ZHANG Cai-Yun, QIU Qin-Tian, MA Bin-Guang

(Hubei Key Laboratory of Agricultural Bioinformatics, College of Informatics, Huazhong Agricultural University, Wuhan 430070, China)

Abstract Objective Deinococcus radiodurans (D. radiodurans) is an extremophile with strong resistance to ultraviolet (UV),ionization, desiccation and chemical reagents. However, the molecular responses of this bacterium in the early recovery stage after UV irradiation are not fully understood. The aim of this work is to reveal the transcriptomic responses of D. radiodurans at this stage.Methods In this study, the transcriptomes of D. radiodurans under normal and UV irradiation culture conditions were determined by using RNA-seq technique. To identify the key genes and their regulatory relationships among the differentially expressed genes(DEGs), functional enrichment analysis was performed. Some key DEGs were selected and validated by real-time quantitative PCR.The transcriptome data from previous studies were adopted to find DEGs common to UV irradiation, ionizing radiation and desiccation stresses. The protein-protein interaction (PPI) network of DEGs was constructed; the hub genes and major modules in the PPI network were identified; functional enrichment analysis was performed for these hubs and modules. Results The results showed that the number of up-regulated genes was more than twice that of down-regulated genes in the early recovery stage after UV irradiation, and most of them were related to stress response and DNA repair. The main repair pathways in the early stage of recovery include single-strand annealing (SSA) pathway (involving genes ddrA-D), nonhomologous end joining (NHEJ) pathway (involving genes ligB and pprA) and nucleotide excision repair (NER) pathway (involving genes uvrA-C), the first two of which are for homologous recombination (HR), while the NER pathway removes pyrimidine dimers caused by UV irradiation. By comparing the transcriptome data under UV irradiation, ionizing radiation and desiccation stresses, it was found that the common responsive DEGs mainly involve Deinococcus-specific genes and the genes related to DNA/RNA metabolism. Several important hub genes and interaction modules were identified from the PPI network of DEGs, whose functions are concentrated in double-strand break repair,DNA topological change and replication. Conclusion These results indicate that in the early recovery stage after UV irradiation, a variety of genes in D. radiodurans undergo responses at transcriptome level, several repair pathways are initiated to cope with this stress, and some repair pathways are common to other stress conditions.

Key words Deinococcus radiodurans, RNA-seq, ultraviolet irradiation, DNA damage repair, protein-protein interaction (PPI)network

Deinococcus radiodurans(D. radiodurans) is an extremophile, whose cells are spherical and usually live in dimer or tetramer form, and the colonies are red[1].D. radioduranshas strong adaptability to various stress conditions, such as ionizing radiation,desiccation, high temperature and chemical reagents[2], which is due to its strong ability to repair DNA damage (such as chromosome double-strand breaks (DSB)). Therefore, this species has become an ideal model organism for studying tolerance and repair mechanisms under extreme stress conditions.The tolerance ofD. radioduransto damage by ionizing radiation, ultraviolet (UV) irradiation and desiccation may be the result of three mechanisms:prevention, tolerance and repair. Studies have found that ultraviolet light can damage DNA of organisms,producing cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs)[3],and such a damage can interfere with the replication and transcription mechanisms of DNA.D. radioduransshows strong resistance to ultraviolet irradiation damage, and its resistance to ultraviolet irradiation is about 20 times that ofEscherichia coliwhen measured from the perspective of survival rate[2].D. radioduranshas a complex damage repair pathway. For example, deletion ofuvsE,uvrA1, anduvrA2in nucleotide excision repair pathways was found to reduce but not completely eliminate the ability of this bacterium to remove CPDs and 6-4PPs from genomic DNA[3], indicating thatD. radioduranshas multiple repair pathways to resist UV damage.What pathways are involved in the repair of UV damage remains to be further explored.

