Transposition of Insertion Sequences Was Triggered by Oxidative Stress in Radiation-Resistant Bacterium Deinococcus Geothermalis

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Article Transposition of Insertion Sequences was Triggered by Oxidative Stress in Radiation-Resistant Bacterium Deinococcus geothermalis

Chanjae Lee †, Nakjun Choi †, Min K. Bae, Kyungsil Choo and Sung-Jae Lee * Department of Biology, Kyung Hee University, Seoul 02447, Korea; [email protected] (C.L.); [email protected] (N.C.); [email protected] (M.K.B.); [email protected] (K.C.) * Correspondence: [email protected]; Tel.: +82-2-961-0406; Fax: +82-2-961-9155 These authors contributed equally to this work. † Received: 29 July 2019; Accepted: 10 October 2019; Published: 12 October 2019

Abstract: During an oxidative stress-response assay on a putative Dps-like gene-disrupted ∆dgeo_0257 mutant strain of radiation-resistant bacterium Deinococcus geothermalis, a non-pigmented colony was observed among the normal reddish color colonies. This non-pigmented mutant cell subsequently displayed higher sensitivity to H2O2. While carotenoid has a role in protecting as scavenger of reactive oxygen species the reddish wild-type strain from radiation and oxidative stresses, it is hypothesized that the carotenoid biosynthesis pathway has been disrupted in the mutant D. geothermalis cell. Here, we show that, in the non-pigmented mutant cell of interest, phytoene desaturase (Dgeo_0524, crtI), a key enzyme in carotenoid biosynthesis, was interrupted by transposition of an ISDge7 family member insertion sequence (IS) element. RNA-Seq analysis between wild-type and ∆dgeo_0257 mutant strains revealed that the expression level of ISDge5 family transposases, but not ISDge7 family members, were substantially up-regulated in the ∆dgeo_0257 mutant strain. We revealed that the non-pigmented strain resulted from the genomic integration of ISDge7 family member IS elements, which were also highly up-regulated, particularly following oxidative stress. The transposition path for both transposases is a replicative mode. When exposed to oxidative stress in the absence of the putative DNA binding protein Dgeo_0257, a reddish D. geothermalis strain became non-pigmented. This transformation was facilitated by transposition of an ISDge7 family IS element into a gene encoding a key enzyme of carotenoid biosynthesis. Further, we present evidence of additional active transposition by the ISDge5 family IS elements, a gene that was up-regulated during the stationary phase regardless of the presence of oxidative stress.

Keywords: Insertion sequences; transposase; transposition; genomic plasticity; oxidative stress; Deinococcus geothermalis

1. Introduction Genus Deinococcus is an aerobic Gram-positive bacterium capable of surviving in several extreme and/or harmful conditions (e.g., high levels of radiation, desiccation, oxidative stress, and starvation) [1–3]. The primary mechanisms mediating resistance to cellular stress are related to the ability of Deinococcus strains to protect damaged DNA and their proteome and other factors as follows: (1) structural organization of cell wall, (2) genome packaging, (3) active removal of toxic compounds, and (4) regulation of gene expression [4,5]. There are many known proteins for the protection of double strand DNA breakage and the DNA repair (eg, RecA, Ddr, and Ppr) [1,6,7]. Despite many studies using a myriad of molecular approaches to detect genus-speciﬁc DNA Damage Repair (DDR) machinery in Deinococcus, the protection and repair mechanisms of this genus remain largely unclear.

Microorganisms 2019, 7, 446; doi:10.3390/microorganisms7100446 www.mdpi.com/journal/microorganisms Microorganisms 2019, 7, 446 2 of 10

