteins are entirely down-regulated [25]. The increased intracellular thiol concentration strongly repressed the expression level of these DNA repair-related unique genes, excluding recA gene in Deinococcus through overexpressed cystine transporter. Therefore, unique DNA repair proteins in Deinococcus are controlled by redox potential levels. If there is a direct controlling system for unique DNA repair genes, maybe it is repressed by the reduced redox potential through the cystine transporter’s overexpression.

In general, the primary antioxidant enzyme, e.g. catalase, is highly induced by an oxidative stress condition. It is positively controlled by a global transcriptional regulator OxyR [16]. In D. radiodurans, the redox sensor OxyR has a single cysteine residue in the active site. It controls the expression of catalase and iron/manganese uptake proteins positively [21]. However, in D. geothermalis wild-type and Δdgeo_1986-87 cystine importer disrupted strain, expression level of oxyR is strongly induced. OxyR is not proportionally affected on catalase expression level. Thus, OxyR is not a positive regulator of catalase.

The strain’s cystine transport has been found to be dependent on the growth phase. In other words, some features are often expressed in the latter half of the exponential phase. In a mutant with the importer gene removed, it reacts relatively sensitive to oxidative stress. However, if the importer is overexpressed, its resistance to hydrogen peroxide is increased. A mutant that artificially over-expression the importer shows increased resistance to hydrogen peroxide without being affected by catalase expression, which results from an increase in the content of total thiol entering the cell through the cystine importer [15]. Therefore, the intracellular reduction state through enhancing thiol contents is a primary defense system of D. geothermalis against oxidative stress without induction of enzymatic ROS defense factors.

2.2 Hints from transcriptomic analysis

We performed transcriptomic analysis using RNA-Seq technology to define functional roles of bacterial TrmB (Dgeo_1985), Dps (Dgeo_0257), a cystine importer (Dgeo_1986-87), and LysR family regulator (Dgeo_2840). We constructed target gene disrupted mutants. Expression levels of all genes at OD₆₀₀ 4.0 as a late exponential growth phase in mutants were then compared to those in wild-type D. geothermalis [55]. Data have been deposited in NCBI’s Gene Expression Omnibus. They are accessible through GEO series accession number GSE151903.

First, a transcriptomic study was done to compare gene expression levels between wild-type and cystine importer deleted mutant of D. geothermalis. Genes up-regulated more than 3.0-fold of log value are listed in Table 1. Both CRISPR-Cas system gene clusters, Dgeo_0233-38 gene cluster and Dgeo_0956-65 gene cluster, were up-regulated 35.1-105.5-fold and 10.3-65.2-fold, respectively. Iron transporter Dgeo_2443 and 2444 genes were up-regulated 4.69 and 12.42-fold, respectively. Three gene clusters for GCN5, Dgeo_0369-70, 2125, and 2313, were up-regulated 12.53, 3.73, and 11.2-fold, respectively. Four MFS transporters, Dgeo_0249, 0530, 1968, and 2330, were up-regulated 3.75, 6.41, 5.57, and 3.22-fold, respectively. Four ABC transporters, Dgeo_0543, 0647, 1805, and 2581, were up-regulated 3.34, 6.96, 8.03, and 3.6-fold, respectively.

Gene clusters	Genes	△dgeo_1986-87	△dgeo_0257	△dgeo_2840
CRISPR-Cas	Dgeo_0233-38	35.1–105.6	—	3.36
	Dgeo_0956-65	10.3–65.2	—	4.59–5.46
Iron; transporter	Dgeo_1370	—	—	3.35
	Dgeo_2443-44	4.69–12.42	—	—
GCN5	Dgeo_0369-70	12.53	—	—
	Dgeo_2125	3.73	—	—
	Dgeo_2313	11.2	—	—
MFS; transporter	Dgeo_0249	3.75	—	—
	Dgeo_0530	6.41	—	—
	Dgeo_1968	5.57	—	—
	Dgeo_2330	3.22	—	—
ABC; transporter	Dgeo_0543	3.34	0.35	1.62
	Dgeo_0647	6.96	1.54	—
	Dgeo_1805	8.03	—	—
	Dgeo_2581	3.60	0.69	—
RpiR family	Dgeo_2822	—	—	3.20
	Dgeo_2619	0.20	0.29	0.28

Table 1.

Transcriptomics analysis for some target genes among wild-type and mutants.

Δdgeo_0257 and Δdgeo_2840 mutant strains were revealed many no effect and several fluctuated patterns. The CRISPR-Cas system’s slightly upregulated expression was also found in the LysR family regulator Dgeo_2840 disrupted mutant, but not in a putative Dps gene Dgeo_0257 disrupted mutant. In the case of Δdgeo_2840 mutant, a different iron transporter dgeo_1370 was up-regulated 3.35-fold. However, gene expression levels of GCN5 and MFS transporter gene clusters were not affected in Δdgeo_0257 or Δdgeo_2840 mutant strain. When the intracellular redox potential was reduced through disruption of a cystine importer, why these gene clusters with several distinct physiological functions showed dramatic overexpression? Do they somehow have a relationship with antioxidant responses? These questions are interesting. Future studies in this field of antioxidation research are needed. We focused on two antioxidant biosynthesis pathways for bacillithiol and mycothiol because these pathways are related to up-regulated GCN5 gene clusters.

