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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.

The Ninth Symposium on Polar Science/Special session: [S] Antarctic ice-ocean interaction ～observation, reconstruction, and modeling～, Tue. 4 Dec. / Entrance Hall (1st floor) at National Institute of Polar Research

An isolation strategy, exploring novel microorganisms in frozen enrichment cultures (ENFE), which uses a combination of enrichment culture and 16S rRNA gene clone analysis, was evaluated for isolating uncultured thermophiles from a terrestrial acidic hot spring. The procedure comprised (a) multiple enrichment cultures under various conditions, (b) cryostorage of all enrichments, (c) microbial community analyses of the enrichments using 16S rRNA gene sequences, and (d) purification of microorganisms from enrichments containing previously uncultured microorganisms. The enrichments were performed under a total of 36 conditions, and 16 of these enrichments yielded positive microbial growth with the detection of three previously uncultured archaea. Two of the three previously uncultured archaea, strains HS-1 and HS-3, were successfully isolated. Strain HS-1 and HS-3 represented a novel lineage of the order Sulfolobales and novel species of the genus Sulfolobus, respectively. Although innovative isolation methods play strategic roles in isolating previously uncultured microorganisms, the ENFE strategy showed potential for characterizing and isolating such microorganisms using conventional media and techniques.

A novel hyperthermophilic, acidophilic and facultatively anaerobic archaeon, strain KD-1T, was isolated from an acidic hot spring in Indonesia and characterized with the phylogenetically related species Sulfurisphaera ohwakuensis Kurosawa et al. 1998, Sulfolobus tokodaii Suzuki et al., 2002 and Sulfolobus yangmingensis Jan et al. 1999. Cells of KD-1T were irregular cocci with diameters of 0.9–1.3 µm. The strain grew at 60–90 °C (optimum 80–85 °C), pH 2.5–6.0 (optimum pH 3.5–4.0) and 0–1.0 % (w/v) NaCl concentration. KD-1T grew anaerobically in the presence of S0 (headspace: H2/CO2) and FeCl3 (headspace: N2). Under aerobic conditions, chemolithoautotrophic growth occurred on S0, pyrite, K2S4O6, Na2S2O3 and H2. This strain utilized various complex substrates, such as yeast extract, but did not grow on sugars and amino acids as the sole carbon source. The main core lipids were calditoglycerocaldarchaeol and caldarchaeol. The DNA G+C content was 30.6 mol%. Analyses of phylogenetic trees based on 16S rRNA and 23S rRNA genes indicated that KD-1T formed an independent lineage near Sulfurisphaera ohwakuensis TA-1T, Sulfolobus tokodaii 7T and Sulfolobus yangmingensis YM1T. On the basis of the results of morphological, physiological, chemotaxonomic and phylogenetic analyses, KD-1T represents a novel species of the genus Sulfurisphaera Kurosawa et al. 1998, for which the name Sulfurisphaera javensis sp. nov. is proposed. The type strain is KD-1T (=JCM 32117T=InaCC Ar81T). Based on the data, we also propose the reclassification of Sulfolobus tokodaii Suzuki et al., 2002 as Sulfurisphaera tokodaii comb. nov. (type strain 7T=JCM 10545T=DSM 16993T).

BACKGROUND AND AIMS: All photosynthetic organisms are faced with photoinhibition, which would lead to death in severe environments. Because light quality and light intensity fluctuate dynamically in natural microenvironments, quantitative and qualitative analysis of photoinhibition is important to clarify how this environmental pressure has impacted ecological behaviour in different organisms. METHODS: We evaluated the wavelength dependency of photoinactivation to photosystem II (PSII) of Prasiola crispa (green alga), Umbilicaria decussata (lichen) and Ceratodon purpureus (bryophyte) harvested from East Antarctica. For evaluation, we calculated reaction coefficients, E(pi)s, of PSII photoinactivation against energy dose using a large spectrograph. Daily fluctuation of the rate coefficient of photoinactivation, k(pi), was estimated from E(pi)s and ambient light spectra measured during the summer season. KEY RESULTS: Wavelength dependency of PSII photoinactivation was different for the three species, although they form colonies in close proximity to each other in Antarctica. The lichen exhibited substantial resistance to photoinactivation at all wavelengths, while the bryophyte showed sensitivity only to UV-B light (

