Cultivation of previously uncultured microorganisms with a continuous-flow down-flow hanging sponge (DHS) bioreactor, using a syntrophic archaeon culture obtained from deep marine sediment as a case study.

In microbiology, cultivation is a central approach for uncovering novel physiology, ecology, and evolution of microorganisms, but conventional methods have left many microorganisms found in nature uncultured. To overcome the limitations of traditional methods and culture indigenous microorganisms, we applied a two-stage approach: enrichment/activation of indigenous organisms by using a continuous-flow down-flow hanging sponge bioreactor and subsequent selective batch cultivation. Here, we provide a protocol for this bioreactor-mediated technique using activation of deep marine sediment microorganisms and downstream isolation of a syntrophic co-culture containing an archaeon closely related to the eukaryote ancestor (Candidatus Promethearchaeum syntrophicum strain MK-D1) as an example. Both stages can easily be tailored to target other environments and organisms by modifying the inoculum, feed solution/gases, attachment material and/or cultivation media. We anaerobically incubate polyurethane sponges inoculated with deep-sea methane seep sediment in a reactor at 10 °C and feed anaerobic artificial seawater medium and methane. Once phylogenetically diverse and metabolically active microorganisms are adapted to synthetic conditions in the reactor, we transition to growing community samples in glass tubes with the above medium, simple substrates and selective compounds (e.g., antibiotics). To accommodate for the slow growth anticipated for target organisms, primary cultures can be incubated for ≥6-12 months and analyzed for community composition even when no cell turbidity is observed. One casamino acid- and antibiotic-amended culture prepared in this way led to the enrichment of uncultured archaea. Through successive transfer in vitro combined with molecular growth monitoring, we successfully obtained the target archaeon with its partner methanogen as a pure syntrophic co-culture.

Isolation of an archaeon at the prokaryote-eukaryote interface.

The origin of eukaryotes remains unclear1-4. Current data suggest that eukaryotes may have emerged from an archaeal lineage known as 'Asgard' archaea5,6. Despite the eukaryote-like genomic features that are found in these archaea, the evolutionary transition from archaea to eukaryotes remains unclear, owing to the lack of cultured representatives and corresponding physiological insights. Here we report the decade-long isolation of an Asgard archaeon related to Lokiarchaeota from deep marine sediment. The archaeon-'Candidatus Prometheoarchaeum syntrophicum' strain MK-D1-is an anaerobic, extremely slow-growing, small coccus (around 550 nm in diameter) that degrades amino acids through syntrophy. Although eukaryote-like intracellular complexes have been proposed for Asgard archaea6, the isolate has no visible organelle-like structure. Instead, Ca. P. syntrophicum is morphologically complex and has unique protrusions that are long and often branching. On the basis of the available data obtained from cultivation and genomics, and reasoned interpretations of the existing literature, we propose a hypothetical model for eukaryogenesis, termed the entangle-engulf-endogenize (also known as E3) model.

Methanofervidicoccus abyssi gen. nov., sp. nov., a hydrogenotrophic methanogen, isolated from a hydrothermal vent chimney in the Mid-Cayman Spreading Center, the Caribbean Sea.

A novel hydrogenotrophic methanogen, strain HHBT, was isolated from a deep-sea hydrothermal vent chimney sample collected from Beebe Vent Field at the Mid-Cayman Spreading Center, Caribbean Sea. The cells were non-motile regular to irregular cocci possessing several flagella. The novel isolate grew at 60-80 °C, pH 5.0-7.4 and with 1-4 % of NaCl (w/v). The isolate utilized H2/CO2 as the only substrates for growth and methane production. The results of phylogenetic analyses of both 16S rRNA and mcrA gene sequences and comparative genome analysis indicated that HHBT represented a member of the order Methanococcales, and was closely related to the members of the genera Methanothermococcus and Methanotorris. The most closely related species were Methanothermococcus okinawensis IH1T and Methanotorris igneus Kol 5T in comparison of 16S rRNA gene sequences (each with 93 % identity), and Methanotorris formicicus Mc-S-70T in the case of deduced amino acid sequence similarity of mcrA genes (92 % similarity). The ANI and AAI values between HHBT and the members of the genera Methanothermococcus and Methanotorris were 69-72 % and 66-70 %, respectively. Although many of the morphological and physiological characteristics were quite similar between HHBT and the species of the genera Methanothermococcus and Methanotorris, they were distinguishable by the differences in susceptibility to antibiotics, formate utilization, growth temperature and NaCl ranges. On the basis of these phenotypic, phylogenetic and genomic properties, we propose that strain HHBT represents a novel species, of a novel genus, Methanofervidicoccus abyssi gen. nov., sp. nov. The type strain is HHBT (=JCM 32161T=DSM 105918T).

Aggregatilinea lenta gen. nov., sp. nov., a slow-growing, facultatively anaerobic bacterium isolated from subseafloor sediment, and proposal of the new order Aggregatilineales ord. nov. within the class Anaerolineae of the phylum Chloroflexi.

