Radiodurans R1 Research Articles

The Construction of an Extreme Radiation-Resistant Perchlorate-Reducing Bacterium Using Deinococcus deserti Promoters

Perchlorate is one of the major inorganic pollutants in the natural environment and the living environment, which is toxic to organisms and difficult to degrade due to its special structure. As previously reported, the Phoenix Mars lander detected approximately 0.6% perchlorate in the Martian soil, indicating challenges for Earth-based life to survive there. Currently, biological approaches using dissimilatory perchlorate-reducing bacteria (DPRB) are the most promising methods for perchlorate degradation. However, the majority of DPRB exhibit limited radiation resistance, rendering them unsuitable for survival on Mars. In this study, we obtained the transcriptome data of Deinococcus deserti, and predicted and identified multiple constitutive expression promoters of D. deserti with varying activities. The top-five most active promoters were separately fused to specific genes involved in the degradation of perchlorate from DPRB Dechloromonas agitata CKB, and transformed into Deinococcus radiodurans R1, forming a novel dissimilatory perchlorate-reducing bacterium, R1−CKB. It exhibited both efficient perchlorate degradation capability and strong radiation resistance, potentially offering a valuable tool for the further enhancement of the Martian atmosphere in the future.

Microbial upcycling of methane to phytoene using metabolically engineered Methylocystis sp. MJC1 strain

Methane, a potent greenhouse gas, requires sustainable mitigation strategies. Here, the microbial upcycling of methane to phytoene, a valuable colorless carotenoid with applications in the cosmeceutical industry was demonstrated. To achieve this goal, a stepwise metabolic engineering approach was employed in Methylocystis sp. MJC1, a methane-oxidizing bacterium. The incorporation of crtE and crtB genes from Deinococcus radiodurans R1 established the phytoene biosynthetic pathway. This pathway was fine-tuned through promoter optimization, resulting in a phytoene production of 450 μg/L from 37 mmol/L methane. Disrupting the ackA gene reduced a by-product, acetate, by 50 % and increased phytoene production by 56 %. Furthermore, overexpressing the dxs gene boosted phytoene titer 3-fold. The optimized strain produced 15 mg/L phytoene from 2 mol/L methane in fed-batch fermentation, a 4-fold increase in phytoene titer and 4-fold in yield. This demonstrates Methylocystis sp. MJC1′s potential for efficient phytoene production and presents a novel approach for greenhouse gas reduction.

Development and Regulation of the Extreme Biofilm Formation of Deinococcus radiodurans R1 under Extreme Environmental Conditions.

To grow in various harsh environments, extremophiles have developed extraordinary strategies such as biofilm formation, which is an extremely complex and progressive process. However, the genetic elements and exact mechanisms underlying extreme biofilm formation remain enigmatic. Here, we characterized the biofilm-forming ability of Deinococcus radiodurans in vitro under extreme environmental conditions and found that extremely high concentrations of NaCl or sorbitol could induce biofilm formation. Meantime, the survival ability of biofilm cells was superior to that of planktonic cells in different extreme conditions, such as hydrogen peroxide stress, sorbitol stress, and high UV radiation. Transcriptome profiles of D. radiodurans in four different biofilm development stages further revealed that only 13 matched genes, which are involved in environmental information processing, carbohydrate metabolism, or stress responses, share sequence homology with genes related to the biofilm formation of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Overall, 64% of the differentially expressed genes are functionally unknown, indicating the specificity of the regulatory network of D. radiodurans. The mutation of the drRRA gene encoding a response regulator strongly impaired biofilm formation ability, implying that DrRRA is an essential component of the biofilm formation of D. radiodurans. Furthermore, transcripts from both the wild type and the drRRA mutant were compared, showing that the expression of drBON1 (Deinococcus radioduransBON domain-containing protein 1) significantly decreased in the drRRA mutant during biofilm development. Further analysis revealed that the drBON1 mutant lacked the ability to form biofilm and DrRRA, and as a facilitator of biofilm formation, could directly stimulate the transcription of the biofilm-related gene drBON1. Overall, our work highlights a molecular mechanism mediated by the response regulator DrRRA for controlling extreme biofilm formation and thus provides guidance for future studies to investigate novel mechanisms that are used by D. radiodurans to adapt to extreme environments.

Artificial Proteins Designed from G3LEA Contribute to Enhancement of Oxidation Tolerance in E. coli in a Chaperone-like Manner.