The long-term effect of alkaline pretreatment on semi-continuous anaerobic digestion (AD) of the lignin-rich submerged macrophyte Potamogeton maackianus was investigated using mesophilic and thermophilic conditions. In pretreated reactors, dissolved lignin accumulated to high levels. CH4 production under the pretreated condition was higher than that of the untreated condition, but decreased from Days 22 (mesophilic) and 42 (thermophilic). However, CH4 production subsequently recovered, although dissolved lignin accumulated. Further, the change in the microbial community was observed between conditions. These results suggest that dissolved lignin temporarily inhibited AD, although acclimatization to dissolved lignin occurred during long-term operation. During the steady state period, mesophilic conditions achieved a 42% increase in the CH4 yield using pretreatment, while thermophilic conditions yielded an 8% increment. Because volatile fatty acids accumulated even after acclimatization during the thermophilic pretreated condition and was discharged with the effluent, improvement of the methanogenic step would enable enhanced CH4 recovery.

In this study, we investigated the clinical effects of long-term administration of the phosphorus (P) binder lanthanum carbonate (LC), which was launched in Japan in 2009. The subjects were 58 dialysis patients who began receiving LC, and we evaluated the clinical effects for up to 36 months after treatment initiation. The average serum P concentration remained low during the 36-month study period, with a significant reduction from 6.25 mg/dL at the start of the study to 4.94 mg/dL after 36 months (P < 0.001). A significant reduction was also observed in the average serum calcium concentration after 36 months (P < 0.05), but not in the serum intact parathyroid hormone concentration. Significant reductions were also observed in the average serum total protein, albumin and potassium concentrations (P < 0.05). The dosages of LC increased by approximately 1.9-fold after 36 months, in contrast, the dosages of concomitantly used sevelamer hydrochloride and Ca carbonate preparations decreased. These results indicate that LC could be used to treat hyperphosphatemia without causing hypercalcemia, and would be useful for long-term treatment with hemodialysis patients.

The distribution of protistan microplankton situated southwest (S09) and northeast (S18) of the Izu Ridge along the Kuroshio Current was revealed by 18S rRNA gene clone analysis. A total of 257 clones were identified, consisting of 65 phylotypes of dinoflagellates, 49 phylotypes of diatoms and 57 phylotypes of other protists affiliated with Ciliophora, Cryptophyta, Cryptophyta nucleomorph, Choanoflagellata, Chlorophyta, Cercozoa, and Hetero- konta. The dinoflagellate phylotypes were affiliated with five genera and 14 uncultured groups, with Gyrodinium as the most frequently detected genus. The diatoms were also well represented and consisted of 13 genera and six uncul- tured groups. The clones belonging to the genus Pseudo-nitzschia were most frequently detected. The frequencies of dinoflagellate clones and phylotypes were higher at station S09 in the south than at station S18 to the north, with the frequency of diatom phylotypes being higher at the latter. The species richness (number of phylotypes) and diversity (Shannon-Wiener) of the protistan microplankton community were slightly higher at S18 compared to S09. When the Kuroshio Current encountered with the Oyashio Current at northwestern Pacific, it affects the water temperature and nutrients of the Kuroshio Current. The clone analysis results showed a difference in the protistan microplankton com- munity at both stations due to the collision of both currents.

Since most studies about community structures of microorganisms in Antarctic terrestrial lakes using molecular biological tools are mainly focused on bacteria, limited information is available about archaeal and eukaryotic microbial diversity. In this study, the biodiversity of microorganisms belonging to all three domains in a typical Antarctic freshwater glacier lake (Yukidori-Ike) was revealed using small subunit ribosomal RNA (SSU rRNA) clone library analysis. The bacterial clones were grouped into 102 operational taxonomic units (OTUs) and showed significant biodiversity. Betaproteobacteria were most frequently detected, followed by Cyanobacteria, Bacteroidetes, and Firmicutes as major lineages. In contrast to the bacterial diversity, much lower archaeal diversity, consisting of only two OTUs of methanogens, was observed. In the eukaryotic microbial community consisting of 20 OTUs, Tardigradal DNA was remarkably frequently detected. Genera affiliated with the phyla Ciliophora, Cryptomycota, Chlorophyta, Bacillariophyta, and Apusozoa were also detected. The biodiversity and species compositions of the whole microbial community of Lake Yukidori-Ike are similar to those of freshwater environments in temperate regions but are different from saline lakes in Antarctica, indicating that the salinity seems to affect the microbial composition more than the temperature.

