Hermann Bothe

Biodiversity of Denitrifying and Dinitrogen-Fixing Bacteria in an Acid Forest Soil

ABSTRACT Isolated soil DNA from an oak-hornbeam forest close to Cologne, Germany, was suitable for PCR amplification of gene segments coding for the 16S rRNA and nitrogenase reductase (NifH), nitrous oxide reductase (NosZ), cytochrome cd1-containing nitrite reductase (NirS), and Cu-containing nitrite reductase (NirK) of denitrification. For each gene segment, diverse PCR products were characterized by cloning and sequencing. None of the 16S rRNA gene sequences was identical to any deposited in the data banks, and therefore each of them belonged to a noncharacterized bacterium. In contrast, the analyzed clones of nifH gave only a few different sequences, which occurred many times, indicating a low level of species richness in the N2-fixing bacterial population in this soil. Identical nifH sequences were also detected in PCR amplification products of DNA of a soil approximately 600 km distant from the Cologne area. Whereas biodiversity was high in the case of nosZ, only a few different sequences were obtained with nirK. With respect to nirS, cloning and sequencing of the PCR products revealed that many false gene segments had been amplified with DNA from soil but not from cultured bacteria. With the 16S rRNA gene data, many sequences of uncultured bacteria belonging to the Acidobacterium phylum and actinomycetes showed up in the PCR products when isolated DNA was used as the template, whereas sequences obtained for nifH and for the denitrification genes were closely related to those of the proteobacteria. Although in such an experimental approach one has to cope with the enormous biodiversity in soils and only a few PCR products can be selected at random, the data suggest that denitrification and N2 fixation are not genetic traits of most of the uncultured bacteria.

Selective Element Deposits in Maize Colonized by a Heavy Metal Tolerance Conferring Arbuscular Mycorrhizal Fungus

Summary The Glomus isolate Br1 from the zinc violet, Viola calaminaria (DC.) Lej., confers heavy metal tolerance to plants including maize, alfalfa, barley and others (see accompanying paper). In the present study, the bulk analysis of maize grown in two different heavy metal soils in greenhouse experiments indicated that roots and shoots contained considerably lower heavy metal concentrations when maize was colonized with the isolate Br1 compared with plants grown with a common Glomus strain or to non-colonized controls. Essential elements like K, P and Mg were enriched in roots in Br1 colonized maize. Since arbuscular mycorrhizal (AM) plants grew much faster until flower and seed formation and had an approximately 25-fold higher dry weight than the controls, a massive acquisition of essential elements has happened. Data from three different microbeam techniques indicated distinct differences in the cellular distribution of heavy metals and essential elements in AM colonized roots compared with the non-mycorrhizal controls. SIMS images showed a selective enrichment of Mg, Ca, Fe, Ni and Zn in the inner cortical cell region containing the fungal structures (arbuscules) and a lower concentration of the heavy metals Fe, Zn and Ni in the stele than in the cortex. EDXA measurements indicated a selective enrichment of Mg and K in the stele. The data from SIMS and LAMMA suggested Al to be more or less evenly distributed in the root cells. The present investigation appears to be the first comprehensive approach to map elemental distribution within root tissues in AM colonized and control maize by three different methods of microbeam analysis. Since the microbeam techniques had to be applied near the detection limit of the methods, the data obtained by the three different approaches were not always uniform. However, the combination of these three techniques showed that the growth of maize in the heavy metal soil was at least partly due to a selective immobilization of heavy metals within those root tissues containing the fungal cells. The measurements also indicated that AM fungi might cope with heavy metal toxicity for each metal individually.