G3LEA is a family of proteins that exhibit chaperone-like activity when under distinct stress. In previous research, DosH was identified as a G3LEA protein from model extremophile-Deinococcus radiodurans R1 with a crucial core HD domain consisting of eight 11-mer motifs. However, the roles of motifs participating in the process of resistance to stress and their underlying mechanisms remain unclear. Here, eight different proteins with tandem repeats of the same motif were synthesized, named Motif1-8, respectively, whose function and structure were discussed. In this way, the role of each motif in the HD domain can be comprehensively analyzed, which can help in finding possibly crucial amino acid sites. Circular dichroism results showed that all proteins were intrinsically ordered in phosphate buffer, and changed into more α-helical ordered structures with the addition of trifluoroethanol and glycerol. Transformants expressing artificial proteins had significantly higher stress resistance to oxidation, desiccation, salinity and freezing compared with the control group; E. coli with Motif1 and Motif8 had more outstanding performance in particular. Moreover, enzymes and membrane protein protection viability suggested that Motif1 and Motif8 had more positive influences on various molecules, demonstrating a protective role in a chaperone-like manner. Based on these results, the artificial proteins synthesized according to the rule of 11-mer motifs have a similar function to wildtype protein. Regarding the sequence in all motifs, there are more amino acids to produce H bonds and α-helices, and more amino acids to promote interaction between proteins in Motif1 and Motif8; in addition, considering linkers, there are possibly more amino acids forming α-helix and binding substrates in these two proteins, which potentially provides some ideas for us to design potential ideal stress-response elements for synthetic biology. Therefore, the amino acid composition of the 11-mer motif and linker is likely responsible for its biological function.

Study on extraction and characterization of new antibiotics violacein from engineered Escherichia coli VioABCDE-SD.

To better understand the characteristic properties of violacein biosynthesized by engineered Escherichia coli VioABCDE-SD, a convenient and simplified method was designed to extract violacein and its stability, antimicrobial activity, and antioxidant capacity were analyzed. Different fromthetraditional extraction methods, ournew method is easierandlesstime consuming and can directly obtain violacein dry powder product with a higher extraction rate. Low temperature, darkcondition, neutral pH, reducing agents, Ba2+ , Mn2+ , Ni2+ , Co2+ , and some food additives of sucrose, xylose, and glucose were conducive tomaintainingitsstability. The violacein also exhibited surprisingly high bacteriostatic action against Gram-positive Bacillus subtilis, Deinococcus radiodurans R1, and Staphylococcus aureus and Gram-negative Pseudomonasaeruginosa, but no effect on E. coli. The violacein of VioABCDE-SD exhibited strong antioxidant activity, with the scavenging rate of 1,1-diphenyl-2-picrylhydrazyl free radicals reaching 60.33%, the scavenging efficiency of hydroxyl radical scavenging reaching 56.34%, and the total antioxidant capacity reaching 0.63U/mL. Violacein from VioABCDE-SD can be synthesized directionally with better stability, antibacterial, and antioxidant properties compared with that from the original strain Janthinobacterium sp. B9-8. Therefore, our study indicated that violacein from engineered E. coli VioABCDE-SD was a kind of new antibiotic with potential biological activities, which may havepotentialutilityinmultiple areas such as pharmacological, cosmetics, and healthy food industries.

Analysis of multipartite bacterial genomes using alignment free and alignment-based pipelines

Since the discovery of second chromosome in Rhodobacter sphaeroides 2.4.1 in 1989, multipartite genomes have been reported in over three hundred bacterial species under nine different phyla. This has shattered the unipartite (single chromosome) genome dogma in bacteria. Since then, many questions on various aspects of multipartite genomes in bacteria have been addressed. However, our understanding of how multipartite genomes emerge and evolve is still lacking. Importantly, the knowledge of genetic factors underlying the differences in multipartite and single-chromosome genomes is lacking. In this work, we have performed comparative evolutionary and functional genomics analyses to identify molecular factors that discriminate multipartite from unipartite bacteria, with the goal to decipher taxon-specific factors, and those that are prevalent across the taxa, underlying these traits. We assessed the roles of evolutionary mechanisms, specifically gene gain, in driving the divergence of bacteria with single and multiple chromosomes. In addition, we performed functional genomic analysis to garner support for our findings from comparative evolutionary analysis. We found genes such as those encoding conserved hypothetical proteins in Deinococcus radiodurans R1, and putative phage phi-C31 gp36 major capsid like and hypothetical proteins in Rhodobacter sphaeroides 2.4.1, which are located on accessory chromosomes in these bacteria but were not found in the inferred ancestral sequences, and on the primary chromosomes, as well as were not found in their closest relatives with single chromosome within the same clade. Our study shines a new light on the potential roles of the secondary chromosomes in helping bacteria with multipartite genomes to adapt to specialized environments or growth conditions.

