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G Muyzer

Publications and source records attributed to G Muyzer.

48 records · Page 3Linked to original sources
PubMed1997

PCR primers to amplify 16S rRNA genes from cyanobacteria.

We developed and tested a set of oligonucleotide primers for the specific amplification of 16S rRNA gene segments from cyanobacteria and plastids by PCR. PCR products were recovered from all cultures of cyanobacteria and diatoms that were checked but not from other bacteria and archaea. Gene segments selectively retrieved from cyanobacteria and diatoms in unialgal but nonaxenic cultures and from cyanobionts in lichens could be directly sequenced. In the context of growing sequence databases, this procedure allows rapid and phylogenetically meaningful identification without pure cultures or molecular cloning. We demonstrate the use of this specific PCR in combination with denaturing gradient gel electrophoresis to probe the diversity of oxygenic phototrophic microorganisms in cultures, lichens, and complex microbial communities.

Cyanobacteria↗
PubMed1996

Skeletal matrices, muci, and the origin of invertebrate calcification.

The sudden appearance of calcified skeletons among many different invertebrate taxa at the Precambrian-Cambrian transition may have required minor reorganization of preexisting secretory functions. In particular, features of the skeletal organic matrix responsible for regulating crystal growth by inhibition may be derived from mucous epithelial excretions. The latter would have prevented spontaneous calcium carbonate overcrusting of soft tissues exposed to the highly supersaturated Late Proterozoic ocean [Knoll, A. H., Fairchild, I. J. & Swett, K. (1993) Palaios 8, 512-525], a putative function for which we propose the term "anticalcification." We tested this hypothesis by comparing the serological properties of skeletal water-soluble matrices and mucous excretions of three invertebrates--the scleractinian coral Galaxea fascicularis and the bivalve molluscs Mytilus edulis and Mercenaria mercenaria. Crossreactivities recorded between muci and skeletal water-soluble matrices suggest that these different secretory products have a high degree of homology. Furthermore, freshly extracted muci of Mytilus were found to inhibit calcium carbonate precipitation in solution.

Journal Article↗
PubMed1996

Molecular identification of bacteria from a coculture by denaturing gradient gel electrophoresis of 16S ribosomal DNA fragments as a tool for isolation in pure cultures.

Molecular information about the bacterial composition of a coculture capable of sulfate reduction after exposure to oxic and microoxic conditions was used to identify and subsequently to isolate the components of the mixture in pure culture. PCR amplification of 16S ribosomal DNA fragments from the coculture, analyzed by denaturing gradient gel electrophoresis, resulted in two distinct 16S ribosomal DNA bands, indicating two different bacterial components. Sequencing showed that the bands were derived from a Desulfovibrio strain and an Arcobacter strain. Since the phylogenetic positions of bacteria are often consistent with their physiological properties and culture requirements, molecular identification of the two components of this coculture allowed the design of specific culture conditions to separate and isolate both strains in pure culture. This approach facilitates the combined molecular and physiological analysis of mixed cultures and microbial communities.

Bacteria↗
PubMed1996

Denaturing gradient gel electrophoresis profiles of 16S rRNA-defined populations inhabiting a hot spring microbial mat community.

Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene segments was used to profile microbial populations inhabiting different temperature regions in the microbial mat community of Octopus Spring, Yellowstone National Park. DGGE allowed a rapid evaluation of the distributions of amplifiable sequence types. Profiles were essentially identical within regions of the mat defined by one temperature range but varied between sites with different temperature ranges. Individual DGGE bands were sequenced, and the sequences were compared with those previously obtained from the mat by cloning and from cultivated Octopus Spring isolates. Two known cyanobacterial populations and one known green nonsulfur bacterium-like population were detected by DGGE, as were many new cyanobacterial and green nonsulfur and green sulfur bacterium-like populations and a novel bacterial population of uncertain phylogenetic affiliation. The distributions of several cyanobacterial populations compared favorably with results obtained previously by oligonucleotide probe analyses and suggest that adaptation to temperature has occurred among cyanobacteria which are phylogenetically very similar.

Bacteria↗
PubMed1996

Distribution of sulfate-reducing bacteria in a stratified fjord (Mariager Fjord, Denmark) as evaluated by most-probable-number counts and denaturing gradient gel electrophoresis of PCR-amplified ribosomal DNA fragments.