Transcriptomics studies the response of organisms to environmental stress from the aspect of gene expression, and its main techniques are microarray and mRNA sequencing (RNA-seq). Up to now, many works have studied various stress responses ofD. radioduransfrom the perspective of transcriptomes, including ionizing radiation[4],desiccation stress[5], heat stress[6], salt stress[7],cadmium stress[8], H2O2stress[9], mitomycin C (MMC)stress,etc.D. radioduranshas strong tolerance to UV irradiation, but there is still a lack of systematic transcriptome study on its response to UV irradiation stress. In this work, we used RNA-seq technology to detect the transcriptome changes ofD. radioduransin response to UV irradiation, and compared and analyzed the differentially expressed genes (DEGs) ofD. radioduransunder normal culture and UV irradiation conditions. Through functional analysis of the DEGs, we found key genes related to UV irradiation stress response, among which many classic damage-repair genes play a role in the early recovery stage from UV irradiation. We analyzed the similarities and differences of DEGs in response to UV irradiation, ionizing radiation and desiccation stresses, and identified common responsive genes.Moreover, the protein-protein interaction (PPI)network of DEGs was constructed and the hub nodes and major modules were identified, which enhanced our understanding on gene functions in UV damage repair. This study is the first one using RNA-seq technology to measure the transcriptomic response ofD. radioduransto UV irradiation, providing new data and insights for further understanding the molecular mechanism of radiation tolerance in this species.

1 Materials and methods

1.1 Strain，growth conditions and treatment

The R1 strain ofD. radioduranswas used in this study, including two culture conditions. (1) Normal culture condition: 30℃, TGY medium, culture to the middle logarithmic stage; (2) UV irradiation condition: the bacterial solution under normal culture condition was irradiated with 2.5 J·m-2·s-1UV (254 nm)for 5 min (namely, irradiation dose of 750 J/m2) and sampled 10 min later (namely, in the early stage of recovery according to the definition in a previous work[4]). For each culture condition, three biological replicate samples were taken for RNA sequencing.

1.2 Transcriptome data analysis

1.2.1 RNA-seq data acquisition and preprocessing

The bacterial samples were sent to Tianjin Nuohe Source Biology Co., Ltd. for RNA extraction,subsequent cDNA library construction and onmachine sequencing. The sequencing standard was double-ended 150 bp, and the sequencing platform was NovA-PE150. Clean RNA-seq data from the company was used for subsequent analysis.

Clean data were checked for sequencing quality before biological analysis. The FastQC program was used for quality check, and Trimmomatic[10]was used for quality control of the data. The genome and annotation information ofD. radioduransR1 strain was downloaded from the NCBI genome database(Genome_Assembly_ID 300483) and used as the reference genome. The sequencing data were aligned to the reference genome by using TopHat[11](which calls Bowtie2[12]). StringTie[13]was used for transcript assembly; the preDE.py script in StringTie was used to extract quantitative results and generate the count matrix.

1.2.2 Screening of differentially expressed genes（DEGs）

The R package DESeq2[14]was used for DEGs analysis. The gene expression levels in FPKM(fragments per kilobase of transcript per million mapped reads) obtained above were merged for the three biological replicates, and the transcriptomes under normal culture and UV irradiation conditions were compared to obtain differentially expressed genes (DEGs). The criteria for DEG identification were:Fold change>2 andFalse Discovery Rate(FDR)<0.05.

1.2.3 GO and KEGG enrichment analysis for DEGs

GO database (http://geneontology.org/) was used for mapping and enrichment analysis of the obtained DEGs before and after UV irradiation. Based on the KEGG database, the R package clusterProfiler[15]was used for KEGG pathway enrichment analysis. In this process, theD. radioduransR1 genome was used as background with threshold ofFDR<0.05.

1.3 Validating RNA-seq results by real-time quantitative PCR （RT-qPCR）

For each of the two above-mentioned culture conditions (normal and UV irradiation), three biological replicate samples were taken for RT-qPCR analysis to validate the RNA sequencing results.Using housekeeping gene DR_1343 (glyceraldehyde 3-phosphate dehydrogenase) as internal reference,three key DEGs (DR_B0100, DR_1262, DR_2275)were selected for RT-qPCR experiments and the designed primer sequences are listed in Table S1. The 2-ΔΔCtmethod was used to calculate the relative expression of DEGs.T-test was used to evaluate the statistical significance (P-value) in data comparison.

