Enumeration of Methanobrevibacter smithii in human feces

A platin medium containing cephalothin and clindamycin was developed for enumeration and isolation of methanogens in human feces. Specimens from nine CH₄-producing subjects had total anaerobe counts of 1–8×10¹¹/g dry weight. Methanogen counts on the antibiotic medium ranged from 0.001–12.6% of the total anaerobe count. There was no correlation between age, sex, or percent dry fecal weight and the ratio of methanogens to total counts. Specimens from eight non-CH₄-producing individuals contained bacteria thay yielded nonmethanogenic colonies on the antibiotic medium. The means±SD of the logarithm of the total counts per gram dry weight were 11.4±0.29 and 11.38±0.44 for the positive and negative groups respectively. Values for the antibiotic-resistant flora were 8.8±1.13 and 7.78±1.08 respectively. Methanogens were isolated from the most dilute inoculum of each specimen from CH₄-producing subjects. All isolates were morphologically, physiologically, and immunologically identical to Methanobrevibacter smithii. Growth of methanogens in media that were essentially extracts of CH₄-negative feces suggested that no nutrients were lacking or inhibitors present in intestinal contents that prevent the growth of methanogens in these individuals.     

Antigenic determinants distinctive of Methanospirillum hungatei and Methanogenium cariaci identified by monoclonal antibodies

Monoclonal antibodies were prepared against two species of Methanomicrobiaceae. Antibody 1A is specific for Methanospirillum hungatei strain JF1 and the determinant it recognizes is expressed on the surface of JF1 cells, where it is exposed and accessible to antibody. The determinant is found in a polypeptide (MW<12,000) in the sheath that covers the bacterial cell; it is not present in Methanospirillum hungatei strain GP1; and it is not expressed on the surface of whole cells of the other 24 methanogenic bacteria tested. It is therefore a marker of strain JF1, consequently, antibody 1A is potentially useful for tracking JF1 and fragments thereof in a variety of samples. Antibody 7A is specific for Methanogenium cariaci JR1c. It did not react with any other methanogen tested, not even with Mg. marisnigri or Ms. hungatei JF1, although these cross-react with Mg. cariaci if tested with polyclonal antisera. Therefore antibody 7A recognizes specifically a marker of Mg. cariaci JR1c.Abbreviations SIA slide immunoenzymatic assay - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis     

Histological analysis of somatic embryogenesis induced in leaf explants ofHelianthus smithii Heiser

Summary According to the hormonal conditions, after one month of culture shoots or somatic embryos could be obtained from leaf explants ofHelianthus smithii. Well-shaped embryos developed on media containing a combination of auxin and cytokinin, while on media containing only cytokinin shoots were observed. The primary leaves of these shoots resembled cotyledons. A detailed histological study of the regeneration process on three media, containing only cytokinin or auxin, or a combination of both, allowed the origin and development of the observed structures to be determined. All three conditions induced somatic embryos, which then developed differently and, within one month, finally gave rise to the two types of structures which were initially observed.Abbrevations BAP 6-benzylaminopurine - MES 2-(N-morpholino)ethane-sulfonic acid - MS medium of Murashige and Skoog - NAA 1 -naphthaleneacetic acid - TDZ thidiazuron     

Occurrence of Sulfolobus acidocaldarius,an extremely thermophilic acidophilic bacterium,in New Zealand hot springs

Several hot springs in the Rotorua-Taupo regions, North Island, New Zealand, were tested for the presence of extremely thermophilic acidophilic bacteria. In the majority of the springs, ranging in temperature from 43–96°C and in pH from 2.1–6.9, direct microscopic observations revealed the presence of both rod-shaped and spherical bacteria. Isolations were attempted at 70°C and pH 2.0 and 7.0, with either yeast extract for heterotrophic growth, or elemental sulfur as the sole source of energy for autotrophic growth. Eight of the samples produced grwoth at pH 2.0 with either yeast extract or sulfur, but none of the samples grew at pH 7.0. All the isolates obtained, resembled Sulfolobus acidocaldarius, a thermophilic acidophilic bacterium which has previously been reported from various regions in the Northern Hemisphere. Immunofluorescence examination of six of these isolates revealed varying degrees of cross reactions with two already characterized Sulfolobus isolates from the Yellowstone National Park, U.S.A. This paper is the first published record of Sulfolobus from the Southern Hemisphere.     

