Metal-ion-induced expression of gene fragments from subseafloor micro-organisms in the Kumano forearc basin, Nankai Trough.

AIMS: Using substrate-induced gene-expression (SIGEX) screening on subseafloor sediment samples from the Nankai Trough, Japan, we identified gene fragments showing an induction response to metal ions. METHODS AND RESULTS: Environmental DNA libraries in Escherichia coli host cells were tested by the addition of metal ions (Ni2+ , Co2+ , Ga3+ or Mo6+ ), followed by cell sorting of clones exhibiting green fluorescence upon co-expression of green fluorescence protein downstream of the inserted gene fragments. One clone displayed Ni2+ -specific induction, three clones displayed Ga3+ -specific induction and three clones displayed an induction response to multiple metal ions. DNA sequence analysis showed that a variety of genes showed induction responses in the screened clones. CONCLUSIONS: Using the SIGEX approach, we retrieved gene fragments with no previously identified response to metal ions that exhibited metal-ion-induced expression. This method has the potential to promote exploration of gene function through gene-induction response. SIGNIFICANCE AND IMPACT OF THE STUDY: We successfully linked gene-induction response with sequence information for gene fragments of previously unknown function. The SIGEX-based approach exhibited the potential to identify genetic function in unknown gene pools from the deep subseafloor biosphere, as well as novel genetic components for future biotechnological applications.

Distribution of anaerobic carbon monoxide dehydrogenase genes in deep subseafloor sediments.

Carbon monoxide (CO) is the simplest oxocarbon generated by the decomposition of organic compounds, and it is expected to be in marine sediments in substantial amounts. However, the availability of CO in the deep subseafloor sedimentary biosphere is largely unknown even though anaerobic oxidation of CO is a thermodynamically favourable reaction that possibly occurs with sulphate reduction, methanogenesis, acetogenesis and hydrogenesis. In this study, we surveyed for the first time the distribution of the CO dehydrogenase gene (cooS), which encodes the catalytic beta subunit of anaerobic CO dehydrogenase (CODH), in subseafloor sediment-core samples from the eastern flank of the Juan de Fuca Ridge, Mars-Ursa Basin, Kumano Basin, and off the Shimokita Peninsula, Japan, during Integrated Ocean Drilling Program (IODP) Expeditions 301, 308 and 315 and the D/V Chikyu shakedown cruise CK06-06, respectively. Our results show the occurrence of diverse cooS genes from the seafloor down to about 390 m below the seafloor, suggesting that microbial communities have metabolic functions to utilize CO in anoxic microbial ecosystems beneath the ocean floor, and that the microbial community potentially responsible for anaerobic CO oxidation differs in accordance with possible energy-yielding metabolic reactions in the deep subseafloor sedimentary biosphere. SIGNIFICANCE AND IMPACT OF THE STUDY: Little is known about the microbial community associated with carbon monoxide (CO) in the deep subseafloor. This study is the first survey of a functional gene encoding anaerobic carbon monoxide dehydrogenase (CODH). The widespread occurrence of previously undiscovered CO dehydrogenase genes (cooS) suggests that diverse micro-organisms are capable of anaerobic oxidation of CO in the deep subseafloor sedimentary biosphere.

Analysis of Low-Biomass Microbial Communities in the Deep Biosphere.

Over the past few decades, the subseafloor biosphere has been explored by scientific ocean drilling to depths of about 2.5km below the seafloor. Although organic-rich anaerobic sedimentary habitats in the ocean margins harbor large numbers of microbial cells, microbial populations in ultraoligotrophic aerobic sedimentary habitats in the open ocean gyres are several orders of magnitude less abundant. Despite advances in cultivation-independent molecular ecological techniques, exploring the low-biomass environment remains technologically challenging, especially in the deep subseafloor biosphere. Reviewing the historical background of deep-biosphere analytical methods, the importance of obtaining clean samples and tracing contamination, as well as methods for detecting microbial life, technological aspects of molecular microbiology, and detecting subseafloor metabolic activity will be discussed.