1.4 Construction of the protein-protein interaction （PPI） network for DEGs

The PPI network ofD. radioduranswas downloaded from STRING database (https://cn.stringdb.org/)[16], and the subnetworks related to DEGs were screened out and imported into Cytoscape V3.9.1[17]for visualization. Unconnected nodes were deleted from the network. In the visualized PPI network,nodes represent proteins, and edges between nodes represent interactions. The color of node shows the regulation direction of a DEG (red for up-regulation,green for down-regulation, and yellow for insignificant change). The node size reflects the connectivity degree of a node.

1.5 Identification of hubs and modules in the PPI network of DEGs

The Centiscape V2.2 plug-in[18]in Cytoscape was used to analyze the PPI network topology. For each node in the network, the values of three network centrality indexes (degree, betweenness, eigenvector)were calculated. For each centrality index, the average value over the whole PPI network was calculated, and the nodes with higher values than the average were selected as high-centrality subset. Therefore, three high-centrality subsets were obtained, corresponding to the above three centrality indexes. The VENNY 2.1 software (https://bioinfogp.cnb.csic.es/tools/venny/) was used to draw the Venn diagram; the genes in the intersection of three high-centrality subsets were selected as the main gene set. The main gene set was uploaded to STRING database again to construct PPI network, and the PPIs with weighted score≥0.4(namely, medium confidence) were screened out as the PPI network of main genes. CytoHubba plug-in[19]was used to calculate the connectivity of each main gene, and the top 15 genes with the highest connectivity were considered as hub nodes in the PPI network. The MCODE plug-in[20]was used to identify the major modules (K-Score>5) from the PPI network of main genes.

2 Results and discussion

2.1 Growth status and viability of D. radiodurans cells under UV irradiation stress

After UV stress, the recovery ofD. radioduransusually goes through three stages: early recovery,middle recovery and late recovery[4]. To investigate the adaptation mechanism ofD. radioduransto UV stress, we measured the survival rates for the Normal and UV irradiation culture conditions (Table 1). The survival rate (UV/Normal) ofD. radioduranscells was about 76.6% after UV irradiation of dose 750 J/m2.

Table 1 The viable counts per 100 μl of broth before and after UV irradiation

2.2 Key functional genes related to UV irradiation stress

Through transcriptome data analysis, we found that a total of 750 genes had significant changes (Fold change>2 andFDR<0.05) in expression level after UV irradiation. Table 2 lists the top 30 most significantly up-and down-regulated DEGs.Interestingly, the fold changes of the top 30 upregulated genes were all more than 8 times(log2FC>3), while the fold changes of the top 30 down-regulated genes were all less than 8 times(log2FC>-3), indicating that positive responses are more likely taken byD. radioduransunder UV irradiation. In addition, many previously reported genes that may be involved in damage repair inD. radioduranswere also identified in our study(Table 3), suggesting that cells utilize many repair processes in response to UV stress. We hope to further investigate the roles of these genes in the early repair of UV damage.

Table 2 The 30 genes with most significant differences in expression after UV irradiation

Table 3 The DEGs involved in the known damage repair pathways

2.2.1 Response of the four replicons to UV irradiation stress

The percentage of responsive genes in each of the four genomic components (replicons) of Chr1,Chr2, pMP1 and pSP1 was compared, and the percentage of responsive genes in pSP1 was the highest (40%). Among the 40 genes of pSP1, the expression levels of 16 genes were significantly changed and all of them were up-regulated, indicating that the small plasmid played an important role in the damage repair after UV irradiation. This point was also found in the repair after ionizing radiation, where most genes on pSP1 were significantly up-regulated in the middle and late stages of post-radiation recovery, and almost all genes were activated in the late stage of recovery[4].