The Zinc Violet and its Colonization by Arbuscular Mycorrhizal Fungi

Summary Among the plants growing on several heavy metal soils, the zinc violets (the yellow Viola calaminaria (DC.) Lej. s. str. of the Aachen/Liege area and the blue Viola guestphalica Nauenburg of Blankenrode/Paderborn) were consistently colonized by arbuscular mycorrhizal (AM) fungi. The degree of AM-colonization apparently correlated with the heavy metal content in soils as indicated by the composition of the plant community. Among diverse violets examined from various non-polluted areas, Viola lutea (DC.) Lej. and some other alpine violets showed high mycorrhizal colonizations of the roots. A specific Glomus Br1 isolate was obtained from the roots of the yellow zinc violet ( V. calaminaria s. str.) of the Breinigerberg area near Aachen. RFLP-analysis indicated the uniformity of this isolate. Incubation with Glomus Br1 allowed plants like maize, barley, alfalfa and zinc violets to grow until flower and seed formation in two different heavy metal soils supplemented with nutrient solutions in greenhouse experiments. Controls (sterilized heavy metal soils not inoculated with Glomus Br1 or yellow lupins as non-mycorrhizal plants) did not grow. The Glomus Br1 isolate from the zinc violet more efficiently supported growth of maize or alfalfa in heavy metal soils than a commonly used Glomus intraradices Schenck and Smith isolate. The potential applications of these findings are discussed.

Nitrogen Fixation and Hydrogen Metabolism in Cyanobacteria

SUMMARY This review summarizes recent aspects of (di)nitrogen fixation and (di)hydrogen metabolism, with emphasis on cyanobacteria. These organisms possess several types of the enzyme complexes catalyzing N2 fixation and/or H2 formation or oxidation, namely, two Mo nitrogenases, a V nitrogenase, and two hydrogenases. The two cyanobacterial Ni hydrogenases are differentiated as either uptake or bidirectional hydrogenases. The different forms of both the nitrogenases and hydrogenases are encoded by different sets of genes, and their organization on the chromosome can vary from one cyanobacterium to another. Factors regulating the expression of these genes are emerging from recent studies. New ideas on the potential physiological and ecological roles of nitrogenases and hydrogenases are presented. There is a renewed interest in exploiting cyanobacteria in solar energy conversion programs to generate H2 as a source of combustible energy. To enhance the rates of H2 production, the emphasis perhaps needs not to be on more efficient hydrogenases and nitrogenases or on the transfer of foreign enzymes into cyanobacteria. A likely better strategy is to exploit the use of radiant solar energy by the photosynthetic electron transport system to enhance the rates of H2 formation and so improve the chances of utilizing cyanobacteria as a source for the generation of clean energy.

Denitrification within the genus Azospirillum and other associative bacteria

Different Azospirillum strains and some other plant growth-promoting rhizobacteria (PGPR) were screened for the occurrence of genes coding for denitrification and nitrogenase reductase (nifH) using polymerase chain reaction (PCR)-based techniques. All PGPR examined were nitrogenasc-positive. Azospirillum strains were remarkably dissimilar with respect to denitrification capabilities, in particular with respect to genes of the dissimilatory nitrite reductase, A. brasilense, A. lipoferum and A. halopraeferens strains possess a cytochrome cd 1 -containing nitritc reductase with low sequence similarities among them. A. irakense and A. doebereinerae have a Cu-containing nitrite reductase and A. amazonense is unable to denitrify. The molecular data were corroborated by activity measurements. The current results indicate that the inability to perform denitrification is unlikely a selective advantage for Azospirillum spp. and other associative bacteria for forming an association with plant roots.

Arbuscular mycorrhizal colonization of halophytes in Central European salt marshes

Abstract Halophytes from both coastal and inland Central European salt marshes were examined for colonization by arbuscular mycorrhizal (AM) fungi. Plants from different families were strongly colonized but the degree of colonization varied with the individual plant and apparently during the vegetation period, too. Members of the typical non-mycorrhizal families like Armeria maritima of the Plumbaginaceae and Salicornia europaea of the Chenopodiaceae were found to be colonized, particularly in the drier salt marshes. High numbers of Glomus spores were found in the saline soils, especially those of the inland locations examined. Approximately 80% of these spores were from Glomus geosporum as shown by a typical restriction fragment length polymorphism (RFLP) pattern of the amplified internal transcribed spacer regions. The present study demonstrates that RFLP analysis is useful when screening habitats for the occurrence of mycorrhizal fungi which can be identified only with difficulty by morphological criteria.

