Unraveling the structure and function of a novel SegC protein interacting with the SegAB chromosome segregation complex in Archaea.

Genome segregation is a fundamental process that preserves the genetic integrity of all organisms, but the mechanisms driving genome segregation in archaea remain enigmatic. This study delved into the unknown function of SegC (SSO0033), a novel protein thought to be involved in chromosome segregation in archaea. Using fluorescence polarization DNA binding assays, we discovered the ability of SegC to bind DNA without any sequence preference. Furthermore, we determined the crystal structure of SegC at 2.8 Å resolution, revealing the multimeric configuration and forming a large positively charged surface that can bind DNA. SegC has a tertiary structure folding similar to those of the ThDP-binding fold superfamily, but SegC shares only 5-15% sequence identity with those proteins. Unexpectedly, we found that SegC has nucleotide triphosphatase (NTPase) activity. We also determined the SegC-ADP complex structure, identifying the NTP binding pocket and relative SegC residues involved in the interaction. Interestingly, images from negative-stain electron microscopy revealed that SegC forms filamentous structures in the presence of DNA and NTPs. Further, more uniform and larger SegC-filaments are observed, when SegA-ATP was added. Notably, the introduction of SegB disrupts these oligomers, with ATP being essential for regulating filament formation. These findings provide insights into the functional and structural role of SegC in archaeal chromosome segregation.

Genome-based classification of the family Haloferacaceae and description of five novel species of Halobaculum.

The taxonomic status of some species of Halobellus, Haloferax, Halogranum, and Haloplanus within the family Haloferacaceae was elucidated by phylogenetic, phylogenomic, and comparative genomic analyses. The relative species of each genus should constitute a single species based on the overall genome-related indexes proposed for species demarcation. The cutoff values of AAI (72.1%), ANI (82.2%), and rpoB' gene similarity (90.7%) were proposed to differentiate genera within the family Haloferacaceae. According to these standards, a novel genus related to the genus Halobaculum was proposed to accommodate Halobaculum halophilum Gai3-2 T and Halobaculum salinum NJ-3-1 T. Five halophilic archaeal strains, DT31T, DT55T, DT92T, SYNS20T, and YSMS11T, isolated from a tidal flat and a marine solar saltern in China, were subjected to polyphasic classification. The phenotypic, phylogenetic, phylogenomic, and comparative genomic analyses revealed that strains DT31T (= CGMCC 1.18923 T = JCM 35417 T), DT55T (= CGMCC 1.19048 T = JCM 36147 T), DT92T (= CGMCC 1.19057 T = JCM 36148 T), SYNS20T (= CGMCC 1.62628 T = JCM 36154 T), and YSMS11T (= CGMCC 1.18927 T = JCM 34912 T) represent five novel species of the genus Halobaculum, for which the names, Halobaculum lipolyticum sp. nov., Halobaculum marinum sp. nov., Halobaculum litoreum sp. nov., Halobaculum halobium sp. nov., and Halobaculum limi sp. nov., are proposed.

Genome-based classification of genera Halosegnis and Salella, and description of four novel halophilic archaea isolated from a tidal flat.

The current species of Halosegnis and Salella within the class Halobacteria are closely related based on phylogenetic, phylogenomic, and comparative genomic analyses. The Halosegnis species showed 99.8-100.0% 16S rRNA and 96.6-99.6% rpoB' gene similarities to the Salella species, respectively. Phylogenetic and phylogenomic analyses showed that Salella cibi CBA1133T, the sole species of Salella, formed a single tight cluster with Halosegnis longus F12-1T, then with Halosegnis rubeus F17-44T. The average nucleotide identity (ANI), digital DNA-DNA hybridization (dDDH), and average amino acid identity (AAI) values between Salella cibi CBA1133T and Halosegnis longus F12-1T were 99.2, 94.2, and 98.6%, respectively, much higher than the thresholds for species demarcation. This genome-based classification revealed that the genus Salella should be merged with Halosegnis, and Salella cibi should be a later heterotypic synonym of Halosegnis longus. Halophilic archaeal strains DT72T, DT80T, DT85T, and DT116T, isolated from the saline soil of a tidal flat in China, were subjected to polyphasic taxonomic characterization. The phenotypic, chemotaxonomic, phylogenetic, and phylogenomic features indicated that strains DT72T (= CGMCC 1.18925T = JCM 35418T), DT80T (= CGMCC 1.18926T = JCM 35419T), DT85T (= CGMCC 1.19049T = JCM 35605T), and DT116T (= CGMCC 1.19045T = JCM 35606T) represent four novel species of the genera Halorussus, Halosegnis and Haloglomus, respectively, for which the names, Halorussus caseinilyticus sp. nov., Halorussus lipolyticus sp. nov., Halosegnis marinus sp. nov., and Haloglomus litoreum sp. nov., are proposed.