2.2.2 Non-homologous end joining (NHEJ) pathway participates in early UV damage repair

DR_A0346 (pprA), DR_B0100 (ligB), DR_B0098 and DR_B0099 were significantly induced after UV irradiation. It was found thatpprAandligBwere involved in the non-homologous end joining(NHEJ) pathway inD. radiodurans.As observed by atomic force microscopy (AFM), PprA protein preferentially binds to DNA double-stranded ends,promotes the ligation of DNA ends catalyzed by ATP-dependent DNA ligase (DR_B0100), and protects it from degradation by exonuclease[21]. DR_B0098-DR_B0100 are three genes encoded in an operon,which may be directly involved in DNA repair[4].DR_B0098 contains a phosphatase domain of the HD hydrolase family and a polynucleotide kinase domain,and resembles a human protein with similar structure that plays an important role in DSB repair[22]. Studies have shown that in the presence of DR_B0098, PprA can stimulate LigB DNA ligase activity several times,and PprA and DR_B0098 proteins are essential for LigB function[23]. At 1.5 to 5 h of ionizing radiation,the expression level of each of these genes was induced 5 to 10 times[4]. Therefore, this pathway plays an important role in both UV damage repair and ionization damage repair.

2.2.3 Single-strand annealing (SSA) pathway participates in early UV damage repair

The genes involved in the single-stranded annealing (SSA) pathway were found to be: DR_0423(ddrA), DR_0070 (ddrB), DR_0003 (ddrC), DR_0326(ddrD), DR_A0346 (pprA) and DR_0100 (ssb). These genes were significantly induced in the early stage of UV irradiation. Some studies have found that the reaction products consistent with annealing mainly appear in the early stage after irradiation (i.e., 1.5 h)[4],which was also proved in this study, namely, genes related to SSA were significantly up-regulated in the early stage after UV irradiation, indicating that the SSA pathway was initiated for repair. DdrB protein can bind to single-stranded DNA and promotes annealing, and single-stranded binding protein (SSB)plays a promoting role in this pathway[24].Alternatively, DdrB may facilitate the accurate assembly of countless small fragments generated by extreme radiation exposureviaSSA, thereby generating suitable substrates for subsequent genomic reconstruction by the ESDSA pathway[25].

2.2.4 Nucleotide excision repair (NER) pathway participates in early UV damage repair

D. radioduranshas a strong tolerance to UV irradiation. Pyrimidine dimers produced by UV damage can interfere with the processes of DNA replication and transcription, while nucleotide excision repair (NER) can remove pyrimidine dimers to ensure normal cell life activities.Mfd,uvrA,uvrB,uvrC,uvrD,polA,ligA,uveE,yejH/rad25andsms/radAare genes involved in the NER pathway, among which DR_1771 (uvrA1), DR_2275 (uvrB) and DR_1354 (uvrC) are significantly different in expression level after UV irradiation. UvrABC complex plays an important role in nucleotide excision repair inD. radiodurans, and the NER pathway has a unique ability to clear DNA damage[26]. This complex recognizes and binds to the damage site and then cleaves, with about 4 nucleotides cleaved at the 3' end and 8 nucleotides cleaved at the 5' end. Finally, DNA is synthesized and the gap is completed under the actions of DNA polymerase and DNA ligase to complete DNA damage repair[27]. Compared with the previous results on the response to ionizing radiation[4], UvrA1 in the NER pathway was expressed at the early stage of UV irradiation and at 1.5 h after ionizing radiation; UvrA2 (DR_A0188)was not expressed at the early stage of UV irradiation,but expressed at 5 h after ionizing radiation; UvrB and UvrC in NER pathway were expressed after UV irradiation and at 3 h after ionizing radiation; UvrD was involved in NER, methylation-dependent mismatch repair (mMM) and SOS repair, and was not expressed after UV irradiation, but was expressed at 1.5 h after ionizing radiation. Two possible pathways for pyrimidine dimer clearance inD. radioduransare ultraviolet damage endonuclease UvsE-dependent excision repair (UVER) and nucleotide excision repair(NER). These two pathways make recovery from UV damage redundant. Some studies have shown that UvrA2 plays a minor role in UV resistance, and NER is more related to UV resistance than UVER[3], which is consistent with our transcriptomic findings, that is,no significant difference was found for UvrA2 and UvsE expression after UV irradiation. One possible explanation is that the contribution of NER and UVER pathways to UV resistance is related to the growth stage[28], which needs to be further investigated.

