Genome-wide transcriptome and antioxidant analyses on gamma-irradiated phases of deinococcus radiodurans R1

Abstract

Adaptation of D. radiodurans cells to extreme irradiation environments requires dynamic interactions between gene expression and metabolic regulatory networks, but studies typically address only a single layer of regulation during the recovery period after irradiation. Dynamic transcriptome analysis of D. radiodurans cells using strand-specific RNA sequencing (ssRNA-seq), combined with LC-MS based metabolite analysis, allowed an estimate of the immediate expression pattern of genes and antioxidants in response to irradiation. Transcriptome dynamics were examined in cells by ssRNA-seq covering its predicted genes. Of the 144 non-coding RNAs that were annotated, 49 of these were transfer RNAs and 95 were putative novel antisense RNAs. Genes differentially expressed during irradiation and recovery included those involved in DNA repair, degradation of damaged proteins and tricarboxylic acid (TCA) cycle metabolism. The knockout mutant crtB (phytoene synthase gene) was unable to produce carotenoids, and exhibited a decreased survival rate after irradiation, suggesting a role for these pigments in radiation resistance. Network components identified in this study, including repair and metabolic genes and antioxidants, provided new insights into the complex mechanism of radiation resistance in D. radiodurans.

Figure 1. Strand-Specific RNA-seq analysis of the genes from D. radiodurans R1.

Strand-specific coverage plot is shown. (Orange indicates the chromosome 1; violet, chromosome 2; green, plasmid 1; red, plasmid 2).

Figure 2. Gene expression patterns in stages of D. radiodurans R1 irradiation and recovery.

A, The differentially expressed genes in stages of D. radiodurans R1 irradiation and recovery. B, The expression pattern of annotated CDS and ncRNA reads. DC: wild type before radiation. D1: wild type with 1000 Gy radiation; D3: wild type with 3000 Gy radiation. DR: wild type one hour after 3000 Gy radiation.

Figure 3. Principal component analysis of transcriptome profiling in D. radiodurans R1.

A, the plot of the principal component 1 (PC1) versus principal component 2 (PC2) was presented. B, Loadings plot of PC1. C, Hierarchical clustering analyses of the selected genes that have a high correlation with PC1. D, Loadings plot of PC2. E, Hierarchical clustering analyses of the selected genes that have a high correlation with PC2.

Figure 4. Expression patterns of selected genes for DNA repair pathway system (* padj<0.1).

A, base excision repair. B, nucleotide excision repair. C, homologous recombination. D. mismatch repair.

Figure 5. The differentially expressed TCA cycle genes during D. radiodurans R1 irradiation and recovery.

The significant level (padj-value <0.1) of genes are indicated by the asterisks.

Figure 6. Analysis of small molecule antioxidants in D. radiodurans R1.

A, Proposed pathway of bacillithiol biosynthesis in D. radiodurans R1. The enzymes involved are marked in red, and their expression level before (DC), during (D1, D3) and after (DR) irradiation are shown by the grids. The bar on the bottom left indicates the relationship between color and gene expression level. The concentration of bacillithiol and cysteine in each treatment is plotted on the top right. The error bars are standard error of the mean (SEM) values based on three biological replicates. B, the survival curves of the wild type (red) and the crtB deficient mutant (blue) during increasing doses of ⁶⁰Co irradiation. An increased sensitivity to acute radiation in the crtB mutant is evident. C, carotenoid variation patterns in LC-MS analysis of the wild type (red) and the mutant strains (yellow).

Figure 7. Overview of network components including genes and antioxidants in D. radiodurans that prevent cell damage from extreme irradiation.

Red arrows and green arrows indicate the up-regulation and down-regulation, respectively. The representative D.radiodurans gene name is shown. The response of the DNA repair system activated in different stages of irradiation is listed. DC: wild type before radiation. D1: wild type with 1000 Gy radiation; D3: wild type with 3000 Gy radiation. DR: wild type one hour after 3000 Gy radiation.