Development of Moloney Murine Leukemia Virus Reverse Transcriptase Fused with Archaeal DNA-binding Protein Sis7a.

INTRODUCTION: Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV RT) is a common enzyme used to convert RNA sequences into cDNA. However, it still has its shortcomings, especially in terms of processivity and thermostability. According to a previous patent, the fusion of polymerase enzyme to an archaeal DNA-binding protein has been proven to enhance its performance. Furthermore, recent studies have also stated that the fusion of a polymerase enzyme to an archaeal DNA-binding protein is predicted to improve its thermostability and processivity. AIM: As an early stage of enzyme development, this study aimed to design, express, and purify enzymatically active MMLV RT fused with archaeal DNA-binding protein. METHODS: RT fusion proteins were designed and evaluated using in silico methods. The RT fusion enzyme was then expressed in Escherichia coli BL21(DE3) and purified. Its reverse transcriptional activity was proved using reverse transcription quantitative polymerase chain reaction (RT-qPCR). RESULTS: This study showed that MMLV RT fusion with Sis7a protein at its C-terminal end using commercial linker (GGVDMI) produced the best in silico evaluation results. The RT fusion was successfully expressed and purified. It was also known that the optimal condition for expression of the RT fusion was using 0.5 mM IPTG with post-induction incubation at room temperature (± 26°C) for 16 hours. In addition, the activity assay proved that the RT fusion has the reverse transcriptional activity. CONCLUSION: This study shows that the designed MMLV RT Sis7a fusion can be expressed and purified, is enzymatically active, and has the potential to be developed as an improved RT enzyme. Further study is still needed to prove its thermostability and processivity, and further characterize, and plan production scale-up of the MMLV RT Sis7a fusion for commercial use.

Natrinema salsiterrestre sp. nov., an extremely halophilic archaeon isolated from a hypersaline soil.

An extremely halophilic archaeal strain, designated S1CR25-10T, was isolated from hypersaline soil sampled in the Odiel Saltmarshes Natural Area in Southwestern Spain (Huelva) and subjected to a polyphasic taxonomic characterization. The cells were Gram-stain-negative, motile and their colonies were pink-pigmented. It was a strictly aerobic haloarchaeon that could grow at 25-55 °C (optimum, 37 °C), at pH 6.0-9.0 (optimum, pH 7.0-8.0) and in the presence of 12-30 % (w/v) total salts (optimum, 20-25 %, w/v). The phylogenetic analysis based on the comparison of the 16S rRNA gene sequences revealed that strain S1CR25-10T belongs to the genus Natrinema, with 98.9 % similarity to Natrinema salinisoli SLN56T. In addition, the values of orthologous average nucleotide identity, digital DNA-DNA hybridization and average amino acid identity were below the threshold limits accepted for prokaryotic species delineation, with N. salinisoli SLN56T showing the highest relatedness values (92.6 % and 48.4 %, respectively). The major polar lipids were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and a glycolipid chromatographically identical to sulfated diglycosyl diether. The DNA G+C content of the isolate was 63.8 mol%. Based on the phylogenetic, phenotypic and chemotaxonomic characterization and the whole genome results, strain S1CR25-10T represents a new species within the genus Natrinema, for which the name Natrinema salsiterrestre sp. nov., with type strain S1CR25-10T (=CECT 30623T=CCM 9251T), is proposed.