One DNA-protection protein involved in stress responses is the DNA-binding protein from starved cells (Dps), a protein controlled by the oxidative stress regulator OxyR [8–11]. Deinococcus radiodurans has two Dps proteins (i.e., Dps1, Dps2) and can survive through DNA protection under a number of extreme stresses (e.g., lengthy desiccation, intensive radiation, harmful chemical environments) [3,12]. Deinococcus geothermalis has been studied less than the genus type strain D. radiodurans [13], but a putative Dps gene dgeo_0257 has been identified; it should be noted this protein has not yet characterized nor matched to D. radiodurans Dps proteins. Importantly, D. radiodurans has one orthologous and hypothetical gene DR0582 with 72% identity. To characterize the functions of a Dps candidate protein of D. geothermalis (Dgeo_0257), a mutant strain with a disrupted target gene (∆dgeo_0257) was constructed and tested for sensitivity to oxidative stress. During oxidative stress response experiments, a non-pigmented colony was observed in the dgeo_0257-disrupted background; we named this mutant strain ∆dgeo_0257 white mutant (aka ∆dgeo_0257w) because a wild-type strain generally is reddish in color. Since the D. geothermalis strain contains carotenoid—a carotene known to play a role in the protection of oxidative stress—it appears likely that this mutant strain has disruptions in the carotenoid-biosynthesis pathway. To support this hypothesis, we report evidence that a key enzyme of carotenoid biosynthesis was disrupted due to transposition of a unique type of transposase. For this reason, we have examined the possibility that DNA-protecting protein Dps controls the transposition of insertion sequence (IS) elements in genomic plasticity. IS elements are frequently identified by their unique sequence features (e.g., transposase-encoding genes, terminal inverted repeats (TIR), and direct repeats (DR)) in the target sequence generated upon insertion [14,15]. These ISs are usually 750~2000 bp and encode enzymes causing transposition. D. geothermalis DSM11300T has 93 transposases with 33 chromosomal, 38 within the plasmid pDGEO01, and 22 in the plasmid pDGEO02. Meanwhile, species within phylum Thermus-Deinococcus, including D. radiodurans, D. deserti, and Thermus thermophiles HB8 genomes, have 52, 17, and 92 ISs, respectively. In archaea, a halophilic archaeaon Halobacterium salinarum NRC-1 has 72 ISs. Amazingly, a crenarchaeon Saccharolobus solfataricus P2 genome has 443 ISs. All data were analyzed using ISfinder (www-is.biotoul.fr). In a recent report, Thermus mobilome for ISs distribution in Thermus spp. was characterized as active transposition of ISs induced by irradiation [16] like the D. radiodurans genome [17]. Here, we identified, for the first time in D. geothermalis, that the non-pigmented phenotypical transition due to the key enzyme of carotene biosynthesis dgeo_0524 (crtI) encoding a phytoene desaturase was inactivated by the transposition of an active IS element under oxidative stress conditions.

2. Materials and Methods

2.1. Bacterial Strains, Culture Conditions, and Construction of the Mutant The strain D. geothermalis DSM 11300T was obtained from the Korean Agricultural strains Collection Center (KACC12208). TGY medium containing 1% tryptone, 0.5% yeast extract, and 0.1% glucose was used for culturing D. geothermalis at 48 ◦C. Escherichia coli DH5α was used for transformation of recombinant DNA and grown on Luria-Bertani medium at 37 ◦C. In order to disrupt a putative dps gene dgeo_0257, a mutant strain with a kanamycin-resistance cassette which was integrated into the target gene was constructed by homologous recombination. For the dgeo_0257 mutant strain, the homologous DNA sequence downstream of dgeo_0257 (roughly 1.0 kb) was amplified from genomic DNA using target-region primers and purified using a PCR purification kit (Bioneer, Korea). The purified DNA fragments and plasmid pKatAPH3 [18] were cleaved by XbaI-PstI and ligated into a plasmid (named pKR0257). The homologous DNA sequence upstream of dgeo_0257 (roughly 1.0 kb) was amplified from genomic DNA using target-region primers and purified using a PCR purification kit (Bioneer, Korea). To yield pKRL0257, a vector which contained upstream and downstream regions of the dgeo_0257 gene, the purified DNA fragments and plasmid pKR0257 were digested with KpnI-SalI, ligated, and propagated in E. coli. Recombinant DNA (ie, pKRL0257) Microorganisms 2019, 7, 446 3 of 10

was purified from E. coli and transformed into D. geothermalis using a CaCl2-dependent technique described previously [19]. Wild-type D. geothermalis cultures were harvested in the exponential growth phase and suspended in TGY broth of 100 mM CaCl2: 100% glycerol (20:8:3, v/v/v). For transformation, 100 µL of the cell suspension and 5 µL of a solution containing pKRL0257 with ca 200 ng/ µL were added. The mixture was incubated on ice for 30 min and then at 32 ◦C for 30 min with gentle shaking. TGY broth (0.9 mL) was added into the mixture and incubated at 37 ◦C for 16 h with gentle shaking. Next, 100 µL of the mixture was spread onto TGY agar containing 8 µL/mL kanamycin for selection. The resulting transformed clone was identified by PCR analysis of the target gene region and named ∆dgeo_0257 mutant strain. Genome sequence of D. geothermalis DSM11300T was used to design the following specific primers: chromosome (GenBank accession number NC_008025.1), plasmid 1 (NC_008010.2), and plasmid 2 (NC_009939.1).

2.2. Viability Test Studies in D. Geothermalis

The wild-type and ∆dgeo_0257 mutant strains of D. geothermalis were grown to an OD600 1.0 in TGY broth at 48 ◦C. Similar number of cells from each culture were treated with 80, 100, and 120 mM hydrogen peroxide and incubated for 1 h. The stressed cells were serially diluted 10-fold in buﬀered 0 5 saline from 10 to 10− . A 5-µL volume from each diluted suspension was spotted on the TGY agar plates and incubated at 48 ◦C.

2.3. PCR Detection of Transposition and Sequence Analysis To detect transposition of ISs, we designed target-gene-encompassing primer sets with similar melting temperatures (Supplementary Table S1). Transposase-integrated sites were noted based on their size following PCR and agarose gel electrophoresis (i.e., enlarged in the non-pigmented mutant compared with wild-type and ∆dgeo_0257 mutant strains). The enlarged PCR products were then sequenced. The type of transposase, integrated sites, and specialized sequences including direct repeat and inverted repeat were determined. Transposition mode was also determined using PCR detection of genome distributed identical IS type genes with primer sets of outside border of the IS element.

