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  • 1
    Electronic Resource
    Electronic Resource
    Whole cell hybridization as a tool to study Frankia populations in root nodules (1997)
    Oxford, UK : Blackwell Publishing Ltd
    Physiologia plantarum 99 (1997), S. 0 
    Details
    ISSN: 1399-3054
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Molecular methods based on DNA or rRNA hybridization are powerful tools in microbial ecology for the specific detection and enumeration of bacteria unbiased by the limitations of culturability. A promising alternative to the analysis of Frankia populations in root nodules by methods based on rRNA extraction or on DNA extraction followed by the polymerase chain reaction (PCR) is the whole cell hybridization technique. This technique includes the microscopic detection of labeled probes hybridized to specific target sequences on marker molecules such as rRNA in fixed microbial cells. The analysis of uncultured Frankia populations in root nodules can reliably be performed on a subgroup level when digoxigenin-labeled oligonucleotide probes or in vitro transcripts directed against an actinomycetes-specific insertion on the 23S rRNA are used. Digoxigenin-labeled probes are more suitable for in situ detection of Frankia than fluorescent probes since the sensitivity is higher and problems arising from the autofluorescence of cells and plant material are avoided. All these strategies, however, require pretreatments to increase the permeability of vesicles, hyhae and spores.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1399-3054.1997.tb05374.x
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  • 2
    Electronic Resource
    Electronic Resource
    Assessing Frankia populations in plants and soil using molecular methods (1999)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology ecology 29 (1999), S. 0 
    Details
    ISSN: 1574-6941
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: In recent years, molecular approaches have increasingly supplemented nodulation-dependent detection methods for studying Frankia populations in nature. The new methods are revealing much about the genetic diversity and distribution of Frankia, as well as refining and expanding knowledge about endophyte-host specificities. PCR-based approaches have been used to unravel the phylogenetic relationships of isolates, as well as of uncultured endophytes in root nodules of many actinorhizal plants from which no isolates have been obtained. A comparative sequence analysis of PCR-amplified 16S ribosomal DNA led to the emendation of the family Frankiaceae to contain only the genus Frankia with four main subdivisions: (i) a large group mainly comprising Frankia alni and other typical nitrogen-fixing strains belonging to the Alnus and the Casuarina host infection groups, respectively, (ii) uncultured endophytes of Dryas, Coriaria and Datisca species, (iii) strains of the Elaeagnus host infection group and (iv) atypical non-nitrogen-fixing strains. A considerable diversity among both cultured Frankia strains and uncultured endophytes in nodules was indicated using RFLP analyses of PCR-amplified fragments of the 16S rRNA gene, the glutamine synthetase II (glnII) gene, the intergenic spacer of the 16S-23S rRNA operon or the intergenic spacer between the nitrogenase nifH and nifD (nifH-D) or the nifD and nifK (nifD-K) genes. The growing database of discriminative target sequences for frankiae is increasingly exploited for studies on the distribution of specific Frankia populations in the environment using PCR or in situ hybridization. Until recently, most studies have focused on the analysis of Frankia populations in root nodules, the natural locale of enrichment for this organism. These populations, however, represent only the fraction of physiologically active, infecting frankiae in soils rather than the total Frankia population. Future approaches to studies of Frankia populations should therefore incorporate the many opportunities for more than just phylogenetic analyses, the description of diversity and studies of Frankia populations in nodules. The molecular approaches open the door to more sophisticated studies of environmental influences on the dynamics of indigenous or introduced Frankia populations in plants and soil. These studies may lead to advancements in the management of actinorhizal plants and Frankia, provided specific Frankia populations can be attributed with silviculturally beneficial features. Such features include persistence and the growth in soil, competition with less efficient Frankia populations for nodule formation, prompt and efficient nodule formation and an ultimately superior nitrogen-fixing capacity.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1574-6941.1999.tb00613.x
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  • 3
    Electronic Resource
    Electronic Resource
    Origins and fate of fungi and bacteria in the gut of Lumbricus terrestris L. studied by image analysis (1999)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology ecology 28 (1999), S. 0 
    Details
    ISSN: 1574-6941