Salinilacihabitans rarus gen. nov., sp. nov., Natrononativus amylolyticus gen. nov., sp. nov., Natronobeatus ordinarius gen. nov., sp. nov., and Halovivax gelatinilyticus sp. nov., halophilic archaea, isolated from a salt lake and soda lakes.

Four halophilic archaea strains, AD-4T, CGA30T, CGA73T, and WLHSJ27T, were isolated from a salt lake and two soda lakes located in different regions of China. The 16S rRNA and rpoB' gene sequence similarities among strains AD-4T, CGA30T, CGA73T, WLHSJ27T, and the current species of the family Natrialbaceae were 90.9-97.5% and 83.1-91.8%, respectively. The phylogenetic and phylogenomic analyses revealed that these four strains separated from existing genera in the family Natrialbaceae and formed distant branches. The ANI, isDDH, and AAI values among these four strains and the current members of the family Natrialbaceae were 72-79%, 20-25%, and 63-73%, respectively, much lower than the threshold values for species demarcation. Strains AD-4T, CGA73T, and WLHSJ27T may represent three novel genera of the family Natrialbaceae according to the cutoff value of AAI (≤ 76%) proposed to differentiate genera within the family Natrialbaceae. These four strains could be distinguished from the related genera according to differential phenotypic characteristics. The major phospholipids of these four strains were identical while their glycolipid profiles were diverse. DGD-1 is a major glycolipid found in strain AD-4T, trace glycolipids, DGD-1, and S-DGD-1, and (or) S-TGD-1 was found in the other three strains. The major respiratory quinones detected in the four strains were menaquinone MK-8 and MK-8(H2). This polyphasic classification indicated that strains AD-4T, CGA73T, and WLHSJ27T represent three novel species of three new genera with the family Natrialbaceae, and strain CGA30T represents a novel species of Halovivax.

Archaeal DNA alkylation repair conducted by DNA glycosylase and methyltransferase.

Alkylated bases in DNA created in the presence of endogenous and exogenous alkylating agents are either cytotoxic or mutagenic, or both to a cell. Currently, cells have evolved several strategies for repairing alkylated base. One strategy is a base excision repair process triggered by a specific DNA glycosylase that is used for the repair of the cytotoxic 3-methyladenine. Additionally, the cytotoxic and mutagenic O6-methylguanine (O6-meG) is corrected by O6-methylguanine methyltransferase (MGMT) via directly transferring the methyl group in the lesion to a specific cysteine in this protein. Furthermore, oxidative DNA demethylation catalyzed by DNA dioxygenase is utilized for repairing the cytotoxic 3-methylcytosine (3-meC) and 1-methyladenine (1-meA) in a direct reversal manner. As the third domain of life, Archaea possess 3-methyladenine DNA glycosylase II (AlkA) and MGMT, but no DNA dioxygenase homologue responsible for oxidative demethylation. Herein, we summarize recent progress in structural and biochemical properties of archaeal AlkA and MGMT to gain a better understanding of archaeal DNA alkylation repair, focusing on similarities and differences between the proteins from different archaeal species and between these archaeal proteins and their bacterial and eukaryotic relatives. To our knowledge, it is the first review on archaeal DNA alkylation repair conducted by DNA glycosylase and methyltransferase. KEY POINTS: • Archaeal MGMT plays an essential role in the repair of O 6 -meG • Archaeal AlkA can repair 3-meC and 1-meA.

The influence of primer choice on archaeal phylogenetic analyses based on 16S rRNA gene PCR / A influência da escolha de iniciadores na filogenia de Archaea em ensaios de PCR para o gene rRNA 16S

Polymerase chain reaction (PCR) assays targeting 16S rRNA genes followed by DNA sequencing are still important tools to characterize microbial communities present in environmental samples. However, despite the crescent number of deposited archaeal DNA sequences in databases, until now we do not have a clear picture of the effectiveness and specificity of the universal primers widely used to describe archaeal communities from different natural habitats. Therefore, in this study, we compared the phylogenetic profile obtained when Cerrado lake sediment DNA samples were submitted to 16S rDNA PCR employing three Archaea-specific primer sets commonly used. Our findings reveal that specificity of primers differed depending on the source of the analyzed DNA. Furthermore, archaeal communities revealed by each primer pair varied greatly, indicating that 16S rRNA gene primer choice affects the community profile obtained, with differences in both taxon detection and operational taxonomic unit (OTU) estimates.

