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Considerable skill is required to identify copepods at the species level based on their morphological characteristics. However, DNA analysis does not require advanced microscopy techniques and provides objective data on the phylogenetic relationships between samples. Therefore, DNA analysis is useful as an alternative method for taxonomic studies of copepods. The lysis buffer method by Lee & Frost (2002) is a simple protocol for extracting DNA from single copepod samples. In this method, a fixative, such as formalin, is first replaced with ethanol and a buffer solution. Then, the copepod sample is lysed in the lysis buffer containing a proteolytic enzyme. Thus far, we have conducted DNA extraction of single copepod samples using this method and performed gene amplification by PCR. However, due to the low success rate of PCR amplification, genetic data could not be obtained for approximately 50% of the formalin-fixed samples. In this study, we improved the lysis buffer method with the aim of enhancing the success rate of DNA extraction and PCR amplification from single copepod samples. In addition, the effect of formalin fixation time on PCR amplification was also examined.Zooplankton samples were collected from Manazuru Port, Sagami Bay on September 14, 2017, using a plankton net with a mesh size of 180 μm and fixed with 5% neutralized formalin-seawater. Adult females of the calanoid copepod Acartia japonica were selected from these samples and stored individually in ethanol. DNA was extracted from these copepods via a modified ethanol removal method, with adjustments made to the dilution of the lysis buffer, and incubation time. The mitochondrial cytochrome b gene was amplified from these DNA samples by PCR. When the concentration of the PCR product was 20 ng μL-1 or more, PCR amplification was considered to be successful. Based on the conditions optimized by the above studies, the effect of the formalin fixation time on the PCR amplification of copepods was also investigated. A higher success rate was obtained when natural drying or vacuum drying was performed to remove ethanol during DNA extraction rather than removal by pipetting. Since there was no significant difference between the results of natural drying and vacuum drying, natural drying, which is easier to perform, was selected as the optimum method. We also confirmed that a high success rate was maintained without diluting the lysis buffer after the inactivation of proteolytic enzyme. Regarding the incubation time for lysis, changing from the conventional 60 minutes to 30 minutes did not result in a significant decrease in the success rate of PCR amplification. Thus, the success rate of PCR increased to approximately 90%. Additionally, compared with the conventional lysis buffer method, the number of steps was reduced by half, and the required time was shortened from 1.5 hours to approximately 50 minutes. Furthermore, we confirmed that this improved lysis buffer method can be applied to single cells of small protozoa such as flagellates and ciliates. The effect of formalin fixation time on PCR amplification of the mitochondrial cytochrome b gene after DNA extraction via this improved lysis buffer method was investigated. As expected, the success rate of PCR amplification decreased with the formalin fixation time. However, when the fixation period was within 1 month, PCR products with a concentration of more than 20 ng μL-1 were obtained in 95% of the individual copepod samples. Furthermore, even after 3 months, similar concentrations of PCR product were obtained in 80% of individuals.Genetic analysis of small zooplankton is increasingly important not only in taxonomy but also for biodiversity and phylogeographic studies. The data presented in this study will be very important and useful in such studies.

The 13th Symposium on Polar Science/Ordinary sessions [OB] Polar biology, Wed. 16 Nov.

The 13th Symposium on Polar Science/Ordinary sessions [OB] Polar biology, Wed. 16 Nov.

The 13th Symposium on Polar Science/Ordinary sessions [OB] Polar biology, Wed. 16 Nov.

The 13th Symposium on Polar Science/Ordinary sessions [OB] Polar biology, Wed. 16 Nov.

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).

The complete genome sequence of the hyperthermophilic archaeon Saccharolobus caldissimus strain HS-3 T was determined. The genome is 3,075,795 bp with a GC content of 32.9%. Genes for the complete semiphosphorylative Entner-Doudoroff pathway, gluconeogenesis, tricarboxylic acid cycle, pentose phosphate pathway, and 3-hydroxypropionate 4-hydroxybutylate cycle were present in the genome.

The genus

The genus Acidianus is composed of facultatively aerobic archaea growing on elemental sulfur as an energy source. Here, we report the 2.58-Mb complete genome sequence of Acidianus sp. strain HS-5, which was isolated from a sulfur hot spring located in Unzen, Japan.