We previously revealed that endogenous bone morphogenetic protein (Bmp) activity is required for lipid accumulation in 3T3-L1 adipocytes. The present study characterized the role of endogenous Bmp activity in preadipocytes. Endogenous Bmp activity was monitored by analyzing the level of phosphorylation of Smad1/5/8, downstream molecules in the Bmp pathway. Higher levels of phosphorylated Smad1/5/8 were detected in adipogenic cells but not in non-adipogenic cells prior to differentiation induction. The inhibition of the Bmp pathway during this period decreased the expression of Pparγ2 and C/ebpα, which are transcription factors responsible for adipocyte differentiation. The expression of these transcription factors were also down-regulated by Bmp4 knockdown. In addition, endogenous Bmp4 was required for the repression of Intrleukin-11 expression. Endogenous Bmp4 in preadipocytes is indispensable for the onset of the adipogenic program, and may help to maintain the preadipocytic state during adipocyte differentiation.

A rod-shaped, endospore-forming, Gram-reaction-positive bacterium, designated strain TC22-2bT, was isolated from compost in Tochigi, Japan. Phylogenetic analysis based on 16S rRNA gene sequences showed that the strain belonged to a cluster comprising species of the genus Paenibacillus and was most closely related to the type strain of Paenibacillus elgii (93.4 % similarity). The major cellular fatty acids were C16:0 (25.5 %), iso-C16:0 (23.6 %) and anteiso-C15:0 (21.5 %). The predominant menaquinone was MK-7. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The diamino acid found in the cell wall peptidoglycan was meso-diaminopimelic acid, and the DNA G+C content was 59.1 mol%. The results of physiological and biochemical tests enabled the phenotypic differentiation of strain TC22-2bT from the most closely related species with validly published names. Phylogenetic and phenotypic evidence reveals that strain TC22-2bT represents a novel species of the genus Paenibacillus , for which the name Paenibacillus thermoaerophilus sp. nov. is proposed. The type strain of the novel species is TC22-2bT ( = DSM 26310T = JCM 18657T).

Ibusuki hot spring is located on the coastline of Kagoshima Bay, Japan. The hot spring water is characterized by high salinity, high temperature, and neutral pH. The hot spring is covered by the sea during high tide, which leads to severe fluctuations in several environmental variables. A combination of molecular- and culture-based techniques was used to determine the bacterial and archaeal diversity of the hot spring. A total of 48 thermophilic bacterial strains were isolated from two sites (Site 1: 55.6°C; Site 2: 83.1°C) and they were categorized into six groups based on their 16S rRNA gene sequence similarity. Two groups (including 32 isolates) demonstrated low sequence similarity with published species, suggesting that they might represent novel taxa. The 148 clones from the Site 1 bacterial library included 76 operational taxonomy units (OTUs; 97% threshold), while 132 clones from the Site 2 bacterial library included 31 OTUs. Proteobacteria, Bacteroidetes, and Firmicutes were frequently detected in both clone libraries. The clones were related to thermophilic, mesophilic and psychrophilic bacteria. Approximately half of the sequences in bacterial clone libraries shared

Archaeal 16S rRNA gene compositions and environmental factors of four distinct solfataric acidic hot springs in Kirishima, Japan were compared. The four ponds were selected by differences of temperature and total dissolved elemental concentration as follows: (1) Pond-A: 93°C and 1679 mg L−1, (2) Pond-B: 66°C and 2248 mg L−1, (3) Pond-C: 88°C and 198 mg L−1, and (4) Pond-D: 67°C and 340 mg L−1. In total, 431 clones of 16S rRNA gene were classified into 26 phylotypes. In Pond-B, the archaeal diversity was the highest among the four, and the members of the order Sulfolobales were dominant. The Pond-D also showed relatively high diversity, and the most frequent group was uncultured thermoacidic spring clone group. In contrast to Pond-B and Pond-D, much less diverse archaeal clones were detected in Pond-A and Pond-C showing higher temperatures. However, dominant groups in these ponds were also different from each other. The members of the order Sulfolobales shared 89% of total clones in Pond-A, and the uncultured crenarchaeal groups shared 99% of total Pond-C clones. Therefore, species compositions and biodiversity were clearly different among the ponds showing different temperatures and dissolved elemental concentrations.

The diversity of protistan microplankton in Sagami Bay was revealed by 18S rRNA gene clone analysis using newly designed PCR primers. PCR amplification consisted of a first reaction targeting the V3–V5 region of the 18S rRNA gene, followed by a nested reaction targeting the V3–V4 region. In total, 629 clones consisting of 108 phylotypes were affiliated with a variety of protistan groups including dinoflagellates, diatoms, prymnesiomonada, chlorophyta, ciliophora, cercozoa, and heterokonta. The dinoflagellate group was detected most frequently and shared approximately 74 % of the total clones. Within this group, approximately half of the clones belonged to the parasitic dinoflagellate Syndiniales group I, which was first reported from Sagami Bay. The genera Woloszynskia, Gonyaulax, Neoceratium, and Karlodinium have not been reported from this bay until now. The second most frequent group was diatoms, which shared approximately 22 % of the total clones. Within this group, highly diverse Thalassiosira phylotypes were detected, and they shared approximately 70 % of the diatom clones. Therefore, highly diverse protists including some candidate groups were successfully detected, indicating that the designed primers and PCR protocol will be useful for molecular diversity analyses of protistan microplankton communities in aquatic environments.