Machine learning-guided prediction of potential engineering targets for microbial production of lycopene

The process of designing streamlined workflows for developing microbial strains using classical methods from vast amounts of biological big data has reached its limits. With the continuous increase in the amount of biological big data, data-driven machine learning approaches are being used to overcome the limits of classical approaches for strain development. Here, machine learning-guided engineering of Deinococcus radiodurans R1 for high-yield production of lycopene was demonstrated. The multilayer perceptron models were first trained using the mRNA expression levels of the key genes along with lycopene titers and yields obtained from 17 strains. Then, the potential overexpression targets from 2,047 possible combinations were predicted by the multilayer perceptron combined with a genetic algorithm. Through the machine learning-aided fine-tuning of the predicted genes, the final-engineered LY04 strain resulted in an 8-fold increase in the lycopene production, up to 1.25 g/L from glycerol, and a 6-fold increase in the lycopene yield.

Probing the sORF-Encoded Peptides of Deinococcus radiodurans in Response to Extreme Stress

Organisms have developed different mechanisms to respond to stresses. However, the roles of small ORF–encoded peptides (SEPs) in these regulatory systems remain elusive, which is partially because of the lack of comprehensive knowledge regarding these biomolecules. We chose the extremophile Deinococcus radiodurans R1 as a model species and conducted large-scale profiling of the SEPs related to the stress response. The integrated workflow consisting of multiple omics approaches for SEP identification was streamlined, and an SEPome of D. radiodurans containing 109 novel and high-confidence SEPs was drafted. Forty-four percent of these SEPs were predicted to function as antimicrobial peptides. Quantitative peptidomics analysis indicated that the expression of SEP068184 was upregulated upon oxidative treatment and gamma irradiation of the bacteria. SEP068184 was conserved in Deinococcus and exhibited negative regulation of oxidative stress resistance in a comparative phenotypic assay of its mutants. Further quantitative and interactive proteomics analyses suggested that SEP068184 might function through metabolic pathways and interact with cytoplasmic proteins. Collectively, our findings demonstrate that SEPs are involved in the regulation of oxidative resistance, and the SEPome dataset provides a rich resource for research on the molecular mechanisms of the response to extreme stress in organisms.

The role of S-layer protein (SlpA) in biofilm-formation of Deinococcus radiodurans.

To investigate the molecular basis of biofilm formation in a recombinant lab strain of Deinococcus radiodurans with a plasmid harbouring gfp and kanR that acquired the biofilm-forming ability. Deinococcus radiodurans R1 is known as a nonbiofilm former bacterium and so far there are no reports on its biofilm-producing capabilities. In this study, we investigated the molecular basis of biofilm formation in a recombinant strain of D. radiodurans using classical biofilm assays, confocal laser scanning microscopy and real-time PCR. Biochemical analysis of D. radiodurans biofilm matrix revealed that it consisted predominantly of protein and carbohydrate complexes with a little amount of extracellular DNA (eDNA). Furthermore, studies showed that D. radiodurans biofilm formation was enhanced in the presence of 25 mM Ca2+ , which enhanced the exopolysaccharide and protein content in the biofilm matrix. Enzymatic treatments with proteinase K, alginate lyase and DNase I indicated the involvement of some proteinaceous components to be critical in the biofilm formation. RT-PCR studies showed that increased expression of a surface layer protein SlpA conferred the biofilm ability to D. radiodurans. Overexpression of SlpA in D. radiodurans conferred the biofilm formation ability to the bacterium, in which a partial role was also played by the recombinant plasmid pKG. It was also shown that the presence of Ca2+ in the growth medium enhanced SlpA production, thus improving biofilm stability and biofilm maturation of D. radiodurans. This study shows how biofilm formation can be augmented in D. radiodurans. The finding has implications for the development of D. radiodurans biofilm-based biotechnological applications.