Significance The DPANN superphylum is a grouping of symbiotic microorganisms categorized based on their genomic contents and a few examples of cultivation experiments. Although the genome information of DPANN archaea is increasing year by year, most of them have remained uncultivated, limiting our knowledge of these organisms. Herein, a thermoacidophilic symbiotic archaeon (ARM-1) from the DPANN superphylum was successfully cultivated and characterized. We determined its physiological, morphological, and genomic characteristics in detail and obtained experimental evidence of the symbiotic lifestyle of this archaeon. Notably, ARM-1 is a symbiotic archaeal strain that showed dependence on a range of host species in a laboratory culture. The results significantly contribute to the true understanding of the physiology and ecology of DPANN archaea., Decades of culture-independent analyses have resulted in proposals of many tentative archaeal phyla with no cultivable representative. Members of DPANN (an acronym of the names of the first included phyla Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanohaloarchaeota, and Nanoarchaeota), an archaeal superphylum composed of at least 10 of these tentative phyla, are generally considered obligate symbionts dependent on other microorganisms. While many draft/complete genome sequences of DPANN archaea are available and their biological functions have been considerably predicted, only a few examples of their successful laboratory cultivation have been reported, limiting our knowledge of their symbiotic lifestyles. Here, we investigated physiology, morphology, and host specificity of an archaeon of the phylum “Candidatus Micrarchaeota” (ARM-1) belonging to the DPANN superphylum by cultivation. We constructed a stable coculture system composed of ARM-1 and its original host Metallosphaera sp. AS-7 belonging to the order Sulfolobales. Further host-switching experiments confirmed that ARM-1 grew on five different archaeal species from three genera—Metallosphaera, Acidianus, and Saccharolobus—originating from geologically distinct hot, acidic environments. The results suggested the existence of DPANN archaea that can grow by relying on a range of hosts. Genomic analyses showed inferred metabolic capabilities, common/unique genetic contents of ARM-1 among cultivated micrarchaeal representatives, and the possibility of horizontal gene transfer between ARM-1 and members of the order Sulfolobales. Our report sheds light on the symbiotic lifestyles of DPANN archaea and will contribute to the elucidation of their biological/ecological functions.

Homologous recombination (HR) is thought to be important for the repair of stalled replication forks in hyperthermophilic archaea. Previous biochemical studies identified two branch migration helicases (Hjm and PINA) and two Holliday junction (HJ) resolvases (Hjc and Hje) as HJ-processing proteins; however, due to the lack of genetic evidence, it is still unclear whether these proteins are actually involved in HR in vivo and how their functional relation is associated with the process. To address the above questions, we constructed hjc-, hje-, hjm-, and pina single-knockout strains and double-knockout strains of the thermophilic crenarchaeon Sulfolobus acidocaldarius and characterized the mutant phenotypes. Notably, we succeeded in isolating the hjm- and/or pina-deleted strains, suggesting that the functions of Hjm and PINA are not essential for cellular growth in this archaeon, as they were previously thought to be essential. Growth retardation in Δpina was observed at low temperatures (cold sensitivity). When deletion of the HJ resolvase genes was combined, Δpina Δhjc and Δpina Δhje exhibited severe cold sensitivity. Δhjm exhibited severe sensitivity to interstrand crosslinkers, suggesting that Hjm is involved in repairing stalled replication forks, as previously demonstrated in euryarchaea. Our findings suggest that the function of PINA and HJ resolvases is functionally related at lower temperatures to support robust cellular growth, and Hjm is important for the repair of stalled replication forks in vivo.

The study of DNA repair in hyperthermophiles has the potential to elucidate the mechanisms of genome integrity maintenance systems under extreme conditions. Previous biochemical studies have suggested that the single-stranded DNA-binding protein (SSB) from the hyperthermophilic crenarchaeon Sulfolobus is involved in the maintenance of genome integrity, namely, in mutation avoidance, homologous recombination (HR), and the repair of helix-distorting DNA lesions. However, no genetic study has been reported that elucidates whether SSB actually maintains genome integrity in Sulfolobus in vivo. Here, we characterized mutant phenotypes of the ssb-deleted strain Δssb in the thermophilic crenarchaeon S. acidocaldarius. Notably, an increase (29-fold) in mutation rate and a defect in HR frequency was observed in Δssb, indicating that SSB was involved in mutation avoidance and HR in vivo. We characterized the sensitivities of Δssb, in parallel with putative SSB-interacting protein-encoding gene-deleted strains, to DNA-damaging agents. The results showed that not only Δssb but also Δalhr1 and ΔSaci_0790 were markedly sensitive to a wide variety of helix-distorting DNA-damaging agents, indicating that SSB, a novel helicase SacaLhr1, and a hypothetical protein Saci_0790, were involved in the repair of helix-distorting DNA lesions. This study expands our knowledge of the impact of SSB on genome integrity and identifies novel and key proteins for genome integrity in hyperthermophilic archaea in vivo.