[2+2+1] cyclization of allenes.

The [2+2+1] cyclization of an alkyne, an alkene and carbon monoxide, i.e., the Pauson-Khand reaction, is one of the most powerful tools for constructing a five-membered ring. In place of the alkene or alkyne part, the use of an allene functionality has proven to make this reaction more valuable for organic synthesis. This review focuses on the origin and progress of the allenic [2+2+1] cyclocarbonylation, including the chirality transfer of the allene and its synthetic applications.

Rapid metabolism fosters microbial survival in the deep, hot subseafloor biosphere.

A fourth of the global seabed sediment volume is buried at depths where temperatures exceed 80 °C, a previously proposed thermal barrier for life in the subsurface. Here, we demonstrate, utilizing an extensive suite of radiotracer experiments, the prevalence of active methanogenic and sulfate-reducing populations in deeply buried marine sediment from the Nankai Trough subduction zone, heated to extreme temperature (up to ~120 °C). The small microbial community subsisted with high potential cell-specific rates of energy metabolism, which approach the rates of active surface sediments and laboratory cultures. Our discovery is in stark contrast to the extremely low metabolic rates otherwise observed in the deep subseafloor. As cells appear to invest most of their energy to repair thermal cell damage in the hot sediment, they are forced to balance delicately between subsistence near the upper temperature limit for life and a rich supply of substrates and energy from thermally driven reactions of the sedimentary organic matter.

A bacterium lipopolysaccharide that elicits Guillain-Barré syndrome has a GM1 ganglioside-like structure.

There is a strong association between Guillain-Barré syndrome (GBS) and Penner's serotype 19 (PEN 19) of Campylobacter jejuni. Sera from patients with GBS after C. jejuni infection have autoantibodies to GM1 ganglioside in the acute phase of the illness. Our previous work has suggested that GBS results from an immune response to cross-reactive antigen between lipopolysaccharide (LPS) of the Gram-negative bacterium and membrane components of peripheral nerves. To clarify the pathogenesis of GBS, we have investigated whether GM1-oligosaccharide structure is present in the LPS of C. jejuni (PEN 19) that was isolated from a GBS patient. After extraction of the LPS, the LPS showing the binding activity of cholera toxin, that specifically recognizes the GM1-oligosaccharide was purified by a silica bead column chromatography. Gas-liquid chromatography-mass spectrometric analysis has shown that the purified LPS contained Gal, GalNAc, and NeuAc, which are sugar components of GM1 ganglioside. 1H NMR methods [Carr-Purcell-Meiboom-Gill (CPMG), total correlation spectroscopy (TOCSY), and nuclear Overhauser effect spectroscopy (NOESY)] have revealed that the oligosaccharide structure [Gal beta 1-3 GalNAc beta 1-4(NeuAc alpha 2-3)Gal beta] protrude from the LPS core. This terminal structure [Gal beta 1-3GalNAc beta 1-4(NeuAc alpha 2-3)Gal beta] is identical to the terminal tetrasaccharide of the GM1 ganglioside. This is the first study to demonstrate the existence of molecular mimicry between nerve tissue and the infectious agent that elicits GBS.

Thermus thermophilus TMY isolated from silica scale taken from a geothermal power plant.

AIMS: To identify an extreme thermophile, strain TMY, isolated from silica scale from the geothermal electric power plant and to examine microdiversity of Thermus thermophilus strains. MATERIALS AND RESULTS: The isolated strain TMY was identified by morphological, biochemical and physiological tests. Phylogenetic comparison of the strain and other Thermus strains with 16S rDNA analysis, RAPD and ERIC-PCR fingerprinting were performed. Strain TMY was closely related to strain which was isolated from a hot spring in New Zealand and shown to belong to the Japanese Thermus cluster. However, there were considerable genetic differences between strain TMY and other Thermus species using DNA fingerprinting. CONCLUSIONS: Based on morphological, physiological and genetic properties, strain TMY could be a strain of T. thermophilus. The distinct properties of strain TMY suggest that microdiversity of T. thermophilus strains should be considered. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study have demonstrated genetic diversity within T. thermophilus strains, which were previously masked by an almost identical 16S rDNA sequence. RAPD and ERIC-PCR could be potential methods for distinguishing between Thermus strains.