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: The effect of the passage through the gut of the earthworm Lumbricus terrestris L. on fungi and bacteria ingested with decomposing leaves of Taraxacum officinale and with soil was quantified using image analysis tools. Both leaf and soil material were labeled with fluorescent latex microbeads to allow a quantification of the food sources in the fore-, mid-, and hindgut of the earthworms. The content of leaf material in the gut varied in a range between 4 and 59% of the total gut content in different earthworms and the different parts of the intestine of individual animals. Filamentous fungi in the gut compartments were found to originate mainly from leaf material (7700±1800 μg (g leaf (dry wt.))−1), however, the major part was disrupted before arriving in the intestine. Remaining hyphae in the foregut with a biomass of up to 900±150 μg (g gut content (dry wt.))−1 were completely digested during passage through the earthworm gut. Spores of fungi were not detected in our studies. Bacterial cell numbers in the gut compartments ranged from 63±5×108 to 327±16×108 (g gut content (dry wt.))−1 and were significantly higher than the numbers found in the soil (50±1×108 cells (g soil (dry wt.))−1). Cell numbers usually increased from fore- to hindgut. This increase was not correlated to contents of organic material and only partially due to a multiplication of bacterial cells. Numbers of dividing cells accounted in total for approximately 12% of all bacteria, increasing significantly from fore- to hindgut, counts were from 10±1×108 to 25±2×108 (g gut content (dry wt.))−1, respectively. Average cell volumes of bacteria calculated from cell size distributions in leaf and soil material differed significantly, being 0.197 and 0.063 μm3, respectively. In the gut compartments, average cell volumes ranged from 0.043 to 0.070 μm3, which may indicate the disruption of large cells originating from the leaves before arriving in the foregut.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1574-6941.1999.tb00579.x
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  • 4
    Electronic Resource
    Electronic Resource
    Analysis of Frankia populations in three soils devoid of actinorhizal plants (1999)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology ecology 28 (1999), S. 0 
    Details
    ISSN: 1574-6941
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Frankia populations were analyzed in three soils devoid of actinorhizal plants but containing monocultures of birch (Betula pendula Roth), pine (Pinus sylvestris L.) or spruce (Picea abies (L.) Karsten). Bioassays using seedlings of Alnus incana as capture plants resulted in nodulation capacities of 3160±7, 2267±13, and 2747±6 nodulation units g−1 of these soils, respectively. Comparative sequence analysis of an actinomycetes-specific insertion in domain III of the 23S rRNA allowed a grouping of isolates obtained from nodules of the capture plants into three distinct groups of the Alnus host infection group. This separation was confirmed by the analysis of genomic fingerprints of the isolates generated by rep-PCR fingerprinting with the BOX primer. Genomic fingerprints also demonstrated that all isolates differed from each other. The isolates accounted for a significant proportion of the Frankia population in root nodules of the capture plants as shown by in situ hybridization with specific probes. However, only those Frankia strains isolated from soil of the birch stand via Alnus seemed to represent the total Frankia population in root nodules. Nodules induced after inoculation with soil from the pine or spruce stand also contained Frankia populations which were not isolated during this study and which could not be identified by in situ hybridization. Depending upon whether the soil originated from a birch, pine or spruce stand, different Frankia populations were found in the nodules of the capture plants. Because a nested PCR on nucleic acids extracted from these different soils did not indicate differences in the diversity of the total Frankia populations, it was concluded that Frankia populations in nodules of the capture plants represent the fraction of physiologically active, infecting frankiae in the soils rather than the total Frankia population.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1574-6941.1999.tb00556.x
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  • 5
    Electronic Resource
    Electronic Resource
    Molecular identification of an uncultured bacterium (“morphotype R”) in meromictic Lake Cadagno, Switzerland (2005)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology ecology 53 (2005), S. 0 
    Details
    ISSN: 1574-6941
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Comparative sequence analysis of almost complete 16S rRNA genes of members of the Desulfobacteriaceae retrieved from two gene clone libraries of uncultured bacteria of the chemocline of Lake Cadagno, Switzerland, resulted in the molecular identification of nine sequences, with a tight cluster of five sequences that represented at least three different populations of bacteria with homology values of 95% and 93% to their closest cultured relatives Desulfomonile tiedjei and Desulfomonile limimaris, respectively. In situ hybridization with probes DsmA455 targeting two subpopulations and DsmB455 targeting one subpopulation, detected bacteria with a peculiar morphology previously described as “morphotype R”. The individual probes detected about the same number of cells in all samples and together added up to represent all cells of “morphotype R” suggesting that the basic ecophysiological requirements of the subpopulations might be similar. In the monimolimnion, “morphotype R” cells accounted for up to 29% of all Bacteria and entirely represented the Desulfobacteriaceae, the most prominent sulfate-reducing bacteria. In the sediment, “morphotype R” was similarly prominent in the upper cm only where it represented all Desulfobacteriaceae and up to 50% of all Bacteria. Numbers and importance within the Desulfobacteriaceae and Bacteria declined significantly with depth in sediments suggesting potential effects of changing environmental conditions on the fate of “morphotype R”.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1016/j.femsec.2004.12.012
