A Rare Mono-Rhamnolipid Congener Efficiently Produced by Recombinant Pseudomonas aeruginosa YM4 via the Expression of Global Transcriptional Regulator irrE.

Rhamnolipids (RLs) are widely used biosurfactants produced mainly by Pseudomonas aeruginosa and Burkholderia spp. in the form of mixtures of diverse congeners. The global transcriptional regulator gene irrE from radiation-tolerant extremophiles has been widely used as a stress-resistant element to construct robust producer strains and improve their production performance. A PrhlA-irrE cassette was constructed to express irrE genes in the Pseudomonas aeruginosa YM4 of the rhamnolipids producer strain. We found that the expression of irrE of Deinococcus radiodurans in the YM4 strain not only enhanced rhamnolipid production and the strain's tolerance to environmental stresses, but also changed the composition of the rhamnolipid products. The synthesized rhamnolipids reached a maximum titer of 26 g/L, about 17.9% higher than the original, at 48 h. The rhamnolipid production of the recombinant strain was determined to be mono-rhamnolipids congener Rha-C10-C12, accounting for 94.1% of total products. The critical micelle concentration (CMC) value of the Rha-C10-C12 products was 62.5 mg/L and the air-water surface tension decreased to 25.5 mN/m. The Rha-C10-C12 products showed better emulsifying activity on diesel oil than the original products. This is the first report on the efficient production of the rare mono-rhamnolipids congener Rha-C10-C12 and the first report that the global regulator irrE can change the components of rhamnolipid products in Pseudomonas aeruginosa.

Deinococcus arenicola sp. nov., a novel radiation-resistant bacterium isolated from sandy soil in Antarctica.

A Gram-stain-positive, aerobic, non-mobile and spherical strain, designated ZS9-10T, belonging to the genus Deinococcus was isolated from soil sampled at the Chinese Zhong Shan Station, Antarctica. Growth was observed in the presence of 0-4 % (w/v) NaCl, at pH 7.0-8.0 and at 4-25 °C. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain ZS9-10T formed a lineage in the genus Deinococcus. It exhibited highest sequence similarity (97.4 %) to Deinococcus marmoris DSM 12784T. The major phospholipids of ZS9-10T were unidentified phosphoglycolipid, unidentified glycolipids and unidentified lipids. The major fatty acids were summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), C16 : 0 and C16 : 1 ω7c. MK-8 was the predominant respiratory quinone. The digital DNA-DNA hybridization and average nucleotide identity values between strain ZS9-10T and its close relative D. marmoris DSM 12784T were 27.4 and 83.9 %, respectively. Based on phenotypic, phylogenetic and genotypic data, a novel species, named Deinococcus arenicola sp. nov., is proposed. The type strain iis ZS9-10T (=CCTCC AB 2019392T=KCTC43192T).

Susceptibility of extremophiles to far-UVC light for bioburden reduction in spacecraft assembly facilities.

The prevention and reduction of microbial species entering and leaving Earth's biosphere is a critical aspect of planetary protection research. While various decontamination methods exist and are currently utilized for planetary protection purposes, the use of far-UVC light (200-230 nm) as a means for microbial reduction remains underexplored. Unlike conventional germicidal ultraviolet at 254 nm, which can pose a health risk to humans even with small exposure doses, far-UVC light poses minimal health hazard making it a suitable candidate for implementation in occupied areas of spacecraft assembly facilities. This study investigates the efficacy of far-UVC 222-nm light to inactivate bacteria using microbial species which are relevant to planetary protection either in vegetative cell or spore form. All the tested vegetative cells demonstrated susceptibility to 222-nm exposure, although susceptibility varied among the tested species. Notably, Deinococcus radiodurans, a species highly tolerant to extreme environmental conditions, exhibited the most resistance to far-UVC exposure with a dose of 112 mJ/cm2 required for a 1-log reduction in survival. While spore susceptibility was similar across the species tested, Bacillus pumilus spores were the most resistant of the tested spores when analyzed with a bi-exponential cell killing model (D90 of 6.8 mJ/cm2). Overall, these results demonstrate the efficacy of far-UVC light for reducing microbial bioburden to help ensure the success and safety of future space exploration missions.