The patient was a 76-year-old male who developed nephrotic syndrome. Idiopathic membranous nephropathy was diagnosed by renal biopsy and clinical findings. The patient had been refractory to predonisolone and cyclosporine A therapies, and overhydration recurred repeatedly during the clinical course. One year after an initial hospitalization, he had to be hospitalized a second time because of overhydration. During the hospitalization, he underwent fluid removal by the extracorporeal ultrafiltration method (ECUM), as his response to diuretics was too weak to permit the control of cardiac insufficiency. The ECUM alleviated his overhydration, but no remission of nephrotic syndrome was achieved. The patient was then discharged temporarily, but overhydration developed again 2 months later. Peritoneal dialysis (PD) using an overnight dwell of a single dose of icodextrin was initiated to obtain stable fluid removal. This promptly alleviated the refractory subcutaneous edema, and type I incomplete remission of nephrotic syndrome was achieved about 2 weeks after the start of PD. The patient could be withdrawn from the PD therapy 4 months later. Subsequently, the urinary volume was maintained and the serum creatinine level was stabilized at about 2 mg/dl. In our patient, the protein leakage into the drainage was small enough to permit remission of the nephrotic syndrome with stable fluid removal. On this basis, we believe that PD using icodextrin is considered as one of the options for the treatment of refractory nephrotic syndrome with poor water control.

The diversity and short-term changes in the protistan microplankton community from April to June 2006 in Sagami Bay were revealed by 18S rRNA gene clone analysis. A total of 1,076 clones consisted of 68 phylotypes of dinoflagellates, 96 phylotypes of diatoms and 27 phylotypes of other protists affiliated with Ciliophora, Prymnesio- monada, Chlorophyta, Cercozoa, Chytridiomycota, and Heterokonta. Approximately half of all dinoflagellate phylo- types were affiliated with the following genera: Ceratium, Gonyaulax, Gymnodinium, Gyrodinium, Lepidodinium, Neoceratium, Prorocentrum, and Woloszynskia. The other half was classified into seven uncultured groups. These di- noflagellate clones were mostly detected in May, in contrast to the diatom clones, which were detected frequently throughout the study period. Diatoms were diverse and consisted of 14 genera and three uncultured groups. The gen- era Discostella, Thalassiosira and Skeletonema were dominant in April, May and June, respectively. Species richness (number of phylotypes) and diversity (Shannon-Weiner) of the whole protistan microplankton community were high- est in May. This is the first example of a comprehensive molecular biological analysis of protistan microplankton community structure, and the results clearly showed a dynamic shift in the protistan community in coastal waters from April to June in Sagami Bay. The results of a direct comparison between the clone analysis and microscopic ob- servations indicated that the clone analysis had the great advantage of enabling identification of plankton that were morphologically indistinguishable, and to reveal detailed information on the biodiversity of protistan microplankton. This advancement in molecular biological analysis will assist in our understanding of the biodiversity of protistan mi

Twelve strains of moderately thermophilic bacteria were isolated from saline hot springs (55°C, pH 8.3) located in Odaito, Hokkaido, Japan. Based on their 16S rRNA gene sequences, all the strains were closely related phylogenetically each other, indicating that the strains belong to a single species. However, maximal growth temperature and enzymatic characteristics of individual strains were slightly different each other. Some of them grew well at 55°C but others did not. The β-galactosidase activity was also different among the strains. Therefore, the representative strains BEK6 and BEK11 were chosen from individual phenotype groups, and were used for further characterization. The nucleotide sequence of full-length 16S rRNA genes of the representative strains showed 96.6% similarity with Bacillus alveayuensis indicating that the strains belong to a novel species of the genus Bacillus . The cells were Gram positive, and showed both catalase and oxidase activities. The optimal growth temperature and pH of strain BEK6 and BEK11 were around 50°C and 7-8, respectively. They were able to grow in the medium containing 10% NaCl in contrast to B. alveayuensis which can grow in the medium with up to 4% NaCl. The strain BEK11 showed relatively strong protease and amylase activities implying the potential industrial uses of these enzymes under the saline condition.