Flavoenzyme dye-linked l-lactate dehydrogenase (Dye-LDH) is primarily involved in energy generation through electron transfer and exhibits potential utility in electrochemical devices. In this study, a gene encoding a Dye-LDH homolog was identified in a hyperthermophilic archaeon, Sulfurisphaera tokodaii. This gene was part of an operon that consisted of four genes that were tandemly arranged in the Sf. tokodaii genome in the following order: stk_16540, stk_16550 (dye-ldh homolog), stk_16560, and stk_16570. This gene cluster was expressed in an archaeal host, Sulfolobus acidocaldarius, and the produced enzyme was purified to homogeneity and characterized. The purified recombinant enzyme exhibited Dye-LDH activity and consisted of two different subunits (products of stk_16540 (α) and stk_16550 (β)), forming a heterohexameric structure (α3β3) with a molecular mass of approximately 253 kDa. Dye-LDH also exhibited excellent stability, retaining full activity upon incubation at 70 °C for 10 min and up to 80% activity after 30 min at 50 °C and pH 6.5–8.0. A quasi-direct electron transfer (DET)-type Dye-LDH was successfully developed by modification of the recombinant enzyme with an artificial redox mediator, phenazine ethosulfate, through amine groups on the enzyme’s surface. This study is the first report describing the development of a quasi-DET-type enzyme by using thermostable Dye-LDH.

The complete genome sequences of Aureimonas sp. strain SA4125 and its native plasmid pSA4125 were determined. The genome sequence comprises 4,968,066 bp, with a GC content of 66.0%, and contains 4,691 coding DNA sequences (CDSs), 3 rRNA operons, and 50 tRNAs. The native plasmid comprises 131,777 bp, with a GC content of 62.3%, and contains 138 CDSs.

The complete genome sequences of Aureimonas sp. strain SA4125 and its native plasmid pSA4125 were determined. The genome sequence comprises 4,968,066 bp, with a GC content of 66.0%, and contains 4,691 coding DNA sequences (CDSs), 3 rRNA operons, and 50 tRNAs. The native plasmid comprises 131,777 bp, with a GC content of 62.3%, and contains 138 CDSs.

The complete genome sequence of the thermoacidophilic crenarchaeon Stygiolobus sp. strain KN-1 was determined and annotated. The genome was 2,958,410 bp in size, with a GC content of 40.1%. It contained 2,973 coding sequences, a single copy of the 16S-23S rRNA operon, 47 tRNA genes, and 9 CRISPR repeat sequences.

The complete genome sequence of the thermoacidophilic crenarchaeon Stygiolobus sp. strain KN-1 was determined and annotated. The genome was 2,958,410 bp in size, with a GC content of 40.1%. It contained 2,973 coding sequences, a single copy of the 16S-23S rRNA operon, 47 tRNA genes, and 9 CRISPR repeat sequences.

The order Sulfolobales includes thermoacidophilic archaea that thrive in acidic geothermal environments. A novel Sulfolobales archaeon strain, HS-7, which may represent a novel genus, was isolated from an acidic hot spring in Japan. We report the 2.15-Mb complete genome sequence of strain HS-7.

The order Sulfolobales includes thermoacidophilic archaea that thrive in acidic geothermal environments. A novel Sulfolobales archaeon strain, HS-7, which may represent a novel genus, was isolated from an acidic hot spring in Japan. We report the 2.15-Mb complete genome sequence of strain HS-7.

Background: Extra-abdominal desmoid tumors often occur in the necks, shoulder, chest wall, back, arm, buttock, thigh and leg. Multicentric extra-abdominal desmoids are rather rare and seem to have other clinical features. The aim of our study was to investigate clinical features, especially multicentric occurrence of extra-abdominal desmoid tumors. Patients and Methods: A total of 135 patients diagnosed with extra-abdominal desmoid were enrolled in this study from January 2005 to December 2019 at the Cancer Institute Hospital of The Japanese Foundation for Cancer Research. The operative procedure was principally wide excision. The clinicopathological factors [e.g., age, gender, pain, restriction of range of motion (ROM), tumor site, tumor size, surgical margin, multicentric occurrence, local recurrence, tumoral regression] were collected and assessed by univariate analysis. We assessed how multicentric occurrence influenced clinicopathological factors of desmoid tumors. Results: The median follow-up was 39.9 months (range=0.29-259 months). Among 135 patients, 20 had multicentric occurrence. Multicentric extra-abdominal desmoids occurred in the neck in six cases, shoulder in four, chest wall in three, back in three, thigh in two and leg in two. In the case of multicentric occurrence on thighs and legs, tumors arose not in the anterior compartment but in the posterior compartment. Univariate analysis showed association of multicentric extra-abdominal desmoids with high local recurrence (p=0.0003), restriction of ROM (p=0.0012) and tumor size larger than 5 cm (p=0.04) but surgical margins were not correlated with local recurrence (p=0.37). Conclusion: Surgery should be performed in those who have severe pain or restriction of ROM. A 'Wait and see' policy is a first-line management, especially for those with multicentric extra-abdominal desmoids.