2.2.5 Superhelix related genes play a role in the repair of early UV damage

GyrA and GyrB are two DNA-gyrase subunits inD. radiodurans, both of which are significantly induced after UV irradiation, indicating that the regulation of DNA supercoiling is important for DNA repair, and GyrA and GyrB may be nucleoid associated proteins (NAPs) ofD. radiodurans[29].Both subunits were also found to be induced after ionizing radiation[4], in which GyrA protein is essential for DNA replication and genome reconstruction after severe ionizing radiation exposure[30].

2.2.6 Mismatch repair (MMR) system participates in the repair of early UV damage

MutS2 and MutS1 are involved in the base mismatch repair system and are significantly induced after UV irradiation. MutS recognizes and binds to base mismatch[31]. MutS1 is a key protein involved in mismatch repair system, which can ensure the replication and recombination fidelity ofD. radiodurans[32], while MutS2 is involved in RecA-independent repair mechanism inD. radiodurans,which enhances the resistance of cells to oxidative stress-induced DNA damage, so as to make organisms exhibit remarkable DNA repair capabilities[33].

2.2.7 Homologous recombination (HR) repair is involved in DNA repair

RecA protein is a recombinant enzyme with unwinding ability, which plays an important role in homologous recombination (HR) and extended synthesis-dependent strand annealing (ESDSA). RecA is essential for genome recovery after irradiation, and its induction is considered to be a major marker for the initiation of homologous recombination[34]. In the present study, RecA protein was only induced more than two-fold at the early stage of UV irradiation(Table 3). Previous studies have found that RecA-dependent homologous recombination occurs 5 h or more after irradiation[4], so it is possible that this protein is not sufficiently induced in the early stage of UV irradiation, and other genes related to homologous recombination are also rarely induced. In addition,there are RuvA/B/C proteins inD. radiodurans,whose functions are similar to the DNA branch transferase and dissociase involved in homologous recombination of DNA in human cells, and among them two proteins (RuvB, RuvC) were significantly induced (Table 3).

2.2.8 Other key functional genes related to UV irradiation stress

In addition, our study also found that DR_1262(rsr), an important gene against UV stress, was significantly up-regulated. Rsr protein is an RNA-binding protein. Studies have shown that Rsr protein can bind some small RNA produced in cells after UV irradiation and improve the resistance of cells to UV irradiation[35].

DR_0928 in the base excision repair (BER)pathway, which is expressed in the early and middle stages of ionizing radiation[4], is also induced after UV irradiation (Table 3). RecA and LexA proteins are involved in SOS repair[22], which are induced at 1.5 h after ionizing radiation[4], are also induced after UV irradiation (Table 3). However,D. radioduransdoes not possess a functional SOS response system, so this remains to be investigated.

ABC transporters (DR_1356-DR_1359) are highly induced and may be involved in the transshipment of damaged products, and these transporters are also highly induced after ionizing radiation[4]. Up-regulated kinases of unknown characteristics (DR_2467, DR_0394) may be involved in a variety of cellular processes, and when expressed inEscherichia coli, these uncharacterized kinases affect DNA topology[22]. Several genes encoding proteins from the extended family of stress response(DinB family) were also induced in early UV irradiation, including DR_1263 and two members of this family (DR_0053, DR_0841), which were strongly induced in a RecA-like manner, supporting their important role in UV irradiation response.

