Methanococcus thermolithotrophicus,a novel thermophilic lithotrophic methanogen

An autotrophic thermophilic motile coccoid methanogen was isolated from geothermally heated sea sediments close to Naples, Italy. Growth occurs on H₂/CO₂ and on formate between 30 and 70°C with an optimum at 65°C. The optimal doubling time is only 55 min. The NaCl-concentration ranges from 1.3% to 8.3% with an optimum around 4%. By its G+C-content of 31.3 mol%, its subunit envelope, and by DNA-RNA hybridization the new isolate is clearly defined to be a member of the genusMethanococcus. We name itMethanococcus thermolithotrophicus.Abbreviations G+C Guanine + Cytosine - SDS Sodium dodecylsulfate (Sodium lauryl sulfate)     

Sequence divergence of an archaebacterial gene cloned from a mesophilic and a thermophilic methanogen

Summary A 1.6-kb fragment of DNA from the thermophilic, methane-producing, anaerobic archaebacteriumMethanobacterium thermoautotrophicum H has been cloned and sequenced. This DNA complements mutations in both the purE₁ and purE₂ loci ofEscherichia coli. The sequence of theM. thermoautotrophicum DNA predicts that complementation inE. coli results from the synthesis of a polypeptide with a molecular weight of 36,249. A polypeptide apparently of this molecular weight is synthesized inE. coli minicells containing recombinant plasmids that carry the cloned fragment of methanogen DNA. We have previously cloned and sequenced a purE-complementing gene from the mesophilic methanogenMethanobrevibacter smithii. The two methanogen-derived purE-complementing genes are 53% homologous and encode polypeptides that are 45% homologous in their amino acid sequences but would be 74% homologous if conservative amino acid substitutions were considered as maintaining sequence homology. The genome ofM. thermoautotrophicum has a molar G+C content of 49.7%, whereas the genome ofM. smithii is 30.6% G+C. Conservation of encoded amino acids while accommodating the very different G+C contents is accomplished by use of different codons that encode the same amino acid. The majority of base changes occur at the third codon position. The intergenic regions of the clonedM. thermoautotrophicum DNA contain sequences previously identified as ribosome binding sites and as putative methanogen promoters. Although the two purE-complementing genes are apparently derived from a common ancestor, only the gene fromM. smithii maintains a codon usage that conforms to the RNY rule.     

Isolation and characterization of Methanocorpusculum parvum,gen. nov., spec. nov., a new tungsten requiring,coccoid methanogen

A new mesophilic, monotrichously flagellated methane-producing coccus of 1m in diameter was isolated from an anaerobic sour whey digester, originally inoculated with sewage sludge. Growth and methane production were observed with H₂/CO₂, formate and — less effectively — with 2-propanol/CO₂. The isolate grew at temperatures between 15° C and 45° C with the optimum at around 37° C. Acetate, yeast extract and tungstate were required in the medium. Clarified rumen fluid stimulated growth.The DNA of the new methanogen has a G+C content of 48.5 mol%. Comparative 16 S rRNA oligonucleotide cataloguing allows to define the new isolate as a member of a new genus of the order Methanomicrobiales. Further evidence for this is provided by the antigenic crossreactivity with anti-S probes and by metabolic features.Because of its small size the new methanogen is named Methanocorpusculum parvum.This work was supported by a grant of the Deutsche Forschungsgemeinschaft DFG to J. W. and E. S. Immunologic studies were supported in part by grants No. DE-FGO2-84 R 13197 from the U.S. Department of Energy, and No. 261.81/82 from the North Atlantic Treaty Organization (NATO)     

Isolation and characterization of a thermophilic acetotrophic strain of Methanothrix

An enrichment culture which converted acetate to methane at 60°C was obtained from a thermophilic anaerobic bioreactor. The predominant morphotype in the enrichment was a sheathed gas-vacuolated rod with marked resemblence to the mesophile Methanothrix soehngenii. This organism was isolated using vancomycin treatments and serial dilutions and was named Methanothrix sp. strain CALS-1. Strain CALS-1 grew as filaments typically 2–5 cells long, and cultures showed opalescent turbidity rather than macroscopic clumps. The cells were enclosed in a striated subunit-type sheath and there were distinct cross-walls between the cells, similar to M. soehngenii. The gas vesicles in cells were typically 70 nm in diameter and up to 0.5 m long, and were collapsed by pressures over 3 atm (ca. 300 kPa). Stationary-phase cells tended to have a higher vesicle content than did growing cells, and occasionally bands of cells were seen floating at the top of the liquid in stationary-phase cultures. Acetate was the only substrate of those tested which was used for methanogenesis by strain CALS-1, and acetate was decarboxylated by the aceticlastic reaction. The optimum temperature for growth of strain CALS-1 was near 60°C (doubling time=24–26 h), with no growth occurring at 70°C and 37°C. The optimum pH value for growth was near 6.5 in bicarbonate/CO₂ buffered medium and no growth occurred at pH 5.5 or pH 8.4. No growth was obtained below pH 7 when the medium was buffered with 20 mM phosphate. Strain CALS-1 grew in a chemically defined medium and required biotin. Sulfide concentrations over 1 mM were inhibitory to the culture, and growth was more rapid with 1 mM 2-mercaptoethane sulfonate (coenzyme M) or 1 mM titanium citrate as an accessory reductant than with 1 mM cysteine. It is likely that strain CALS-1 represents a new species in the genus Methanothrix.     

