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M Wagner

Publications and source records attributed to M Wagner.

At least 271 records · Page 15Linked to original sources
PubMed1999

Phylogeny and in situ identification of a morphologically conspicuous bacterium, Candidatus Magnospira bakii, present at very low frequency in activated sludge.

A morphologically conspicuous bacterium that constituted a very small fraction (< 0.01%) of the total microbial community of activated sludge was enriched and analysed phylogenetically by a combination of cultivation-independent molecular and physical methods. The large, corkscrew-shaped, filamentous bacteria were first detected in municipal activated sludge by light microscopy owing to their unusual rotating gliding motility. Various attempts at microbiological enrichment and pure culture isolation with traditional techniques failed, as did attempts to retrieve the morphotype of interest by micromanipulation. In situ hybridization with the group-specific, rRNA-targeted oligonucleotide probe CF319a indicated a phylogenetic affiliation to the Cytophaga-Flexibacter group of the Cytophaga-Flavobacterium-Bacteroides phylum. Based on strong morphological resemblance to members of the genus Saprospira, additional 16S rRNA-targeted oligonucleotides with more narrow specificity were designed and evaluated for in situ hybridization to the morphotype of interest. Flow cytometric cell sorting based on the fluorescence conferred by probe SGR1425 and forward scatter enabled a physical enrichment of the helical coiled cells. Subsequent polymerase chain reaction (PCR) amplification of 16S rDNA fragments from whole fixed sorted cells with a primer pair based on probes CF319a and SGR1425 resulted in the retrieval of 12 almost identical partial 16S rDNA fragments with sequence similarities among each other of more than 99.2%. In situ hybridizations proved that the sequences that showed the highest similarity (88.4%) to the 16S rRNA of Saprospira grandis were indeed retrieved from the corkscrew-shaped filaments. The bacterium is likely to be a member of a genus of which no species has been cultured hitherto. It was consequently tentatively named 'Magnospira bakii' and has the taxonomic rank of Candidatus Magnospira bakii, as the ultimate taxonomic placement has to await its cultivation. In this study, it was demonstrated that even bacteria occurring at very low frequencies in highly complex environmental samples can be retrieved selectively without cultivation for further molecular analysis.

Cloning, Molecular↗
PubMed1999

Novel bacterial endosymbionts of Acanthamoeba spp. related to the Paramecium caudatum symbiont Caedibacter caryophilus.

Acanthamoebae are increasingly being recognized as hosts for obligate bacterial endosymbionts, most of which are presently uncharacterized. In this study, the phylogeny of three Gram-negative, rod-shaped endosymbionts and their Acanthamoeba host cells was analysed by the rRNA approach. Comparative analyses of 16S rDNA sequences retrieved from amoebic cell lysates revealed that the endosymbionts of Acanthamoeba polyphaga HN-3, Acanthamoeba sp. UWC9 and Acanthamoeba sp. UWE39 are related to the Paramecium caudatum endosymbionts Caedibacter caryophilus, Holospora elegans and Holospora obtusa. With overall 16S rRNA sequence similarities to their closest relative, C. caryophilus, of between 87% and 93%, these endosymbionts represent three distinct new species. In situ hybridization with fluorescently labelled endosymbiont-specific 16S rRNA-targeted probes demonstrated that the retrieved 16S rDNA sequences originated from the endosymbionts and confirmed their intracellular localization. We propose to classify provisionally the endosymbiont of Acanthamoeba polyphaga HN-3 as 'Candidatus Caedibacter acanthamoebae', the endosymbiont of Acanthamoeba sp. strain UWC9 as 'Candidatus Paracaedibacter acanthamoebae' and the endosymbiont of Acanthamoeba sp. strain UWE39 as 'Candidatus Paracaedibacter symbiosus'. The phylogeny of the Acanthamoeba host cells was analysed by comparative sequence analyses of their 18S rRNA. Although Acanthamoeba polyphaga HN-3 clearly groups together with most of the known Acanthamoeba isolates (18S rRNA sequence type 4), Acanthamoeba sp. UWC9 and UWE39 exhibit <92% 18S rRNA sequence similarity to each other and to other Acanthamoeba isolates. Therefore, we propose two new sequence types (T13 and T14) within the genus Acanthamoeba containing, respectively, Acanthamoeba sp. UWC9 and Acanthamoeba sp. UWE39.

Acanthamoeba↗
PubMed1999

Alpha-melanocyte-stimulating hormone inhibits NF-kappaB activation in human melanocytes and melanoma cells.