Sequence, structure, and function of the Dps DNA-binding protein from Deinococcus wulumuqiensis R12

Deinococcus wulumuqiensis R12, which was isolated from arid irradiated soil in Xinjiang province of China, belongs to a genus that is well-known for its extreme resistance to ionizing radiation and oxidative stress. The DNA-binding protein Dps has been studied for its great contribution to oxidative resistance. To explore the role of Dps in D. wulumuqiensis R12, the Dps sequence and homology-modeled structure were analyzed. In addition, the dps gene was knocked out and proteomics was used to verify the functions of Dps in D. wulumuqiensis R12. Docking data and DNA binding experiments in vitro showed that the R12 Dps protein has a better DNA binding ability than the Dps1 protein from D. radiodurans R1. When the dps gene was deleted in D. wulumuqiensis R12, its resistance to H2O2 and UV rays was greatly reduced, and the cell envelope was destroyed by H2O2 treatment. Additionally, the qRT-PCR and proteomics data suggested that when the dps gene was deleted, the catalase gene was significantly down-regulated. The proteomics data indicated that the metabolism, transport and oxidation–reduction processes of D. wulumuqiensis R12 were down-regulated after the deletion of the dps gene. Overall, the data conformed that Dps protein plays an important role in D. wulumuqiensis R12.

Deinococcus radiodurans R1 Lysate Induces Tolerogenic Maturation in Lipopolysaccharide-Stimulated Dendritic Cells and Protects Dextran Sulfate Sodium-Induced Colitis in Mice.

Deinococcus radiodurans is an extremophilic bacterium that can thrive in harsh environments. This property can be attributed to its unique metabolites that possess strong antioxidants and other pharmacological properties. To determine the potential of D. radiodurans R1 lysate (DeinoLys) as a pharmacological candidate for inflammatory bowel disease (IBD), we investigated the anti-inflammatory activity of DeinoLys in bone marrow-derived dendritic cells (BMDCs) and a colitis mice model. Lipopolysaccharide (LPS)-stimulated BMDCs treated with DeinoLys exhibited alterations in their phenotypic and functional properties by changing into tolerogenic DCs, including strongly inhibited proinflammatory cytokines (TNF-α and IL-12p70) and surface molecule expression and activated DC-induced T cell proliferation/activation with high IL-10 production. These phenotypic and functional changes in BMDCs induced by DeinoLys in the presence of LPS were abrogated by IL-10 neutralization. Furthermore, oral administration of DeinoLys significantly reduced clinical symptoms against dextran sulfate sodium-induced colitis, including body weight loss, disease activity index, histological severity in colon tissue, and lower myeloperoxidase level in mice. Our results establish DeinoLys as a potential anti-inflammatory candidate for IBD therapy.

Genome-wide lone strand adenine methylation in Deinococcus radiodurans R1: Regulation of gene expression through DR0643-dependent adenine methylation.

DNA methylation is a covalent modification of adenine or cytosine in the genome of an organism and is found in diverse microbes including the radiation resistant bacterium Deinococcus radiodurans R1. Although earlier findings have confirmed repression or de-repression of certain genes in adenine methyltransferase (DR_0643/Dam1DR) deficient D. radiodurans mutant however, the overall regulatory aspects of Dam1DR-mediated adenine methylation remain mostly unexplored. In the present study, we compared the genome-wide methylome and the corresponding transcriptome of D. radiodurans WT and Δdam1 mutant to explore the correlation between methylation and gene expression. In D. radiodurans, deletion of DR_0643 ORF (Δdam1) led to hypomethylation of 512 genes resulting in differential expression of 168 genes (99 genes are upregulated and 69 genes are downregulated). The modification patterns deduced for Dam1DR (DR_0643) and Dam2DR (DR_2267) were non-palindromic and atypical. Moreover, we observed methylation at opportunistic sites that show adenine methylation only in D. radiodurans Δdam1 and not in D. radiodurans WT. Correlation between the methylome and transcriptome suggests that hypomethylation at Dam1DR specific sites had both negative as well as a positive effects on gene expression. Pathways such as amino acid metabolism, transport, oxidative phosphorylation, quorum sensing, signal transduction, two-component system, glycolysis/gluconeogenesis, TCA cycle, glyoxylate and dicarboxylate metabolism were modulated by Dam1DR-mediated adenine methylation in D. radiodurans. Processes such as DNA repair, recombination, ATPase and transmembrane transporter activity were enriched when Dam1DR mutant was subjected to radiation stress. We further evaluated the molecular interactions and mode of binding between Dam1DR protein and S-adenosyl methionine using molecular docking followed by MD simulation. To get a better insight into the methylation mechanism, the Dam1DR-SAM complex was also docked with a DNA molecule to elucidate DNA-Dam1DR structural interaction during methyl-group transfer reaction. In summary, our work presents comprehensive and integrative approaches to investigate both functional and structural aspects of DNA adenine methyltransferase (Dam1DR) in D. radiodurans biology.