Isolation of a novel rod shaped methanogen growing on methanol and 2-propanol

A methanogen utilizing methanol was isolated from a reactor anaerobically degrading leaves of Leucaena leucocephala. The cells are rods of length 2.2 m with diameter 0.46 m also occurring in short chains. The cells stain gram-positive and the G+C content is 52.8 mol%. Growth occurred optimally with methanol (0.1 M) at 37°C and pH 7.4 with a doubling time of 7.5 h. The preferred sulfur source was glutathione. In addition to methanol, the isolate utilized 2-propanol as carbon and hydrogen source and also grew effectively on H₂+CO₂. Electron microscopy revealed the cell enveloped in triple layers consisting of a cell membrane and a cell wall separated by an electron transparent space with the nuclear zone at the centre of the cytoplasm. Presence of cytochromes was evident in the membrane fractions. The isolate possesses novel features.     

pS10147-2, a3.7 kb multicopy plasmid isolated from Streptomyces coelicolor

The following putative precursors of the pseudomurein were isolated from trichloroacetic acid extracts of Methanobacterium thermoautotrophicum: a uridine diphosphate activated derivative of glutamic acid and the uridine diphosphate activated peptides (see text). The activated glutamic acid residue and the three activated pepetides lack the glycan components N-acetylglucosamine and N-acetyltalosaminuronic acid present in the intact pseudomurein. In this case uridine diphosphate should be directly linked to the amino group of a glutamic acid residue, which represents a new mode of amino acid and peptide activation.     

Isolation and characterization of a new coccoid methanogen,Methanogenium tatii spec. nov. from a solfataric field on Mount Tatio

A new coccoid methanogen, Methanogenium tatii, was isolated and characterized. The mesophilic isolate can grow on and produce methane from H₂:CO₂ and formate. For growth acetate is strictly required. The cell shape, the G+C content of 54 mol% and DNA-DNA homology data suggest it to be a Methanogenium species.Abbreviations G+C Guanine+Cytosine - SDS Sodium dodecylsulfate     

Methanogenium liminatans spec. nov., a new coccoid,mesophilic methanogen able to oxidize secondary alcohols

A new mesophilic, coccoid methanogen, assigned as Methanogenium liminatans spec. nov. strain DSM 4140, was isolated from effluent of a reactor for the anaerobic treatment of industrial waste water. Cells of M. Liminatans formed irregular cocci, about 1.5 m in size, and occurred singly. The cell envelope was an S-layer with hexagonally arranged glycoprotein subunits (M_r=118000). The center-to-center spacings were 15.4 nm. The polar lipid pattern was similar to that of Methanogenium tationis, the polyamine content similar to that found in several Methanogenium species. Strain DSM 4140 grew with H₂/CO₂, formate, 2-propanol/CO₂, 2-butanol/CO₂ and cyclopentanol/CO₂. For growth with the different substrates acetate was required as an additional carbon source. Growth on H₂/CO₂ was stimulated by the addition of tungstate. The optimal concentration was 1–2 M Na₂WO₄. ¹⁸⁵WO _inf4 ^sup2- was incorporated into cells. Growth was not influenced by 0–600 mM NaCl, but no growth occurred in the presence of 800 mM NaCl. Increasing concentrations of KCl up to 100 mM were slightly inhibitory for growth. The optimal growth temperature was around 40°C. The G+C content of the DNA was 59.3 mol% (T_m) or 60.5 mol% (HPLC).Abbreviations HPLC = high performance liquid chromatography - PAS = periodic acid-Schiff reagent - sADH = secondary alcohol dehydrogenase - F₄₂₀ = 7,8-didemethyl-8-hydroxy-5-deazariboflavin     

Isolation and characterization of a thermophilic Methanobacterium able to use formate,the strain FTF