Efficient biotransformation of naringenin to naringenin α-glucoside, a novel α-glucosidase inhibitor, by amylosucrase from Deinococcus wulumuquiensis.

Amylosucrase (ASase) efficiently biosynthesizes α-glucoside using flavonoids as acceptor molecules and sucrose as a donor molecule. Here, ASase from Deinococcus wulumuqiensis (DwAS) biosynthesized more naringenin α-glucoside (NαG) with sucrose and naringenin as donor and acceptor molecules, respectively, than other ASases from Deinococcus sp. The biotransformation rate of DwAS to NαG was 21.3% compared to 7.1-16.2% for other ASases. Docking simulations showed that the active site of DwAS was more accessible to naringenin than those of others. The 217th valine in DwAS corresponded to the 221st isoleucine in Deinococcus geothermalis AS (DgAS), and the isoleucine possibly prevented naringenin from accessing the active site. The DwAS-V217I mutant had a significantly lower biosynthetic rate of NαG than DwAS. The kcat/Km value of DwAS with naringenin as the donor was significantly higher than that of DgAS and DwAS-V217I. In addition, NαG inhibited human intestinal α-glucosidase more efficiently than naringenin.

Plasmid expression of Deinococcus radiodurans RecA confers UV-A protection to Escherichia coli with an inverse protein dose dependence, which does not exceed conspecific RecA protection.

Low level expression in Escherichia coli of the RecA protein from the radiation resistant bacterium Deinococcus radiodurans protects a RecA deficient strain of E. coli from UV-A irradiation by up to ∼160% over basal UV-A resistance. The protection effect is inverse protein dose dependent: increasing the expression level of the D. radiodurans RecA (DrRecA) protein decreases the protection factor. This inverse protein dose dependence effect helps resolve previously conflicting reports of whether DrRecA expression is protective or toxic for E. coli. In contrast to the D. radiodurans protein effect, conspecific plasmid expression of E. coli RecA protein in RecA deficient E. coli is consistently protective over several protein expression levels, as well as consistently more protective to higher levels of UV-A exposure than that provided by the D. radiodurans protein. The results indicate that plasmid expression of D. radiodurans RecA can modestly enhance the UV resistance of living E. coli, but that the heterospecific protein shifts from protective to toxic as expression is increased.

A novel ionizing radiation-induced small RNA, DrsS, promotes the detoxification of reactive oxygen species in Deinococcus radiodurans.

A plethora of gene regulatory mechanisms with eccentric attributes in Deinoccocus radiodurans confer it to possess a distinctive ability to survive under ionizing radiation. Among the many regulatory processes, small RNA (sRNA)-mediated regulation of gene expression is prevalent in bacteria but barely investigated in D. radiodurans. In the current study, we identified a novel sRNA, DrsS, through RNA-seq analysis in D. radiodurans cells while exposed to ionizing radiation. Initial sequence analysis for promoter identification revealed that drsS is potentially co-transcribed with sodA and dr_1280 from a single operon. Elimination of the drsS allele in D. radiodurans chromosome resulted in an impaired growth phenotype under γ-radiation. DrsS has also been found to be upregulated under oxidative and genotoxic stresses. Deletion of the drsS gene resulted in the depletion of intracellular concentration of both Mn2+ and Fe2+ by ~70% and 40%, respectively, with a concomitant increase in carbonylation of intracellular protein. Complementation of drsS gene in ΔdrsS cells helped revert its intracellular Mn2+ and Fe2+ concentration and alleviated carbonylation of intracellular proteins. Cells with deleted drsS gene exhibited higher sensitivity to oxidative stress than wild-type cells. Extrachromosomally expressed drsS in ΔdrsS cells retrieved its oxidative stress resistance properties by catalase-mediated detoxification of reactive oxygen species (ROS). In vitro binding assays indicated that DsrS directly interacts with the coding region of the katA transcript, thus possibly protecting it from cellular endonucleases in vivo. This study identified a novel small RNA DrsS and investigated its function under oxidative stress in D. radiodurans. IMPORTANCE: Deinococcus radiodurans possesses an idiosyncratic quality to survive under extreme ionizing radiation and, thus, has evolved with diverse mechanisms which promote the mending of intracellular damages caused by ionizing radiation. As sRNAs play a pivotal role in modulating gene expression to adapt to altered conditions and have been delineated to participate in almost all physiological processes, understanding the regulatory mechanism of sRNAs will unearth many pathways that lead to radioresistance in D. radiodurans. In that direction, DrsS has been identified to be a γ-radiation-induced sRNA, which is also induced by oxidative and genotoxic stresses. DrsS appeared to activate catalase under oxidative stress and detoxify intracellular ROS. This sRNA has also been shown to balance intracellular Mn(II) and Fe concentrations protecting intracellular proteins from carbonylation. This novel mechanism of DrsS identified in D. radiodurans adds substantially to our knowledge of how this bacterium exploits sRNA for its survival under stresses.