2.4. RNA-Seq Analysis To extract total RNA for RNA-Seq analysis, wild-type and the ∆dgeo_0257 mutant D. geothermalis strains were harvested at OD600 4.0 in TGY broth at 48 ◦C. We manually used RIDOEx reagent (GeneAll, Korea) for extraction of total RNA. The extracted total RNA was puriﬁed using an RNeasy Mini Puriﬁcation Kit and RNase-Free DNase I Set (Qiagen, Hilden, Germany). We commissioned D. geothermalis bacterial RNA-Seq and results analysis in e-biogen Co (Seoul, Korea). Total RNA quality measured by Agilent’s 2100 Bioanalyzer system and sequenced by Illumina HiSeq 2500 platform. Total raw read counts of wild-type and ∆dgeo_0257 mutant strains were 20,705,663 and 19,722,792, except plasmid pDGEO02, respectively. RNA-Seq analysis was performed using ExDEGA analysis tool of e-biogen Co.

2.5. Quantitative Reverse Transcriptase (qRT) PCR To determine the expression levels of target transposase genes and related genes, we performed qRT-PCR as previously reported [20]. After cells were harvested at OD 4.0: (1) total RNA was extracted using a phenol-based RNA extraction procedure, (2) cDNAs were synthesized by PCR using reverse transcriptase, and (3) the synthesized cDNA were quantiﬁed and stored at –70 ◦C until real time PCR. Quantitative PCR was conducted using the Bio-Rad RT-PCR model CFX96TM Optics Module. The data were calculated using the qRT-PCR formula. Microorganisms 2019, 7, 446 4 of 10

3. Results Microorganisms 2019, 7, x FOR PEER REVIEW 4 of 10 3.1. Construction of the D. Geothermalis ∆dgeo_0257 Mutant Strain To identify physiological roles (e.g., oxidative stress resistance, DNA protection) of Dgeo_0257, a putativeTo identify Dps physiologicalprotein, we built roles (e.g.,a recombinant oxidative stressplasmid resistance, carrying DNA the protection)dgeo_0257 gene-disrupted of Dgeo_0257, aconstruct putative Dpsin the protein, cloning we vector built apKatAPH3 recombinant (Figure plasmid 1A). carrying This construct the dgeo was_0257 generated gene-disrupted by replacing construct the indgeo the_0257 cloning locus vector with pKatAPH3 the kanamycin-resistant (Figure1A). This aph construct gene via was homologous generated recombination by replacing the of dgeoa roughly_0257 locus1 kb segment with the kanamycin-resistantin both border regionsaph ofgene the viatarget homologous gene. The recombination selected mutant of aclones roughly with 1kb kanamycin segment inresistance both border were regions confirmed of the by target PCR gene. (based The on selected an increase mutant in clonesamplicon with size). kanamycin When growth resistance patterns were conﬁrmedwere compared by PCR between (based wild-type on an increase and mutant in amplicon strain in size). TGY When medium growth at optimum patterns temperature, were compared both betweenstrains revealed wild-type identical and mutant growth strain curves in TGY (data medium not shown), at optimum confirming temperature, that dgeo both_0257 strains is not revealedessential identicalfor cell growth. growth curves (data not shown), conﬁrming that dgeo_0257 is not essential for cell growth.

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Figure 1. Scheme of construction of the ∆dgeo_0257-disrupted mutant strain (A) and viability assay

