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W Ludwig

Publications and source records attributed to W Ludwig.

At least 91 records · Page 5Linked to original sources
PubMed1996

Sequence heterogeneities of genes encoding 16S rRNAs in Paenibacillus polymyxa detected by temperature gradient gel electrophoresis.

Sequence heterogeneities in 16S rRNA genes from individual strains of Paenibacillus polymyxa were detected by sequence-dependent separation of PCR products by temperature gradient gel electrophoresis (TGGE). A fragment of the 16S rRNA genes, comprising variable regions V6 to V8, was used as a target sequence for amplifications. PCR products from P. polymyxa (type strain) emerged as a well-defined pattern of bands in the gradient gel. Six plasmids with different inserts, individually demonstrating the migration characteristics of single bands of the pattern, were obtained by cloning the PCR products. Their sequences were analyzed as a representative sample of the total heterogeneity. An amount of 10 variant nucleotide positions in the fragment of 347 bp was observed, with all substitutions conserving the relevant secondary structures of the V6 and V8 regions in the RNA molecules. Hybridizations with specifically designed probes demonstrated different chromosomal locations of the respective rRNA genes. Amplifications of reverse-transcribed rRNA from ribosome preparations, as well as whole-cell hybridizations, revealed a predominant representation of particular sequences in ribosomes of exponentially growing laboratory cultures. Different strains of P. polymyxa showed not only remarkably differing patterns of PCR products in TGGE analysis but also discriminative whole-cell labeling with the designed oligonucleotide probes, indicating the different representation of individual sequences in active ribosomes. Our results demonstrate the usefulness of TGGE for the structural analysis of heterogeneous rRNA genes together with their expression, stress problems of the generation of meaningful data for 16S rRNA sequences and probe designs, and might have consequences for evolutionary concepts.

Bacillus↗
PubMed1996

Case of fatal systemic infection with an Aureobacterium sp.: identification of isolate by 16S rRNA gene analysis.

The case of a 75-year-old man who succumbed to a disseminated infection most likely caused by a species of the genus Aureobacterium is reported. Identification of the isolate was achieved by comparative 16S rRNA gene analysis. Aureobacteria are commonly found in the environment. However, only recently have they been recognized as a cause of infections including septicemia and soft tissue infections. To our knowledge, this is the first documentation of a fatal infection caused by an Aureobacterium sp.

Aged↗
PubMed1995

Phylogenetic position of Gluconobacter species as a coherent cluster separated from all Acetobacter species on the basis of 16S ribosomal RNA sequences.

The 16S rRNA sequences from the Gluconobacter species G. asaii, G. cerinus and G. frateurii were determined and compared with homologous sequences from published databases and sequences of G. oxydans and Acetobacter species previously described [Sievers, M., Ludwig, W. and Teuber, M. (1994) System. Appl. Microbiol. 17, 189-196]. The Gluconobacter species have unique 16S rRNA sequences and exhibit sequence similarity values of 97.4 to 99.1%, corresponding to 36 to 14 base differences. The phylogenetic tree inferring methods (distance matrix, maximum parsimony and maximum likelihood) show that the species of Gluconobacter form a coherent, closely related cluster. Based on the distance matrix method including Rhodopila globiformis as an outgroup reference organism, Gluconobacter is well separated from Acetobacter.

Acetobacter↗
PubMed1995

A new obligately chemolithoautotrophic, nitrite-oxidizing bacterium, Nitrospira moscoviensis sp. nov. and its phylogenetic relationship.

A gram-negative, non-motile, non-marine, nitrite-oxidizing bacterium was isolated from an enrichment culture initiated with a sample from a partially corroded area of an iron pipe of a heating system in Moscow, Russia. The cells were 0.9-2.2 microns x 0.2-0.4 microns in size. They were helical- to vibroid-shaped and often formed spirals with up to three turns 0.8-1.0 micron in width. The organism possessed an enlarged periplasmic space and lacked intracytoplasmic membranes and carboxysomes. The cells tended to excrete extracellular polymers, forming aggregates. The bacterium grew optimally at 39 degrees C and pH 7.6-8.0 in a mineral medium with nitrite as sole energy source and carbon dioxide as sole carbon source. The optimal nitrite concentration was 0.35 mM. Nitrite was oxidized to nitrate stoichiometrically. The doubling time was 12 h in a mineral medium with 7.5 mM nitrite. The cell yield was low; only 0.9 mg protein/l was formed during oxidation of 7.5 mM nitrite. Under anoxic conditions, hydrogen was used as electron donor with nitrate as electron acceptor. Organic matter (yeast extract, meat extract, peptone) supported neither mixotrophic nor heterotrophic growth. At concentrations as low as 0.75 g organic matter/l or higher, growth of nitrite-oxidizing cells was inhibited. The cells contained cytochromes of the b- and c-type. The G+C content of DNA was 56.9 +/- 0.4 mol%. The chemolithoautotrophic nitrite-oxidizer differed from the terrestrial members of the genus Nitrobacter with regard to morphology and substrate range and equaled Nitrospira marina in both characteristics. The isolated bacterium is designated as a new species of the genus Nitrospira.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗
PubMed1995

Molecular genetic evidence of bacterial colonization of cholesterol gallstones.