From Table 3, it can be found that among the 20 DEGs, there are 6 genes involved in the SSA pathway,3 genes involved in the NER pathway, 2 genes involved in the NHEJ pathway and 4 genes involved in the HR pathway. Among the top 30 up-regulated genes (Table 2), in addition to genes with known functions, some genes with unknown functions expressed, including DR_1143, DR_RS02180,DR_RS05900, DR_1977, DR_0206, and DR_RS05895, indicating that these genes also play an important role in the response to UV irradiation,which should be the focus of future research.

2.2.9 PprI/DdrO mediated regulatory relationship

A large number of proteins inD. radioduranswere significantly up-regulated in the early recovery from UV irradiation, among which PprI/DdrO mediated regulatory proteins were the most prominent. RDRM is a conserved palindromic sequence that exists upstream of many radiationinduced genes (e.g.,ddrO, ddrA, ddrB, ddrC, ddrD,ddrF, ddrR, recA, pprA, gyrA, gyrB, ssb, recQ, ruvB,uvrA, uvrB, uvrD). It has been found thatddrOacts as a repressor by binding to RDRM, the promoter that appears to be located at or very close to radiationinduced genes, includingddrOitself. However, PprI(IrrE) is a metalloproteinase required for the hydrolytic inactivation of DdrO protein to deactivate the suppressor gene, a process that is somehow activated after exposure ofD. radioduransto radiation[36]. IrrE-dependent cleavage of the DdrO,and hence up-regulation of RNA-dependent RNA polymerase (RDR), is essential for radiation tolerance.The genes that were significantly induced include theDeinococcus-specific genes (ddrO, ddrA, ddrB, ddrC,ddrD, ddrF, ddrR, pprA), DNA repair genes (recA,recQ, ruvB, uvrA, uvrB, uvrD, ssb), DNA superhelix genes (gyrA, gyrB) (Figure 1), among whichpprAis inhibited bypprMandlexA2[37-38], andrecAis inhibited byrecX[39].

Fig. 1 PprI/DdrO mediated regulatory relationships in D. radiodurans

2.3 Functional enrichment analysis of DEGs under UV irradiation

For the selected criteria (log2(Fold change)>1 andFDR<0.05), there were 750 DEGs (Figure 2a),among which 544 genes were up-regulated and 206 genes were down-regulated, and the number of upregulated genes was more than twice that of downregulated genes. It seems that the tolerance ofD. radioduransto UV irradiation mainly depends on the enhancement of most stress response activities under irradiation stress to maintain cell survival and repair damage, rather than the slowing down or even stagnation of normal physiological activities. To determine the biological functions of DEGs under UV irradiation stress, we performed KEGG (Figure 2b)and GO (Figure 2c, d) functional enrichment analysis.To ensure the accuracy of the results, the hypothetical proteins in the protein functional annotation ofD. radioduranswere removed before enrichment analysis.

Fig. 2 Differentially expressed genes and their enrichment

According to the GO enrichment results, damage repair responses, including SOS response and DNA recombination repair, appeared inD. radioduranswhen exposed to UV irradiation stress. The translation process was enhanced, the translocation of translationrelated carboxylic acid and amino acid was enhanced,and the ribosome, rRNA, tRNA and other related genes were significantly up-regulated, presumably due to the massive synthesis of damage repair related proteins. ATP-binding and ATP-dependent activities were enhanced to maintain normal physiological status and to supply more energy for damage repair pathways. DNA binding is enhanced, due to the proteins in the damage repair pathway, such as DNA ligase LigB, DNA damage repair protein DdrB, singlestrand binding protein SSB, UvrABC component protein, and mismatch repair protein MutS, need to bind to DNA for damage repair. When exposed to UV irradiation, the number of down-regulated genes was significantly less than that of up-regulated genes, and mainly concentrated on the reduction of redox reactions: oxidoreductase activity in redox reactions decreased, cytochrome complex assembly decreased,and heme synthesis decreased. Cytochrome is involved in redox reactions with heme as a prosthetic group. Redox reactions provide energy for life activities, which is essentially the transfer of electrons and an important way of energy transfer. Therefore,UV irradiation has an impact on the energy metabolism ofD. radiodurans.