Precise CRISPR/Cpf1 genome editing system in the Deinococcus radiodurans with superior DNA repair mechanisms.

Deinococcus radiodurans, with its high homologous recombination (HR) efficiency of double-stranded DNA breaks (DSBs), is a model organism for studying genome stability maintenance and an attractive microbe for industrial applications. Here, we developed an efficient CRISPR/Cpf1 genome editing system in D. radiodurans by evaluating and optimizing double-plasmid strategies and four Cas effector proteins from various organisms, which can precisely introduce different types of template-dependent mutagenesis without off-target toxicity. Furthermore, the role of DNA repair genes in determining editing efficiency in D. radiodurans was evaluated by introducing the CRISPR/Cpf1 system into 13 mutant strains lacking various DNA damage response and repair factors. In addition to the crucial role of RecA-dependent HR required for CRISPR/Cpf1 editing, D. radiodurans showed higher editing efficiency when lacking DdrB, the single-stranded DNA annealing (SSA) protein involved in the RecA-independent DSB repair pathway. This suggests a possible competition between HR and SSA pathways in the CRISPR editing of D. radiodurans. Moreover, off-target effects were observed during the genome editing of the pprI knockout strain, a master DNA damage response gene in Deinococcus species, which suggested that precise regulation of DNA damage response is critical for a high-fidelity genome editing system.

Deinococcus wulumuqiensis R12 synthesized silver nanoparticles with peroxidase-like activity for synergistic antibacterial application.

The use of a combination of several antibacterial agents for therapy holds great promise in reducing the dosage and side effects of these agents, improving their efficiency, and inducing potential synergistic therapeutic effects. Herein, this study provides an innovative antibacterial treatment strategy by synergistically combining R12-AgNPs with H2O2 therapy. R12-AgNPs were simply produced with the supernatant of an ionizing radiation-tolerant bacterium Deinococcus wulumuqiensis R12 by one-step under room temperature. In comparison with chemically synthesized AgNPs, the biosynthesized AgNPs presented fascinating antibacterial activity and peroxidase-like properties, which endowed it with the capability to catalyze the decomposition of H2O2 to generate hydroxyl radical. After the combination of R12-AgNPs and H2O2, an excellent synergistic bacteriostatic activity was observed for both Escherichia coli and Staphylococcus aureus, especially at low concentrations. In addition, in vitro cytotoxicity tests showed R12-AgNPs had good biocompatibility. Thus, this work presents a novel antibacterial agent that exhibits favorable synergistic antibacterial activity and low toxicity, without the use of antibiotics or a complicated synthesis process.

Characterization of a unique pH-dependent amylosucrase from Deinococcus cellulosilyticus.