BACKGROUND/AIMS: Cholesterol gallstone formation is believed to be unrelated to the presence of bacteria because attempts to culture potentially causative bacteria from surgically removed cholesterol stones have failed. However, the formation of gallbladder gallstones takes years. Embedded bacteria may be damaged or killed. The aim of this study was to search for bacterial DNA sequences in cholesterol stones with negative bacterial culture. METHODS: Bacterial gene fragments were amplified in vitro from DNA extracted from cholesterol gallbladder stones. Comparative 16S ribosomal RNA sequence analysis was used for identification. RESULTS: Gallstones with cholesterol content between 70% to 90% harbored bacterial DNA (16 of 17 patients). No bacterial DNA was found in the gallstones with cholesterol content of > 90% (3 patients). Three bacterial groups typical for gallstone colonization were identified. Propionibacteria-related DNA was found in the stones of 9 patients (45%). Enterobacterial type sequences were obtained in 5 patients (25%). A more heterogenous sequence collection was retrieved from 7 patients (35%) and could be assigned to the major bacterial line of gram-positive bacteria with a low DNA guanine and cytosine content. CONCLUSIONS: Most cholesterol gallstones harbor bacterial DNA. It is important to determine the actual role of these microorganisms in gallstone formation.

Adult↗
PubMed1995

Taxonomic position of aromatic-degrading denitrifying pseudomonad strains K 172 and KB 740 and their description as new members of the genera Thauera, as Thauera aromatica sp. nov., and Azoarcus, as Azoarcus evansii sp. nov., respectively, members of the beta subclass of the Proteobacteria.

In the past workers have isolated several pseudomonad strains which have been used for studies of anaerobic aromatic metabolism. The best studied of these strains are strains KB 740T (T = type strain) and K 172T. The taxonomic positions of these two organisms were determined by classical methods, including experiments to determine substrate spectrum, quinone type, and total fatty acid composition. Our results clearly excluded these strains from the authentic genus Pseudomonas, which belongs to the gamma subclass of the Proteobacteria. Instead, the properties of these organisms indicated that they belong to the beta subclass of the Proteobacteria. The sequences of the 16S ribosomal DNA genes confirmed this conclusion and indicated that strain K 172T represents a new species of the genus Thauera, Thauera aromatica, and that strain KB 740T represents a new species of the genus Azoarcus, Azoarcus evansii.

Base Sequence↗
PubMed1995

Lactosphaera gen. nov., a new genus of lactic acid bacteria, and transfer of Ruminococcus pasteurii Schink 1984 to Lactosphaera pasteurii comb. nov.

The phylogenetic position and physiology of strain KoTa2T (T = type strain), which was previously classified as a Ruminococcus pasteurii strain, were studied. A determination of the 16S ribosomal DNA sequence of this taxon revealed its position within the radiation of the gram-positive lactic acid bacteria having low DNA G+C contents and that it is closely related to the genus Carnobacterium. L-Lactic acid was produced from glucose by a fructose-1,6-bisphosphate-activated lactate dehydrogenase, and oxygen tolerance was observed, characteristics which are consistent with assignment to this group. On the basis of its phenotypic characteristics and unique signature nucleotides, we propose that strain KoTa2 (= DSM 2381 = ATCC 35945) should be transferred to a new genus, Lactosphaera gen. nov., as the type strain of the species Lactosphaera pasteurii comb. nov.

Anaerobiosis↗
PubMed1995

Phylogenetic identification and in situ detection of individual microbial cells without cultivation.