Several species of Sulfolobales have been used as model organisms in the study of response mechanisms to ultraviolet (UV) irradiation in hyperthermophilic crenarchaea. To date, the transcriptional responses of genes involved in the initiation of DNA replication, transcriptional regulation, protein phosphorylation, and hypothetical function have been observed in Sulfolobales species after UV irradiation. However, due to the absence of knockout experiments, the functions of these genes under in situ UV irradiation have not yet been demonstrated. In the present study, we constructed five gene knockout strains (cdc6-2, tfb3, rio1, and two genes encoding the hypothetical proteins, Saci_0951 and Saci_1302) of Sulfolobus acidocaldarius and examined their sensitivities to UV irradiation. The knockout strains exhibited significant sensitivities to UV-B irradiation, indicating that the five UV-regulated genes play an important role in responses to UV irradiation in vivo. Furthermore, Δcdc6-2, Δrio1, ΔSaci_0951, and Δtfb3 were sensitive to a wide variety of helix-distorting DNA lesions, including UV-induced DNA damage, an intra-strand crosslink, and bulky adducts. These results reveal that cdc6-2, tfb3, rio1, and Saci_0951 are play more important roles in broad responses to helix-distorting DNA damage than in specific responses to UV irradiation.

DNA polymerase B1 (PolB1) is a member of the B-family DNA polymerase family and is a replicative DNA polymerase in Crenarchaea. PolB1 is responsible for the DNA replication of both the leading and lagging strands in the thermophilic crenarchaeon Sulfolobus acidocaldarius. Recently, two subunits, PolB1-binding protein (PBP)1 and PBP2, were identified in Saccharolobus solfataricus. Previous in vitro studies suggested that PBP1 and PBP2 influence the core activity of apoenzyme PolB1 (apo-PolB1). PBP1 contains a C-terminal acidic tail and modulates the strand-displacement synthesis activity of PolB1 during the synthesis of Okazaki fragments. PBP2 modestly enhances the DNA polymerase activity of apo-PolB1. These subunits are present in Sulfolobales, Acidilobales, and Desulfurococcales, which belong to Crenarchaea. However, it has not been determined whether these subunits are essential for the activity of apo-PolB1. In this study, we constructed a pbp1 deletion strain in S. acidocaldarius and characterized its phenotypes. However, a pbp2 deletion strain was not obtained, indicating that PBP2 is essential for replication by holoenzyme PolB1. A pbp1 deletion strain was sensitive to various types of DNA damage and exhibited an increased mutation rate, suggesting that PBP1 contribute to the repair or tolerance of DNA damage by holoenzyme PolB1. The results of our study suggest that PBP1 is important for DNA repair by holoenzyme PolB1 in S. acidocaldarius.

Lignocellulosic biomass comprises cellulose, hemicellulose, and lignin and is a potential source of fuels and chemicals. Although this complex biomass is persistent, it can be cooperatively decomposed by a microbial consortium in nature. In this study, a coculture of the moderately thermophilic bacteria Thermobifida fusca and Ureibacillus thermosphaericus was used for biodegradation of rice chaff. The bacterial strains were incubated in modified Brock’s basal salt medium (pH 8.0) supplemented with yeast extract and rice chaff at 50 °C for 7 days. The concentration of reducing sugars and the enzymatic activities of laccase, lignin peroxidase, cellulase, and xylanase in the supernatant of the culture medium were measured every day. The concentrations of reducing sugars in solo cultures of T. fusca and U. thermosphaericus and a mixed culture of the two strains after 7 days of incubation were 0.047, 0.040, and 0.195 mg/mL, respectively, indicating that the decomposition of rice chaff was enhanced in the coculture. Based on the results, it is thought that the lignin surrounding the cellulose was decomposed by laccase and lignin peroxidase secreted from U. thermosphaericus, resulting in cellulose and hemicellulose in the rice chaff being easily decomposed by enzymes from T. fusca.

Significantly high eicosapentaenoic acid (EPA) and fucoxanthin contents with high production rate were achieved in semi continuous culture of marine diatom. Effects of dilution rate on the production of biomass and high value biocompounds such as EPA and fucoxanthin were evaluated in semi-continuous cultures of Chaetoceros gracilis under high light condition. Cellular dry weight increased at lower dilution rate and higher light intensity conditions, and cell size strongly affected EPA and fucoxanthin contents. The smaller microalgae cells showed significantly higher (p < 0.05) value of 17.1 mg g-dw–1 fucoxanthin and 41.5% EPA content per total fatty acid compared to those observed in the larger cells. Chaetoceros gracilis can accumulate relatively higher EPA and fucoxanthin than those reported previously. In addition, maintenance of small cell size by supplying sufficient nutrients and light energy can be the key for the increase production of valuable biocompounds in C. gracilis.