Biology of inorganic nitrogen and sulfur

General Introduction.- Microorganisms Involved in the Nitrogen and Sulfur Cycles (With 2 Figures).- Nitrogen Metabolism in General.- Nitrogen Metabolism in Plants (With 5 Figures).- The Assimilatory Reduction of Nitrate.- Dissimilatory Nitrate Reduction (With 10 Figures).- Dinitrogen Fixation.- Genetics of Dinitrogen Fixation (With 1 Figure).- Rhizobium Genetics (With 7 Figures).- Some Aspects of the Physiology of Dinitrogen Fixation (With 3 Figures).- The Biochemistry of Dinitrogen Fixation (With 3 Figures).- Pathways and Regulatory Aspects of N2 and NH4+ Assimilation in N2 -Fixing Bacteria (With 1 Figure).- The Hydrogenase-Nitrogenase Relationship in Nitrogen-Fixing Organisms (With 1 Figure).- Sulfur Metabolism in General.- Assimilatory Sulfate Reduction (With 7 Figures).- Ecology and Physiology of Some Anaerobic Bacteria from the Microbial Sulfur Cycle (With 1 Figure).- Dissimilatory Sulfate Reduction, Mechanistic Aspects (With 2 Figures).- Dissimilatory Sulfate Reduction, Energetic Aspects (With 9 Figures).- Photolithotrophic Sulfur Oxidation.- Oxidation of Ammonia by Nitrosomonas and of Inorganic Sulfur by Thiobacilli (With 3 Figures).- Contributed Papers: Nitrogen Metabolism.- Enzymatic Mechanism and Regulation of Nitrate Reduction in Rhodopseudomonas capsulata.- HCN and the Control of Nitrate Reduction. The Regulation of the Amount of Active Nitrate Reductase Present in Chlorella Cells (With 4 Figures).- Regulation of Nitrate Uptake in Green Algae and Duckweeds. Effect of Starvation and Induction(With 6 Figures).- Nitrate Uptake and Reduction in Chlorella - Characterisation of Nitrate Uptake in Nitrate-Grown and Nitrogen-Starved Chlorella sorokiniana (With 9 Figures).- Nitrogen Metabolism in Photosynthetically Inhibited Plants (With 5 Figures).- Investigations on the Reduction of Aliphatic and Aromatic Nitro Compounds by Clostridium Species and Enzyme Systems (With 6 Figures).- Fine Structure Analysis of the Gene Region for N2-Fixation (nif) of Klebsiella pneumoniae (With 7 Figures).- In vitro Associations Between Non-legumes and Rhizobium (With 13 Figures).- Limitations of Symbiotic and Associative Nitrogen Fixation by Developmental Stages in the Systems Rhizobium japonicum with Glycine max and Azospirillum brasilense with grasses e.g. Triticum aestivum (With 9 Figures).- Regulation of Nitrogenase Biosynthesis in Free-Living and Symbiotic N2-Fixing Bacteria: a Comparison (With 4 Figures).- Characterization of the Nitrite-Oxidizing System in Nitrobacter (With 8 Figures).- Contributed Papers: Sulfur.- The Role of Thioredoxins for Enzyme Regulation in Cyanobacteria (With 4 Figures).- Recent Results on the Assimilatory Sulfate Reduction: APS-Kinase and the reduction of APS to Cysteine in Higher Plants (With 1 Figure).- Aspects of S- and N-Metabolism in Tissue Cultures (With 4 Figures).- Regulation of Adenosine 5?-Phosphosulfate Sulfotransferase in Higher Plants (With 3 Figures).- On the Enzymatic System Thiosulfate-Cytochrome c-Oxidoreductase (With 6 Figures).