ofFigure the wild-type, 1. Scheme∆ ofdgeo construction_0257, and of∆dgeo the _0257wΔdgeo_0257-disrupted mutant strains mutant when treated strain ( withA) and H2 viabilityO2 (B). ( Cassay) PCR of detectionthe wild-type, was done Δdgeo by_0257, encompassing and Δdgeo primer_0257w sets mutant for dgeo strains_0257. when Lanes: treated 1, wild-type; with H2O 2,2 (∆B).dgeo (C_0257) PCR mutant;detection 3, ∆wasdgeo done_0257w by mutant.encompassing primer sets for dgeo_0257. Lanes: 1, wild-type; 2, Δdgeo_0257 mutant; 3, Δdgeo_0257w mutant. 3.2. The Effect of H2O2 on the Viability of the ∆dgeo_0257 Mutant Strain 3.2. TheTo characterize Effect of H2O any2 on potential the Viability diff erencesof the Δdgeo_0257 between the Mutant viability Strain of wild-type and ∆dgeo_0257 mutant strains,To cellscharacterize were grown any underpotential oxidative differences stress between conditions the (i.e., viability treatment of wild-type with H2O and2). AfterΔdgeo H_02572O2 treatmentmutant strains, (80, 100, cells 120 were mM grown final concentration) under oxidative for 1stress h, the conditions viability of (i.e., the wild-typetreatment strainwith H was2O2 similar). After toH the2O2 ∆treatmentdgeo_0257 (80, mutant 100, 120 strain mM at allfinal concentrations concentration) (Figure for 11 h,B). the Therefore, viability we of concludethe wild-type that straindgeo_0257 was genesimilar products to the Δ aredgeo not_0257 directly mutant involved strain in at cell all viability concentrations by a DNA (Figure protection 1B). Therefore, procedure we under conclude oxidative that stressdgeo_0257 conditions. gene products are not directly involved in cell viability by a DNA protection procedure underThe oxidative results of stress the oxidative conditions. stress assay of the ∆dgeo_0257 mutant and wild-type strains revealed a non-pigmentedThe results of colony the oxidative on diluted stress spread assay plate of the after Δdgeo H2_0257O2 treatment. mutant and The wild-type white colony, strains which revealed we nameda non-pigmented∆dgeo_0257w, colony grew on well diluted grown spread at 48 plate◦C. Using after H 16S2O2 rRNA treatment. sequence The white analysis, colony, we confirmed which we thatnamed the Δ sequencedgeo_0257w, of the grew 16S rRNAwell grown subunit at 48 is identical°C. Using to 16SD. geothermalisrRNA sequence(i.e., analysis, 100% homology we confirmed to the 16Sthat rRNA the sequence sequence of of the the 16S∆dgeo rRNA_0257 subunit mutant). is identical This datum to D. suggestsgeothermalis that (i.e., the non-pigmented100% homology strainto the ∆16Sdgeo rRNA_0257w sequence originated of the from Δdgeo∆dgeo_0257_0257 mutant). mutant butThis not datum from suggests the wild-type that the (Figure non-pigmented1C). Interestingly, strain theΔdgeo non-pigmented_0257w originated strain revealedfrom Δ slightdgeo_0257 sensitivity mutant on oxidativebut not stressfrom (Figurethe wild-type1B). (Figure 1C). Interestingly, the non-pigmented strain revealed slight sensitivity on oxidative stress (Figure 1B).

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3.3. Detection of Transposition in Non-Pigmented Strain In general, Deinococcus strains are orange in color resulting from carotenoid production, a pigment which is also involved in the oxidative stress response. The non-pigmented strain is likely the result of dysfunctionMicroorganisms of a key 2019,carotenoid 7, x FOR PEER pathwayREVIEW enzyme [21] (Figure2A). We examined the corresponding 5 of 10 pathway at KEGG database (https://www.genome.jp/kegg/) and chose the four genes (dgeo_0523, corresponding pathway at KEGG database (https://www.genome.jp/kegg/) and chose the four genes 0524, 0857, and 2309) to search for possible transposition among these genes by PCR with target (dgeo_0523, 0524, 0857, and 2309) to search for possible transposition among these genes by PCR with gene–encompassingtarget gene–encompassing primers. Interestingly, primers. Interestingly,dgeo_0524 dgeo gene_0524 (i.e., gene phytoene (i.e., phytoene desaturase) desaturase) was disrupted was by integrationdisrupted of by an integration IS element of (Figure an IS element2B). Thus, (Figure as hypothesized, 2B). Thus, as hypothesized, disruption of disruption a key enzyme of a key in the carotenoidenzyme synthetic in the carotenoid pathway synthetic created thepathway observed created non-pigmented the observed non-pigmented phenotype. phenotype.