A amplificação de genes que codificam o rRNA 16S por reação em cadeia da polimerase (PCR) e o seu sub sequentesequenciamento consistem em uma ferramenta importante na caracterização de comunidades microbianas presentes em amostras ambientais. No entanto, apesar do crescente número de sequências de DNA de Archaea depositadas em bancos de dados, a especificidade e efetividade dos iniciadores de PCR descritos como universais e amplamente utilizados na descrição desse grupo ainda não está clara. Neste estudo foram comparados os perfis filogenéticos de comunidades de arqueias obtidos a partir amostras de DNA de sedimentos lacustres do Cerrado submetidas a ensaios de PCR empregando três pares de iniciadores específicos para Archaea, comumente utilizados neste tipo de estudo. Nossos resultados indicam que as comunidades de arqueias detectadas com cada par de iniciadores apresentaram grande variação filogenética, sugerindo que a escolha de iniciadores dirigidos ao gene de rRNA 16S tem efeito significativo no perfil da comunidade descrita, com diferenças tanto em relação aos táxons detectados, como nas estimativas de unidades taxonômicas operacionais (OTU).

Dissection of Functional Domains of Orc1-2, the Archaeal Global DNA Damage-Responsive Regulator.

Orc1-2 is a non-initiator ortholog of archaeal/eukaryotic Orc1 proteins, which functions as a global regulator in DNA damage-responsive (DDR) expression. As for Orc1 initiators, the DDR regulator harbors an AAA+ ATPase domain, an Initiator-Specific Motif (ISM) and a winged-helix (wH) DNA-binding domain, which are also organized in a similar fashion. To investigate how Orc1-2 mediates the DDR regulation, the orc1-2 mutants inactivating each of these functional domains were constructed with Saccharolobus islandicus and genetically characterized. We found that disruption of each functional domain completely abolished the DDR regulation in these orc1-2 mutants. Strikingly, inactivation of ATP hydrolysis of Orc1-2 rendered an inviable mutant. However, the cell lethality can be suppressed by the deficiency of the DNA binding in the same protein, and it occurs independent of any DNA damage signal. Mutant Orc1-2 proteins were then obtained and investigated for DNA-binding in vitro. This revealed that both the AAA+ ATPase and the wH domains are involved in DNA-binding, where ISM and R381R383 in wH are responsible for specific DNA binding. We further show that Orc1-2 regulation occurs in two distinct steps: (a) eliciting cell division inhibition at a low Orc1-2 content, and this regulation is switched on by ATP binding and turned off by ATP hydrolysis; any failure in turning off the regulation leads to growth inhibition and cell death; (b) activation of the expression of DDR gene encoding DNA repair proteins at an elevated level of Orc1-2.

Methanofollis propanolicus sp. nov., a novel archaeal isolate from a Costa Rican oil well that uses propanol for methane production.

A novel methanogenic strain, CaP3V-MF-L2AT, was isolated from an exploratory oil well from Cahuita National Park, Costa Rica. The cells were irregular cocci, 0.8-1.8 µm in diameter, stained Gram-negative and were motile. The strain utilized H2/CO2, formate and the primary and secondary alcohols 1-propanol and 2-propanol for methanogenesis, but not acetate, methanol, ethanol, 1-butanol or 2-butanol. Acetate was required as carbon source. The novel isolate grew at 25-40 °C, pH 6.0-7.5 and 0-2.5% (w/v) NaCl. 16S rRNA gene sequence analysis revealed that the strain is affiliated to the genus Methanofollis. It shows 98.8% sequence similarity to its closest relative Methanofollis ethanolicus. The G + C content is 60.1 mol%. Based on the data presented here type strain CaP3V-MF-L2AT (= DSM 113321T = JCM 39176T) represents a novel species, Methanofollis propanolicus sp. nov.

Stygiolobus caldivivus sp. nov., a facultatively anaerobic hyperthermophilic archaeon isolated from the Unzen hot spring in Japan.