2.3 Mycothiol as a major under oxidation state

D. geothermalis genome contains 28 GNAT proteins [56]. Four GNAT genes, dgeo_0369-0370, 2125, and 2313, contribute to its redox-balancing regulation. In Δdgeo_1986-87 mutant, these four GNAT genes were up-regulated over 3.0-fold (Table 1). Dgeo_2125 is an acetyltrans_3 family member. However, its function has not been characterized yet. Dgeo_0370 is a putative phosphinothricin acetyltransferase, a broad-spectrum herbicide that acts as a competitive inhibitor of glutamine synthetase. Dgeo_0369 is a putative RimI which is a S18 ribosomal protein acetylation enzyme. Dgeo_2313 is a putative mycothiol synthase MshD (Table 2). Thus, Dgeo_2313 is a gene direct-related to redox potential because mycothiol acts as a total thiol balance. Mycothiol is the main LMW thiol in most Actinomycetes, including Mycobacterium tuberculosis [31]. MshD and MshC were strongly induced in Δdgeo_1986-87 mutant with the absence of hydrogen peroxide. However, when hydrogen peroxide was present, MshD expression was strongly down-regulated to be under 0.3-fold.

Genes	△dgeo_1986-87	△dgeo_0257	△dgeo_2840
MshA (Dgeo_2307)	0.79	0.98	0.69
MshB (Dgeo_1021)	1.14	1.00	1.10
MshC (Dgeo_1714)	10.78	0.88	1.36
MshD (Dgeo_2313)	11.2	1.21	0.99

Table 2.

Expression levels of MSH biosynthesis-related genes.

Somehow, intracellular redox potential affects these GNAT regulations. If two artificial conditions such as oxidation and reduction are provided, the expression levels of redox potential-dependent GNAT genes would be detected. These variable expression levels of GNAT genes will provide stress response control. At the moment, the physiological roles of these four GNAT proteins remain unclear. In general, proteins in the GNAT superfamily have broad-spectrum physiological functions. Their amino acid sequence identities are very low. Thus, predicting their functional roles through protein sequence similarities is difficult.

How about expression levels of bacillithiol (BSH) biosynthesis-related genes in the transcriptome of D. geothermalis? The genome of D. geothermalis contains BSH biosynthesis enzymes BshA (Dgeo_1099; BSH biosynthesis glycosyltransferase), BshB1 (Dgeo_2305; BSH biosynthesis deacetylase), BshC (Dgeo_1276; BSH biosynthesis cysteine-adding enzyme), and BstA (Dgeo_1829; BSH transferase) as Drad BSH-related genes. It also contains BSH reductase (Dgeo_2331; YpdA) and bacilliredoxin (Dgeo_1464; YtxJ). Despite all genes involved in BSH biosynthesis and degradation pathway, expression levels of these genes were not affected in the intracellular oxidation state of Δdgeo_1986-87 strain. However, they might be affected by other stressors such as heat shock and hydrogen peroxide treatment.

2.4 Dps and its mysterious roles

D. radiodurans have two paralogous Dps proteins, each known to play a different role. DrDps1 (DR2263) binds to both linear and coiled DNA. However, DrDps2 (DRB0092) preferentially binds to coiled DNA, forming different conformation of protein-DNA complexes to protect DNA against ROS, although its protection occurs at different iron to protein ratios. The difference between two DrDps could result from the fact that DrDps1 has higher iron oxidation rate in the presence of hydrogen peroxide and higher affinity to bind DNA than DrDps2 [37]. In summary, DrDps1 may function in DNA metabolism, while DrDps2 may protect against exogenously derived ROS [57].

D. geothermalis has two Dps proteins homologous to Dps proteins of D. radiodurans. Dgeo_0281 is homologous to DrDps1 (DR_2263). The novel Dgeo_0257 has been proposed to be one of Dps DNA-binding proteins in D. geothermalis. It probably has different roles from DrDps1 homologous protein, Dgeo_0281. Dgeo_0257 shares 72% amino acid sequence identity to DR_0528 of D. radiodurans, suggesting the need for research as another candidate protein of DrDps. The DrDps2 (DR_B0092) did not share any significant sequence identities with proteins of D. geothermalis. Dgeo_0281 and Dgeo_0257 share only 11.5% amino acid sequence identity, lower than 16% amino acid sequence identity shared between DrDps1 and DrDps2.