Alpha-melanocyte-stimulating hormone is produced by several different cell types including neural cells, endothelial cells, monocytes, and keratinocytes. A biologic role in melanocyte pigmentation is widely recognized, but more recent studies describe a part in modulating inflammatory and immune responses. The aim of the this study was to investigate the mechanism by which alpha-melanocyte-stimulating hormone antagonizes proinflammatory cytokine action. We report that alpha-melanocyte-stimulating hormone (10-9 M) was effective in opposing a tumor necrosis factor-alpha stimulated increase in NF-kappaB DNA binding activity in: (i) normal ocular melanocytes; (ii) cells cultured from ocular melanoma tumors; and (iii) two cutaneous melanoma cell lines. NF-kappaB is activated by many inflammatory mediators and controls transcription of genes required for immune and inflammatory responses. The transcription factor complex was positively identified as the p50/p65 heterodimer, recognized to have transcriptional activating potential. Maximum reduction of NF-kappaB DNA binding activity with alpha-melanocyte-stimulating hormone was detected 2 h after cellular stimulation and varied from between 53% and 18% depending on cell type. Whereas the acute inhibitory effects could be mimicked by elevating cyclic adenosine monophosphate, alpha-melanocyte-stimulating hormone was not found to have any effect on the relative level of IkappaBalpha protein expression over 24 h. These data show that alpha-melanocyte-stimulating hormone has a pronounced effect on NF-kappaB activity in melanocytes and melanoma cells, identifying a specific dimeric complex, and suggest this to be a key pathway by which immunomodulation/anti-inflammation may operate. The results may also be considered in the broader context of general inflammatory pathologies concerning cells which express alpha-melanocyte-stimulating hormone receptors and utilize the NF-kappaB signaling pathway.

Cells, Cultured↗
PubMed1999

Occult dysplasia is disclosed by Lugol chromoendoscopy in alcoholics at high risk for squamous cell carcinoma of the esophagus.

BACKGROUND AND STUDY AIMS: Squamous cell carcinoma of the esophagus (SCCE) becomes symptomatic at a late stage when the disease is already advanced, and this contributes to its poor prognosis. Esophagoscopy of asymptomatic individuals exposed to known risk factors associated with the development of this cancer may facilitate the diagnosis of early cancerous or precancerous lesions; however, conventional esophagoscopy is not accurate enough. The aim of this study was to measure the value of Lugol chromoendoscopy of the esophagus (LCE) as an endoscopic technique to detect dysplasia in patients at risk. PATIENTS AND METHODS: We studied 190 male patients older than 35 attending an outpatient unit for alcoholics who consumed more than 80g of alcohol, more than 10 cigarettes and more than 500 ml 'maté' (a hot infusion of herbs) per day over 10 years. All underwent conventional upper gastrointestinal endoscopy followed by LCE, a spraying of Lugol 3% on the entire esophagus. All patients denied dysphagia. Biopsies were obtained from any unstained areas larger than 5mm and also from stained areas in all individuals. Biopsies were analyzed independently by two pathologists unaware of the biopsy sites. All conventional esophagoscopies showed normal mucosa, except for two suspicious small elevated lesions, confirmed histologically to be SCCE. These two cases were excluded from the statistical analysis. RESULTS: The LCE found unstained areas in 23 patients and a uniformly stained esophageal mucosa in the remaining 165. Biopsies taken from these 23 unstained areas showed dysplasia in six (two high grade and four low grade), and the ones from the 165 stained areas taken at the middle esophagus showed low-grade dysplasia in seven. There was a high prevalence (6.9%) of dysplastic lesions in these individuals and occult dysplasia was significantly more frequent in unstained than stained areas (p = 0.0017). LCE showed a sensitivity of 46%, a specificity of 90%, a positive predictive value of 26% and a negative predicitve value of 96% when unstained areas were compared to stained ones. Agreement between two independent pathologists was high, with a kappa coefficient of 0.64. CONCLUSION: We concluded that individuals who abuse alcohol, smoke and consume 'maté' have a high prevalence of dysplastic lesions that can be better detected by LCE. Esophagi with unstained areas had an eight-fold higher chance of revealing dysplasia than the uniformly stained ones. LCE is an easy and inexpensive method. It improves the detection of dysplasia and should be added to conventional upper GI endoscopy in patients at risk for SCCE.

Alcoholism↗
PubMed1999

Functional magnetic resonance imaging of the human motor cortex before and after whole-hand afferent electrical stimulation.