The sulfate-reducing bacterial populations of a stratified marine water column, Mariager Fjord, Denmark, were investigated by molecular and culture-dependent approaches in parallel. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA and DNA encoding rRNA (rDNA) isolated from the water column indicated specific bacterial populations in different water column layers and revealed a highly differentiated pattern of rRNA- and rDNA-derived PCR amplificates, probably reflecting active and resting bacterial populations. Hybridization of DGGE patterns with rRNA probes indicated the increased presence and activity (by at least 1 order of magnitude) of sulfate-reducing bacteria within and below the chemocline. Parallel to this molecular approach, an approach involving most-probable-number (MPN) counts was used, and it found a similar distribution of cultivable sulfate-reducing bacteria in the water column of Mariager Fjord, Approximately 25 cells and 250 cells per ml above and below the chemocline, respectively, were found. Desulfovibrio- and Desulfobulbus-related strains occurred in the oxic zone. DGGE bands from MPN cultures were sequenced and compared with those obtained from nucleic acids extracted from water column samples. The MPN isolates were phylogenetically affiliated with sulfate-reducing delta subdivision proteobacteria (members of the genera Desulfovibrio, Desulfobulbus, and Desulfobacter), whereas the molecular isolates constituted an independent lineage of the delta subdivision proteobacteria. DGGE of PCR-amplified nucleic acids with general eubacterial PCR primers conceptually revealed the general bacterial population, whereas the use of culture media allowed cultivable sulfate-reducing bacteria to be selected. A parallel study of Mariager Fjord biogeochemistry, bacterial activity, and bacterial counts complementing this investigation has been presented elsewhere (N.B. Ramsing, H. Fossing, T. G. Ferdelman, F. Andersen, and B. Thamdrup, Appl. Environ.

Bacteria↗
PubMed1996

Identification of bacteria in a biodegraded wall painting by denaturing gradient gel electrophoresis of PCR-amplified gene fragments coding for 16S rRNA.

Medieval wall paintings are often affected by biodecay. An inventory of the existing microorganisms associated with the damage to the paintings is not yet an integral part of the restoration process. This stems from the lack of effective means for such a stocktaking. Nevertheless, fungi and bacteria cause severe damage through mechanical processes from growth into the painting and its grounding and through their metabolism. Detailed information on the bacterial colonization of ancient wall paintings is essential for the protection of the paintings. We used a molecular approach based on the detection and identification of DNA sequences encoding rRNA (rDNA) to identify bacteria present on an ancient wall painting without prior cultivation of the organisms, since it has been shown that most of these bacteria cannot be cultivated under laboratory conditions. To trace the noncultivated fraction of bacteria, total DNA from a biodegraded wall painting sample from a 13th century fresco was extracted and 194-bp fragments of the 16S rDNA were amplified with eubacterial primers. The 16S rDNA fragments of uniform length obtained from the different bacterial species were separated according to their sequence differences by denaturing gradient gel electrophoresis (DGGE). By sequencing excised and reamplified individual DNA bands, we characterized the phylogenetic affiliation of the corresponding bacteria. Using this approach, we identified members or close relatives of the genera Halomonas, Clostridium, and Frankia. To our knowledge, these groups of bacteria have not yet been isolated and implicated by conventional microbiological techniques as contributing to the biodegradation of wall paintings.

Bacteria↗
PubMed1996

Phenotypic and phylogenetic analyses show Microcoleus chthonoplastes to be a cosmopolitan cyanobacterium.

We used micromanipulation to isolate from their environment representative samples of seven geographically distant field populations fitting the description of Microcoleus chthonoplastes (a cyanobacterium) and obtained seven corresponding cultured strains. Samples of both field populations and cultures were phenotypically characterized by microscale techniques, and their partial 16S rRNA gene sequences were compared by denaturing gradient gel electrophoresis and in some cases by sequencing. All field populations and strains were phenotypically extremely coherent, and their 16S rRNA sequences were indistinguishable by DGGE. The sequences determined were identical or virtually identical. Thus, M. chthonoplastes represents a single, well-delimited taxon with a truly cosmopolitan distribution. Comparison with three culture collection strains originally assigned to M. chthonoplastes revealed that strain PCC 7420 belongs to the same tightly delimited group, both phenotypically and in 16S rRNA gene sequence, but that strains SAG 3192 and 10mfx do not.

Base Sequence↗
PubMed1995

Phylogenetic relationships of Thiomicrospira species and their identification in deep-sea hydrothermal vent samples by denaturing gradient gel electrophoresis of 16S rDNA fragments.

Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA fragments was used to explore the genetic diversity of hydrothermal vent microbial communities, specifically to determine the importance of sulfur-oxidizing bacteria therein. DGGE analysis of two different hydrothermal vent samples revealed one PCR band for one sample and three PCR bands for the other sample, which probably correspond to the dominant bacterial populations in these communities. Three of the four 16S rDNA fragments were sequenced. By comparison with 16S rRNA sequences of the Ribosomal Database Project, two of the DGGE-separated fragments were assigned to the genus Thiomicrospira. To identify these 'phylotypes' in more detail, a phylogenetic framework was created by determining the nearly complete 16S rRNA gene sequence (approx. 1500 nucleotides) from three described Thiomicrospira species, viz., Tms. crunogena, Tms. pelophila, Tms. denitrificans, and from a new isolate, Thiomicrospira sp. strain MA2-6. All Thiomicrospira species except Tms. denitrificans formed a monophyletic group within the gamma subdivision of the Proteobacteria. Tms. denitrificans was assigned as a member of the epsilon subdivision and was distantly affiliated with Thiovulum, another sulfur-oxidizing bacterium. Sequences of two dominant 16S rDNA fragments obtained by DGGE analysis fell into the gamma subdivision Thiomicrospira. The sequence of one fragment was in all comparable positions identical to the 16S rRNA sequence of Tms. crunogena. Identifying a dominant molecular isolate as Tms. crunogena indicates that this species is a dominant community member of hydrothermal vent sites. Another 'phylotype' represented a new Thiomicrospira species, phylogenetically in an intermediate position between Tms. crunogena and Tms. pelophila. The third 'phylotype' was identified as a Desulfovibrio, indicating that sulfate-reducing bacteria, as sources of sulfide, may complement sulfur- and sulfide-oxidizing bacteria ecologically in these sulfide-producing hydrothermal vents.

Base Sequence↗
PubMed1995

Genetic diversity of Desulfovibrio spp. in environmental samples analyzed by denaturing gradient gel electrophoresis of [NiFe] hydrogenase gene fragments.

The genetic diversity of Desulfovibrio species in environmental samples was determined by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified [NiFe] hydrogenase gene fragments. Five different PCR primers were designed after comparative analysis of [NiFe] hydrogenase gene sequences from three Desulfovibrio species. These primers were tested in different combinations on the genomic DNAs of a variety of hydrogenase-containing and hydrogenase-lacking bacteria. One primer pair was found to be specific for Desulfovibrio species only, while the others gave positive results with other bacteria also. By using this specific primer pair, we were able to amplify the [NiFe] hydrogenase genes of DNAs isolated from environmental samples and to detect the presence of Desulfovibrio species in these samples. However, only after DGGE analysis of these PCR products could the number of different Desulfovibrio species within the samples be determined. DGGE analysis of PCR products from different bioreactors demonstrated up to two bands, while at least five distinguishable bands were detected in a microbial mat sample. Because these bands most likely represent as many Desulfovibrio species present in these samples, we conclude that the genetic diversity of Desulfovibrio species in the natural microbial mat is far greater than that in the experimental bioreactors.

Base Sequence↗
PubMed1993

Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA.

We describe a new molecular approach to analyzing the genetic diversity of complex microbial populations. This technique is based on the separation of polymerase chain reaction-amplified fragments of genes coding for 16S rRNA, all the same length, by denaturing gradient gel electrophoresis (DGGE). DGGE analysis of different microbial communities demonstrated the presence of up to 10 distinguishable bands in the separation pattern, which were most likely derived from as many different species constituting these populations, and thereby generated a DGGE profile of the populations. We showed that it is possible to identify constituents which represent only 1% of the total population. With an oligonucleotide probe specific for the V3 region of 16S rRNA of sulfate-reducing bacteria, particular DNA fragments from some of the microbial populations could be identified by hybridization analysis. Analysis of the genomic DNA from a bacterial biofilm grown under aerobic conditions suggests that sulfate-reducing bacteria, despite their anaerobicity, were present in this environment. The results we obtained demonstrate that this technique will contribute to our understanding of the genetic diversity of uncharacterized microbial populations.

Base Sequence↗
PubMed1987

A Combined Immunofluorescence-DNA-Fluorescence Staining Technique for Enumeration of Thiobacillus ferrooxidans in a Population of Acidophilic Bacteria.

An antiserum raised against whole cells of Thiobacillus ferrooxidans was allowed to react with a variety of acidophilic and nonacidophilic bacteria in an enzyme-linked immunosorbent assay and an indirect immunofluorescence assay. Both experiments demonstrated that the antiserum was specific at the species level. This preparation was used to evaluate the role of T. ferrooxidans in the microbial desulfurization process. Leaching experiments were performed, and the numbers of T. ferrooxidans cells and other bacteria were estimated by using a combined immunofluorescence-DNA-fluorescence staining technique that was adapted for this purpose. Nonsterile coal samples inoculated with T. ferrooxidans yielded high concentrations of soluble iron after 16 days. After this period, however, T. ferrooxidans cells could no longer be detected by the immunofluorescence assay, whereas the DNA-fluorescence staining procedure demonstrated a large number of microorganisms on the coal particles. These results indicate that T. ferrooxidans is removed by competition with different acidophilic microorganisms that were originally present on the coal.

Journal Article↗