The thermophilic crenarchaeonSulfolobus acidocaldariushas four DNA polymerases (DNAPs): PolB1, PolB2, PolB3, and Dbh (PolY). Previousin vitrostudies suggested that PolB1 is the main replicative DNAP ofSulfolobaleswhereas PolB2 and Y-family polymerases Dpo4 (Saccharolobus solfataricus) or Dbh are involved in DNA repair and translesion DNA synthesis. On the other hand, there are various opinions about the role of PolB3, which remains to be clearly resolved. In order to examine the roles of the DNAPs ofS. acidocaldariusthroughin vivoexperiments, we constructedpolB2,polB3, anddbhdeletion strains and characterized their phenotypes. Efforts to construct apolB1deletion strain were not successful; in contrast, it was possible to isolate triple gene-deletion strains lackingpolB2,polB3, anddbh. The growth of these strains was nearly the same as that of the parent strains under normal growth conditions. ThepolB2,polB3, anddbhsingle-deletion strains were sensitive to some types of DNA-damaging treatments, but exhibited normal sensitivity to UV irradiation and several other damaging treatments. Overall, the genotype which exhibited the greatest sensitivity to the DNA-damaging treatments we tested was the ΔpolB2ΔpolB3combination, providing the first evidence of overlapping function for these two DNAPsin vivo. The results of our study strongly suggest that PolB1 is responsible for the DNA replication of both the leading and lagging strands and is sufficient to complete the repair of most DNA damage under normal growth conditions inS. acidocaldarius.

The 11th Symposium on Polar Science/Ordinary sessions: [OB] Polar Biology, Poster presentations, Mon. 16 Nov. - Fri. 18 Dec.

The 11th Symposium on Polar Science/Ordinary sessions: [OB] Polar Biology, Poster presentations, Mon. 16 Nov. - Fri. 18 Dec.

The 11th Symposium on Polar Science/Ordinary sessions: [OB] Polar Biology, Poster presentations, Mon. 16 Nov. - Fri. 18 Dec.

The 11th Symposium on Polar Science/Ordinary sessions: [OB] Polar Biology, Poster presentations, Mon. 16 Nov. - Fri. 18 Dec.

The thermophilic crenarchaeon Sulfolobus acidocaldarius has four DNA polymerases (DNAPs): PolB1, PolB2, PolB3, and Dbh (PolY). Previous in vitro studies suggested that PolB1 is the main replicative DNAP of Sulfolobales whereas PolB2 and Y-family polymerases Dpo4 (Saccharolobus solfataricus) or Dbh are involved in DNA repair and translesion DNA synthesis. On the other hand, there are various opinions about the role of PolB3, which remains to be clearly resolved. In order to examine the roles of the DNAPs of S. acidocaldarius through in vivo experiments, we constructed polB2, polB3, and dbh deletion strains and characterized their phenotypes. Efforts to construct a polB1 deletion strain were not successful; in contrast, it was possible to isolate triple gene-deletion strains lacking polB2, polB3, and dbh. The growth of these strains was nearly the same as that of the parent strains under normal growth conditions. The polB2, polB3, and dbh single-deletion strains were sensitive to some types of DNA-damaging treatments, but exhibited normal sensitivity to UV irradiation and several other damaging treatments. Overall, the genotype which exhibited the greatest sensitivity to the DNA-damaging treatments we tested was the ΔpolB2 ΔpolB3 combination, providing the first evidence of overlapping function for these two DNAPs in vivo. The results of our study strongly suggest that PolB1 is responsible for the DNA replication of both the leading and lagging strands and is sufficient to complete the repair of most DNA damage under normal growth conditions in S. acidocaldarius.

A gene encoding a dye-linked d -amino acid dehydrogenase (Dye-DADH) homologue was found in a hyperthermophilic archaeon, Sulfurisphaera tokodaii. The predicted amino acid sequence suggested that the gene product is a membrane-bound type enzyme. The gene was overexpressed in Escherichia coli, but the recombinant protein was exclusively produced as an inclusion body. In order to avoid production of the inclusion body, an expression system using the thermoacidophilic archaeon Sulfolobus acidocaldarius instead of E. coli as the host cell was constructed. The gene was successfully expressed in Sulfolobus acidocaldarius, and its product was purified to homogeneity and characterized. The purified enzyme catalyzed the dehydrogenation of various d -amino acids, with d -phenylalanine being the most preferred substrate. The enzyme retained its full activity after incubation at 90 °C for 30 min and after incubation at pH 4.0–11.0 for 30 min at 50 °C. This is the first report on membrane-bound Dye-DADH from thermophilic archaea that was successfully expressed in an archaeal host.