Electrical stimulation of the whole hand using a mesh-glove has been shown to improve volitional movement of the hand and arm, and decrease muscle hypertonia after hemispherical stroke in patients who have reached a recovery plateau. The goal of this study was to investigate the effect of stimulation of the nerve afferents of the hand on brain cortical activity elicited by whole-hand subthreshold stimulation for sensation in humans with intact nervous systems. Brain cortical activity in 6 healthy subjects (30-45 years) was studied using blood oxygenation level-dependent functional Magnetic Resonance Imaging during a test motor task, finger-to-thumb tapping and after 20 minutes of mesh-glove stimulation of the resting hand prior to performance of an identical motor task, to test the changes in the conditioned motor task established after 20 minutes of mesh-glove stimulation. Fifteen contiguous echo-planar sequences parallel to the bicommissural plane were acquired for functional magnetic resonance. Post-processing of image data included correction of motion artefacts and calculation of correlation coefficients between the signal intensity of pixels during rest and finger tapping and a rectangular reference wave function. The functional Magnetic Resonance Imaging examinations revealed a signal increase in the primary and secondary motor and somatosensory areas when comparing the number of activated pixels during test and conditioned motor tasks. Our preliminary study indicated that change occurred in a definite pattern in the region of the regional cerebral blood flow of the brain cortex after mesh-glove whole-hand stimulation at the subthreshold level for sensation. We assumed that this increase in regional cerebral blood flow also reflected augmented neuronal activity.

Adult↗
PubMed1999

Altered copper status in adult men with cystic fibrosis.

OBJECTIVES: To measure indices of copper status in adult men with cystic fibrosis (CF). A previous study in children showed changes in copper homeostasis compared to controls. This study was designed to investigate whether this observation persisted into adulthood. METHODS: This was a case-control age-matched study using seven men with CF and six healthy men. Blood samples were drawn into metal free tubes and fractionated into plasma, polymorphonuclear cells, mononuclear cells and erythrocytes. Cell fractions were assayed for copper and CuZn-superoxide dismutase; plasma was assayed for ceruloplasmin. RESULTS: The men with cystic fibrosis had significantly greater plasma copper and ceruloplasmin activity, yet had significantly lower copper-zinc superoxide dismutase activity in mononuclear and polymorphonuclear cells. Furthermore, the mononuclear cells of the cystic fibrosis subjects had about 45% percent less copper-zinc superoxide dismutase protein. Cellular copper levels were not statistically different between the two groups. A significant correlation was found between lung function and copper-zinc superoxide dismutase activity in the polymorphonuclear cells. Iron status was normal. CONCLUSIONS: The results indicate that individuals with cystic fibrosis have altered copper distribution compared to control individuals. Some aspects are characteristic of an inflammatory response; however, other measures suggest that copper homeostasis may be abnormal. It is not known whether the deviation in copper homeostasis in these individuals is a result of poor copper absorption, inadequate dietary intake, a result of their chronic inflammation or a direct effect due to the defect in ion transport caused by the disease. However, this research suggests that the severity of the disease and the activity of a copper dependent enzyme may be related. Further work will be necessary to determine the cause of the abnormal copper homeostasis and whether correcting it has any bearing on the course of the disease.

Adult↗
PubMed1999

IMAGEne I: clustering and ranking of I.M.A.G.E. cDNA clones corresponding to known genes.

MOTIVATION: To enhance the usefulness of the I.M.A.G.E. Consortium (Lennon et al., 1996, Genomics, 33, 151-152) cDNA clone collection by directed analysis and organization of their associated Expressed Sequence Tags (ESTs), thus enabling effective mining of the immense amounts of public cDNA information. RESULTS: This paper introduces the IMAGEne suite of tools, which clusters ESTs around known genes, then ranks each clone within a cluster. IMAGEne filters data from known gene sequence databases and the GenBank's EST database (Boguski and Shuler, 1995, Nature Genet., 10, 369-371). It applies biological criteria in connection with judicious use of the BLAST (Altschul et al., 1990, J. Mol. Biol., 215), FASTA (Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA, 85, 2444-2448; Pearson, 1990, Methods Enzymol., 183, 63-98; Gusfield, 1997, Algorithms on Strings, Trees, and Sequences, Cambridge University Press), and SIM (Huang et al., 1990, Comput. Appl. Biosci., 6, 373-381) tools to form known gene clusters. It then applies criteria derived from experienced biologists to select the best representative I.M.A.G.E. clone for a gene. The tool provides an intuitive Java interface for query and display of the gene and its associated clones, thus directing researchers in selecting a clone that will best enhance their research. An important product is a listing of clones that best represent all known genes. The listing will be used for re-arraying clones into minimally redundant Master Arrays. Both the listings and Master Arrays will be made available to the public, which will be a valuable resource to the genomic community in furthering discovery in the area of gene function.