Solution structure and ligand-binding site of the carboxy-terminal SH3 domain of GRB2.

BACKGROUND: Growth factor receptor-bound protein 2 (GRB2) is an adaptor protein with three Src homology (SH) domains in the order SH3-SH2-SH3. Both SH3 domains of GRB2 are necessary for interaction with the protein Son of sevenless (Sos), which acts as a Ras activator. Thus, GRB2 mediates signal transduction from growth factor receptors to Ras and is thought to be a key molecule in signal transduction. RESULTS: The three-dimensional structure of the carboxy-terminal SH3 domain of GRB2 (GRB2 C-SH3) was determined by NMR spectroscopy. The SH3 structure consists of six beta-strands arranged in two beta-sheets that are packed together perpendicularly with two additional beta-strands forming the third beta-sheet. GRB2 C-SH3 is very similar to SH3 domains from other proteins. The binding site of the ligand peptide (VPP-PVPPRRR) derived from the Sos protein was mapped on the GRB2 C-SH3 domain indirectly using 1H and 15N chemical shift changes, and directly using several intermolecular nuclear Overhauser effects. CONCLUSIONS: Despite the structural similarity among the known SH3 domains, the sequence alignment and the secondary structure assignments differ. We therefore propose a standard description of the SH3 structures to facilitate comparison of individual SH3 domains, based on their three-dimensional structures. The binding site of the ligand peptide on GRB2 C-SH3 is in good agreement with those found in other SH3 domains.

Ganglioside composition of GH3 cells: enhancement of fucoganglioside expression by estradiol, epidermal growth factor and insulin.

The GH3 cell line, a bipotential cell line secreting both prolactin (PRL) and growth hormone (GH), is a useful model for investigating GH/PRL cell lineage differentiation and anterior pituitary adenoma formation. In this study, we investigated the ganglioside composition of GH3 cells and identified two fucogangliosides as the major gangliosides expressed by these cells. Analyses by DEAE-Sephadex A-25 and thin-layer chromatography (TLC) revealed that the GH3 cells contained two major gangliosides, designated FG1 and FG2, respectively. Their structures were identified by fast atom bombardment mass spectrometry and proton nuclear magnetic resonance spectrometry: FG1 is IV2FUc alpha,II3NeuAc-GgOse4Cer and FG2 is IV2FUc alpha,IV3Gal alpha,II3NeuAc-GgOse4Cer. Expression of these fucogangliosides was enhanced by chronic treatment with 17 beta-estradiol (1 nM), epidermal growth factor (10 nM) and insulin (300 nM), which induced differentiation of GH3 cells to normal PRL-secreting cells. Interestingly, immunocytochemistry and flow cytometry revealed that the increased expression of these gangliosides reflected a quantitative change inside the cells but not on the cell surface. These results suggest that the intracellular distribution of fucogangliosides is closely related to the differentiation of GH3 cells.

Structural elucidation of two novel amphoteric glycosphingolipids from the earthworm, Pheretima hilgendorfi.