The frequent discrepancy between direct microscopic counts and numbers of culturable bacteria from environmental samples is just one of several indications that we currently know only a minor part of the diversity of microorganisms in nature. A combination of direct retrieval of rRNA sequences and whole-cell oligonucleotide probing can be used to detect specific rRNA sequences of uncultured bacteria in natural samples and to microscopically identify individual cells. Studies have been performed with microbial assemblages of various complexities ranging from simple two-component bacterial endosymbiotic associations to multispecies enrichments containing magnetotactic bacteria to highly complex marine and soil communities. Phylogenetic analysis of the retrieved rRNA sequence of an uncultured microorganism reveals its closest culturable relatives and may, together with information on the physicochemical conditions of its natural habitat, facilitate more directed cultivation attempts. For the analysis of complex communities such as multispecies biofilms and activated-sludge flocs, a different approach has proven advantageous. Sets of probes specific to different taxonomic levels are applied consecutively beginning with the more general and ending with the more specific (a hierarchical top-to-bottom approach), thereby generating increasingly precise information on the structure of the community. Not only do rRNA-targeted whole-cell hybridizations yield data on cell morphology, specific cell counts, and in situ distributions of defined phylogenetic groups, but also the strength of the hybridization signal reflects the cellular rRNA content of individual cells. From the signal strength conferred by a specific probe, in situ growth rates and activities of individual cells might be estimated for known species. In many ecosystems, low cellular rRNA content and/or limited cell permeability, combined with background fluorescence, hinders in situ identification of autochthonous populations. Approaches to circumvent these problems are discussed in detail.

Bacteria↗
PubMed1994

16S rDNA sequence and phylogenetic position of an uncultivated spirochete from the hindgut of the termite Mastotermes darwiniensis Froggatt.

We have analyzed the 16S rDNA sequence and the phylogenetic position of an uncultivated spirochete from the hindgut contents of the Australian termite Mastotermes darwiniensis Froggatt. The 16S rRNA genes of bacteria from the hindgut contents of Mastotermes darwiniensis were amplified by polymerase chain reaction. The amplification products were cloned and sequenced. The sequences were compared to known homologous primary structures. Two of the clones (MDS1 and MDS3) had an insert of 1498 nucleotides showing typical signatures of spirochete 16S rRNA sequences. The sequences of the two clones were most similar to the 16S rRNA sequence of Spirochaeta stenostrepta (89.8%) and Treponema sp. strain H1 (90.7%). Phylogenetical analysis positioned the hindgut spirochete sequence with that of the free-living anaerobic Spirochaeta stenostrepta and Treponema sp. strain H1 as its nearest relatives within the cluster of the spirochetes. We conclude that analyzed SSU rDNA sequences originate from a spirochete related to the genus Treponema. It is possibly one of the uncultivated unique spirochetes symbiotic in termite hindguts.

Animals↗
PubMed1994

Reverse dot blot hybridization: a useful method for the direct identification of lactic acid bacteria in fermented food.

A rapid method for a reliable and simultaneous identification of different lactic acid bacteria in fermented food has been developed. Various 16S and 23S rRNA-targeted, species-specific oligonucleotides were applied as capture probes in a non-radioactive reverse dot blot hybridization. A simple and fast DNA extraction method in combination with in vitro amplification of rRNA gene fragments enables the direct detection of typical starter organisms without any preceding enrichment or cultivation steps. Various lactic acid bacteria occurring in cheese, yogurt, sausages, sauerkraut and sourdough could be identified at the species level within 1 day.

Base Sequence↗
PubMed1994

Identification of Bacillus strains isolated from milk and cream with classical and nucleic acid hybridization methods.

A total of 529 bacterial strains have been isolated from milk and cream sampled at different sites in a dairy production plant under conditions selective for aerobic sporeforming bacteria. Identification with classical methods based on morphological, physiological and biochemical criteria showed Bacillus licheniformis to be the most frequently occurring Bacillus sp. The investigation also revealed 62 unidentified strains. Classical identification methods were time consuming (3-7 d), lacked specificity and--because of their dependence on phenotypic gene expression--sometimes produced ambiguous results. Consequently, a colony hybridization method developed for the identification of B. licheniformis strains and using nonradioactive labelled 23S rRNA targeted oligonucleotide probes was also used. Identification of B. licheniformis with both classical and hybridization methods revealed diverging identification results for 70 strains.

Animals↗
PubMed1994

Identification of lactobacilli from sourdough and description of Lactobacillus pontis sp. nov.

The microflora of sourdough preparations was investigated by examining the physiological characteristics, whole-cell protein patterns, and 16S rRNA sequences of Lactobacillus isolates. Strains isolated from sourdough were placed in the species Lactobacillus brevis, Lactobacillus sanfrancisco, and Lactobacillus reuteri. 16S rRNA sequences were determined for L. brevis, Lactobacillus fructivorans, Lactobacillus fermentum, L. sanfrancisco, and L. reuteri, and oligonucleotide probes for fast specific identification of these sourdough lactobacilli were deduced. The physiological characteristics, protein patterns, and 16S rRNA sequences of these organisms were compared with data for other sourdough lactobacilli and additional reference strains. Strains of a Lactobacillus species were isolated from rye sourdough; these strains may account for most of the flora in sourdough made from wheat or rye. These organisms were differentiated from other sourdough lactobacilli by their protein pattern, 16S rRNA sequence, G + C content, and physiological properties. The 16S rRNA sequence of this species was determined, and we constructed a phylogenetic tree which reflected the relationships of this species to other lactobacilli. This organism is closely related to L. reuteri. A new Lactobacillus species, Lactobacillus pontis, is proposed. The type strain is L. pontis LTH 2587 (= DSM 8475 = LMG 14187). We describe a general strategy in which a polyphasic approach was used to characterize a new species.