KEGG pathway enrichment analysis was performed on DEGs to investigate the involved metabolic pathways (Figure 2b). Genes related to the ABC transporters pathway were significantly upregulated, while genes related to the biosynthesis of ubiquinone and other terpenoid-quinone and phenylalanine metabolism were significantly downregulated. Studies have found that ABC transporters inD. radioduransplay an important role in the supply of exogenous amino acids, and ABC transporters can be used for the uptake of amino acids and peptides[40].When adapting to irradiation pressure, bacterial cells need various ions and amino acids to synthesize corresponding proteins, which can explain the significant up-regulation of this metabolic pathway.On the other hand, it also indicates thatD. radioduranshas a certain ability to adapt to UV irradiation pressure. Ubiquitin, also known as coenzyme Q, is a hydrogen transporter in the respiratory chain and plays a role in electron transport. The decrease of ubiquitin synthesis corresponds to the decrease of redox reactions in the GO enrichment analysis, which jointly indicates that UV irradiation has an impact on the energy metabolism ofD. radiodurans.

2.4 Validation of DEGs by RT-qPCR

To further validate the expression level changes of DEGs in response to UV irradiation, 3 key functional genes inD. radiodurans, namely,DR_B0100 (RNA ligase family protein LigB),DR_1262 (TROVE domain-containing protein Rsr)and DR_2275 (excinuclease ABC subunit UvrB),were selected for RT-qPCR analysis. The results showed that the changes in expression level of the 3 genes after UV irradiation are consistent with the transcriptome sequencing results (Figure 3),indicating that the transcriptome data are reliable.

Fig. 3 RT-qPCR results of 3 DEGs in response to UV irradiation

2.5 Similarities and differences of DEGs under the three stress conditions of UV irradiation，ionizing radiation and desiccation

The intersection (Figure 4) of DEGs (Fold change>3) under UV irradiation with ionizing radiation and desiccation stresses was taken to obtain the genes listed in Table S2. These genes can be broadly classified into the following categories: DNA metabolism, energy acquisition, putative regulatory proteins, RNA metabolism, protein synthesis,transport, specific genes of theDeinococcusgenus,and other genes[41]. We identified the common responsive genes (shown in bold fonts in Table S2)under UV irradiation, ionizing radiation and desiccation stresses. These genes are mainly concentrated in DNA metabolism, RNA metabolism andDeinococcus-specific genes. It seems that these are common molecular responses ofD. radioduransto UV, ionization and desiccation stresses. DNA metabolism includes some repair genes and superhelix related genes. Some genes were common response genes of UV irradiation and ionizing radiation but did not participate in the response to desiccation stress,while others were common response genes of UV irradiation and desiccation but did not participate in the response to ionizing radiation. These genes need to be further analyzed.

Fig. 4 Venn diagram of DEGs in response to UV irradiation，ionizing radiation and desiccation stresses

It has been reported that there is a strong correlation between desiccation resistance and γ-radiation resistance inD. radiodurans[5]; both radiation (3 kGy) and desiccation stresses induced 5 genes, and we found that UV irradiation induced high expression of the same 5 genes: DR_0423 (ddrA),DR_0070 (ddrB), DR_0003 (ddrC), DR_0326 (ddrD)and DR_A0346 (pprA). These 5 genes correspond to GO terms: “Response to Gamma radiation (GO:0010332)” and “Cellular response to Desiccation(GO:0071465)”. This indicates a strong correlation between UV irradiation and resistance to desiccation and γ-radiation, and since these five proteins are involved in the SSA pathway, it is speculated that the common properties of resistance to desiccation,γ-radiation and UV irradiation may be related to the SSA pathway.

2.6 Hub and module analysis in the PPI network of DEGs

The protein-protein interaction (PPI) network ofD. radioduranswas downloaded from the STRING database[16], and the DEGs related subnetwork was screened out. Unconnected nodes were deleted from the network. The initial PPI network consisted of 2 747 nodes and 100 965 edges (Figure 5). From the initial network, we can see that there are many genes,although they are not DEGs themselves, but they interact with DEGs, and these genes may still play an important role in the response to UV irradiation.