We exposed mesophilic up-flow anaerobic sludge blanket (UASB) reactors to high concentrations of NaCl to elucidate the saline adaptation capacity of their granular sludge. We operated 10 lab-scale UASB reactors at 37 °C and added NaCl to the influent either abruptly or gradually. With abrupt addition, NaCl concentrations were increased from 0 g L−1 to 20, 30, 35, 40, 45, or 50 g L−1. With gradual addition, the NaCl concentrations were gradually increased from 0 to 64 g L−1 or 0 to 40 g L−1. We successfully saline-adapted the granules up to 32 g NaCl L−1, while maintaining high reactor performance, suggesting that 32 g NaCl L−1 is a practical level for system operation. In the UASB reactors gradually exposed to 32 g L−1 NaCl, methane production decreased by only 13%. We also learned that combining abrupt and gradual salinity increases could shorten the adaptation period. Thus we were able to shorten the adaptation period to only 30 days by increasing the salinity abruptly to 20 g L−1, followed by gradual adaptation to 30 g NaCl L−1.

Prokaryotic community structures in the anoxic sediment of the Antarctic meromictic Lake Nurume-Ike were revealed by sequence analysis of 16S rRNA gene clones. The archaeal clones obtained (205 total) consisted of only three phylotypes, and were dominantly affiliated with uncultured euryarchaeotes. Specifically, 93% of the clones were identified as marine benthic group-D archaeal phylotype. In contrast to the limited archaeal diversity, 53 phylotypes were detected within 312 bacterial clones. Major bacterial phylotypes were affiliated with α-Proteobacteria (20% of clones), d-Proteobacteria (9%), Planctmycetales (7%), and Cyanobacteria (7%). A small numbers of clones belonging to γ-Proteobacteria, Actinobacteria, Spirochaetes, Flavobacteria, and Verrucomicrobia were also found. A total of 53% of the bacterial clones, consisting of 13 phylotypes, could not be classified into any known group. These results indicated that the bacterial community of Lake Nurume-Ike sediment consisted of numerous phylogenetic groups and had a diversity comparable to the diversity of other Antarctic lakes communities previously reported. Interestingly, however, there were very few phylotypes shared between the communities of lakes Nurume-Ike and five other lakes located in the Vestfold Hills area. This is the first comprehensive study to analyze more than 500 16S rDNA clones for microbial community analysis of an Antarctic lake sediment sample, and the results significantly expand current views of bacterial diversity in Antarctic lakes.

Composting is an efficient and cost-effective process for organic waste treatment. In order to expand our knowledge regarding microorganisms and their roles in the composting process, bacterial community structures in the personal-use composting reactor were examined by isolation and 16S rDNA clone analysis (cultivation-independent method). The results of 16S rDNA clone analysis showed that populations of the Bacillaceae family (such as Bacillus spp., Cerasibacillus spp., Gracilibacillus spp.), dominate (98%). By using cultivation method, a total of four species including one novel species (Ureibacillus thermosphaericus, Geobacillus thermoglucosidasius, G. toebii and Thermobacillus composti) were isolated, and were classified into the order Bacillales corresponding to the result of 16S rDNA clone analysis. However, most species detected by clone analysis have not been cultivated, and may be viable but non-culturable VBNC species implying symbiotic interactions among the microorganisms.

Bacterial community succession in the start-up of a large-scale, completely-mixed composting reactor was analyzed by 16S rRNA gene (16S rDNA) clone analysis and denaturing gradient gel electrophoresis (DGGE) combined with measurements of temperature, pH, moisture contents, and decomposing rate. DGGE analysis and physicochemical parameters showed that bacterial community succession occurred in four phases; (1) at the start of operation and pH decreasing period (day 0–3), (2) pH decreased and increased period (day 4–11), (3) middle term, moisture content decreasing and maximum temperature increased period (day 12–16) and (4) latter term, temperature decreasing period (day 17–24). Lactobacillus spp. and Bacillus coagulans were detected from the initial phase and middle term, respectively. 16S rDNA clone analysis showed that the dominant bacteria shifted from the order “Lactobacillales” to Bacillales and Actinomycetales. The order “Lactobacillales” was unique which may be caused by using the plastic bottle flakes (polyethylene terephthalate, PET) as bulking agent.