The response of mycorrhizal colonization to elevated CO2 and climate change in Pascopyrum smithii and Bouteloua gracilis

Large intact soil cores of nearly pure stands of Pascopyrum smithii (western wheatgrass, C₃) and Bouteloua gracilis (blue grama, C₄) were extracted from the Central Plains Experimental Range in northeastern Colorado, USA and transferred to controlled environment chambers. Cores were exposed to a variety of water, temperature and CO₂ regimes for a total of four annual growth cycles. Root subsamples were harvested after the completion of the second and fourth growth cycles at a time corresponding to late winter, and were examined microscopically for the presence of mycorrhizae. After two growth cycles in the growth chambers, 54% of the root length was colonized in P. smithii, compared to 35% in blue grama. Field control plants had significantly lower colonization. Elevation of CO₂ increased mycorrhizal colonization in B. gracilis by 46% but had no effect in P. smithii. Temperatures 4° C higher than normal decreased colonization in P. smithii by 15%. Increased annual precipitation decreased colonization in both species. Simulated climate change conditions of elevated CO₂, elevated temperature and lowered precipitation decreased colonization in P. smithii but had less effect on B. gracilis. After four growth cycles in P. smithii, trends of treatments remained similar, but overall colonization rate decreased.     

Synthesis and assembly of flagellar surface antigens during zoosporogenesis inPhytophthora cinnamomi

Summary Cryomicrotomy and immunofluorescence microscopy employing three different categories of monoclonal antibody (MAb) that label antigens on the surface of one or both flagella ofPhytophthora dnnamomi have been used to follow the synthesis and assembly of flagellar surface components. MAb Zf 1 binds to the surface of both the anterior tinsel and posterior whiplash flagella, as well as to a nuclear component. The labeling of the flagella is punctate in nature, is brighter at the flagellar base, and does not always extend to the distal tip of the flagella. MAbs in the Zt group recognise an antigen that is located along the sides of the tinsel flagellum and may be associated with the base of the mastigonemes. Immunodot-blot analysis has shown that binding of Zt MAbs is abolished by pretreatment with either pronase or periodate oxidation indicating that the antigen is a glycoprotein. MAbs in the Zg group bind to the mastigonemes on the tinsel flagellum and to packets of mastigonemes in the cytoplasm of zoospores. Zt and Zg antigens increase in abundance during zoosporogenesis and are present throughout the life cycle of the fungus, whereas the non-nuclear localisation of the Zf antigen appears only during sporulation. Prior to association with the flagellar surface, all three components become clustered in the groove region of zoospores. They do not become associated with the flagellar surface until at least 15 min after the flagellar axoneme has formed.Abbreviations BSA bovine serum albumin - DAPI 4,6-diamidino-2-phenylindole - DMF dimethylformamide - lgG₁ immunoglobulin G₁ - MAbs monoclonal antibodies - NIM non-immune mouse antibodies - PBS phosphate-buffered saline - PBST phosphate-buffered saline with 0.5% Tween 20 - PIPES 1,4-piperazinediethanesulfonic acid - PPD paraphenylenediamine dihydrochloride - RT room temperature - TBS tris-buffered saline - TEST tris-buffered saline with 0.05% Tween 20     

A reappraisal of antigenic determinants for nitrogenase from Azotobacter and nitrate reductase from Escherichia coli

Previous work based on double immunodiffusion assays had shown that there are common antigenic determinants for nitrate reductase from Escherichia coli and component I of nitrogenase from Azotobacter vinelandii. Further work reported herein using a variety of immunoelectrophoretic techniques indicates that the cross-reaction between nitrate reductase and antiserum to component I of nitrogenase results from a contaminant antigen co-purified with nitrate reductase.     

Common antigenic determinants in the glycoproteins of plants,molluscs and insects

An antiserum raised against -fructosidase isolated from the cell walls of suspension-cultured carrot cells cross-reacts with many plant proteins and hemocyanin ofHelix pomatia. The shared epitope appears to be a small complex glycan with a (1–2)-linked xylose residue attached to the -linked mannose residue of the core of an asparagine-linked oligosaccharide. There is strong cross-reactivity with the proteins of many seed plants, molluscs and insects, and no cross-reactivity with the proteins of fungi, algae, mosses, ferns, or any of the vertebrates tested. Xylose-containing glycans appear to increase the immunogenicity of the proteins to which they are attached, and we suggest that they may be responsible for some allergic responses of people that are repeatedly exposed to plant or insect proteins.     