A novel hyperthermophilic, acidophilic and facultatively anaerobic archaeon, strain KN-1T, was isolated from Unzen hot spring in Japan and characterized. The cells of KN-1T were irregular cocci with a diameter of 1.0-3.0 µm that grew at 55-87.5 °C (optimum: 75 °C) and pH 1.0-5.5 (optimum: 3.0). Chemolithoautotrophic growth of KN-1T occurred in the presence of S0 or H2 under oxic conditions. Under anoxic conditions, KN-1T grew with S0, ferric citrate and FeCl3 as electron acceptors. A phylogenetic analysis of 16S rRNA gene sequences showed that the species most closely related to KN-1T was Stygiolobus azoricus JCM 9 021T, with 98.9 % sequence identity, indicating that strain KN-1T belongs to the genus Stygiolobus. This genus has been considered to consist of obligate anaerobes since its description in 1991. However, KN-1T grew under oxic, microoxic and anoxic conditions. Moreover, KN-1Tutilized various complex substrates and some sugars as carbon or energy sources, which is also different from S. azoricus JCM 9 021T. The average nucleotide identity and amino acid identity values between KN-1T and S. azoricus JCM 9 021T were 79.4 and 76.1 %, respectively, indicating that KN-1T represents a novel species. Its main polar lipids were calditoglycerocaldarchaeol and caldarchaeol, and its DNA G+C content was 40.1 mol%. We also found that S. azoricus JCM 9021T grew under microoxic conditions in the presence of H2 as an electron donor, indicating that this genus does not comprise obligate anaerobes. Based on this polyphasic taxonomic analysis, we propose the novel species, Stygiolobus caldivivus sp. nov., whose type strain is KN-1T (=JCM 34 622T=KCTC 4 293T).

Sala cibi gen. nov., sp. nov., an extremely halophilic archaeon isolated from solar salt.

Two novel halophilic archaeal strains, CBA1133T and CBA1134, were isolated from solar salt in South Korea. The 16S rRNA gene sequences of the isolates were identical to each other and were closely related to the genera Natronomonas (92.3-93.5%), Salinirubellus (92.2%), Halomarina (91.3-92.0%), and Haloglomus (91.4%). The isolated strains were coccoid, Gram-stain-negative, aerobic, oxidase-positive, and catalase-negative. Growth occurred under temperatures of 25-50°C (optimum, 45°C), NaCl levels of 10-30% (optimum, 15%), pH levels of 6.0-8.5 (optimum, 7.0), and MgCl2 concentrations of 0-500 mM (optimum, 100 mM). Digital DNA-DNA hybridization values between the strains and related genera ranged from 18.3% to 22.7%. The major polar lipids of the strains were phosphatidyl glycerol, phosphatidyl glycerol phosphate methyl ester, and phosphatidyl glycerol sulfate. Genomic, phenotypic, physiological, and biochemical analyses of the isolates revealed that they represent a novel genus and species in the family Halobacteriaceae. The type strain is CBA1133T (= KACC 22148T = JCM 34265T), for which the name Sala cibi gen. nov., sp. nov. is proposed.

Haloterrigena gelatinilytica sp. nov., a new extremely halophilic archaeon isolated from salt-lake.

Two extremely halophilic strains, designated SYSU A558-1T and SYSU A121-1, were isolated from a saline sediment sample collected from Aiding salt-lake, China. Cells of strains SYSU A558-1T and SYSU A121-1 were Gram-stain-negative, coccoid, and non-motile. The strains were aerobic and grew at NaCl concentration of 10-30% (optimum, 20-22%), at 20-55 °C (optimum, 37-42 °C) and at pH 6.5-8.5 (optimum, 7.0-8.0). Cells lysed in distilled water. The polar lipids were phosphatidyl choline, phosphatidylglycerol phosphate methyl ester, disulfated diglycosyl diether-1 and unidentified glycolipid. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the two strains SYSU A558-1T and SYSU A121-1 were closely related to the membranes of the genus Haloterrigena. Phylogenetic and phylogenomic trees of strains SYSU A558-1T and SYSU A121-1 demonstrated a robust clade with Haloterrigena turkmenica, Haloterrigena salifodinae and Haloterrigena salina. The genomic DNA G + C content of strains SYSU A558-1T and SYSU A121-1 were 65.8 and 65.0%, respectively. Phenotypic, phylogenetic, chemotaxonomic and genome analysis suggested that the two strains SYSU A558-1T and SYSU A121-1 represent a novel species of the genus Haloterrigena, for which the name Haloterrigena gelatinilytica sp. nov. is proposed. The type strain is SYSU A558-1T (= KCTC 4259T = CGMCC 1.15953T).