The amylosucrase (ASase, EC 2.4.1.4) utilizes sucrose as the sole substrate to catalyze multifunctional reactions. It can naturally synthesize α-1,4-linked glucans such as amylose as well as sucrose isomers with more favorable properties than sucrose with a lower intestinal digestibility and non-cariogenic properties. The amino acid sequence of the asase gene from Deinococcus cellulosilyticus (DceAS) exhibits low homology with those of other ASases from other Deinococcus species. In this study, we cloned and expressed DceAS and demonstrated its high activity at pH 6 and pH 8 and maintained stability. It showed higher polymerization activity at pH 6 than at pH 8, but similar isomerization activity and produced more turanose and trehalulose at pH 6 than at pH 8 and produced more isomaltulose at pH 8. Furthermore, the molecular weight of DceAS was 226.6 kDa at pH 6 and 145.5 kDa at pH 8, indicating that it existed as a trimer and dimer, respectively under those conditions. Additionally, circular dichroism spectra showed that the DceAS secondary structure was different at pH 6 and pH 8. These differences in reaction products at different pHs can be harnessed to naturally produce sucrose alternatives that are more beneficial to human health.

Structure predictions and functional insights into Amidase_3 domain containing N-acetylmuramyl-L-alanine amidases from Deinococcus indicus DR1.

BACKGROUND: N-acetylmuramyl-L-alanine amidases are cell wall modifying enzymes that cleave the amide bond between the sugar residues and stem peptide in peptidoglycan. Amidases play a vital role in septal cell wall cleavage and help separate daughter cells during cell division. Most amidases are zinc metalloenzymes, and E. coli cells lacking amidases grow as chains with daughter cells attached to each other. In this study, we have characterized two amidase enzymes from Deinococcus indicus DR1. D. indicus DR1 is known for its high arsenic tolerance and unique cell envelope. However, details of their cell wall biogenesis remain largely unexplored. RESULTS: We have characterized two amidases Ami1Di and Ami2Di from D. indicus DR1. Both Ami1Di and Ami2Di suppress cell separation defects in E. coli amidase mutants, suggesting that these enzymes are able to cleave septal cell wall. Ami1Di and Ami2Di proteins possess the Amidase_3 catalytic domain with conserved -GHGG- motif and Zn2+ binding sites. Zn2+- binding in Ami1Di is crucial for amidase activity. AlphaFold2 structures of both Ami1Di and Ami2Di were predicted, and Ami1Di was a closer homolog to AmiA of E. coli. CONCLUSION: Our results indicate that Ami1Di and Ami2Di enzymes can cleave peptidoglycan, and structural prediction studies revealed insights into the activity and regulation of these enzymes in D. indicus DR1.

Enhanced chaotrope tolerance and (S)-2-hydroxypropiophenone production by recombinant Pseudomonas putida engineered with Pprl from Deinococcus radiodurans.

Pseudomonas putida is a soil bacterium with multiple uses in fermentation and biotransformation processes. P. putida ATCC 12633 can biotransform benzaldehyde and other aldehydes into valuable α-hydroxyketones, such as (S)-2-hydroxypropiophenone. However, poor tolerance of this strain toward chaotropic aldehydes hampers efficient biotransformation processes. To circumvent this problem, we expressed the gene encoding the global regulator PprI from Deinococcus radiodurans, an inducer of pleiotropic proteins promoting DNA repair, in P. putida. Fine-tuned gene expression was achieved using an expression plasmid under the control of the LacIQ /Ptrc system, and the cross-protective role of PprI was assessed against multiple stress treatments. Moreover, the stress-tolerant P. putida strain was tested for 2-hydroxypropiophenone production using whole resting cells in the presence of relevant aldehyde substrates. P. putida cells harbouring the global transcriptional regulator exhibited high tolerance toward benzaldehyde, acetaldehyde, ethanol, butanol, NaCl, H2 O2 and thermal stress, thereby reflecting the multistress protection profile conferred by PprI. Additionally, the engineered cells converted aldehydes to 2-hydroxypropiophenone more efficiently than the parental P. putida strain. 2-Hydroxypropiophenone concentration reached 1.6 g L-1 upon a 3-h incubation under optimized conditions, at a cell concentration of 0.033 g wet cell weight mL-1 in the presence of 20 mM benzaldehyde and 600 mM acetaldehyde. Product yield and productivity were 0.74 g 2-HPP g-1 benzaldehyde and 0.089 g 2-HPP g cell dry weight-1 h-1 , respectively, 35% higher than the control experiments. Taken together, these results demonstrate that introducing PprI from D. radiodurans enhances chaotrope tolerance and 2-HPP production in P. putida ATCC 12633.