Carbohydrate antigens and lectin receptors of the plasma membrane of carrot cells

Summary A monoclonal antibody (JIM 1) has been derived, subsequent to immunization of rats with carrot protoplasts and a hybridoma screen of protoplast immunoagglutination, that recognizes a determinant at the outer face of the plasma membrane of carrot cells. The binding of JIM 1 is readily inhibitable by -D-galactosyl residues. Although weakly cross-reacting with an extracellular arabinogalactan protein, isolated from the conditioned medium of suspension-cultured carrot cells, JIM 1 does not recognize arabinogalactan proteins associated with the plasma membrane. The plasma membrane antigen recognized by JIM 1 was of low molecular weight and was sensitive to both periodate treatment and a protease. JIM 1 therefore defines a new class of galactosyl-residue containing plant cell surface antigen, distinct from the arabinogalactan proteins. However, the extracellular arabinogalactan protein and related plasma membrane-associated glycoproteins are demonstrated to bind the anti-galactose plant lectin peanut agglutinin.Abbrevations AGP arabinogalactan protein - McAb monoclonal antibody - PNA peanut agglutinin     

Defective formation and/or utilization of carbon monoxide in H2/CO2 fermenting methanogens dependent on acetate as carbon source

Autotrophic methanogens reduce CO₂ to CO and assimilate CO in a carbonylation reaction. Heterotrophic species were found not to form CO and/or to incorporate CO into cell matiral. The absence of CO formation correlated with the absence of carbon monoxide dehydrogenase activity. The heterotrophic Methanobrevibacter ruminantium, Methanobrevibacter smithii, Methanococcus voltae and Methanospirillum hungatei (strain GP 1) were investigated.     

Selectivity of food bacteria for the growth of anaerobic ciliate Trimyema compressum

The anaerobic ciliate Trimyema compressum was cultivated on various food bacteria. Significant growth was observed when Lactobacillus sp., Escherichia coli, Enterobacter aerogenes, Desulfovibrio vulgaris, Methanoculleus bourgense, or Pelobacter propionicus cells were fed to the ciliates. The highest cell yield which we obtained was ca. 9,000 cells/ml when feeding D. vulgaris. However, no growth of the ciliates was observed on the culture with Clostridium novyi, Propionibacterium sp., Desulfobulbus propionicus, Methanobrevibacter arboriphilicus, Methanobacterium sp., Methanosarcina barkeri, or Methanothrix soehngenii cells. The ciliates produced acetate and methane as major end products in any cultures and small amounts of propionate, butyrate and hydrogen were also detected in some cultures. Physiological studies on the food bacteria which we tested indicated that the growth of T. compressum depended on the bacterial species, but there was no apparent correlation between the digestibility and the basic properties of those bacteria (i.e. size of the bacteria, gram-staining properties, susceptibility to the known lytic enzymes, Archaea or Bacteria).     

N 5,N 10-Methenyltetrahydromethanopterin cyclohydrolase from the extreme thermophile Methanopyrus kandleri: increase of catalytic efficiency (kcat/K M) and thermostability in the presence of salts

The activity of purified N ⁵,N ¹⁰-methenyltetrahydromethanopterin cyclohydrolase from Methanopyrus kandleri was found to increase up to 200-fold when potassium phosphate was added in high concentrations (1.5 M) to the assay. A 200-fold stimulation was also observed with sodium phosphate (1 M) and sodium sulfate (1 M) whereas stimulation by potassium sulfate (0.8 M), ammonium sulfate (1.5 M), potassium chloride (2.5 M), and sodium chloride (2 M) was maximal 100-fold. A detailed kinetic analysis of the effect of potassium phosphate revealed that this salt exerted its stimulatory effect by decreasing the K _m for N ⁵,N ¹⁰-methenyltetrahydromethanopterin from 2 mM to 40 M and by increasing the V _max from 2000 U/mg (k_cat=1385 s^-1) to 13300 U/mg (k_cat=9200 s^-1). Besides increasing the catalytic efficiency (k_cat/K _m) salts were found to protect the cyclohydrolase from heat inactivation. For maximal thermostability much lower concentrations (0.1 M) of salts were required than for maximal activity.Abbreviations H₄MPT tetrahydromethanopterin - N ⁵,N ¹⁰-methenyl-H₄MPT - CHO-H₄MPT N ⁵-formyl-H₄-MPT - CH₂=H₄MPT N ⁵,N ¹⁰-methylene-H₄MPT - CH₃–H₄-MPT N ⁵-methyl-H₄MPT - MOPS -N-morpholinopropane sulfonic acid - TRICINE N-[Tris(hydroxymethyl)-methyl]glycine - 1 U = 1 mol/min     

The apocytochrome b gene of Chlamydomonas smithii contains a mobile intron related to both Saccharomyces and Neurospora introns.