The contents of fed-batch composting (FBC) reactors often aggregate after prolonged operation. This process leads to irreversible breakdown of the decomposition reaction and possible alteration of the bacterial communities. We compared the structures of bacterial communities in reactors under aggregate and optimal conditions. The results of 16S rRNA gene clone analysis showed that populations of the family Bacillaceae (such as Bacillus spp., Cerasibacillus spp., Gracilibacillus spp.), which dominate (98%) under optimal condition, were significantly decreased under aggregate condition. In contrast, populations of the family Staphylococcaceae considerably increased after aggregation and accounted for 53% of the total. Phylogenetic analysis also showed that anaerobes or facultative anaerobes related to Tetragenococcus halophilus, Atopostipes suicloacalis, Jeotgalicoccus pinnipedialis, and Staphylococcus spp. were dominant in the aggregates. These results suggested that aerobic Gram-positive bacteria mainly contributed to organic degradation and that aggregation created some anaerobic environment, which promoted the growth of bacterial communities usually not found in well-functioning FBC reactors.

Long term fed-batch composting experiments were conducted for 200 days using two types of bulking agents; woodchip and PET flake, with periodic compost withdrawal through a washing process. The bacterial communities of composting materials in the two different bulking agents were also investigated by 16S rRNA gene clone analysis. The decomposition rate in both composting reactors was 86.1% and 88.2% of the total organic load, respectively. The control experiment of dead-end operation without compost withdrawal by washing process could not be maintained for more than 102 days because of its low performance. The reactor with compost withdrawal, however, improved the decomposition rate in the composting process, and could be applied in the long run. There was a significant difference in the bacterial community between the FBC reactor with woodchip and another with PET flake as the bulking agent though the decomposition rates were similar. The reactor with woodchip as the bulking agent consisted of 95% Bacillales while the PET flake reactor contained 54% of total bacteria count. In addition, Lactobacillales was dominant at 38% in the PET flake reactor and the bacterial community in general significantly differed from the woodchip reactor. Furthermore, there was a difference in the species composition in the Bacillales and the bacterial community showed a significant difference at the species level between the two reactors. Although bacterial community differed, the decomposition rates between the two reactors were similar and PET flake showed greater viability than woodchip as a bulking agent due to its high abrasion resistance and non-biodegradability.

第6回極域科学シンポジウム[OB] 極域生物圏11月17日（火） 統計数理研究所 セミナー室1（D305）

第6回極域科学シンポジウム[OB] 極域生物圏11月16日（月） 国立極地研究所１階交流アトリウム

Sulfolobus acidocaldarius is a useful model organism for the genetic study of thermophilic archaea due to its ease of cultivation. Here we describe the development of a host-vector system for S. acidocaldarius consisting of SuaI restriction system-deficient strain SK-1 and shuttle vector pSAV2. The new host strain SK-1 was constructed by pop-out recombination based on the pyrE marker gene. Plasmid pSAV2 was constructed from the S. islandicus native plasmid pRN1, in which selectable markers and functional genes were inserted in suitable locations and orientations followed by the deletion of non-essential open reading frames. SK-1 allowed direct transformation without N(4)-methylation at SuaI restriction sites, so unmethylated vector pSAV2 could be introduced directly into SK-1 by electroporation. The transformants were selected by pyrEF complementation on xyrose-tryptone solid medium without prior liquid culturing. The transformation efficiency was approximately 1.0 × 10(3)/μg DNA. After replication in S. acidocaldarius, pSAV2 was successfully recovered from transformant cultures by the standard alkaline lysis method. Plasmid yield was approximately 40-50 ng/ml from late-log through stationary phase cultures. In addition, pSAV2 was maintained stably and at relatively high copy number in S. acidocaldarius.

In order to quantify recombination between exogenous DNA and the Sulfolobus acidocaldarius chromosome, we electroporated pyrE (uracil-auxtotrophic) recipient strains with functional pyrE sequences and counted Pyr+ transformants by direct plating. Certain culture and post-electroporation conditions increased the yield of Pyr+ recombinants from non-replicating pyrE plasmid, whereas cognate methylation of SuaI restriction sites in the plasmid decreased it. Recombination of linear DNAs with the S. acidocaldarius genome was proportional to the length of a limiting overlap, but even synthetic oligonucleotides produced reasonable numbers of recombinants with appropriate recipient strains. To investigate uses of this latter property, we electroporated an 18-bp pyrE deletion mutant with mixtures of synthetic oligonucleotides altering glycine-55 of the orotate phosphoribosyl transferase encoded by pyrE. Pyr+ transformants were recovered in which this codon was converted to each of the alternatives encoded by the oligonucleotide mixtures, thereby identifying five amino acid substitutions tolerated at this position of the thermostable enzyme.