A thermophilic anaerobic which produced methane from formate and H₂ and CO₂ was isolated from a bench-scale digester treating a mixture of solid wastes at 55°C, after enrichment cultures on sodium acetate. The cells were slightly crooked rods occurring singly or in filaments. The bacterium was not motile, and stained Gram positive. Colonies appearing after 1 week of incubation were white with filamentous edges and 1 mm in diameter. The organism used H₂:CO₂ or formate as an energy source. Yeast extract was not required but stimulated growth significantly. Casamino acids were stimulatory and could serve as a nitrogen source. Cysteine was used as a sulfur source. The optimum pH for growth was 7.5. Growth occurred from 35 to 70°C with an optimum at 55°C. The deoxyribonucleic acid base composition was 49.2 mol% guanine plus cytosine. Though this isolate conforms to Methanobacterium thermoformicium, its proper assignment awaits further studies. It has been deposited in the Deutsche Sammlung von Mikroorganismen as strain DSM 3012.This work was supported in part by the Conseil Régional Nord/Pas-de-Calais     

Diaporthosporellaceae,a novel family of Diaporthales (Sordariomycetes,Ascomycota)

Diaporthosporella cercidicola, collected from diseased branches of Cercis chinensis in China, is herein described and illustrated. Evidence for this new genus and species is provided by both holomorphic morphology and phylogenetic analysis. Diaporthosporella cannot be classified into any existing family. Thus, Diaporthosporellaceae is introduced as a new family to accommodate Diaporthosporella, typified by D. cercidicola. Morphologically, Diaporthosporellaceae can be distinguished from other diaporthalean families by irregularly uniseriate, allantoid or subreniform ascospores, conidiophores acropleurogenous, branched or sympodially branched, cylindrical, aseptate, and ellipsoidal, biguttulate conidia. Phylogenetic analysis using the internal transcribed spacers (ITS) and large subunit (LSU) ribosomal (r) DNA sequence data shows that Diaporthosporellaceae forms a distinct family within Diaporthales.     

Homophilic adhesion mechanism of neurofascin, a member of the L1 family of neural cell adhesion molecules

The L1 family neural cell adhesion molecules play key roles in specifying the formation and remodeling of the neural network, but their homophilic interaction that mediates adhesion is not well understood. We report two crystal structures of a dimeric form of the headpiece of neurofascin, an L1 family member. The four N-terminal Ig-like domains of neurofascin form a horseshoe shape, akin to several other immunoglobulin superfamily cell adhesion molecules such as hemolin, axonin, and Dscam. The neurofascin dimer, captured in two crystal forms with independent packing patterns, reveals a pair of horseshoes in trans-synaptic adhesion mode. The adhesion interaction is mediated mostly by the second Ig-like domain, which features an intermolecular β-sheet formed by the joining of two individual GFC β-sheets and a large but loosely packed hydrophobic cluster. Mutagenesis combined with gel filtration assays suggested that the side chain hydrogen bonds at the intermolecular β-sheet are essential for the homophilic interaction and that the residues at the hydrophobic cluster play supplementary roles. Our structures reveal a conserved homophilic adhesion mode for the L1 family and also shed light on how the pathological mutations of L1 affect its structure and function.     

Planctopirus ephydatiae,a novel Planctomycete isolated from a freshwater sponge

The microbiome of freshwater sponges is rarely studied, and not a single novel bacterial species has been isolated and subsequently characterized from a freshwater sponge to date. A previous study showed that 14.4% of the microbiome from Ephydatia fluviatilis belong to the phylum Planctomycetes. Therefore, we sampled an Ephydatia sponge from a freshwater lake and employed enrichment techniques targeting bacteria from the phylum Planctomycetes. The obtained strain spb1^T was subject to genomic and phenomic characterization and found to represent a novel planctomycetal species proposed as Planctopirus ephydatiae sp. nov. (DSM 106606 = CECT 9866). In the process of differentiating spb1^T from its next relative Planctopirus limnophila DSM 3776^T, we identified and characterized the first phage – Planctopirus phage vB_PlimS_J1 – infecting planctomycetes that was only mentioned anecdotally before. Interestingly, classical chemotaxonomic methods would have failed to distinguish Planctopirus ephydatiae strain spb1^T from Planctopirus limnophila DSM 3776^T. Our findings demonstrate and underpin the need for whole genome-based taxonomy to detect and differentiate planctomycetal species.     