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FigureFigure 2. Scheme 2. Scheme of of carotenoid carotenoid biosynthesis biosynthesis pathway pathway and anddetection detection of transposition of transposition locus. (A) locus.A (A) A simplifiedsimplified carotenoid biosynthetic biosynthetic pathway pathway is illust is illustrated,rated, and andgenes genes encoding encoding metabolic metabolic enzymes enzymes are are indicated.indicated.Symbolized Symbolized compounds: GGPP, GGPP, geranylgeran geranylgeranylyl pyrophosphate; pyrophosphate; a, Phytofluene; a, Phytofluene; b, ζ- b, Carotene; c, Neurosporene; d, β-Zeacarotene; e, δ-Carotene. (B) PCR detection of transposition loci ζ-Carotene; c, Neurosporene; d, β-Zeacarotene; e, δ-Carotene. (B) PCR detection of transposition loci on four target genes for carotenoid biosynthesis. Lanes: 1, 3, 5, and 7 samples and 2, 4, 6, and 8 samples on four target genes for carotenoid biosynthesis. Lanes: 1, 3, 5, and 7 samples and 2, 4, 6, and 8 samples were amplified from Δdgeo_0257 and Δdgeo_0257w genomic DNA, respectively. (C) IS integration were amplified from ∆dgeo_0257 and ∆dgeo_0257w genomic DNA, respectively. (C) IS integration sites sites were determined by sequencing of dgeo_0524 gene amplicons. were determined by sequencing of dgeo_0524 gene amplicons. Inserted DNA sequence analysis revealed the exact insertion site and facilitated the Insertedcharacterization DNA sequence of the inserted analysis DNA revealed sequence the exact (Figure insertion 2C). The site andinsertion facilitated sequence the characterizationis an ISDge7 of thefamily inserted IS element DNA sequence which has (Figure 5′-end 642C). nt Theextension insertion and 3 sequence′-end 16 nt isextension an IS Dge with7 family798 nt ORF, IS element 265 whichamino has 50 -endacids, 64 of nt IS5 extension type transposase. and 30-end This 16 IS ntDge extension7 type IS with element 798 nthas ORF, an 8 265 nt aminoTIR sequence acids, ofof IS5 type transposase.‘GAGGCTGG’ This on both ISDge ends7 type of the IS boarder. element This has sequence an 8 nt TIRwas sequenceintegrated ofat the ‘GAGGCTGG’ 147th nucleotide on of both ends ofdgeo the_0524 boarder. on counter This sequencetranscriptional was directio integratedn with at 2 the nt DR 147th sequence nucleotide of “TA.” of dgeo The _0524D. geothermalis on counter transcriptionalgenome contains direction four with identical 2 nt IS DRDge sequence7 chromosomal of “TA.” family The transposasD. geothermalises, Dgeo_1042,genome 1699, contains 2208, and four identical2276 IS Dge(Supplementary7 chromosomal Table family S2).transposases, The transpos Dgeo_1042,ition pattern 1699, was 2208,determined and 2276 by (Supplementary target-gene amplification using gene-specific primers at outside boarder of IS elements (Supplementary Table Table S2). The transposition pattern was determined by target-gene amplification using gene-specific S1). All four target genes were still located at their original sites (Supplementary Figure S1A). primersTherefore, at outside transposition boarder of of IS IS elementsDge7 family (Supplementary IS element occurred Table via S1).replicative All four mode. target genes were still located at their original sites (Supplementary Figure S1A). Therefore, transposition of ISDge7 family IS element3.4. occurred RNA-Seq viaAnalysis replicative of Δdgeo_0257 mode. and Δdgeo_0257w and Identification of New Transpositions To characterize the gene expression levels and patterns of Δdgeo_0257 and Δdgeo_0257w 3.4. RNA-Seq Analysis of ∆dgeo_0257 and ∆dgeo_0257w and Identification of New Transpositions mutants, RNA-Seq analysis was performed on both mutant and wild-type strains. Interestingly, 10 ToIS characterize elements of thefamily gene ISDge expression5, located levels on the and chromosome patterns of and∆dgeo plasmid_0257 pDGEO01, and ∆dgeo have_0257w identical mutants, RNA-Seqnucleotide analysis sequences was performed contain ona transposase both mutant that and was wild-type up-regulated. strains. The Interestingly, mean expression 10 IS level elements was of family7.48-fold ISDge5, locatedexcept dgeo on_1807 the chromosome of 3.49-fold (Supplementary and plasmid pDGEO01, Table S2). have identical nucleotide sequences contain a transposaseIn the non-pigmented that was up-regulated. colonies, however, The the mean expres expressionsion levels level of four was transposases 7.48-fold except belongingdgeo _1807to IS elements of the ISDge7 family were not affected (i.e., expression level of roughly 1.0). Thus, we of 3.49-fold (Supplementary Table S2). aimed to identify transposition of up-regulated ISDge5 family members of the IS701 type using strictly down-regulated genes with less than 0.3-fold between Δdgeo_0257 and Δdgeo_0257w mutants