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  • 6
    Electronic Resource
    Electronic Resource
    Analysis of bacterial communities in heavy metal-contaminated soils at different levels of resolution (1999)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology ecology 30 (1999), S. 0 
    Details
    ISSN: 1574-6941
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: The impact of heavy metal contamination on soil bacterial communities was studied in soils amended for many years with sewage sludge contaminated with heavy metals to varying extents. At the broad level of resolution, DNA reassociation analysis indicated a dramatic decrease in bacterial diversity from 16 000 bacterial genomes (g soil [wet wt])−1 in the non-contaminated soil to 6400 bacterial genomes (g soil [wet wt])−1 in soil with low metal amendments and only 2000 bacterial genomes (g soil [wet wt])−1 in soil with high metal amendments. No differences between bacterial communities of these soils, however, were displayed in the %G+C profiles analysed by thermal denaturation. At a coarse level of characterisation, in situ hybridisation analysing larger phylogenetic groups of bacteria revealed a general decrease in the percentage of cells detected with probes ARCH915, BET42a, GAM42a, SRB385, CF319a, LGCb and HGC69a with increasing metal amendment. Only cells detected with probe ALF1b increased significantly from 3.1±0.8% of the cells detected by the domain-specific probe EUB338 in the non-contaminated soil to 6.5±1.3% in soil with high metal amendments. These shifts in populations of larger phylogenetic groups were largely confirmed by dot blot analysis of 16S and 23S rDNA clone libraries from bacteria in soil with low metal and high metal amendments, respectively. For a fine-level characterisation, 72 clones of 16S rDNA libraries were identified by comparative sequence analysis. A few sequences could not be assigned to the major taxa described. Most of the sequences were assigned to the Gram-positive bacteria with a high DNA G+C content (45%) and the α-subdivision of Proteobacteria (24%). However, only minor differences were seen between bacterial communities from the low and high metal soils. In the soil with high metal amendment, more sequences clustered to the α-subdivision of Proteobacteria, while in the low metal soil, more sequences clustered to the Gram-positive bacteria with a high DNA G+C content.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1574-6941.1999.tb00652.x
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  • 7
    Electronic Resource
    Electronic Resource
    Analysis of bacterial and protozoan communities in an aquifer contaminated with monoaromatic hydrocarbons (1998)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology ecology 27 (1998), S. 0 
    Details
    ISSN: 1574-6941
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Bacterial and protozoan communities were examined in three cores (A, B and C) from an aquifer located at an abandoned refinery near Hünxe, Germany. Cores were removed along a transect bordering a plume containing various monoaromatic hydrocarbons. Monoaromatic hydrocarbons could not be detected in the unsaturated zone in any core but were present in the saturated zones of core C (between 280 and 42 600 μmol kg−1 of core material [dry wt.]) and cores A and B (between 30 and 190 μmol kg−1 of core material [dry wt.]). Xylene isomers accounted for 50–70% of monoaromatic hydrocarbons in all cores. The number of DAPI-stained bacteria was found to increase from the low-contaminated cores A and B (approx. 0.1×108 cells and 0.2×108 cells g−1 of core material [dry wt.], respectively) to the high-contaminated core C (2.4×108 cells g−1 of core material [dry wt.]). The higher bacterial numbers in core C were found to coincide with a higher detection rate obtained by in situ hybridization using probe Eub338 to target the domain Bacteria (13–42% for core C as compared to 3–25% for cores A and B, respectively). Proteobacteria of the δ-subdivision (which includes many sulfate-reducing bacteria) were the most predominant of the groups investigated (7–15% of DAPI-stained bacteria) and were followed by Proteobacteria of the γ- and β-subdivisions (4% and 1% of DAPI-stained bacteria, respectively). The total numbers of protozoa and bacteria determined by direct counting occurred in a ratio of approx. 1:103, which was independent of depth or core examined. Most probable number analysis combined with a subsequent classification of the culturable protozoa revealed nanoflagellates as the major component of the protozoan community. Naked amoebae became increasingly more encysted with depth, except in the high-contaminated core C where vegetative trophozoites were present in the saturated zone. The co-occurrence of bacteria and protozoa in association with high concentrations of monoaromatic hydrocarbons suggests the involvement of trophic interactions in the process of biodegradation.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1574-6941.1998.tb00532.x
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  • 8
    Electronic Resource
    Electronic Resource
    Isolation and characterization of aggregate-forming sulfate-reducing and purple sulfur bacteria from the chemocline of meromictic Lake Cadagno, Switzerland (2003)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology ecology 45 (2003), S. 0 