The Tenth Symposium on Polar Science/Ordinary sessions : [OB] Polar Biology, Wed. 4 Dec. / Entrance Hall (1st floor) , National Institute of Polar Research

The Tenth Symposium on Polar Science/Ordinary sessions : [OB] Polar Biology, Wed. 4 Dec. / Entrance Hall (1st floor) , National Institute of Polar Research

A novel hyperthermophilic archaeon of strain HS-3T, belonging to the family Sulfolobaceae , was isolated from an acidic terrestrial hot spring in Hakone Ohwaku-dani, Japan. Based on 16S rRNA gene sequence analysis, the closest phylogenetic relatives of strain HS-3T were, first, Sulfolobus solfataricus (96.4 %) and, second, Sulfolobus shibatae (96.2 %), indicating that the strain belongs to the genus Sulfolobus . However, the sequence similarity to the type species of the genus Sulfolobus ( Sulfolobus acidocaldarius ) was remarkably low (91.8 %). In order to determine whether strain HS-3T belongs to the genus Sulfolobus , its morphological, biochemical and physiological characteristics were examined in parallel with those of S. solfataricus and S. shibatae . Although there were some differences in chemolithotrophic growth between strain HS-3T, S. solfataricus and S. shibatae , their temperature, pH and facultatively anaerobic characteristics of growth, and their utilization of various sugars were almost identical. In contrast, the utilization of various sugars by S. acidocaldarius was quite different from that of HS-3T, S. solfataricus and S. shibatae . Phylogenetic evidence based on the 16S and the 23S rRNA gene sequences also clearly distinguished the monophyletic clade composed of strain HS-3T, S. solfataricus , and S. shibatae from S. acidocaldarius . Based on these results, we propose a new genus and species, Saccharolobus caldissimus gen. nov., sp. nov., for strain HS-3T, as well as two reclassifications, Saccharolobus solfataricus comb. nov. and Saccharolobus shibatae comb. nov. The type strain of Saccharolobus caldissimus is HS-3T (=JCM 32116T and InaCC Ar80T). The type species of the genus is Saccharolobus solfataricus.

To develop an effective treatment for the globally invasive Brazilian waterweed Egeria densa , anaerobic digestion was observed at 37 °C, 55 °C, and 65 °C. The average methane production rate at 55 °C was 220 mL L −1 day −1 , which was two-fold that at 37 °C and 65 °C. Volatile fatty acid accumulation was detected under thermophilic conditions; however, although there was methane production, the system did not shutdown. The microbial communities differed between mesophilic (37 °C) and thermophilic (55 °C and 65 °C) conditions. A bacterial community consisting of the phyla Bacteroidetes (43%), Firmicutes (37%), Proteobacteria (9%), Synergistetes (5%), Spirochaetes (1%), and unclassified bacteria (5%) were detected under mesophilic condition. In contrast, the phylum Firmicutes was dominant under thermophilic conditions. In the archaeal community, Methanosaeta concilii (40%), Methanolinea sp. (17%), and unclassified euryarchaeota (43%) were detected under mesophilic condition. Methanosarcina thermophila (87% at 55 °C, 54% at 65 °C) and Methanothermobacter thermautotrophicus (13% at 55 °C, 46% at 65 °C) were detected under thermophilic conditions. At both 37 °C and 55 °C, acetoclastic methanogenesis likely occurred because of the lower abundance of hydrogenotrophic methanogens. At 65 °C, the growth of the acetoclastic methanogen Methanosarcina thermophila was limited by the high temperature, therefore, acetate oxidation and hydrogenotrophic methanogenesis may have occurred.

Despite being an extreme environment, the water temperature of freshwater lakes in Antarctica reaches 10 °C in summer, accelerating biological activity. In these environments, proteolytic microbial decomposers may play a large role in protein hydrolysis. We isolated 71 microbial strains showing proteolytic activity at 4 °C from three Antarctic freshwater lakes. They were classified as bacteria (63 isolates) and eukaryotes (8 isolates). The bacterial isolates were classified into the genera Flavobacterium (28 isolates), Pseudomonas (14 isolates), Arthrobacter (10 isolates), Psychrobacter (7 isolates), Cryobacterium (2 isolates), Hymenobacter (1 isolate), and Polaromonas (1 isolate). Five isolates of Flavobacterium and one of Hymenobacter seemed to belong to novel species. All eukaryotic isolates belonged to Glaciozyma antarctica , a psychrophilic yeast species originally isolated from the Weddell Sea near the Joinville Island, Antarctica. A half of representative strains were psychrophilic and did not grow at temperatures above 25 °C. The protease secreted by Pseudomonas prosekii strain ANS4-1 showed the highest activity among all proteases from representative isolates. The results of inhibitor tests indicated that nearly all the isolates secreted metalloproteases. Proteases from four representative isolates retained more than 30% maximal activity at 0 °C. These results expand our knowledge about microbial protein degradation in Antarctic freshwater lakes.