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MicroorganismsIn the non-pigmented 2019, 7, x FOR PEER colonies, REVIEW however, the expression levels of four transposases belonging 6 of 10 to IS elements of the ISDge7 family were not affected (i.e., expression level of roughly 1.0). Thus, and selected four genes, dgeo_0926, 0927, 1785, and 1365 regions (Table 1). An extended near region we aimed to identify transposition of up-regulated ISDge5 family members of the IS701 type using dgeo_0928 with 6.20-fold was shown. Using a target-gene amplification assay, we identified two dgeo dgeo strictlyadditional down-regulated transposition genes target with sites less on thandgeo_0928 0.3-fold and between dgeo_1785∆ region_0257 but and not ∆on dgeo_0926,_0257w mutants 0927, andand selected 1365 as four down-regulated genes, dgeo_0926, targets 0927, (Figure 1785, 3A & and Table 1365 1). regions Surprisingly, (Table both1). An transpositions extended near involved region dgeoelements_0928 with of the 6.20-fold ISDge5 wasfamily. shown. The IS Using element a target-gene integrated amplificationat 1278th and assay,36th nucleotide we identified of the two additionaldgeo_0928 transposition and dgeo_1785 target genes, sites respectively on dgeo_0928 (Figure and 3B,dgeo C)._1785 The IS region element but of not ISDge on5 dgeo_0926, family has 0927,5′- andend 1365 59 asnt down-regulatedextension and 3′-end targets 1 nt (Figure extension3A &encompassing Table1). Surprisingly, a 1092 nt, both363 amino transpositions acids long, involved IS701- elementslike transposase. of the ISDge Unusually,5 family. Theboth IS elementends of integrated border athave 1278th 16 andnt 36thTIR nucleotidesequence ofof the dgeo‘CTCAGGAGTTGCACCT’._0928 and dgeo_1785 genes, The respectively 3′ end of the (Figure TIR sequence3B,C). The was IS invo elementlved in of transposition IS Dge5 family within has 5 the0-end 59ORF nt extension region containing and 30-end a 1stop nt extension codon. We encompassing also detected a 1092a transposition nt, 363 amino pattern acids by long, target-gene IS701-like transposase.amplification Unusually, using region both specific ends of primers. border haveEight 16target nt TIR genes sequence were still of ‘CTCAGGAGTTGCACCT’.located at their own sites Theexcept 30 end for of dgeo the_2108 TIR sequence and dgeo_2659 was involved (Supplementary in transposition Figure S1B). within These the two ORF genes region were containing not amplified a stop codon.in any We of alsothe three detected strains. a transposition It is possible that pattern these by genes target-gene were wrongly amplification annotated using or lost region within specific the primers.tested strains. Eight target Thus, genes the need were to still further located define at their target own genes sites from except original for dgeo stock_2108 collections and dgeo and_2659 (Supplementarycompare gene Figureloci among S1B). Thesethem remains. two genes From were bo notrder amplified sequence in analysis any of theof IS three insertion, strains. the It is possibleduplicated that theseDR sequences genes were were wrongly 5 nt in annotatedlength with or completely lost within different the tested sequences strains. (Supplementary Thus, the need to furtherTable define S2). DR target sequences genes fromof ISDge original5 family stock transposase collections in and dgeo compare_0928 and gene dgeo loci_1785 among integration them remains. were “TCCGA” and “CTCTC,” respectively. Therefore, the up-regulated ISDge5 family transposases were From border sequence analysis of IS insertion, the duplicated DR sequences were 5 nt in length with also moved by replicative transposition with nonspecific integration within the background of a completely different sequences (Supplementary Table S2). DR sequences of ISDge5 family transposase putative Dps gene dgeo_0257 mutant. in dgeo_0928 and dgeo_1785 integration were “TCCGA” and “CTCTC,” respectively. Therefore, the up-regulatedTable 1. IS ListDge of5 familydown-regulated transposases genes under were also0.3-fold moved on expression by replicative ratio of transposition Δdgeo_0257w/Δ withdgeo_ nonspecific0257 integrationin RNA-Seq within analysis. the background of a putative Dps gene dgeo_0257 mutant.

Table 1. List ofGene down-regulated Δ0257/WT genes under 0.3-foldΔ0257w/WT on expression ratio ofΔ0257w/∆dgeo_0257wΔ0257/ ∆dgeo_0257 in RNA-Seq analysis. dgeo_0927 2.35 0.21 0.09 Gene ∆0257/WT ∆0257w/WT ∆0257w/∆0257 dgeo_0926 2.10 0.28 0.13 dgeo_0927 2.35 0.21 0.09 dgeo_1785 dgeo_09260.61 2.10 0.28 0.09 0.13 0.15 dgeo_1785 0.61 0.09 0.15 dgeo_1365 dgeo_13651.58 1.58 0.45 0.45 0.28 0.28

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FigureFigure 3. 3.Detection Detection of of integrated integrated ISsISs onon substantiallysubstantially down-regulated candidate candidate genes. genes. (A ()A PCR) PCR ampliﬁcationamplification on on three three candidate candidate genes genes (ie, (ie,dgeo dgeo_0928,_0928, 1785, 1365). 1365). Transposition Transposition by by integrated integrated ISs ISs was was detecteddetected in indgeo dgeo_0928_0928 and anddgeo dgeo_1785_1785 regions. regions. Lanes:Lanes: 1, 4, and and 7 7 were were wild-type wild-type strain; strain; 2, 2,5, 5,and and 8 were 8 were ∆dgeo_Δdgeo_0257;0257; 3, 3, 6, 6, and and 9 9 were were∆ Δdgeo_dgeo_0257w0257w genomicgenomic DNA. B-C, B-C, An An IS ISDge5Dge5 familyfamily member member IS ISelement element waswas integrated integrated into into both bothdgeo dgeo_0928_0928 ( B(B)) and anddgeo dgeo_1785_1785 ((C) genes.