To obtain the main PPI network from the initial network, we performed the following analysis. Firstly,nodes whose connectivity degree, betweenness and eigen vector values were higher than the average of the network were selected and their intersection was taken as the main gene set, resulting in a total of 336 nodes (Figure 6a). Next, these 336 main genes were submitted to the STRING database to construct a PPI network. Finally, unconnected nodes were removed from this network and nodes with interaction scores greater than 0.4 (medium confidence) were selected to obtain the main PPI network (Figure 6b), which consists of 314 nodes and 3 924 edges.

Fig. 6 Hubs and modules in the main PPI network of DEGs

To analyze the main PPI network, we used the CytoHubba plug-in[19]for Cytoscape[17]to identify the top 15 hub genes with high connectivity (Figure 6c)and rank them as follows:polA(degree=164),metG(degree=138),guaA(degree=136), DR_0603 (degree=134),dnaK(degree=134),guaB(degree=128),pheT(degree=126), DR_0183 (degree=122), DR_2168(degree=104),RecA(degree=98),fusA(degree=90),rpsG(degree=86),tsf(degree=82),rpoZ(degree=82),secD(degree=82). The names and functions of these hub genes are shown in Table 4. Subsequently, GO functional enrichment analysis was performed for these hub genes (Figure 7b), and according to the results, the most enriched GO biological processes(BP) include: GMP biosynthetic process, doublestrand break repair, translation; the most enriched GO molecular functions (MF) include: catalytic activity,acting on a nucleic acid, nucleic acid binding.

Fig. 7 Function enrichment analysis

Four major modules were discovered through the MCODE plug-in of Cytoscape (Figure 6d).Subsequently, GO function and KEGG pathway enrichment analysis were performed for genes in these four modules (Figure 7a), and according to the results, the most enriched GO biological processes include: translational initiation, DNA topological change, macromolecule biosynthetic process, cellular nitrogen compound biosynthetic process; the most enriched KEGG pathways include: RNA degradation,biosynthesis of cofactors, porphyrin metabolism,bacterial secretion system, DNA replication, protein export.

3 Conclusion

D. radioduransis the most radiation-resistant organism ever discovered on earth. Our study found thatD. radioduransnot only has the common DNA damage repair genes of general prokaryotes and eukaryotes, but also has some special multidomain gene families in the repair of UV irradiation damage.Through transcriptomic analysis, according to different DNA repair pathways, we explored genes or enzymes related to DNA repair in the early response to UV irradiation, especially the genes that may play a key role in DNA repair, such aspprA,ddrA-D,rsr,etc. Our results show pathways (SSA, NHEJ, NER,HR) that may be involved in early damage repair and genes that play important roles, and suggest that these genes may be regulated by PprI/DdrO. By comparing with ionizing radiation and desiccation stresses, the common molecular responses to the three stresses were found, which mainly involved DNA metabolism,RNA metabolism andDeinococcus-specific genes.The PPI network of DEGs was constructed, and hub genes includingpolA,metG,guaA, DR_0603,dnaK,guaB,pheT, DR_0183, DR_2168,recA,fusA,rpsG,tsf,rpoZ,secDwere identified. These genes also played important roles in the response to UV irradiation stress. At the same time, some highly expressed genes with unknown functions may also play a key role in UV irradiation resistance.Therefore, further elucidation of the functions and regulatory mechanisms of these genes is crucial for understanding the extreme resistance and damage repair mechanisms ofD. radiodurans.

SupplementaryAvailable online (http://www.pibb.ac.cn or http://www.cnki.net):

PIBB_20220471_Table S1.pdf

PIBB_20220471_Table S2.pdf

Data AccessibilityThe transcriptome data generated in this work were deposited in the National Genomics Data Center (NGDC) with accession number CRA009109. The data were also uploaded to the National Center for Biotechnology Information(NCBI) Sequence Read Archive (SRA) under the accession numbers SRR22460685 to SRR22460690.