A novel thermoacidophilic archaeon, strain HS-1T, was isolated from the Hakone Ohwaku-dani hot spring in Japan. Cells of strain HS-1T in exponential phase were cocci to irregular cocci with a diameter of 0.8–1.5 µm. The strain grew within a temperature range of 50–70 °C (optimal: 65–70 °C), a pH range of pH 1.4–5.5 (optimal: pH 3.0–3.5) and a NaCl concentration range of 0–2.5 % (w/v). The novel strain grew in aerobic conditions but did not grow anaerobically. Moreover, this strain utilized various complex substrates (beef extract, casamino acids, peptone, tryptone and yeast extract) and sugars (arabinose, xylose, galactose, glucose, maltose, sucrose, raffinose and lactose) as sole carbon sources. No chemolithoautotrophic growth occurred on elemental sulfur, pyrite, K2S4O6, Na2S2O3 or FeSO4 . 7H2O; however, growth by the oxidation of hydrogen occurred weakly. The core lipids were calditoglycerocaldarchaeol (CGTE) and caldarchaeol (DGTE). The DNA G+C content of the strain was 52.0 mol%, which was remarkably higher than those of known species of the order Sulfolobales (31–46.2 %). The growth of the strain was significantly inhibited in the presence of elemental sulfur. Analyses of 16S rRNA and 23S rRNA gene sequences showed that HS-1T belonged to the order Sulfolobales ; however, it was distantly related to all known species of the order Sulfolobales (less than 89 % sequence similarity). On the basis of these results, we propose the novel genus, Sulfodiicoccus, in the order Sulfolobales (in the family Sulfolobaceae ). The type species of the genus is Sulfodiicoccus acidiphilus sp. nov., and the type strain of the species is HS-1T (=JCM 31740T=InaCC Ar79T).

Multiple gene knockout systems developed in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius are powerful genetic tools. However, plasmid construction typically requires several steps. Alternatively, PCR tailing for high-throughput gene disruption was also developed in S. acidocaldarius, but repeated gene knockout based on PCR tailing has been limited due to lack of a genetic marker system. In this study, we demonstrated efficient homologous recombination frequency (2.8 × 104 ± 6.9 × 103 colonies/μg DNA) by optimizing the transformation conditions. This optimized protocol allowed to develop reliable gene knockout via double crossover using short homologous arms and to establish the multiple gene knockout system with one-step PCR (MONSTER). In the MONSTER, a multiple gene knockout cassette was simply and rapidly constructed by one-step PCR without plasmid construction, and the PCR product can be immediately used for target gene deletion. As an example of the applications of this strategy, we successfully made a DNA photolyase- (phr-) and arginine decarboxylase- (argD-) deficient strain of S. acidocaldarius. In addition, an agmatine selection system consisting of an agmatine-auxotrophic strain and argD marker was also established. The MONSTER provides an alternative strategy that enables the very simple construction of multiple gene knockout cassettes for genetic studies in S. acidocaldarius.

To maximize the production rate of Chlorella zofingiensis, a lumostatic continuous culture was operated under light intensities of 250–1510 μE m− 2 s− 1. The cell density and volumetric biomass production rate were increased without photo inhibition and reached 13.5 g-dry weight (dw) L− 1 on day 21.5 and 2.41 g-dw L− 1 day− 1 on day 10.5, respectively. These maximum values were higher than any previous photoautotrophic culture study with C. zofingiensis. The specific growth rate was maintained at a high level > 0.5 day− 1 until the light intensity per dry weight decreased below 28 μE g-dw− 1 s− 1, which coincided with the value estimated in our previous study, verifying the reliability of this estimated value. There was a strong relationship between the photosynthetic efficiency and light intensity per dry weight for C. zofingiensis. This relationship may be useful for evaluating species-specific productivity to select productive species.

Objective Sulfodiicoccus acidiphilus HS-1T is the type species of the genus Sulfodiicoccus, a thermoacidophilic archaeon belonging to the order Sulfolobales (class Thermoprotei; phylum Crenarchaeota). While S. acidiphilus HS-1T shares many common physiological and phenotypic features with other Sulfolobales species, the similarities in their 16S rRNA gene sequences are less than 89%. In order to know the genomic features of S. acidiphilus HS-1T in the order Sulfolobales, we determined and characterized the genome of this strain. Results The circular genome of S. acidiphilus HS-1T is comprised of 2353,189 bp with a G+C content of 51.15 mol%. A total of 2459 genes were predicted, including 2411 protein coding and 48 RNA genes. The notable genomic features of S. acidiphilus HS-1T in Sulfolobales species are the absence of genes for polB3 and the autotrophic carbon fixation pathway, and the distribution pattern of essential genes and sequences related to genomic replication initiation. These insights contribute to an understanding of archaeal genomic diversity and evolution.