A novel slow-growing, facultatively anaerobic, filamentous bacterium, strain MO-CFX2T, was isolated from a methanogenic microbial community in a continuous-flow bioreactor that was established from subseafloor sediment collected off the Shimokita Peninsula of Japan. Cells were multicellular filamentous, non-motile and Gram-stain-negative. The filaments were generally more than 20 µm (up to approximately 200 µm) long and 0.5-0.6 µm wide. Cells possessed pili-like structures on the cell surface and a multilayer structure in the cytoplasm. Growth of the strain was observed at 20-37 °C (optimum, 30 °C), pH 5.5-8.0 (pH 6.5-7.0), and 0-30 g l-1 NaCl (5 g l-1 NaCl). Under optimum growth conditions, doubling time and maximum cell density were estimated to be approximately 19 days and ~105 cells ml-1, respectively. Strain MO-CFX2T grew chemoorganotrophically on a limited range of organic substrates in anaerobic conditions. The major cellular fatty acids were saturated C16 : 0 (47.9 %) and C18 : 0 (36.9 %), and unsaturated C18 : 1ω9c (6.0 %) and C16 : 1ω7 (5.1 %). The G+C content of genomic DNA was 63.2 mol%. 16S rRNA gene-based phylogenetic analysis showed that strain MO-CFX2T shares a notably low sequence identity with its closest relatives, which were Thermanaerothrix daxensis GNS-1T and Thermomarinilinea lacunifontana SW7T (both 85.8 % sequence identity). Based on these phenotypic and genomic properties, we propose the name Aggregatilinea lenta gen. nov., sp. nov. for strain MO-CFX2T (=KCTC 15625T, =JCM 32065T). In addition, we also propose the associated family and order as Aggregatilineaceae fam. nov. and Aggregatilineales ord. nov., respectively.

Anti-Semaphorin 3A neutralization monoclonal antibody prevents sepsis development in lipopolysaccharide-treated mice.

Semaphorin 3A (Sema3A), originally identified as a potent growth cone collapsing factor in developing sensory neurons, is now recognized as a key player in immune, cardiovascular, bone metabolism and neurological systems. Here we established an anti-Sema3A monoclonal antibody that neutralizes the effects of Sema3A both in vitro and in vivo. The anti-Sema3A neutralization chick IgM antibodies were screened by combining an autonomously diversifying library selection system and an in vitro growth cone collapse assay. We further developed function-blocking chick-mouse chimeric and humanized anti-Sema3A antibodies. We found that our anti-Sema3A antibodies were effective for improving the survival rate in lipopolysaccharide-induced sepsis in mice. Our antibody is a potential therapeutic agent that may prevent the onset of or alleviate symptoms of human diseases associated with Sema3A.

Semaphorin3A signaling mediated by Fyn-dependent tyrosine phosphorylation of collapsin response mediator protein 2 at tyrosine 32.

Collapsin response mediator protein 2 (CRMP2) is an intracellular protein that mediates signaling of Semaphorin3A (Sema3A), a repulsive axon guidance molecule. Fyn, a Src-type tyrosine kinase, is involved in the Sema3A signaling. However, the relationship between CRMP2 and Fyn in this signaling pathway is still unknown. In our research, we demonstrated that Fyn phosphorylated CRMP2 at Tyr(32) residues in HEK293T cells. Immunohistochemical analysis using a phospho-specific antibody at Tyr(32) of CRMP showed that Tyr(32)-phosphorylated CRMP was abundant in the nervous system, including dorsal root ganglion neurons, the molecular and Purkinje cell layer of adult cerebellum, and hippocampal fimbria. Overexpression of a nonphosphorylated mutant (Tyr(32) to Phe(32)) of CRMP2 in dorsal root ganglion neurons interfered with Sema3A-induced growth cone collapse response. These results suggest that Fyn-dependent phosphorylation of CRMP2 at Tyr(32) is involved in Sema3A signaling.

Cell surface N-glycans mediated isolation of mouse neural stem cells.

The isolation of neural stem cells (NSCs) from the brain has been hampered by the lack of valid cell surface markers and the requirement for long-term in vitro cultivation that may lead to phenotype deterioration. However, few suitable specific cell surface antigens are available on NSCs that could be used for their prospective isolation. The present study demonstrated that the expression of complex type asparagine-linked oligosaccharide (N-glycans) was detected on brain cells dissociated from embryonic and adult brain using Phaseolus vulgaris erythroagglutinating lectin (E-PHA) which binds to biantennary complex type N-glycans, and demonstrated that E-PHA bound preferentially to purified NSCs, but not to neurons, microglia, or oligodendrocyte precursor cells. The labeling of dissociated mouse embryonic brain cells or adult brain cells with E-PHA enabled the enrichment of NSCs by 25-fold or 9-fold of the number of neurosphere-forming cells in comparison to that of unsorted cells, respectively. Furthermore, a lectin blot analysis revealed the presence of several glycoproteins which were recognized by E-PHA in the membrane fraction of the proliferating NSCs, but not in the differentiated cells. These results indicate that complex type N-glycans is a valuable cell surface marker for living mouse NSCs from both the embryonic and adult brain.