We prepared both dps genes disrupted mutant strains, Δdgeo_0257 and Δdgeo_0281, and they were more susceptible to H₂O₂ than the wild-type strain. The novel putative Dps Dgeo_0257 might play a role in DNA protection and antioxidant reactions such as Dgeo_0281. DNA-binding capacities of purified Dgeo_0257 and Dgeo_0281 proteins were then determined by electrophoretic mobility shift assay (EMSA). Gel filtration assay was also performed for conformational determination [58]. Dgeo_0257 protein has a 5-fold higher DNA-binding affinity than Dgeo_0281. Interestingly, both Dps proteins were found to have similar metal-sensing behavior (Figure 2). When ferrous ion was present, Dps proteins could not bind to DNA. Their DNA-binding activity was found to be non-specific for DNA sequence. To determine the physiological functions of these two Dps proteins, we performed quantitative real-time (qRT)-PCR analysis for both dgeo_0257 and dgeo_0281 genes in wild-type, Δdgeo_0257, and Δdgeo_0281 mutant strains at different growth phase in a time-course study. Surprisingly, the dgeo_0281 gene was early expressed at OD₆₀₀ 2.0. Its expression then gradually reduced at OD₆₀₀ 4.0 and 8.0. However, dgeo_0257 was dramatically induced in a stationary phase at OD₆₀₀ 8.0. Thus, we predicted that both Dps proteins of D. geothermalis had growth phase-dependent specificity.

Figure 2.

Illustration of DNA protection and iron detoxification roles of two Dps proteins in D. geothermalis.

2.5 Active transposition of insertion sequences under oxidative stress condition

Various selectable approaches have detected transposition events of ISs. For example, ISDra2 was induced by irradiation, causing the thyA (thymidylate synthase) gene to be destroyed in D. radiodurans. As a result, thyA mutant became resistant to trimethoprim [47, 59]. In the case of antibiotic-resistant phenotype, a certain IS element was integrated into rsmG gene disrupted by ISTth7 of IS5 family in Thermus thermophilus, resulting in streptomycin-resistance [60].

The genome of D. geothermalis contains a total of 73 ISs. Deinococcus species were found to have pink or reddish colored colonies. However, D. geothermalis wild-type, Dps-like gene disrupted mutant (Δdgeo_0257 mutant), and LysR gene disrupted mutant (Δdgeo_2840 mutant) were found to have white-colored colonies under an oxidative stress condition. The reason was that phytoene desaturase function of Dgeo_0524 as a carotenoid pathway-related gene was interrupted by the transposition of each IS element: ISDge6 for Δdgeo_2840 mutant, ISDge7 for Δdgeo_0257 mutant, and ISDge11 for wild-type (Figure 3) [32, 61, 62]. Among down-regulated genes in RNA-seq, two genes (dgeo_0928 and dgeo_1785) were disturbed by ISDge5 in the Δdgeo_0257 mutant strain. A new biomarker for finding transposition loci with antibiotic streptomycin-resistance was also used easily to selecting colonies on streptomycin contained media. When the ISDge6 element was inserted into the rsmG gene (dgeo_2335) encoding ribosomal RNA small subunit methyltransferase and a point mutation or frameshift mutation on rsmG gene occurred, mutant strains were resistant to 50 μg/ml streptomycin (prepared manuscript). In the current discovery, ISDge5, ISDge6, ISDge7, and ISDge11 were all replicating transposition modes through PCR detection of target genes. We found that each IS element was transposed, explicitly depending on DNA-binding proteins from these active transposition events. There is an open question. Although the genome of D. geothermalis contains a high copy number of IS elements such as ISDge2, ISDge3, ISDge4, and ISDge13, transposition events have not been found yet. Nevertheless, in the case of ISDge2, its transposase gene expression was strongly induced by oxidative stress. Thus, it is a big challenge to detect DNA-binding protein-dependent IS transposition occurrence. We can imagine that when the environmental factor is changed from oxidative stress to others such as other source radiations, gravity, pressure, and certain chemicals, specialized IS elements might be transposed into other loci in the genome.

Figure 3.

Brief scheme of metabolic pathway (A) and the ISs integrated loci in the gene cluster for carotenoid biosynthesis (B).

3. Conclusion

As a model for oxidative stress response, Deinococcus species is a beneficial model organism to understand its survival strategies in the presence of harsh environmental stressors such as ionizing radiation, desiccation, and ultraviolet light. It is also a useful model organism to understand DNA damage repair mechanisms and industrial application such as bioremediation of toxic substances. For these reasons, many researchers are interested in applying extreme conditions, including microgravity and universe exposure outside the international space station, to a type strain of D. radiodurans recently. Here, we focused on several aspects of oxidative stress defense systems dependent on our research destination, for example, intracellular redox balance through a cystine importer, antioxidant substance carotenoid biosynthesis, DNA protecting and iron detoxification protein Dps, and transposition of IS elements under oxidative stress. We hope this chapter will provide an opportunity to open up a new horizon in traditional research as we learn about the phenomena linked differently to known antioxidant response mechanisms in radiation-resistant strains.

Acknowledgments

This study was supported by the National Research Foundation of Korea Grant funded by the Korean government (MSIT) (2020R1F1A1070779) and SJL thanks Kyung Hee University’s support.

Conflict of interest

The authors declare no conflict of interest.