In this study, we investigated the clinical effects of long-term administration of the phosphorus (P) binder lanthanum carbonate (LC), which was launched in Japan in 2009. The subjects were 58 dialysis patients who began receiving LC, and we evaluated the clinical effects for up to 36 months after treatment initiation. The average serum P concentration remained low during the 36-month study period, with a significant reduction from 6.25 mg/dL at the start of the study to 4.94 mg/dL after 36 months (P0.001). A significant reduction was also observed in the average serum calcium concentration after 36 months (P0.05), but not in the serum intact parathyroid hormone concentration. Significant reductions were also observed in the average serum total protein, albumin and potassium concentrations (P0.05). The dosages of LC increased by approximately 1.9-fold after 36 months, in contrast, the dosages of concomitantly used sevelamer hydrochloride and Ca carbonate preparations decreased. These results indicate that LC could be used to treat hyperphosphatemia without causing hypercalcemia, and would be useful for long-term treatment with hemodialysis patients.

Paenibacillus thermoaerophilus strain TC22-2b, a thermophilic bacterium with an optimum growth temperature of 50–55 °C isolated from compost (55 °C) in Japan, secreted a chitinase into culture medium in the presence of colloidal chitin. Adding glucose, lactose, mannose, or sucrose to culture medium decreased the amount of chitinase in culture supernatants. This chitinase was purified by ammonium sulfate precipitation, colloidal chitin adsorption, and ion exchange chromatography. The apparent molecular mass of this enzyme was approximately 48 kDa, and its N-terminal amino acid sequence was determined to be Ala–Val–Ser–Thr–Gly–Lys–Lys. The optimum temperature and pH for chitinase activity were 60 °C and pH 4, respectively. The chitinase retained 68 % of its initial activity after incubation at 50 °C for 2 h. Using p-nitrophenyl N,N′-diacetyl-β-d-chitobioside [pNP-(GlcNAc)2] as a substrate, the K m, V max, and k cat values for this enzyme were 1.4 mM, 0.058 mM min−1, and 9.6 s−1, respectively. Analysis of hydrolysis products showed that the chitinase digested N-acetyl-chitooligosaccharides in an endo manner. N-acetylglucosamine dimers were not degraded by the enzyme. When colloidal chitin was used as the substrate, N-acetylglucosamine dimers, -trimers, and -tetramers were detected as hydrolysis products. Thus, the thermophilic chitinase may prove useful for industrial applications in chitooligosaccharide production from chitin biomass.

A spherical thermoacidophilic archaeon, strain TA-2, was obtained from acidic hot springs located in Ohwaku Valley, Hakone, Japan. This isolate is an obligate aerobic chemoorganoheterotroph that grows optimally at about 75 degrees C, pH 2.8. The G + C content of DNA from TA-2 is 47 mol%. The 16S rRNA gene from TA-2 showed more than 99% similarity with those of Metallosphaera sedula and Metallosphaera prunae and less than 92% similarity with other members of the order Sulfolobales. DNA-DNA hybridization experiments showed more than 93% genomic DNA homology among TA-2, M. sedula DSM5348T, and M. prunae DSM10039T. However, TA-2 lacks calditoglycerocaldarchaeol derivatives, which are usually found in the membrane lipids of members of the order Sulfolobales. Therefore, calditoglycerocaldarchaeol may not be essential for survival in thermophilic and acidophilic environments. The isolate was deposited as Metallosphaera sedula TA-2 (JCM 9064, IFO 15160).

Proliferating cell nuclear antigen (PCNA) is an essential component of the DNA replication and repair machinery in the domain Eucarya. Eukaryotes and euryarchaeotes, which belong to one subdomain of Archaea, possess a single PCNA homologue, whereas two distinct PCNA homologues have been identified from Sulfolobus solfataricus, which belongs to the other archaeal subdomain, Crenarchaeota. We have cloned and sequenced two genes of PCNA homologues from the thermoacidophilic crenarchaeon Sulfurisphaera ohwakuensis. These genes, referred to as the Soh PCNA A gene and the Soh PCNA B gene, were found to encode 245 amino acids (aa) (27 kDa) and 248 aa (27 kDa), respectively. In deduced amino acid sequences of both PCNA homologues, the motif L/I-A-P-K/R, implicated in binding of PCNA with replication factor C (RFC), was identified. Phylogenetic analysis of all available archaeal PCNA homologues suggests that crenarchaeal homologues are divided into two groups. Group A consists of Soh PCNA A, one of the S. solfataricus PCNA homologues, and one of the Aeropyrum pernix PCNA homologues. The other crenarchaeal homologues form group B. Crenarchaeal PCNA homologues constitute a monophyletic subfamily. These results suggest that the evolution of crenarchaeal PCNA homologues has been characterized by one or two gene duplication events, which are assumed to have occurred after the split of the crenarchaeal and euryarchaeal lineages.