The novel amphoteric glycosphingolipids containing a choline phosphate were purified from whole tissues of the earthworm, Pheretima hilgendorfi. Their chemical structures were completely characterized as cholinephosphoryl-->6(Man alpha 1-4)Gal beta 1-6Gal beta 1-1Cer (cholinephosphorylmannosylneogalabiaosylceramide, named PGL3a) and cholinephosphoryl-->6Gal beta 1-6Gal beta 1-6Gal beta 1-1Cer (cholinephosphorylneogalatriaosylceramide, named PGL3b) by compositional sugar, fatty acid and sphingoid analyses, hydrogen fluoride degradation, partial acid hydrolysis, methylation analysis, exoglycosidase degradation, proton magnetic resonance spectroscopy and fast atom bombardment mass spectrometry. The ceramide moieties of these lipids consisted of 22:0, 23:0 and 24:0 acids as major fatty acids, and branched octadeca- and nonadeca-4-sphingenines and octadeca-4-sphingenine as main sphingoids. Since the oligosaccharides and the ceramide moieties of PGL3a and PGL3b were identical with those of neutral glycosphingolipids found in this organism, the biosynthesis of the amphoteric glycolipids may occur by the addition of a choline phosphate residue to the corresponding neutral glycolipids, Man alpha 1-4Gal beta 1-6Gal beta 1-1Cer or Gal beta 1-6Gal beta 1-6Gal beta 1-1Cer.

Structural characterization of a novel glycoinositolphospholipid from the parasitic nematode, Ascaris suum.

A novel glycosphingolipid containing inositol phosphate as an acidic group has been demonstrated in whole tissues of the porcine roundworm, Ascaris suum. The thin layer chromatographic pattern of the total acidic glycolipid revealed the presence of several components, of which a major component (named AGL) with positive reactions toward both orcinol-sulfuric acid (sugar) and molybdate (phosphate) spray reagents was isolated and purified by the use of successive column chromatography on DEAE-Sephadex and silicic acid (latrobeads). From structural studies including compositional sugar analysis, hydrogen fluoride degradation, methylation analysis, periodate oxidation, proton magnetic resonance spectroscopy and fast atom bombardment mass spectrometry, the structure of AGL was deduced to be Gal alpha 1-2Ins(1-->)-P-Cer. Aliphatic constituents were lignoceric acid and its 2-hydroxy homologue as the principal fatty acids, and octadecasphinganine and branched heptadecasphinganine as the major sphingoids.

Solution structure of ferredoxin from the thermophilic cyanobacterium Synechococcus elongatus and its thermostability.

The three-dimensional structure of ferredoxin, purified from the thermophilic cyanobacterium Synechococcus elongatus, was determined in aqueous solution by two-dimensional proton nuclear magnetic resonance. In addition to the 946 distance constraints from nuclear Overhauser effect connectivities, we added 241 distance constraints derived from the crystal structure of Spirulina platensis ferredoxin to the 19 residues close to the [2Fe-2S] iron-sulfur center, where crosspeaks disappeared due to paramagnetic effects. The atomic root-mean-square difference of the ten converged structures from the mean structure was 0.61(+/-0.12) A for backbone atoms (N, C(alpha), C'). The main-chain structure was almost the same as the crystal structures of other mesophile ferredoxins, but comparison of the side-chain structures revealed an extension of the hydrophobic core, a unique hydrophobic patch on the surface of the large beta-sheet, and two unique charge networks in this thermostable ferredoxin structure, some of which might contribute to thermostability.

Molecular conformation of alpha-bungarotoxin as studied by nuclear magnetic resonance and circular dichroism.

We have examined the circular dichroism and nuclear magnetic resonance spectra of a long neurotoxin, alpha-bungarotoxin, over a wide range of pH values and temperatures, and under high salt conditions. The observations are interpreted partly in terms of the known crystal structure of this polypeptide. We support earlier findings of a greater degree of beta-sheet structure in solution than has been reported by X-ray crystallography and, importantly, the invariant residue associated with neurotoxicity, Trp29, is shown to be in a similar environment to that found in alpha-cobratoxin and LS III from Laticauda semifasciata. The implications of this observation for structure/function relationships are outlined.

Solution structure of the SH2 domain of Grb2 complexed with the Shc-derived phosphotyrosine-containing peptide.