High Proportions of Radiation-Resistant Strains in Culturable Bacteria from the Taklimakan Desert.

Simple SummaryRadiation-resistant extremophiles have frequently been found in the Taklimakan Desert, which is known for its harsh conditions. However, there is no systemic study investigating the diversity and proportion of radiation-resistant strains among culturable bacteria. The results of this study revealed the distribution of culturable bacteria in the Taklimakan Desert and indicated high proportions of radiation-resistant strains in the culturable bacteria. The study helps to better understand the ecological origin of radio-resistance and to quantitatively describe the desert as a common habitat for radiation-resistant extremophiles.The Taklimakan Desert located in China is the second-largest shifting sand desert in the world and is known for its harsh conditions. Types of γ-rays or UV radiation-resistant bacterial strains have been isolated from this desert. However, there is no information regarding the proportions of the radiation-resistant strains in the total culturable microbes. We isolated 352 bacterial strains from nine sites across the Taklimakan Desert from north to south. They belong to Actinobacteria, Firmicutes, Proteobacteria, and Bacteroidetes. The phylum Actinobacteria was the most predominant in abundance and Firmicutes had the highest species richness. Bacteroidetes had the lowest abundance and was found in four sites only, while the other three phyla were found in every site but with different distribution profiles. After irradiating with 1000 J/m2 and 6000 J/m2 UV-C, the strains with survival rates higher than 10% occupied 72.3% and 36.9% of all culturable bacteria, respectively. The members from Proteobacteria had the highest proportions, with survival rates higher than 10%. After radiation with 10 kGy γ-rays, Kocuria sp. TKL1057 and Planococcus sp. TKL1152 showed higher radiation-resistant capabilities than Deinococcus radiodurans R1. Besides obtaining several radiation-resistant extremophiles, this study measured the proportions of the radiation-resistant strains in the total culturable microbes for the first time. This study may help to better understand the origin of radioresistance, especially by quantitatively comparing proportions of radiation-resistant extremophiles from different environments in the future.

Metabolic engineering of Deinococcus radiodurans for pinene production from glycerol

BackgroundThe objective of this work was to engineer Deinococcus radiodurans R1 as a microbial cell factory for the production of pinene, a monoterpene molecule prominently used for the production of fragrances, pharmaceutical products, and jet engine biofuels. Our objective was to produce pinene from glycerol, an abundant by-product of various industries.ResultsTo enable pinene production in D. radiodurans, we expressed the pinene synthase from Abies grandis, the geranyl pyrophosphate (GPP) synthase from Escherichia coli, and overexpressed the native 1-deoxy-d-xylulose 5-phosphate synthase. Further, we disrupted the deinoxanthin pathway competing for the substrate GPP by either inactivating the gene dr0862, encoding phytoene synthase, or substituting the native GPP synthase with that of E. coli. These manipulations resulted in a D. radiodurans strain capable of producing 3.2 ± 0.2 mg/L pinene in a minimal medium supplemented with glycerol, with a yield of 0.13 ± 0.04 mg/g glycerol in shake flask cultures. Additionally, our results indicated a higher tolerance of D. radiodurans towards pinene as compared to E. coli.ConclusionsIn this study, we successfully engineered the extremophile bacterium D. radiodurans to produce pinene. This is the first study demonstrating the use of D. radiodurans as a cell factory for the production of terpenoid molecules. Besides, its high resistance to pinene makes D. radiodurans a suitable host for further engineering efforts to increase pinene titer as well as a candidate for the production of the other terpenoid molecules.

Characterization of the Radiation Desiccation Response Regulon of the Radioresistant Bacterium Deinococcus radiodurans by Integrative Genomic Analyses.