Microorganisms 2019, 7, x FOR PEER REVIEW 7 of 10

Microorganisms 2019, 7, 446 7 of 10 3.5. Detection of Expression Levels of Transposases on Oxidative Stress Condition To characterize the expression levels of ISDge5 family and ISDge7 family transposases under 3.5. Detection of Expression Levels of Transposases on Oxidative Stress Condition oxidative stress conditions (i.e., treatment with 50 mM H2O2), quantitative RT-PCR was conducted for selectedTo characterize candidate the IS expression members of levels ISDge of7 ISandDge IS5Dge family5 family and in IS wild-typeDge7 family and transposases Δdgeo_0257 mutant under oxidativestrains. Relative stress conditions expression (i.e., levels treatment of ISDge with7 and 50 mMISDge H52 Ofamily2), quantitative transposases RT-PCR were was indicated conducted 4.20 forand selected0.60-fold candidate in Δdgeo IS_0257 members mutant of ISstrainDge7 under and IS HDge2O52 familyabsent incondition. wild-type When and ∆ treateddgeo_0257 with mutant 50 mM strains. H2O2, Relativeexpression expression levels of levels ISDge of7 ISandDge IS7 andDge5 IS transposasesDge5 family transposasesindicated 0.49- were and indicated 1.29-fold 4.20 in andwild-type 0.60-fold but instrongly∆dgeo_0257 increased mutant 304.99- strain underand 19.96-fold H2O2 absent in the condition. Δdgeo_0257 When mutant treated strain, with 50 respectively mM H2O2, expression (Figure 4). levelsThus, of the ISDge expression7 and ISDge of 5both transposases ISDge5 and indicated ISDge 0.49-7 family and 1.29-foldtransposases in wild-type substantially but strongly increased increased under 304.99-oxidative and stress 19.96-fold conditions in the ∆indgeo the_0257 Δdgeo mutant_0257 strain. strain, It respectively is possible (Figurethat the4). induction Thus, the of expression transposable of bothelement ISDge insertion5 and IS Dgesequences7 family depends transposases on regulatory substantially machineries increased via under a certain oxidative DNA-binding stress conditions protein in(e.g., the ∆Dps).dgeo _0257The up-regulated strain. It ispossible transposases that the were induction more integrated of transposable into other element sites within insertion thesequences dgeo_0257- dependsdisrupted on mutant regulatory strain machineries genome compared via a certain with DNA-binding the wild-type protein strain. (e.g., Therefore, Dps). The the up-regulatedputative Dps transposasesprotein Dgeo_0257 were more may integrated protect against into other induction sites withinof ISs theanddgeo integration_0257-disrupted into genomic mutant DNA strain by genomereplicative compared transposition. with the wild-type strain. Therefore, the putative Dps protein Dgeo_0257 may protect against induction of ISs and integration into genomic DNA by replicative transposition.

(A) (B)

Figure 4. Comparison of relative expression levels of ISDge5 & ISDge7 family transposases upon Figure 4. Comparison of relative expression levels of ISDge5 & ISDge7 family transposases upon treatment with/without H2O2 by qRT-PCR. Relative expression levels of ISDge5 family member treatment with/without H2O2 by qRT-PCR. Relative expression levels of ISDge5 family member transposasestransposases (A (A) and) and the the IS ISDge7Dge7family family member member transposase transposase (B) ( inB) wild-typein wild-type and and∆dgeo_ Δdgeo_02570257 strains strains in in the presence or absence of 50 mM H2O2. The bars indicate relative expression levels compared to the presence or absence of 50 mM H2O2. The bars indicate relative expression levels compared to those those of the wild-type-strain in the absence of H2O2. of the wild-type-strain in the absence of H2O2.

4.4. Discussion Discussion and and Conclusions Conclusions InsertionInsertion sequences sequences (ISs) (ISs) are are the the smallest smallest and and most most ubiquitous ubiquitous transposable transposable element element of of mobile mobile geneticgenetic elements elements within within bacterial bacterial genomes genomes [ 13[13,22].,22]. ISs ISs can can be be categorized categorized into into a a family family by by combining combining severalseveral properties properties or or their their characteristics characteristics and and have have been been recently recently classified classified [ 23[23,24].,24]. Briefly,Briefly, there there are are fourfour major major classification classification criteria: criteria: (1) (1) the the length length and and sequence sequence of of the the perfect perfect TIRs TIRs carried carried by by many many ISs ISs atat their their ends, ends, (2) (2) the the length length and and sequence sequence of of the the short short flanking flanking DRs DRs often often generated generated within within target target insertioninsertion sites, sites, (3) (3) the the organization organization of theirof their open open reading reading frame frame of transposases of transposases (e.g., DDE,(e.g., DEDD,DDE, DEDD, HuH motifs,HuH motifs, tyrosine tyrosine or serine or specificserine specific recombinase recombinase relation), relation), or (4) theor (4) target the target sequences sequences into which into which they insertthey insert [24–26 [24–26].]. Nevertheless, Nevertheless, there arethere miss-matched are miss-matched classifications classifications of ISs of type ISs betweentype between GenBank GenBank and specializedand specialized program program for analyzing for analyzing ISs [eg, ISs ISfinder [eg, (IShttp:finder//www-is.biotoul.fr (http://www-is.biotoul.fr)])] [15]. For this[15]. analysis, For this weanalysis, followed we IS followed classification IS classification using the ISfinderusing the program ISfinder for program ISs of D. for geothermalis ISs of D. geothermalis. . ISsISs act act directly directly on on the the genome genome by by moving moving within within a specific a specific genome genome or from or from cell to cell cell to through cell through gene transfergene transfer machinery. machinery. Therefore, Therefore, ISs affect geneISs affect expression, gene genomeexpression, shaping, genome and genomeshaping, evolution and genome [27]. Organismsevolution with[27]. highOrganisms transposition with activityhigh transposition could often leadactivity to severe could down-regulation often lead to orsevere disorder down- of generegulation expression. or disorder Because transpositionof gene expression. of mobile Bec geneticause elements,transposition including of mobile ISs and transposons,genetic elements, may causeincluding serious ISs harm and totransposons, the host, there may is cause a mechanism serious toha preventrm to the it. host, Inactive there elements is a mechanism could also to produce prevent inactiveit. Inactive transposases elements that could can delayalso theproduce transposition inactive induced transposases by active that elements. can delay DNA the methylation transposition or RNAinduced interference by active are elements. additional DNA methods methylation to reduce or RN transpositionA interference activity are additional [28]. The ISmethods families to within reduce transposition activity [28]. The IS families within E. coli were analyzed by the 50-mutation