Algorithms↗
PubMed1999

Distinct steps in yeast spore morphogenesis require distinct SMK1 MAP kinase thresholds.

The SMK1 mitogen-activated protein kinase is required for spore morphogenesis in Saccharomyces cerevisiae. In contrast to the multiple aberrant spore wall assembly patterns seen even within a single smk1 null ascus, different smk1 missense mutants block in a coordinated fashion at intermediate stages. One smk1 mutant forms asci in which the four spores are surrounded only by prospore wall-like structures, while another smk1 mutant forms asci in which the spores are surrounded by inner but not outer spore wall layers. Stepwise increases in gene dosage of a hypomorphic smk1 allele allow for the completion of progressively later morphological and biochemical events and for the acquisition of distinct spore-resistance phenotypes. Furthermore, smk1 allelic spore phenotypes can be recapitulated by reducing wild-type SMK1 expression. The data demonstrate that SMK1 is required for the execution of multiple steps in spore morphogenesis that require increasing thresholds of SMK1 activity. These results suggest that quantitative changes in mitogen-activated protein kinase signaling play a role in coordinating multiple events of a single cellular differentiation program.

Alleles↗
PubMed1999

Visualization of collagenase-induced cartilage degradation using NMR microscopy.

RATIONALE AND OBJECTIVES: There is an ongoing discussion about the role of collagenase as a specific cartilage-degrading enzyme in the course of osteoarthritis. Using NMR microscopy, the influence of collagenase on the cartilage structure was investigated. METHODS: High-resolution MR imaging investigations were performed at 7.1 Tesla to study the influence of collagenase on the cartilage structure of pig femoral condyles. Two different contrast agents (polylysine-Gd-DTPA and liposome-entrapped contrast agents) were tested to improve the NMR microscopic visualization of the damaged cartilage regions. RESULTS: The NMR microscopic investigations showed that collagenase affects only the cartilage surface. Changes within the cartilage layer were not detected. However, after the application of specific contrast agents, it was possible to detect with great sensitivity collagenase-induced damage to the collagen network on the cartilage surface and the superficial cartilage zones. The application of liposome-entrapped contrast agents improved the visualization of the collagenase-degraded superficial cartilage zone. CONCLUSIONS: These findings could be of great importance when estimating the state of the articular cartilage. The degree of fibrillation of the cartilage surface in the course of osteoarthritic disease can be visualized by MR tomography with a high degree of sensitivity.

Animals↗
PubMed1999

Linear and nonlinear current density reconstructions.

Minimum norm algorithms for EEG source reconstruction are studied in view of their spatial resolution, regularization, and lead-field normalization properties, and their computational efforts. Two classes of minimum norm solutions are examined: linear least squares methods and nonlinear L1-norm approaches. Two special cases of linear algorithms, the well known Minimum Norm Least Squares and an implementation with Laplacian smoothness constraints, are compared to two nonlinear algorithms comprising sparse and standard L1-norm methods. In a signal-to-noise-ratio framework, two of the methods allow automatic determination of the optimum regularization parameter. Compensation methods for the different depth dependencies of all approaches by lead-field normalization are discussed. Simulations with tangentially and radially oriented test dipoles at two different noise levels are performed to reveal and compare the properties of all approaches. Finally, cortically constrained versions of the algorithms are applied to two epileptic spike data sets and compared to results of single equivalent dipole fits and spatiotemporal source models.

Algorithms↗
PubMed1999

Enhanced expression of the type II transforming growth factor-beta receptor is associated with decreased survival in human pancreatic cancer.