The solution structure of growth factor receptor-bound protein 2 (Grb2) SH2 complexed with a Shc-derived phosphotyrosine (pTyr)-containing peptide was determined by nuclear magnetic resonance (NMR) spectroscopy. The pTyr binding site of Grb2 SH2 was similar to those of other SH2 domains. In contrast, the amino acid residues C-terminal to pTyr did not form an extended structure because of steric hindrance caused by a bulky side-chain of Trp121 (EF1). As a result, the peptide formed a turn-structure on the surface of Grb2 SH2. The asparagine residue at the pTyr+2 position of the Shc-peptide interacted with the main-chain carbonyl groups of Lys109 and Leu120. The present solution structure was similar to the crystal structure reported for Grb2 SH2 complexed with a BCR-Abl-derived phosphotyrosine-containing peptide. Finally, the structure of Grb2 SH2 domain was compared with those of the complexes of Src and phospholipase C-gamma1 with their cognate peptides, showing that the specific conformation of the peptide was required for binding to the SH2 domains.

Tertiary structure of erabutoxin b in aqueous solution as elucidated by two-dimensional nuclear magnetic resonance.

The three-dimensional structure of erabutoxin b, a short-chain neurotoxic peptide purified from the venom of the sea snake Laticauda semifasciata, was determined in aqueous solution by two-dimensional proton nuclear magnetic resonance and simulated annealing-based calculations. On the basis of 883 assigned nuclear Overhauser effect (NOE) connectivities, 676 final distance constraints were derived and used together with 38 torsion angle (phi, chi 1) constraints, four distance constraints derived from disulfide bridges and 30 distance constraints derived from hydrogen bonds. A total of 14 converged structures were obtained from 50 runs of calculations. The atomic root-mean-square difference about the mean coordinate positions (excluding the residues 18 to 22) is 0.60 A for backbone atoms (N, C alpha and C'). The protein consists of a core region from which three finger-like loops emerge outwards. It includes a short, two-stranded antiparallel beta-sheet of residues 2 to 5 and 13 to 16, a three-stranded antiparallel beta-sheet involving residues 23 to 30, 35 to 41 and 50 to 56, and four disulfide bridges in the core region. Comparison with two crystal structures of erabutoxin b at 1.4 A and 1.7 A resolution indicated that the solution and the crystal structures were very similar, but less defined regions were observed at the localized region of the tip of the central loop and the outside of the third loop in solution. Other short-chain alpha-neurotoxins showed structural characteristics similar to those of erabutoxin b.

Identification of the receptor-recognition surface of bombyxin-II, an insulin-like peptide of the silkmoth Bombyx mori: critical importance of the B-chain central part.

Bombyxin-II, a brain-secretory peptide of the silkmoth Bombyx mori, shares 40% sequence identify and the characteristics core structure with human insulin. In spite of the structural similarity, no cross-activity is observed between them. To localize the active region of bombyxin-II, we have synthesized chimeric molecules of bombyxin-II and human insulin, and examined their bombyxin activity. Two chimeric molecules, which were sequentially identical except for the B-chain central part, showed significantly different potencies in bombyxin activity. Solution structure determination of these chimeric molecules revealed that their B-chain central parts took similar main-chain conformation, but formed dissimilar patches on their molecular surfaces. Therefore, the surface patch formed by the central part of the bombyxin-II B-chain is of critical importance for recognition of the bombyxin receptor. The above results, together with other data on the structure-activity relationships of bombyxin, indicate that the receptor-recognition surface of bombyxin-II includes the A-chain N and C, termini in addition to the B-chain central part. Though bombyxin-II, human insulin and human relaxin 2 use the common surface as their receptor-recognition sites, each of the surface patches is characterized by the variety of involved side-chains. Insulin and relaxin involve additional parts for receptor recognition, particularly the B-chain C-terminal part and the extended A-chain N-terminal helix, respectively. In conclusion, these ligands have evolved their own specific mechanisms for receptor recognition while retaining the major recognition surface.

Three-dimensional solution structure of bombyxin-II an insulin-like peptide of the silkmoth Bombyx mori: structural comparison with insulin and relaxin.