Halorubrum salinarum sp. nov., an extremely halophilic archaeon isolated from a saturated brine pond of a saltern.

A novel extremely halophilic archaeon, strain RHB-CT, was isolated from a saturated brine pond of a solar saltern in Bolinao, Pangasinan, Philippines. Colonies were orange-red-pigmented, smooth, convex and round on a solid modified growth medium containing 25 % (w/v) of total salts. Cells of strain RHB-CT on the solid modified growth medium were ovoid-shaped (0.89-2.66 µm long), while the cells in a liquid modified growth medium were rod-shaped (1.53-5.65 µm long and 0.45-1.03 µm wide). The strain was Gram-stain-negative, motile and strictly aerobic. Strain RHB-CT grew with NaCl concentrations ranging from 10 to 30 % (w/v; optimum, 20-25 %), at pH 6.5-8.5 (optimum, pH 7.0-7.5) and at 20-55 °C (optimum, 40-45 °C). Furthermore, the strain grew even in the absence of Mg2+; however, when supplemented with Mg2+, growth was observed optimally at 0.2-0.4 M Mg2+. The 16S rRNA gene phylogeny inferred that the strain is a member of the genus Halorubrum and was related to Halorubrum xinjiangense CGMCC 1.3527T (99.0 %), Halorubrum sodomense DSM 3755T (98.8 %), Halorubrum coriense Ch2T (98.8 %), Halorubrum trapanicum NRC 34021T (98.4 %) and Halorubrum distributum JCM 9100T (98.1 %). The rpoB' gene sequences also showed that strain RHB-CT is related to Hrr. xinjiangense JCM 12388T (97.1 %), Hrr. distributum JCM 9100T (97.1 %), Hrr. coriense JCM 9275T (96.5 %), Hrr. californiense JCM 14715T (96.5 %), Hrr. trapanicum JCM 10477T (96.3%), Hrr. sodomense JCM 8880T (96.2%) and Hrr. tebenquichense DSM 14210T (95.6 %). The DNA G+C content of strain RHB-CT was 68.7 mol% (genome). Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between strain RHB-CT and the closely related species of Halorubrum were below 40 and 90 %, respectively, which are far below the thresholds to delineate a new species. The polar lipids of strain RHB-CT were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulphate and sulfated mannosyl glycosyl diether. Based on dDDH and ANI values, and the significant morphological and physiological differences from known taxa, it is hereby suggested that strain RHB-CT represents a novel species of the genus Halorubrum, for which the name Halorubrum salinarum sp. nov. is proposed. The type strain is RHB-CT (=KCTC 4274T=CMS 2103T).

Chromosome segregation in Archaea: SegA- and SegB-DNA complex structures provide insights into segrosome assembly.

Genome segregation is a vital process in all organisms. Chromosome partitioning remains obscure in Archaea, the third domain of life. Here, we investigated the SegAB system from Sulfolobus solfataricus. SegA is a ParA Walker-type ATPase and SegB is a site-specific DNA-binding protein. We determined the structures of both proteins and those of SegA-DNA and SegB-DNA complexes. The SegA structure revealed an atypical, novel non-sandwich dimer that binds DNA either in the presence or in the absence of ATP. The SegB structure disclosed a ribbon-helix-helix motif through which the protein binds DNA site specifically. The association of multiple interacting SegB dimers with the DNA results in a higher order chromatin-like structure. The unstructured SegB N-terminus plays an essential catalytic role in stimulating SegA ATPase activity and an architectural regulatory role in segrosome (SegA-SegB-DNA) formation. Electron microscopy results also provide a compact ring-like segrosome structure related to chromosome organization. These findings contribute a novel mechanistic perspective on archaeal chromosome segregation.