Microorganisms 2019, 7, 446 8 of 10

E. coli were analyzed by the 50-mutation accumulation (MA) method through generations without stressing E. coli. As generation progressed, no further transposition was caused by auto-regulation [29]. In general, the preferential target of IS insertion is in plasmids. Indeed, IS density is significantly higher in bacterial plasmids than in their host chromosomes [16,23]. In the D. geothermalis, IS density of plasmid was condensed with 66 copies/Mbp compared to 13 copies/Mbp of chromosome. There are several reports summarizing IS transposition in D. radiodurans. The first identified IS2621 element of Deinococcus was found within the uvrA gene [30]. Later, IS8301 was detected in the pprI gene [31], and in mutagenesis via IS transposition, multiple ISs were integrated within the thyA gene [32]. One of them, ISDra2 is a member of IS200/IS605 family transposase and ISDra2 transition is triggered approximately 100-fold by γ-irradiation [17,32]. Dgeo_0257 is a putative Dps protein and annotated as a ferritin-like protein, and in this study, we first characterized the transposition of ISDge5 and ISDge7 family transposases in the ∆dgeo_0257-disrupted mutant in D. geothermalis. The ∆dgeo_0257-disrupted mutant has been constructed to evaluate the physiological roles of this protein. When ∆dgeo_0257 mutant strain was treated with H2O2, its viability was not affected in the same way as the wild-type strain. After one of H2O2 treatments on the ∆dgeo_0257 mutant strain, we detected a non-pigmented colony. The white clone presented higher sensitivity under oxidative stress conditions. Consequentially, transposition of ISDge7 occurred on phytoene desaturase, dgeo_0524, a key enzyme of carotenoid biosynthesis. Phytoene desaturase is an enzyme that converts the 40-carbon phytoene to lycopene. Nevertheless, these ISDge7 type elements were not induced in the absence of H2O2 according to the RNA-Seq analysis data sets. Interestingly, ISDge5 family transposases were upregulated in the same data sets. Thus, we tried to identify new transposition events on strictly down regulated genes. ISDge5 transposase integrated into both dgeo_0928 and dgeo_1785 genes by replicative transposition with sequence non-specificity. At the moment, we are unable to explain why both dgeo_0926 and 0927 genes are substantially down-regulated in the ∆dgeo_0257w mutant strain (Table1). Our results indicate that active transposition of IS Dge5 and ISDge7 family IS elements occurred by oxidative stress (ie, H2O2 treatment) in the absence of the putative Dps protein Dgeo_0257 which resulted in disruption of functional genes. These transpositions are replicative action on genomic DNA. The Dgeo_0257 protein acts as a DNA protector against transposition of ISs. In the absence of the DNA-protection protein, a reddish D. geothermalis strain became non-pigmented by transposition of an IS element into a gene encoding phytoene desaturase, a key enzyme of carotenoid biosynthesis. In the future, the procedures used in this work will be further applicable to the study of transposition by other stress conditions (e.g., high γ-irradiation, temperature variations [33]) and other DNA damage agents). We also plan to determine the transposition rate of ISs in accumulated generations under oxidative stress conditions [29]. Those experiments may lead to a better understanding of the physiological pros and cons of transposition phenomena. Furthermore, we will investigate more exciting challenges, including whether the transposition induction of unique ISs was controlled in a special DNA-binding-protein-dependent manner.

Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/2076-2607/7/10/446/s1. Author Contributions: C.L., N.C., and S.-J.L. designed the study and carried out the experimental work. C.L., M.K.B., K.C., and S.-J.L. analyzed data, wrote the manuscript and corrected the manuscript. Funding: This study was supported by the National Research Foundation of Korea (NRF 2015R1D1A1A02062106). S.J.L. also thanks a grant from Kyung Hee University (20151262). Acknowledgments: The authors thank D. Lee for selection of non-pigmented mutant clone and several anonymous reviewers for fruitful comments and suggestions. Conﬂicts of Interest: The authors declare no conﬂict of interest. Microorganisms 2019, 7, 446 9 of 10

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