The three-dimensional solution structure of bombyxin-II, an insulin-like two-chain peptide produced by the brain of the silkworm Bombyx mori, has been determined by simulated annealing calculations based on 535 distance constraints and 24 torsion-angle constraints derived from NMR data and three distance constraints of the disulfide bonds. To our knowledge, this is the first three-dimensional structure determined for an invertebrate insulin-related peptide. The root-mean-square deviations between the best 10 structures and the mean structure are 0.58(+/- 0.15) A for the backbone heavy atoms (N, C alpha, C) and 1.03(+/- 0.18) A for all non-hydrogen atom if less well-defined N and C termini (A1, A20, B(-2) to B4 and B23 to B25) are excluded. The overall main-chain structure of bombyxin-II is similar to that of insulin. However, there are significant conformational and functional differences in their B-chain C-terminal parts. The B-chain C-terminal part of bombyxin-II adopts an extension of the B-chain central helix like that of relaxin and is not required for bombyxin activity, while the corresponding part of insulin adopts a sharp turn and a beta-strand and is essential for insulin activity. This structure demonstrates that bombyxin-II is more closely related to relaxin than to insulin, and suggests that insulin might have evolved the additional receptor-recognition site in the B-chain C-terminal beta-strand to distinguish itself from bombyxin and relaxin. The structure of bombyxin-II thus provides novel insights into the receptor recognition and divergent molecular evolution of insulin-superfamily peptides.

Solution structure of Grb2 reveals extensive flexibility necessary for target recognition.

Grb2 is an adaptor protein composed of a single SH2 domain flanked by two SH3 domains. Grb2 functions as an important evolutionary conserved link between a variety of cell membrane receptors and the Ras/MAP kinase-signaling cascade. Here, we describe the solution structure of Grb2 as revealed by NMR and small angle X-ray scattering measurements. We demonstrate that Grb2 is a flexible protein in which the C-terminal SH3 domain is connected to the SH2 domain via a flexible linker. This is in contrast to the previously described Grb2 crystal structure, which showed a compact structure with intramolecular contact between two SH3 domains. Binding experiments on Grb2 and peptides containing two different proline-rich sequences indicate that Grb2 adapts the relative position and orientation of the two SH3 domains to bind bivalently to the target peptide sequences.

Analysis of human IgE epitope of Der f 2 with anti-Der f 2 mouse monoclonal antibodies.

Der f 2 is one of the major mite allergens recognized by human IgE antibodies of allergic patients. Using five anti-Der f 2 mouse monoclonal antibodies, human IgE epitopes of Der f 2 were analyzed. Among them, two monoclonal antibodies 15E11 and 13A4 inhibited the binding between Der f 2 and human IgE antibodies. To determine major IgE epitopes of Der f 2, epitopes for the monoclonal IgG antibodies were analyzed using 43 single site Der f 2 mutants constructed by site-directed mutagenesis. Binding ability of 13A4 and 15E11 was decreased by the amino acid replacement around the C-terminus, and around 73rd, respectively. These results suggest that the C-terminal portion and the central portion around 73rd of Der f 2 were recognized by human IgE antibodies as major epitopes. The location of the putative IgE epitopes on 3-D structure of Der f 2 is also discussed.

DEEP BIOSPHERE. Exploring deep microbial life in coal-bearing sediment down to ~2.5 km below the ocean floor.

Microbial life inhabits deeply buried marine sediments, but the extent of this vast ecosystem remains poorly constrained. Here we provide evidence for the existence of microbial communities in ~40° to 60°C sediment associated with lignite coal beds at ~1.5 to 2.5 km below the seafloor in the Pacific Ocean off Japan. Microbial methanogenesis was indicated by the isotopic compositions of methane and carbon dioxide, biomarkers, cultivation data, and gas compositions. Concentrations of indigenous microbial cells below 1.5 km ranged from <10 to ~10(4) cells cm(-3). Peak concentrations occurred in lignite layers, where communities differed markedly from shallower subseafloor communities and instead resembled organotrophic communities in forest soils. This suggests that terrigenous sediments retain indigenous community members tens of millions of years after burial in the seabed.