Take a Break to Repair: A Dip in the World of Double-Strand Break Repair Mechanisms Pointing the Gaze on Archaea.

All organisms have evolved many DNA repair pathways to counteract the different types of DNA damages. The detection of DNA damage leads to distinct cellular responses that bring about cell cycle arrest and the induction of DNA repair mechanisms. In particular, DNA double-strand breaks (DSBs) are extremely toxic for cell survival, that is why cells use specific mechanisms of DNA repair in order to maintain genome stability. The choice among the repair pathways is mainly linked to the cell cycle phases. Indeed, if it occurs in an inappropriate cellular context, it may cause genome rearrangements, giving rise to many types of human diseases, from developmental disorders to cancer. Here, we analyze the most recent remarks about the main pathways of DSB repair with the focus on homologous recombination. A thorough knowledge in DNA repair mechanisms is pivotal for identifying the most accurate treatments in human diseases.

Haloferax litoreum sp. nov., Haloferax marinisediminis sp. nov., and Haloferax marinum sp. nov., low salt-tolerant haloarchaea isolated from seawater and sediment.

Three novel halophilic archaea were isolated from seawater and sediment near Yeoungheungdo Island, Republic of Korea. The genome size and G + C content of the isolates MBLA0076T, MBLA0077T, and MBLA0078T were 3.56, 3.48, and 3.48 Mb and 61.7, 60.8, and 61.1 mol%, respectively. The three strains shared 98.5-99.5 % sequence similarity of the 16 S rRNA gene, whereas their sequence similarity to the 16 S rRNA gene of type strains was below 98.5 %. Phylogenetic analysis based on sequences of the 16 S rRNA and RNA polymerase subunit beta genes indicated that the isolates belonged to the genus Haloferax. The orthologous average nucleotide identity, average amino-acid identity, and in silico DNA-DNA hybridization values were below species delineation thresholds. Pan-genomic analysis indicated that the three novel strains and 11 reference strains had 8981 pan-orthologous groups in total. Fourteen Haloferax strains shared 1766 core pan-genome orthologous groups, which were mainly related to amino acid transport and metabolism. Cells of the three isolates were gram-negative, motile, red-pink pigmented, and pleomorphic. The strains grew optimally at 30 °C (MBLA0076T) and 40 °C (MBLA0077T, MBLA0078T) in the presence of 1.28 M (MBLA0077T) and 1.7 M (MBLA0076T, MBLA0078T) NaCl and 0.1 M (MBLA0077T), 0.2 M (MBLA0076T), and 0.3 M (MBLA0078T) MgCl2·6H2O at pH 7.0-8.0. Cells of all isolates lysed in distilled water; the minimum NaCl concentration necessary to prevent lysis was 0.43 M. The major polar lipids of the three strains were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, and sulphated diglycosyl archaeol-1. Based on their phenotypic and genotypic properties, MBLA0076T, MBLA0077T, and MBLA0078T were described as novel species of Haloferax, for which we propose the names Haloferax litoreum sp. nov., Haloferax marinisediminis sp. nov., and Haloferax marinum sp. nov., respectively. The respective type strains of these species are MBLA0076T (= KCTC 4288T = JCM 34,169T), MBLA0077T (= KCTC 4289T = JCM 34,170T), and MBLA0078T (= KCTC 4290T = JCM 34,171T).

The regulation of DNA supercoiling across evolution.

DNA supercoiling controls a variety of cellular processes, including transcription, recombination, chromosome replication, and segregation, across all domains of life. As a physical property, DNA supercoiling alters the double helix structure by under- or over-winding it. Intriguingly, the evolution of DNA supercoiling reveals both similarities and differences in its properties and regulation across the three domains of life. Whereas all organisms exhibit local, constrained DNA supercoiling, only bacteria and archaea exhibit unconstrained global supercoiling. DNA supercoiling emerges naturally from certain cellular processes and can also be changed by enzymes called topoisomerases. While structurally and mechanistically distinct, topoisomerases that dissipate excessive supercoils exist in all domains of life. By contrast, topoisomerases that introduce positive or negative supercoils exist only in bacteria and archaea. The abundance of topoisomerases is also transcriptionally and post-transcriptionally regulated in domain-specific ways. Nucleoid-associated proteins, metabolites, and physicochemical factors influence DNA supercoiling by acting on the DNA itself or by impacting the activity of topoisomerases. Overall, the unique strategies that organisms have evolved to regulate DNA supercoiling hold significant therapeutic potential, such as bactericidal agents that target bacteria-specific processes or anticancer drugs that hinder abnormal DNA replication by acting on eukaryotic topoisomerases specialized in this process. The investigation of DNA supercoiling therefore reveals general principles, conserved mechanisms, and kingdom-specific variations relevant to a wide range of biological questions.