Improved Assessment of Denitrifying, N2-Fixing, and Total-Community Bacteria by Terminal Restriction Fragment Length Polymorphism Analysis Using Multiple Restriction Enzymes

ABSTRACT A database of terminal restriction fragments (tRFs) of the 16S rRNA gene was set up utilizing 13 restriction enzymes and 17,327 GenBank sequences. A computer program, termed TReFID, was developed to allow identification of any of these 17,327 sequences by means of polygons generated from the specific tRFs of each bacterium. The TReFID program complements and exceeds in its data content the Web-based phylogenetic assignment tool recently described by A. D. Kent, D. J. Smith, B. J. Benson, and E. W. Triplett (Appl. Environ. Microb. 69:6768-6766, 2003). The method to identify bacteria is different, as is the region of the 16S rRNA gene employed in the present program. For the present communication the software of the tRF profiles has also been extended to allow screening for genes coding for N2 fixation (nifH) and denitrification (nosZ) in any bacterium or environmental sample. A number of controls were performed to test the reliability of the TReFID program. Furthermore, the TReFID program has been shown to permit the analysis of the bacterial population structure of bacteria by means of their 16S rRNA, nifH, and nosZ gene content in an environmental habitat, as exemplified for a sample from a forest soil. The use of the TReFID program reveals that noncultured denitrifying and dinitrogen-fixing bacteria might play a more dominant role in soils than believed hitherto.

Towards Growth of Arbuscular Mycorrhizal Fungi Independent of a Plant Host

ABSTRACT When surface-sterilized spores of the arbuscular mycorrhizal fungus (AMF) Glomus intraradices Sy167 were germinated on agar plates in the slightly modified minimum mineral medium described by G. Bécard and J. A. Fortin (New Phytol. 108:211-218, 1988), slime-forming bacteria, identified as Paenibacillus validus, frequently grew up. These bacteria were able to support growth of the fungus on the agar plates. In the presence of P. validus, hyphae branched profusely and formed coiled structures. These were much more densely packed than the so-called arbuscule-like structures which are formed by AMF grown in coculture with carrot roots transformed with T-DNA from Agrobacterium rhizogenes. The presence of P. validus alone also enabled G. intraradices to form new spores, mainly at the densely packed hyphal coils. The new spores were not as abundant as and were smaller than those formed by AMF in the monoxenic culture with carrot root tissues, but they also contained lipid droplets and a large number of nuclei. In these experiments P. validus could not be replaced by bacteria such as Escherichia coli K-12 or Azospirillum brasilense Sp7. Although no conditions under which the daughter spores regerminate and colonize plants have been found yet, and no factor(s) from P. validus which stimulates fungal growth has been identified, the present findings might be a significant step forward toward growth of AMF independent of any plant host.

Quantification of vesicular-arbuscular mycorrhiza by biochemical parameters

Summary The present study compares three different biochemical parameters (yellow pigment, chitin, sterols) to quantify vesicular-arbuscular mycorrhiza. Plants like onion and corn form a yellow pigment upon mycorrhizal colonization which can easily be extracted in water by autoclaving roots and be quantified spectrophotometrically. The method is, however, not very sensitive and not applicable to all VA-mycorrhizal symbioses. A new method is described to assess mycorrhizal colonization by the chitin content. However, it is probably of limited value as the chitin content apparently does not well correlate with the different VAM structures (external and internal hyphae, vesicles and arbuscules). Among the free sterols, ergosterol content cannot be taken as a measure for VAM colonization because it occurs at best in minute amounts in this symbiosis and does not correlate with the mycorrhizal colonization. In contrast, campesterol (24-methylcholesterol) and 24-methylenecholesterol are formed at significant higher percentages of free sterols upon mycorrhizal colonization with all plants and fungal isolates assayed. The two sterols could not easily be separated from each other but readily from other sterols by gas chromatography under the conditions employed. A close correspondance was observed between the relative concentrations of both campesterol and 24-methylenecholesterol and the degree of mycorrhizal colonization determined by microscopic counting. Thus, quantifying the campesterol and 24-methylenecholesterol content by gas chromatographic analysis appears to be the method of choice to assess mycorrhizal colonization. The two sterols can quantitatively be separated from each other by combining HPLC and gas chromatography which is, however, unnecessary for routine analysis. Sitosterol, stigmasterol and cholesterol as well as total sterol content do not change very much in parallel with mycorrhizal colonization and can, therefore, not be taken as measures to assess VAM.