Besides continuous progress in molecular biological techniques, metagenomic approach has become one of the routine experiments to examine the microbial diversity, ecology, and enzymes. This approach basically does not require the cultivation of microbes, which has a strong bias to a population of microbes and allows to have a glimpse of uncultured microbes and to find novel biomolecules. In the last 15 years, nearly 20 papers describing properties of thermostable enzymes discovered from hot environments by metagenomic strategies have been published. Most of them attempted to find and analyze hydrolytic enzymes such as cellulases, amylase, xylanase, esterase, lipase, and others because such enzymes are relatively easy to detect their activities in the function-based screening procedure. On the other hand, sequencing-based approach does not rely on the activity measurement and aids in discovering novel thermostable enzymes employing recently improved DNA sequencing technology and cloning vectors. The spectacular progress made in molecular biological techniques facilitates comprehensive metagenomic analysis for discovering novel biomolecules from the major fraction of the uncultured microbes in nature.

To determine the orthology of sterol 14-demethylase (P45014DM), the only known P450 enzyme distributed widely in eukaryotes with a conserved metabolic role, the full-length amino acid sequences of rat and human P45014DMs were determined from the cloned cDNA sequences, and compared with those of the corresponding fungal proteins (CYP51). The amino acid identity value between given pairs of P45014DMs ranged from 93% (human/rat) to 39% (human or rat/Saccharomyces cerevisiae). All the P45014DMs formed a single cluster in a phylogenetic tree constructed from representative P450 protein sequences currently available. The nearest neighbors to the P45014DM cluster in the phylogenetic tree were CYP7 (cholesterol 7 alpha-hydroxylase) and CYP8 (prostacyclin synthase), and the divergence point of fungal and mammalian P45014DMs was clearly more recent than that of P45014DM and CYP7/CYP8. These lines of evidence show that fungal and mammalian P45014DMs are really orthologous. This is the first example of orthologous P450s occurring in distinct kingdoms. P45014DM may be an ancient P450 which arose before the divergence of major eukaryotic branches and has been conserved throughout evolution. The amino acid identity value (93%) between human and rat P45014DMs was comparable to those observed for some housekeeping enzymes. In addition, a processed pseudogene of P45014DM was found in a rat genomic DNA library, suggesting the expression of P45014DM in germ line cells. These facts suggest that P45014DM may be a housekeeping enzyme essential for the viability of mammals.

Three isolates of spherical thermoacidophilic archaebacteria (strains TA-1, TA-2 and N-8) were obtained from acidic hot springs at Hakone and Noboribetsu in Japan. We have studied the isolates using electron microscopy, Gram staining, sensitivity to lysozyme, growth conditions, lipid compositions, SDS-PAGE patterns of whole-cell proteins, DNA base composition and partial 16S rRNA sequences. These characteristics of the isolates were compared with those of the type strains, Sulfolobus acidocaldarius, Sulfolobus solfataricus, Acidianus brierleyi, Acidianus infernus, Metallosphaera sedula and Desulfurolobus ambivalens. Comparative studies indicated that the isolate TA-1 was novel species of either Sulfolobus or Acidianus, the isolate TA-2 belonged to the genus Metallosphaera, and the isolate N-8 belonged to the same species of S. acidocaldarius isolated from Yellowstone.

Examination was made to determine the structure of a major neutral glycolipid (GL-la) from Sulfolobus acidocaldarius N-8. NMR indicated GL-la to have a structure in which a glucose is linked via a glycosidic bond at the β-position to the ring of calditol, 2-hydroxymethy1-1- (2, 3-dihydroxypropoxy) -2, 3, 4, 5-cyclopentanetetraol, in calditoglycerocaldarchaeol (caldityl-glyceryl-dialkyl-tetraether core lipid).

Birds have several advantages in the study of memory formation, as imprinting and passive avoidance behaviors in chick are often used as model systems. However, the primary structure of the bird N-methyl-D-aspartate (NMDA) responsive glutamate receptor, which is assumed to play a critical role in memory formation, has not been determined. In this report we describe the cDNA cloning of a subunit of NMDA receptors (NMDA-R1) from duck and analysis of its structure and distribution in the brain. The N-terminal 898 amino acids of the NMDA-R1 were well conserved between duck and mammals, but the homology was completely lost in the C-terminus. In situ hybridization showed that the duck NMDA-R1 gene was expressed throughout the brain as it is in mammals.