Structural and Functional Characterization of a Gene Cluster Responsible for Deglycosylation of C-glucosyl Flavonoids and Xanthonoids by Deinococcus aerius.

Plant C-glycosylated aromatic polyketides are important for plant and animal health. These are specialized metabolites that perform functions both within the plant, and in interaction with soil or intestinal microbes. Despite the importance of these plant compounds, there is still limited knowledge of how they are metabolized. The Gram-positive aerobic soil bacterium Deinococcus aerius strain TR0125 and other Deinococcus species thrive in a wide range of harsh environments. In this work, we identified a C-glycoside deglycosylation gene cluster in the genome of D. aerius. The cluster includes three genes coding for a GMC-type oxidoreductase (DaCGO1) that oxidizes the glucosyl C3 position in aromatic C-glucosyl compounds, which in turn provides the substrate for the C-glycoside deglycosidase (DaCGD; composed of α+ß subunits) that cleaves the glucosyl-aglycone C-C bond. Our results from size-exclusion chromatography, single particle cryo-electron microscopy and X-ray crystallography show that DaCGD is an α2ß2 heterotetramer, which represents a novel oligomeric state among bacterial CGDs. Importantly, the high-resolution X-ray structure of DaCGD provides valuable insights into the activation of the catalytic hydroxide ion by Lys261. DaCGO1 is specific for the 6-C-glucosyl flavones isovitexin, isoorientin and the 2-C-glucosyl xanthonoid mangiferin, and the subsequent C-C-bond cleavage by DaCGD generated apigenin, luteolin and norathyriol, respectively. Of the substrates tested, isovitexin was the preferred substrate (DaCGO1, Km 0.047 mM, kcat 51 min-1; DaCGO1/DaCGD, Km 0.083 mM, kcat 0.42 min-1).

Spatial arrangement and density variations in the cell envelope of Deinococcus radiodurans.

The cell envelope of the poly-extremophile bacterium Deinococcus radiodurans is renowned for its highly organized structure and unique functional characteristics. In this bacterium, a precise regularity characterizes not just the S-layer, but it also extends to the underlying cell envelope layers, resulting in a dense and tightly arranged configuration. This regularity is attributed to a minimum of three protein complexes located at the outer membrane level. Together, they constitute a recurring structural unit that extends across the cell envelope, effectively tiling the entirety of the cell body. Nevertheless, a comprehensive grasp of the vacant spaces within each layer and their functional roles remains limited. In this study, we delve into these aspects by integrating the state of the art with structural calculations. This approach provides crucial evidence supporting an evolutive pressure intricately linked to surface phenomena depending on the environmental conditions.

The Deinococcus protease PprI senses DNA damage by directly interacting with single-stranded DNA.

Bacteria have evolved various response systems to adapt to environmental stress. A protease-based derepression mechanism in response to DNA damage was characterized in Deinococcus, which is controlled by the specific cleavage of repressor DdrO by metallopeptidase PprI (also called IrrE). Despite the efforts to document the biochemical, physiological, and downstream regulation of PprI-DdrO, the upstream regulatory signal activating this system remains unclear. Here, we show that single-stranded DNA physically interacts with PprI protease, which enhances the PprI-DdrO interactions as well as the DdrO cleavage in a length-dependent manner both in vivo and in vitro. Structures of PprI, in its apo and complexed forms with single-stranded DNA, reveal two DNA-binding interfaces shaping the cleavage site. Moreover, we show that the dynamic monomer-dimer equilibrium of PprI is also important for its cleavage activity. Our data provide evidence that single-stranded DNA could serve as the signal for DNA damage sensing in the metalloprotease/repressor system in bacteria. These results also shed light on the survival and acquired drug resistance of certain bacteria under antimicrobial stress through a SOS-independent pathway.

DNA Polymerase I Large Fragment from Deinococcus radiodurans, a Candidate for a Cutting-Edge Room-Temperature LAMP.