Numerous genes are overexpressed in the radioresistant bacterium Deinococcus radiodurans after exposure to radiation or prolonged desiccation. It was shown that the DdrO and IrrE proteins play a major role in regulating the expression of approximately twenty genes. The transcriptional repressor DdrO blocks the expression of these genes under normal growth conditions. After exposure to genotoxic agents, the IrrE metalloprotease cleaves DdrO and relieves gene repression. At present, many questions remain, such as the number of genes regulated by DdrO. Here, we present the first ChIP-seq analysis performed at the genome level in Deinococcus species coupled with RNA-seq, which was achieved in the presence or not of DdrO. We also resequenced our laboratory stock strain of D. radiodurans R1 ATCC 13939 to obtain an accurate reference for read alignments and gene expression quantifications. We highlighted genes that are directly under the control of this transcriptional repressor and showed that the DdrO regulon in D. radiodurans includes numerous other genes than those previously described, including DNA and RNA metabolism proteins. These results thus pave the way to better understand the radioresistance pathways encoded by this bacterium and to compare the stress-induced responses mediated by this pair of proteins in diverse bacteria.

Study of the properties of carotenoids and key carotenoid biosynthesis genes from Deinococcus xibeiensis R13.

To investigate the properties of carotenoids from the extremophile Deinococcus xibeiensis R13, the factors affecting the stability of carotenoids extracted from D. xibeiensis R13, including temperature, illumination, pH, redox chemicals, metal ions, and food additives, were investigated. The results showed that low temperature, neutral pH, reducing agents, Mn2+ , and food additives (xylose and glucose) can effectively improve the stability of Deinococcus carotenoids. The carotenoids of D. xibeiensis R13 exhibited strong antioxidant activity, with the scavenging rate of hydroxyl radicals reaching 71.64%, which was higher than the scavenging efficiency for 1,1-diphenyl-2-picrylhydrazyl free radicals and 2,2'-azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid) free radicals (44.55 and 27.65%, respectively). In addition, the total antioxidant capacity reached 0.60 U/ml, which was 2.61-fold that of carotenoids from the model strain Deinococcus radiodurans R1. Finally, we predicted the gene clusters encoding carotenoid biosynthesis pathways in the genome of R13 and identified putative homologous genes. The key enzyme genes (crtE, crtB, crtI, crtLm, cruF, crtD, and crtO) in carotenoid synthesis of D. xibeiensis R13 were cloned to construct the multigene coexpression plasmids pET-EBI and pRSF-LmFDO. The carotenoid biosynthesis pathway was heterologously introduced into engineered Escherichia coli EBILmFDO, which exhibited a higher yield (7.14mg/L) than the original strain. These analysis results can help us to better understand the metabolic synthesis of carotenoids in extremophiles.

Functional and structural characterization of Deinococcus radiodurans R1 MazEF toxin-antitoxin system, Dr0416-Dr0417.

In prokaryotes, toxin-antitoxin (TA) systems are commonly found. They likely reflect the adaptation of pathogenic bacteria or extremophiles to various unfavorable environments by slowing their growth rate. Genomic analysis of the extremophile Deinococcus radiodurans R1 revealed the presence of eight type II TA systems, including the genes dr0417, dr0660, dr1530, dr0690, and dr1807. Expression of these toxin genes led to inhibition of Escherichia coli growth, whereas their antidote antitoxins were able to recover the growth defect. Remarkably, Dr0417 (DrMazF) showed endoribonuclease activity toward rRNAs as well as mRNAs, as determined by in vivo and in vitro RNA cleavage assays, and this activity was inhibited by Dr0416 (DrMazE). It was also found that the expression of dr0416-0417 module is directly regulated by the DrMazE-MazF complex. Furthermore, this TA module was induced under stress conditions and plays an important role in survival. To understand the regulatory mechanism at the molecular level, we determined the first high-resolution structures of DrMazF alone and of the DrMazE-MazF complex. In contrast with the hetero-hexameric state of typical MazE-MazF complexes found in other species, DrMazE-MazF crystal structure consists of a hetero-trimer, with the DNA-binding domain of DrMazE undergoing self-cleavage at the flexible linker loop. Our structure revealed that the unique residue R54 provides an additional positive charge to the substrate-binding pocket of DrMazF, its mutation significantly affects the endonuclease activity. Thus, our work reports the unique structural and biochemical features of the DrMazE-MazF system.