Summary The mitochondrial DNA of the two interfertile algal species Chlamydomonas smithii and Chlamydomonas reinhardtii are co-linear with the exception of ca. 1 kb insertion (the a insert) present in C. smithii DNA only. In vegetative diploids resulting from interspecific crosses, mitochondrial genomes are transmitted biparentally except for the a insert which is transmitted to all C. reinhardtii molecules in a manner reminiscent of the intron-mediated conversion event that occurs at the omega locus in yeast mitochondria, under the action of the I-SceI endonuclease. Here we report that the insert corresponds to a typical group I intron of 1075 bp, inserted within the gene for apocytochrome b and containing a 237 codon open reading frame (ORF). We also report the complete sequence of the apocytochrome b gene of C. smithii. Comparison with the sequence of the same gene in C. reinhardtii reveals the precise intron insertion site. These data, together with the previous genetic data provide the first example of intron mobility in mitochondria of the plant kingdom. The product of the intronic ORF shows 36% amino acid identity with the I-SceI endonuclease whereas the intron ribozyme shows a 60% identity at the nucleotide level with the Neurospora crassa cob · 1 intron. The possibility of a recent horizontal transfer of introns between fungi and algae is discussed.     

Immunolocalization of arabinogalactan proteins inAmaranthus hypochondriacus L. ovules

Summary Amaranthus hypochondriacus ovules are of the crassinucellate type, having several layers of nucellus cells between the micropyle and the embryo sac through which pollen tubes have to penetrate. The ultrastructural features of the micropylar nucellus cells appear to reflect cells with high metabolic activity. With the monoclonal antibodies MAC207 and JIM8 (against arabinogalactan proteins) we have shown that the presence of the two epitopes was different in the gametophytic tissues and embryo sac. The young embryo and suspensor cells are reactive only to Mab JIM8. The selective presence and localization of these two epitopes was also demonstrated in the micropylar nucellus cells. The expression of these arabinogalactan proteins in this ovule seems to be closely aligned with the pathway of the pollen tube, possibly providing directional guides for tube growth inside the ovule.     

Unusual distribution of a glycolipid antigen in the flagella ofChlamydomonas

Summary Mouse hybridomas were obtained that secrete monoclonal antibodies recognizing glycolipid antigens located in the flagellar membrane of the biflagellate alga,Chlamydomonas reinhardtii. The antigen is an acidic lipid that migrates slightly slower than a GM1 ganglioside on thin layer chromotography. The binding of the antibodies to the thin layer plate was inhibited by periodate oxidation suggesting that the antibodies are recognizing a carbohydrate epitope. In a variety ofChlamydomonas strains, these antibodies were found to stain the flagella of only a sub-set of the cells in the population, generally varying from 50% to 75% of the cells. Even after cloning, the population of cells continued to express this variability in staining, and presumably, expression of the glycolipid epitope. Although most cells showed either strong staining of both flagella or no detectable staining of both flagella, a subset of the cells in the culture exhibited differential antibody labeling of the two flagella, suggesting that an individualChlamydomonas can exhibit a different glycolipid composition in each of its two flagellar membranes and even differential expression along the length of an individual flagellum.     

Methanobacterium formicicum,an endosymbiont of the anaerobic ciliateMetopus striatus McMurrich

The Gram-positive methanogenic endosymbiont of the sapropelic ciliateMetopus striatus was isolated and identified asMethanobacterium formicicum. In the ciliate cell the methanogens are in close association with microbody-like organelles. No mitochondria could be detected. The nature of the microbodies and the physiological background of the observed association are discussed.     

Functional integration of Methanobacterium formicicum into the anaerobic ciliate Trimyema compressum

Monoxenic cultures of the anaerobic, endosymbiont-free ciliate Trimyema compressum were incubated with low numbers of Bacteroides sp. strain WoCb15 as food bacteria and two strains (DSM 3636 and 3637) of Methanobacterium formicicum, which originally had been isolated from the anaerobic protozoa Metopus striatus and Pelomyxapalustris. The ciliate which had lost its original endosymbiotic methanogens ingested both strains of M. formicicum. The methanogenic bacteria were found intact in large vacuoles in contrast to the food bacteria which were digested. Single methanogens were separated from the vacuoles and appeared surrounded by a membrane in the cytoplasm of the ciliate. After 2 months of incubation, the methanogenic bacteria still exhibited the typical bluish fluorescence and the new symbiotic association of M. formicicum and T. compressum excreted methane. Increasing the growth rate of the ciliates by large numbers of food bacteria resulted in a loss of the methanogenic bacteria, due to statistical outgrowth.     