In recent years, the loop-mediated isothermal amplification (LAMP) technique, designed for microbial pathogen detection, has acquired fundamental importance in the biomedical field, providing rapid and precise responses. However, it still has some drawbacks, mainly due to the need for a thermostatic block, necessary to reach 63 °C, which is the BstI DNA polymerase working temperature. Here, we report the identification and characterization of the DNA polymerase I Large Fragment from Deinococcus radiodurans (DraLF-PolI) that functions at room temperature and is resistant to various environmental stress conditions. We demonstrated that DraLF-PolI displays efficient catalytic activity over a wide range of temperatures and pH, maintains its activity even after storage under various stress conditions, including desiccation, and retains its strand-displacement activity required for isothermal amplification technology. All of these characteristics make DraLF-PolI an excellent candidate for a cutting-edge room-temperature LAMP that promises to be very useful for the rapid and simple detection of pathogens at the point of care.

Characterization of a Novel N4-Methylcytosine Restriction-Modification System in Deinococcus radiodurans.

Deinococcus radiodurans is an extremophilic microorganism that possesses a unique DNA damage repair system, conferring a strong resistance to radiation, desiccation, oxidative stress, and chemical damage. Recently, we discovered that D. radiodurans possesses an N4-methylation (m4C) methyltransferase called M.DraR1, which recognizes the 5'-CCGCGG-3' sequence and methylates the second cytosine. Here, we revealed its cognate restriction endonuclease R.DraR1 and recognized that it is the only endonuclease specially for non-4C-methylated 5'-CCGCGG-3' sequence so far. We designated the particular m4C R.DraR1-M.DraR1 as the DraI R-M system. Bioinformatics searches displayed the rarity of the DraI R-M homologous system. Meanwhile, recombination and transformation efficiency experiments demonstrated the important role of the DraI R-M system in response to oxidative stress. In addition, in vitro activity experiments showed that R.DraR1 could exceptionally cleave DNA substrates with a m5C-methlated 5'-CCGCGG-3' sequence instead of its routine activity, suggesting that this particular R-M component possesses a broader substrate choice. Furthermore, an imbalance of the DraI R-M system led to cell death through regulating genes involved in the maintenance of cell survival such as genome stability, transporter, and energy production. Thus, our research revealed a novel m4C R-M system that plays key roles in maintaining cell viability and defending foreign DNA in D. radiodurans.

Deinococcus rhizophilus sp.nov., isolated from rhizosphere soil.

A spherical, pink, aerobic, Gram-stain-positive bacterial strain (MIMF12T) was isolated from rhizosphere soil collected in the Inner Mongolia Autonomous Region, PR China. Cellular growth of the strain was observed at pH 6.0-8.0 (optimum, pH 7.0), at 20-37 °C (optimum, 28 °C) and with 0-1 % (w/v) NaCl (optimum, 0 %). Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain MIMF12T was most closely related to Deinococcus terrestris SDU3-2T with a similarity value of 96.0 %. The respiratory quinone was menaquinone 8, the major fatty acids were C15 : 1 ω6c and C17 : 1 ω8c, and the major polar lipids were composed of two aminophospholipids, one phospholipid and four unidentified lipids. The G+C content of the genomic DNA was 70.1 mol%. The average nucleotide identity and digital DNA-DNA hybridization values between strain MIMF12T and the closest related type strain SDU3-2T were 88.1 and 52.1 %, respectively. The discovery that MIMF12T differs not only from validly named species in the genus Deinococcus, but also from currently unnamed species in the GDTB, gives us new insights into the genus. Based on phenotypic, chemotaxonomic and phylogenetic characteristics, strain MIMF12T represents a novel species of the genus Deinococcus, for which the name Deinococcus rhizophilus sp. nov. is proposed. The type strain is MIMF12T (=CGMCC 1.61579T=KCTC 43572T).

Biochemical, Biophysical, and Structural Analysis of an Unusual DyP from the Extremophile Deinococcus radiodurans.