Human EML4, a novel member of the EMAP family, is essential for microtubule formation

Human EML4 (EMAP-like protein 4) is a novel microtubule-associated WD-repeat protein of 120 kDa molecular weight, which is classified as belonging to the conserved family of EMAP-like proteins. Cosedimentation assays demonstrated that EML4 associates with in vitro polymerized microtubules. Correspondingly, immunofluorescence stainings and transient expression of EGFP-labeled EML4 revealed a complete colocalization of EML4 with the interphase microtubule array of HeLa cells. We present evidence that the amino-terminal portion of EML4 (amino acids 1-249) is essential for the association with microtubules. Immunoprecipitation experiments revealed that EML4 is hyperphosphorylated on serine/threonine residues during mitosis. In addition, immunofluorescence stainings demonstrated that hyperphosphorylated EML4 is associated with the mitotic spindle, suggesting that the function of EML4 is regulated by phosphorylation. siRNA-mediated knockdown of EML4 in HeLa cells led to a significant decrease in the number of cells. In no case mitotic figures could be observed in EML4 negative HeLa cells. Additionally, we observed a significant reduction of the proliferation rate and the uptake of radioactive [3H]-thymidine as a result of EML4 silencing. Most importantly, EML4 negative cells showed a completely modified microtubule network, indicating that EML4 is necessary for correct microtubule formation.     

Characterization of a new mesophilic,secondary alcohol-utilizing methanogen,Methanobacterium palustre spec. nov. from a peat bog

The isolation and characterization of a new methanogen from a peat bog, Methanobacterium palustre spec. nov., strain F, is described. Strain F grew on H₂/CO₂ and formate in complex medium. It also grew autotrophically on H₂/CO₂. Furthermore, growth on 2-propanol/CO₂ was observed. Methane was formed from CO₂ by oxidation of 2-propanol to acetone or 2-butanol to 2-butanone, but growth on 2-butanol plus CO₂ apparently was too little to be measurable. Similarly, Methanobacterium bryantii M. o. H. and M. o. H. G formed acetone and 2-butanone from 2-propanol and 2-butanol, but no growth was measurable.On the basis of morphological and biochemical features strain F could be excluded from the genus Methanobrevibacter. Due to its cell morphology, lipid composition and polyamine pattern it belonged to the genus Methanobacterium. From known members of this genus strain F could be distinguished either by a different G+C content of the DNA, low DNA-DNA homology with reference strains, lacking serological reactions with anti-S probes and differences in the substrate spectrum.An alcohol dehydrogenase activity, specific for secondary alcohols and its substrate specificity was determined in crude extracts of strain F. NADP⁺ was the only electron carrier that was utilized. No reaction was found with NAD⁺, F₄₂₀, FMN and FAD.Abbreviations NAD⁺ nicotinamide adenine dinucleotide - NADH₂ reduced form of NAD⁺ - NADP⁺ nicotinamide adenine dinucleotide phosphate - NADPH₂ reduced form of NADP⁺ - FMN flavin adenine mononucleotide - FAD flavin adenine dinucleotide - ADH alcohol dehydrogenase - F₄₂₀ 8-hydroxy-7,8-didemethyl-5-deazaflavin - SSC standard saline citrate (0.15 M NaCl, 0.015 M trisodium citrate, pH 7.5)     

Cloning and characterization of CD300d, a novel member of the human CD300 family of immune receptors

Herein we present the cloning and molecular characterization of CD300d, a member of the human CD300 family of immune receptors. CD300d cDNA was cloned from RNA obtained from human peripheral blood mononuclear cells, and RT-PCR revealed the gene to be expressed in cells of myeloid lineage. The cloned cDNA encoded for a type I protein with a single extracellular Ig V-type domain and a predicted molecular mass of 21.5 kDa. The short cytoplasmic tail is lacking in any known signaling motif, but there is a negatively charged residue (glutamic acid) within the transmembrane domain. CD300d forms complexes with the CD300 family members, with the exception of CD300c. Contrary to other activating members of the CD300 family of receptors, surface expression of CD300d in COS-7-transfected cells required the presence of an immunoreceptor tyrosine-based activating motif-bearing adaptor (FcεRγ). Accordingly, we found that CD300d was able to recruit FcεRγ. Unexpectedly, we could not detect CD300d on the surface of cells expressing FcεRγ, suggesting the existence of unknown mechanisms regulating the trafficking of this molecule. The presence of other CD300 molecules also did not modify the intracellular expression of CD300d. In fact, the presence of CD300d decreased the levels of surface expression of CD300f but not CD300c. Our data suggest that the function of CD300d would be related to the regulation of the expression of other CD300 molecules and the composition of CD300 complexes on the cell surface.     

Teichoic acid choline esterase, a novel hydrolytic activity in Streptococcus oralis

Streptococcus oralis contains an enzyme that can remove a limited amount of choline residues when tested on purified cell walls. This activity has been identified as an esterase that exhibits some biochemical properties similar to those previously found for several lytic enzymes of S. pneumoniae and its bacteriophages.