Thermococcus henrietii sp. nov., a novel extreme thermophilic and piezophilic sulfur-reducing archaeon isolated from a deep-sea hydrothermal chimney.

A novel extreme thermophilic and piezophilic chemoorganoheterotrophic archaeon, strain EXT12cT, was isolated from a hydrothermal chimney sample collected at a depth of 2496 m at the East Pacific Rise 9° N. Cells were strictly anaerobic, motile cocci. The strain grew at NaCl concentrations ranging from 1 to 5 % (w/v; optimum, 2.0%), from pH 6.0 to 7.5 (optimum, pH 6.5-7.0), at temperatures between 60 and 95 °C (optimum, 80-85 °C), and at pressures from 0.1 to at least 50 MPa (optimum, 30 MPa). Strain EXT12cT grew chemoorganoheterotrophically on complex proteinaceous substrates. Its growth was highly stimulated by the presence of elemental sulphur or l-cystine, which were reduced to hydrogen sulfide. The DNA G+C content was 54.58 mol%. Phylogenetic analyses based on 16S rRNA gene sequences and concatenated ribosomal protein sequences showed that strain EXT12cT falls into the genus Thermococcus and is most closely related to Thermococcus nautili strain 30-1T. Overall genome relatedness index analyses (average nucleotide identity scores and in silico DNA-DNA hybridizations) showed a sufficient genomic distance between the new genome and the ones of the Thermococcus type strains for the delineation of a new species. On the basis of genotypic and phenotypic data, strain EXT12cT is considered to represent a novel species, for which the name Thermococcus henrietii sp. nov. is proposed, with the type strain EXT12cT (=UBOCC M-2417T=DSM 111004T).

Thermococcus camini sp. nov., a hyperthermophilic and piezophilic archaeon isolated from a deep-sea hydrothermal vent at the Mid-Atlantic Ridge.

A coccoid-shaped, strictly anaerobic, hyperthermophilic and piezophilic organoheterotrophic archaeon, strain Iri35cT, was isolated from a hydrothermal chimney rock sample collected at a depth of 2300 m at the Mid-Atlantic Ridge (Rainbow vent field). Cells of strain Iri35cT grew at NaCl concentrations ranging from 1-5 % (w/v) (optimum 2.0 %), from pH 5.0 to 9.0 (optimum 7.0-7.5), at temperatures between 50 and 90 °C (optimum 75-80 °C) and at pressures from 0.1 to at least 50 MPa (optimum: 10-30 MPa). The novel isolate grew on complex organic substrates, such as yeast extract, tryptone, peptone or beef extract, preferentially in the presence of elemental sulphur or l-cystine; however, these molecules were not necessary for growth. Its genomic DNA G+C content was 54.63 mol%. The genome has been annotated and the metabolic predictions are in accordance with the metabolic characteristics of the strain and of Thermococcales in general. Phylogenetic analyses based on 16S rRNA gene sequences and concatenated ribosomal protein sequences showed that strain Iri35cT belongs to the genus Thermococcus, and is closer to the species T. celericrescens and T. siculi. Average nucleotide identity scores and in silico DNA-DNA hybridization values between the genome of strain Iri35cT and the genomes of the type species of the genus Thermococcus were below the species delineation threshold. Therefore, and considering the phenotypic data presented, strain Iri35cT is suggested to represent a novel species, for which the name Thermococcus camini sp. nov. is proposed, with the type strain Iri35cT (=UBOCC M-2026T=DSM 111003T).