Effect of coculture of anaerobic fungi isolated from ruminants and non-ruminants with methanogenic bacteria on cellulolytic and xylanolytic enzyme activities

Neocallimastix strain N1, an isolate from a ruminant (sheep), was cocultured with three Methanobacterium formicicum strains, Methanosarcina barkeri, and Methanobrevibacter smithii. The coculture with Methanobacterium formicicum strains resulted in the highest production of cellulolytic and xylanolytic enzymes. Subsequently four anaerobic fungi, two Neocallimastix strains (N1 and N2) from a ruminant and two Piromyces species from non-ruminants (E2 and R1), were grown in coculture with Methanobacterium formicicum DSM 3637 on filter paper cellulose and monitored over a 7-day period for substrate utilisation, fermentation products, and secretion of cellulolytic and xylanolytic enzymes. Methanogens caused a shift in fermentation products to more acetate and less ethanol, lactate and succinate. Furthermore the cellulose digestion rate increased by coculture. For cocultures of Neoallimastix strains with Methanobacterium formicicum strains the cellulolytic and xylanolytic enzyme production increased. Avicelase, CMCase and xylanase were almost completely secreted into the medium, while 40–60% of the -glucosidase was found to be cell bound. Coculture had no significant effect on the location of cellulolytic and xylanolytic enzymes.     

Inhibition of methanogenesis by sulphate reducing bacteria competing for transferred hydrogen

A methanogenic bacterial consortium was obtained after inoculation of benzoate medium under N₂/CO₂ atmosphere with intertidal sediment. A hydrogen donating organotroph andMethanococcus mazei were isolated from this enrichment. H₂-utilising sulphate reducing bacteria were isolated under H₂/CO₂ in the absence of organic electron donors. TheMethanococcus was able to produce methane in yeast extract medium under N₂/CO₂ if the H₂ donating organism was present, and sulphate reduction occurred if the hydrogen utilising sulphate reducing bacteria were grown with the H₂ donating organism. The ability of the H₂ utilising sulphate reducing bacteria to inhibitMethanococcus competitively was shown in cultures containing both of these H₂ utilising bacteria.Abbreviations HDO hydrogen donating organism - SRB sulphate reducing bacteria - HSRB hydrogen utilising sulphate reducing bacteria     

Total synthesis of acetyl coenzyme a involved in autotrophic CO2 fixation inAcetobacterium woodii

The Gram positive anaerobeAcetobacterium woodii is able to grow autotrophically with a mixture of H₂ and CO₂ as the energy and carbon source. The question, by which pathway CO₂ is assimilated, was studied using long term isotope labeling.Autotrophically growing cultures produced acetate parallel to cell proliferation, and, when U-[¹⁴C]acetate was present as tracer, incorporated radioactivity into all cell fractions. The specific radioactivity and the label positions were determined for those representative cell compounds which biosynthetically originated directly from acetyl CoA (N-acetyl groups), pyruvate (alanine), oxaloacetate (aspartate), -ketoglutarate (glutamate), and hexosephosphates (glucosamine). Per mol compound the same amount of labeled acetate was incorporated into N-acetyl groups, alanine (C-2, C-3), aspartate (C-2, C-3), and twice the amount into glutamate (C-2, C-3, C-4, C-5) and into glucosamine. Consequently, the unlabeled carbon atoms of the C₃–C₆ compounds must have been derived from CO₂ by carboxylation subsequent to acetyl CoA synthesis. When 0.2 mM 2-[¹⁴C]pyruvate was added to autotrophically growing cultures, also a substantial amount of radioactivity was incorporated. Two important differences in comparison to the acetate experiment were observed: The N-acetyl groups were almost unlabeled and glutamate contained the same specific radioactivity as alanine or aspartate.These data showed that acetyl CoA is the central intermediate for biosynthesis and excluded the operation of the Calvin cycle inA. woodii. The results were consistent with the operation of a different autotrophic CO₂ fixation pathway in which CO₂ is converted into acetyl CoA by total synthesis via methyltetrahydrofolate; acetyl CoA is then further reductively carboxylated to pyruvate.