Sulfide inhibition to nitrifying bacteria has prevented the integration of digestate nitrification and biogas desulfurization to simplify anaerobic digestion systems. In this study, liquid digestate with NaHS solution was treated using nitrifying sludge in a sequential-batch reactor with a long fill period, with an ammonium loading rate of 293 mg-N L−1 d−1 and a stepwise increase in the sulfide loading rate from 0 to 32, 64, 128, and 256 mg-S L−1 d−1. Batch bioassays and microbial community analysis were also conducted with reactor sludge under each sulfide loading rate to quantify the microbial acclimatization to sulfide. In the reactor, sulfide was completely removed. Complete nitrification was maintained up to a sulfide load of 128 mg-S L−1 d−1, which is higher than that in previous reports and sufficient for biogas treatment. In the batch bioassays, the sulfide tolerance of NH4+ oxidizing activity (the 50% inhibitory sulfide concentration) increased fourfold over time with the compositional shift of nitrifying bacteria to Nitrosomonas nitrosa and Nitrobacter spp. However, the sulfur removal rate of the sludge slightly decreased, although the abundance of the sulfur-oxidizing bacteria Hyphomicrobium increased by 30%. Therefore, nitrifying sludge was probably acclimatized to sulfide not by the increasing sulfide removal rate but rather by the increasing nitrifying bacteria, which have high sulfide tolerance. Successful simultaneous nitrification and desulfurization were achieved using a sequential-batch reactor with a long fill period, which was effective in facilitating the present acclimatization.

Canonical single-stranded DNA-binding proteins (SSBs) are universally conserved helix-destabilizing proteins that play critical roles in DNA replication, recombination and repair. Many biochemical and genetic studies have demonstrated the importance of functional SSBs for all life forms. Herein, we report successful deletion of the gene encoding the only canonical SSB of the thermophilic crenarchaeon Sulfolobus acidocaldarius. Genomic sequencing of the ssb-deficient strain using illumina sequencing revealed that the canonical ssb gene is completely deleted from the genome of S. acidocaldarius. Phenotypic characterization demonstrated robust growth of the thermophilic archaeal cells lacking a canonical SSB, thereby demonstrating tolerance to the loss of a universal protein that is generally considered to be essential. Therefore, our work provides evidence that canonical SSBs are not essential for all life forms. Furthermore, on the basis of universal distribution and essentiality pattern of canonical SSBs, our findings can provide a conceptual understanding of the characteristics of early life forms before the last universal common ancestor.

The DNA repair mechanisms of hyperthermophiles can provide important insights for understanding how genetic information is maintained under extreme environments. Recent biochemical studies have identified a novel endonuclease in hyperthermophilic archaea, NucS/EndoMS, that acts on branched DNA substrates and mismatched bases. NucS/EndoMS is thought to participate in the DNA repair of helix-distorting DNA lesions, including UV-induced DNA damage and DNA adducts, and mismatched bases; however, the specific in vivo role of NucS/EndoMS in hyperthermophilic archaeal DNA repair has not been reported. To explore the role of this protein, we knocked out the nucS/endoMS gene of the thermophilic crenarchaeon Sulfolobus acidocaldarius and characterized the mutant phenotypes. While the nucS/endoMS-deleted strain exhibited sensitivity to DNA adducts, it did not have high mutation rates or any sensitivity to UV irradiation. It has been proposed that the XPF endonuclease is involved in homologous recombination-mediated stalled-fork DNA repair. The xpf-deficient strain exhibited sensitivity to helix-distorting DNA lesions, but the sensitivity of the nucS/endoMS and xpf double knockout strain did not increase compared to that of the single knockout strains. We conclude that the endonuclease NucS/EndoMS works with XPF in homologous recombination-mediated stalled-fork DNA repair for the removal of helix-distorting DNA lesions in S. acidocaldarius.

Objective Sulfodiicoccus acidiphilus HS-1T is the type species of the genus Sulfodiicoccus, a thermoacidophilic archaeon belonging to the order Sulfolobales (class Thermoprotei; phylum Crenarchaeota). While S. acidiphilus HS-1T shares many common physiological and phenotypic features with other Sulfolobales species, the similarities in their 16S rRNA gene sequences are less than 89%. In order to know the genomic features of S. acidiphilus HS-1T in the order Sulfolobales, we determined and characterized the genome of this strain. Results The circular genome of S. acidiphilus HS-1T is comprised of 2353,189 bp with a G+C content of 51.15 mol%. A total of 2459 genes were predicted, including 2411 protein coding and 48 RNA genes. The notable genomic features of S. acidiphilus HS-1T in Sulfolobales species are the absence of genes for polB3 and the autotrophic carbon fixation pathway, and the distribution pattern of essential genes and sequences related to genomic replication initiation. These insights contribute to an understanding of archaeal genomic diversity and evolution. Electronic supplementary material The online version of this article (10.1186/s13104-019-4488-5) contains supplementary material, which is available to authorized users.