Enhanced Lycopene Production by UV-C Irradiation in Radiation-Resistant Deinococcus radiodurans R1.

Although classical metabolic engineering strategies have succeeded in developing microbial strains capable of producing desired bioproducts, metabolic imbalance resulting from extensive genetic manipulation often leads to decreased productivity. Thus, abiotic strategies for improving microbial production performance can be an alternative to overcome drawbacks arising from intensive metabolic engineering. Herein, we report a promising abiotic method for enhancing lycopene production by UV-C irradiation using a radiation-resistant ΔcrtLm/crtB+dxs+ Deinococcus radiodurans R1 strain. First, the onset of UV irradiation was determined through analysis of the expression of 11 genes mainly involved in the carotenoid biosynthetic pathway in the ΔcrtLm/crtB+dxs+ D. radiodurans R1 strain. Second, the effects of different UV wavelengths (UV-A, UV-B, and UV-C) on lycopene production were investigated. UV-C irradiation induced the highest production, resulting in a 69.9% increase in lycopene content [64.2 ± 3.2 mg/g dry cell weight (DCW)]. Extended UV-C irradiation further enhanced lycopene content up to 73.9 ± 2.3 mg/g DCW, a 95.5% increase compared to production without UV-C irradiation (37.8 ± 0.7 mg/g DCW).

Crystal structure of the AhpD-like protein DR1765 from Deinococcus radiodurans R1

Deinococcus radiodurans is well known for its extreme resistance to ionizing radiation (IR). Since reactive oxygen species generated by IR can damage various cellular components, D. radiodurans has developed effective antioxidant systems to cope with this oxidative stress. dr1765 from D. radiodurans is predicted to encode an alkyl hydroperoxidase-like protein (AhpD family), which is implicated in the reduction of hydrogen peroxide (H2O2) and organic hydroperoxides. In this study, we constructed a dr1765 mutant strain (Δdr1765) and examined the survival rate after H2O2 treatment. Δdr1765 showed a significant decrease in the H2O2 resistance compared to the wild-type strain. We also determined the crystal structure of DR1765 at 2.27 Å resolution. DR1765 adopted an all alpha helix protein fold representative of the AhpD-like superfamily. Structural comparisons of DR1765 with its structural homologues revealed that DR1765 possesses the Glu74-Cys86-Tyr88-Cys89-His93 signature motif, which is conserved in the proton relay system of AhpD. Complementation of Δdr1765 with dr1765 encoding C86A or C89A mutation failed to restore the survival rate to wild-type level. Taken together, these results suggest that DR1765 might function as an AhpD to protect cells from oxidative stress.

Construction, analysis and validation of co-expression network to understand stress adaptation in Deinococcus radiodurans R1.

Systems biology based approaches have been effectively utilized to mine high throughput data. In the current study, we have performed system-level analysis for Deinococcus radiodurans R1 by constructing a gene co-expression network based on several microarray datasets available in the public domain. This condition-independent network was constructed by Weighted Gene Co-expression Network Analysis (WGCNA) with 61 microarray samples from 9 different experimental conditions. We identified 13 co-expressed modules, of which, 11 showed functional enrichments of one or more pathway/s or biological process. Comparative analysis of differentially expressed genes and proteins from radiation and desiccation stress studies with our co-expressed modules revealed the association of cyan with radiation response. Interestingly, two modules viz darkgreen and tan was associated with radiation as well as desiccation stress responses. The functional analysis of these modules showed enrichment of pathways important for adaptation of radiation or desiccation stress. To decipher the regulatory roles of these stress responsive modules, we identified transcription factors (TFs) and then calculated a Biweight mid correlation between modules hub gene and the identified TFs. We obtained 7 TFs for radiation and desiccation responsive modules. The expressions of 3 TFs were validated in response to gamma radiation using qRT-PCR. Along with the TFs, selected close neighbor genes of two important TFs, viz., DR_0997 (CRP) and DR_2287 (AsnC family transcriptional regulator) in the darkgreen module were also validated. In our network, among 13 hub genes associated with 13 modules, the functionality of 5 hub genes which are annotated as hypothetical proteins (hypothetical hub genes) in D. radiodurans genome has been revealed. Overall the study provided a better insight of pathways and regulators associated with relevant DNA damaging stress response in D. radiodurans.