Transforming growth factor-betas (TGF-betas) bind to the type II TGF-beta receptor (TbetaRII), which then heterodimerizes with the type I TGF-beta receptor (TbetaRI), thereby initiating a signaling cascade. TGF-betas are overexpressed in pancreatic cancer, and this overexpression is associated with more aggressive disease. Although TbetaRII also is overexpressed in pancreatic ductal adenocarcinoma cells in vivo, the biologic significance of this overexpression is not completely known. Therefore in this study, we characterized TbetaRII expression by Northern blot analysis in 32 normal and 42 cancerous pancreatic tissues and correlated the survival of the cancer patients with TbetaRII messenger RNA (mRNA) levels. Northern blot analysis revealed that, by comparison with the normal controls, TbetaRII expression was increased in 19 (45%) of 42 cancer samples. Densitometric analysis of all the pancreatic tissues revealed a 3.4-fold increase (p < 0.01) in TbetaRII mRNA levels in the cancer tissues by comparison with normal controls. There was a strong correlation between the expression of TbetaRII and the levels of two invasion-promoting genes, plasminogen-activator-inhibitor-1 (PAI-1) and matrix-metalloproteinase-9 (MMP9). Log-rank analysis of the Kaplan-Meier survival curves indicated that patients whose tumors overexpressed TbetaRII had a significantly shorter survival period than did patients whose cancers expressed low levels of TbetaRII. It is suggested that TbetaRII overexpression may be a marker that correlates with disease progression in pancreatic ductal adenocarcinoma.

Adolescent↗
PubMed1999

A comparative study of the effect of pigment on drug toxicity in human choroidal melanocytes and retinal pigment epithelial cells.

The aim of this study was to investigate whether the presence of pigment affects the sensitivity of pigmented cells of the eye, retinal pigment epithelium (RPE) and choroidal melanocytes (CMs) to the cytotoxic effects of xenobiotic drugs. Two approaches were used to compare pigmented versus unpigmented cells: RPE cells were repigmented by phagocytosis of synthetic melanin; UVB irradiation was used to induce an increase in pigment in both RPE and CMs. Three drugs known to induce toxicity in the eye, tamoxifen, chloroquine and thioridazine, were used to assess the sensitivity of cells to xenobiotic drugs. RPE cells were more resistant than CMs to the cytotoxic effects of all three drugs by a factor of 5-fold for tamoxifen, 7-fold for thioridazine and 30-fold for chloroquine. When RPE cells were repigmented using synthetic melanin, their sensitivity to tamoxifen was unchanged, they showed a slightly improved response to thioridazine (after 3 days of incubation with this drug), but they showed greatly increased toxicity to chloroquine (after 1 and 3 days of exposure to the drug), suggesting accumulation of this latter drug on the synthetic melanin. UVB irradiation was used to achieve an increase in the pigment content of both RPE and CMs. CMs were much more sensitive to UVB than RPE cells. CMs appeared to synthesise pigment via DOPA oxidase activity; RPE cells showed an increase in fluorescent material independent of any detectable DOPA oxidase activity. Irrespective of the nature of the pigment that UVB induced in melanocytes and RPE cells, their subsequent response to thioridazine and chloroquine was unchanged by the presence of this pigment.

Antimalarials↗
PubMed1999

Morphological and biochemical properties of a Sphaerotilus sp. Isolated from paper mill slimes.

Four strains of filamentous bacteria were isolated from slimes collected in different paper mill factories. Morphological and physiological characterization of the isolates indicated an affiliation with the genus Sphaerotilus. However, while the physiological properties of the isolates were almost identical, pronounced physiological differences between the isolates and Sphaerotilus natans DSM 6575(T), DSM 565, and DSM 566 with respect to their ability to metabolize complex polysaccharides, sugars, polyalcohols, or organic acids as carbon sources were detected. In contrast to the analyzed culture collection strains of S. natans, all paper mill isolates were able to grow at elevated temperatures of up to 40 degrees C. Comparative sequence analysis of nearly complete 16S ribosomal DNA (rDNA) sequences from the four new isolates demonstrated that the retrieved sequences were highly similar to each other (99.6 to 99.8% similarity) and to previously published partial 16S rDNA sequences of S. natans DSM 6575(T) and ATCC 15291. Polyphasic characterization of the isolated Sphaerotilus strains revealed interesting adaptations of the strains to the environmental paper mill conditions with regard to temperature tolerance and utilization of cellulose and starch.

Base Sequence↗
PubMed1999

In situ detection of novel bacterial endosymbionts of Acanthamoeba spp. phylogenetically related to members of the order Rickettsiales.