Dye-decolorizing peroxidases (DyPs) are heme proteins with distinct structural properties and substrate specificities compared to classical peroxidases. Here, we demonstrate that DyP from the extremely radiation-resistant bacterium Deinococcus radiodurans is, like some other homologues, inactive at physiological pH. Resonance Raman (RR) spectroscopy confirms that the heme is in a six-coordinated-low-spin (6cLS) state at pH 7.5 and is thus unable to bind hydrogen peroxide. At pH 4.0, the RR spectra of the enzyme reveal the co-existence of high-spin and low-spin heme states, which corroborates catalytic activity towards H2O2 detected at lower pH. A sequence alignment with other DyPs reveals that DrDyP possesses a Methionine residue in position five in the highly conserved GXXDG motif. To analyze whether the presence of the Methionine is responsible for the lack of activity at high pH, this residue is substituted with a Glycine. UV-vis and RR spectroscopies reveal that the resulting DrDyPM190G is also in a 6cLS spin state at pH 7.5, and thus the Methionine does not affect the activity of the protein. The crystal structures of DrDyP and DrDyPM190G, determined to 2.20 and 1.53 Å resolution, respectively, nevertheless reveal interesting insights. The high-resolution structure of DrDyPM190G, obtained at pH 8.5, shows that one hydroxyl group and one water molecule are within hydrogen bonding distance to the heme and the catalytic Asparagine and Arginine. This strong ligand most likely prevents the binding of the H2O2 substrate, reinforcing questions about physiological substrates of this and other DyPs, and about the possible events that can trigger the removal of the hydroxyl group conferring catalytic activity to DrDyP.

Natural transformation-specific DprA coordinate DNA double-strand break repair pathways in heavily irradiated D. radiodurans.

Deinococcus radiodurans exhibits remarkable survival under extreme conditions, including ionizing radiation, desiccation, and various DNA-damaging agents. It employs unique repair mechanisms, such as single-strand annealing (SSA) and extended synthesis-dependent strand annealing (ESDSA), to efficiently restore damaged genome. In this study, we investigate the role of the natural transformation-specific protein DprA in DNA repair pathways following acute gamma radiation exposure. Our findings demonstrate that the absence of DprA leads to rapid repair of gamma radiation-induced DNA double-strand breaks primarily occur through SSA repair pathway. Additionally, our findings suggest that the DprA protein may hinder both the SSA and ESDSA repair pathways, albeit in distinct manners. Overall, our results highlight the crucial function of DprA in the selection between SSA and ESDSA pathways for DNA repair in heavily irradiated D. radiodurans.IMPORTANCEDeinococcus radiodurans exhibits an extraordinary ability to endure and thrive in extreme environments, including exposure to radiation, desiccation, and damaging chemicals, as well as intense UV radiation. The bacterium has evolved highly efficient repair mechanisms capable of rapidly mending hundreds of DNA fragments in its genome. Our research indicates that natural transformation (NT)-specific dprA genes play a pivotal role in regulating DNA repair in response to radiation. Remarkably, we found that DprA is instrumental in selecting DNA double-strand break repair pathways, a novel function that has not been reported before. This unique regulatory mechanism highlights the indispensable role of DprA beyond its native function in NT and underscores its ubiquitous presence across various bacterial species, regardless of their NT proficiency. These findings shed new light on the resilience and adaptability of Deinococcus radiodurans, opening avenues for further exploration into its exceptional survival strategies.

Structural characterization and functional insights into the type II secretion system of the poly-extremophile Deinococcus radiodurans.

The extremophile bacterium D. radiodurans boasts a distinctive cell envelope characterized by the regular arrangement of three protein complexes. Among these, the Type II Secretion System (T2SS) stands out as a pivotal structural component. We used cryo-electron microscopy to reveal unique features, such as an unconventional protein belt (DR_1364) around the main secretin (GspD), and a cap (DR_0940) found to be a separated subunit rather than integrated with GspD. Furthermore, a novel region at the N-terminus of the GspD constitutes an additional second gate, supplementing the one typically found in the outer membrane region. This T2SS was found to contribute to envelope integrity, while also playing a role in nucleic acid and nutrient trafficking. Studies on intact cell envelopes show a consistent T2SS structure repetition, highlighting its significance within the cellular framework.