    Details
    ISSN: 1574-6941
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: In situ hybridization with specific oligonucleotide probes was used to monitor enrichment cultures of yet uncultured populations of sulfate-reducing and small-celled purple sulfur bacteria found to associate into aggregates in the chemocline of meromictic Lake Cadagno, Switzerland, and to select potential isolates. Enrichment and isolation conditions resembled those of their nearest cultured relatives, the sulfate-reducing bacterium Desulfocapsa thiozymogenes and small-celled purple sulfur bacteria belonging to the genus Lamprocystis, respectively. Based on comparative 16S rRNA analysis and physiological characterization, isolate Cad626 was found to resemble D. thiozymogenes although it differed from the type strain by its ability to grow on lactate and pyruvate. Like D. thiozymogenes, isolate Cad626 was able to disproportionate inorganic sulfur compounds (sulfur, thiosulfate, sulfite) and to grow, although growth on sulfur required a sulfide scavenger (FeOOH). Isolate Cad16 represented small-celled purple sulfur bacteria that belonged to a previously detected, but uncultured population designated F and was related to Lamprocystis purpurea as evidenced by comparative 16S rRNA analysis and the presence of bacteriochlorophyll a and the carotenoid okenone. Mixed cultures of isolates Cad626 and Cad16 resulted in their association in aggregates similar to those observed in the chemocline of Lake Cadagno. Concomitant growth enhancement of both isolates in mixed culture suggested synergistic interactions that presumably resemble a source–sink relationship for sulfide between the sulfate-reducing bacterium growing by sulfur disproportionation and the purple sulfur bacteria acting as biotic scavenger.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1016/S0168-6496(03)00107-7
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  • 9
    Electronic Resource
    Electronic Resource
    Spatio-temporal distribution of phototrophic sulfur bacteria in the chemocline of meromictic Lake Cadagno (Switzerland) (2003)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology ecology 43 (2003), S. 0 
    Details
    ISSN: 1574-6941
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: In situ hybridization was used to study the spatio-temporal distribution of phototrophic sulfur bacteria in the permanent chemocline of meromictic Lake Cadagno, Switzerland. At all four sampling times during the year the numerically most important phototrophic sulfur bacteria in the chemocline were small-celled purple sulfur bacteria of two yet uncultured populations designated D and F. Other small-celled purple sulfur bacteria (Amoebobacter purpureus and Lamprocystis roseopersicina) were found in numbers about one order of magnitude lower. These numbers were similar to those of large-celled purple sulfur bacteria (Chromatium okenii) and green sulfur bacteria that almost entirely consisted of Chlorobium phaeobacteroides. In March and June when low light intensities reached the chemocline, cell densities of all populations, with the exception of L. roseopersicina, were about one order of magnitude lower than in August and October when light intensities were much higher. Most populations were evenly distributed throughout the whole chemocline during March and June, while in August and October a microstratification of populations was detected suggesting specific eco-physiological adaptations of different populations of phototrophic sulfur bacteria to the steep physico-chemical gradients in the chemocline of Lake Cadagno.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1574-6941.2003.tb01048.x
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  • 10
    Electronic Resource
    Electronic Resource
    Detection of mRNA of nprM in Bacillus megaterium ATCC 14581 grown in soil by whole-cell hybridization (1995)
    Springer
    Archives of microbiology 163 (1995), S. 235-241 
    Details
    ISSN: 1432-072X
    Keywords: In situ detection of mRNA ; Bacillus megaterium ; Extracellular neutral protease ; nprM ; In vitro transcripts ; Whole-cell hybridization
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Transcripts of nprM, the gene encoding the major extracellular protease of Bacillus megaterium ATCC 14581, were detected by both Northern blot analysis and whole-cell hybridization with digoxigenin-labeled in vitro ranscripts throughout the exponential growth phase and the early stationary phase. In cells of the late stationary phase, only low amounts of transcripts were observed with the two techniques. No transcripts could be detected in spores. In soil the presence of mRNA of nprM could be demonstrated by whole-cell hybridization in growing cells germinated from heat-activated spores until they reached the late transition state. No transcripts of nprM were detected in cells containing forespores. Both cells grown in pure culture and in soil had to be permeabilized with lysozyme to allow hybridization with digoxigeninlabeled probes. These results demonstrate the applicability of nucleic-acid probing techniques to localize microbial processes in soil. The approach described of detecting mRNA in fixed bacterial cells should facilitate in situ studies of gene transcription and specific activities in individual cells in heterogeneous environmental systems.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00393374
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