Acanthamoebae are ubiquitous soil and water bactivores which may serve as amplification vehicles for a variety of pathogenic facultative bacteria and as hosts to other, presently uncultured bacterial endosymbionts. The spectrum of uncultured endosymbionts includes gram-negative rods and gram-variable cocci, the latter recently shown to be members of the Chlamydiales. We report here the isolation from corneal scrapings of two Acanthamoeba strains that harbor gram-negative rod endosymbionts that could not be cultured by standard techniques. These bacteria were phylogenetically characterized following amplification and sequencing of the near-full-length 16S rRNA gene. We used two fluorescently labelled oligonucleotide probes targeting signature regions within the retrieved sequences to detect these organisms in situ. Phylogenetic analyses demonstrated that they displayed 99.6% sequence similarity and formed an independent and well-separated lineage within the Rickettsiales branch of the alpha subdivision of the Proteobacteria. Nearest relatives included members of the genus Rickettsia, with sequence similarities of approximately 85 to 86%, suggesting that these symbionts are representatives of a new genus and, perhaps, family. Distance matrix, parsimony, and maximum-likelihood tree-generating methods all consistently supported deep branching of the 16S rDNA sequences within the Rickettsiales. The oligonucleotide probes displayed at least three mismatches to all other available 16S rDNA sequences, and they both readily permitted the unambiguous detection of rod-shaped bacteria within intact acanthamoebae by confocal laser-scanning microscopy. Considering the long-standing relationship of most Rickettsiales with arthropods, the finding of a related lineage of endosymbionts in protozoan hosts was unexpected and may have implications for the preadaptation and/or recruitment of rickettsia-like bacteria to metazoan hosts.

Acanthamoeba↗
PubMed1999

Diversity of sulfate-reducing bacteria in oxic and anoxic regions of a microbial mat characterized by comparative analysis of dissimilatory sulfite reductase genes.

Sequence analysis of genes encoding dissimilatory sulfite reductase (DSR) was used to identify sulfate-reducing bacteria in a hypersaline microbial mat and to evaluate their distribution in relation to levels of oxygen. The most highly diverse DSR sequences, most related to those of the Desulfonema-like organisms within the delta-proteobacteria, were recovered from oxic regions of the mat. This observation extends those of previous studies by us and others associating Desulfonema-like organisms with oxic habitats.

Anaerobiosis↗
PubMed1999

Detection and differentiation of Listeria spp. by a single reaction based on multiplex PCR.

The iap gene encodes the protein p60, which is common to all Listeria species. A previous comparison of the DNA sequences indicated conserved and species-specific gene portions. Based on these comparisons, a combination consisting of only five different primers that allows the specific detection and differentiation of Listeria species with a single multiplex PCR and subsequent gel analysis was selected. One primer was derived from the conserved 3' end and is specific for all Listeria species; the other four primers are specific for Listeria monocytogenes, L. innocua, L. grayi, or the three grouped species L. ivanovii, L. seeligeri, and L. welshimeri, respectively. The PCR method, which also enables the simultaneous detection of L. monocytogenes and L. innocua, was evaluated against conventional biotyping with 200 food hygiene-relevant Listeria strains. The results indicated the superiority of this technique. Thus, this novel type of multiplex PCR may be useful for rapid Listeria species confirmation and for identification of Listeria species for strains isolated from different sources.

Bacterial Proteins↗
PubMed1999

Combination of fluorescent in situ hybridization and microautoradiography-a new tool for structure-function analyses in microbial ecology.

A new microscopic method for simultaneously determining in situ the identities, activities, and specific substrate uptake profiles of individual bacterial cells within complex microbial communities was developed by combining fluorescent in situ hybridization (FISH) performed with rRNA-targeted oligonucleotide probes and microautoradiography. This method was evaluated by using defined artificial mixtures of Escherichia coli and Herpetosiphon aurantiacus under aerobic incubation conditions with added [3H]glucose. Subsequently, we were able to demonstrate the potential of this method by visualizing the uptake of organic and inorganic radiolabeled substrates ([14C]acetate, [14C]butyrate, [14C]bicarbonate, and 33Pi) in probe-defined populations from complex activated sludge microbial communities by using aerobic incubation conditions and anaerobic incubation conditions (with and without nitrate). For both defined cell mixtures and activated sludge, the method proved to be useful for simultaneous identification and analysis of the uptake of labeled substrates under the different experimental conditions used. Optimal results were obtained when fluorescently labeled oligonucleotides were applied prior to the microautoradiographic developing procedure. For single-cell resolution of FISH and microautoradiographic signals within activated sludge flocs, cryosectioned sample material was examined with a confocal laser scanning microscope. The combination of in situ rRNA hybridization techniques, cryosectioning, microautoradiography, and confocal laser scanning microscopy provides a unique opportunity for obtaining cultivation-independent insights into the structure and function of bacterial communities.

Aerobiosis↗