Bacterial Proteins↗
PubMed1994

Sequence determination of rRNA genes of pathogenic Vibrio species and whole-cell identification of Vibrio vulnificus with rRNA-targeted oligonucleotide probes.

A comparative analysis of seven new 16S rRNA gene sequences of pathogenic Vibrio species with previously published vibrio sequences confirmed that Vibrio vulnificus represents a group that is not closely related to the core organisms of the genus Vibrio. In addition, we found that V. vulnificus, Listonella (Vibrio) anguillarum and Vibrio diazotrophicus branch off separately from the core group. A comparison of the 16S rRNA gene sequences of V. vulnificus strains belonging to biotypes 1 and 2 revealed that the sequences of all but four biotype 1 strains were identical to each other but slightly different (17 bases) from the sequences of the rest of the V. vulnificus strains investigated. In addition, the sequences of variable regions of the 23S rRNA genes of Vibrio fluvialis, Vibrio furnissii, Vibrio harveyi, Vibrio cholerae, and V. vulnificus C7184 and TW1 were determined, aligned, and compared with all available bacterial 23S rRNA sequences in order to search for specific target sites. As a result, four oligonucleotide probes specific for V. vulnificus were synthesized, and the specificities of these probes were evaluated by dot blot hybridization to membrane-bound RNAs from 21 V. vulnificus strains, 13 strains belonging to other Vibrio species, 61 strains belonging to species that are members of the alpha, beta, and gamma subclasses of the Proteobacteria, and 3 eucaryotic microorganisms. Two probes hybridized with all of the V. vulnificus strains tested, and the other two probes distinguished V. vulnificus biotype 1 strains from all other organisms. In situ identification of V. vulnificus by using tetramethylrhodamine- or fluorescein-labelled oligonucleotides is now possible.

Bacterial Typing Techniques↗
PubMed1994

In situ probing of gram-positive bacteria with high DNA G + C content using 23S rRNA-targeted oligonucleotides.

23S-rRNA-targeted oligonucleotide probes were designed for the phylogenetic group 'Gram-positive bacteria with high G + C content of DNA' (GPBHGC). A sequence idiosyncrasy in two adjacent base pairs in the stem of helix 69 in domain IV of the 23S rRNA is present in all hitherto analysed strains of GPBHGC. An oligonucleotide probe targeted to this region hybridized only with strains of GPBHGC and was successfully used for in situ monitoring of these cells in activated sludge. Another unique feature of the 23S rRNA molecules of GPBHGC is a large insertion in domain III. Fluorescent oligonucleotides targeted to the highly variable regions of the rRNA within the insertions of Corynebacterium glutamicum DSM 20300, Aureobacterium testaceum DSM 20166 and Brevibacterium sp. DSM 20165 hybridized specifically to their target strains, whereas probing with oligonucleotides complementary to the rRNA-coding strand of the 23S rDNA and to the spacer between 16S and 23S rRNA of C. glutamicum did not result in detectable fluorescence. This confirmed that the large 23S insertions are indeed present in 23S rRNAs of GPBHGC and provide potential target sites for highly specific nucleic acid probes.

Base Sequence↗
PubMed1994

Bacterial phylogeny based on 16S and 23S rRNA sequence analysis.

Molecular phylogeny increasingly supports the understanding of organismal relationships and provides the basis for the classification of microorganisms according to their natural affiliations. Comparative sequence analysis of ribosomal RNAs or the corresponding genes currently is the most widely used approach for the reconstruction of microbial phylogeny. The highly and less conserved primary and higher order structure elements of rRNAs document the history of microbial evolution and are informative for definite phylogenetic levels. An optimal alignment of the primary structures and a careful data selection are prerequisites for reliable phylogenetic conclusions. rRNA based phylogenetic trees can be reconstructed and the significance of their topologies evaluated by applying distance, maximum parsimony and maximum likelihood methods of phylogeny inference in comparison, and by fortuitous or directed resampling of the data set. Phylogenetic trees based on almost equivalent data sets of bacterial 23S and 16S rRNAs are in good agreement and their overall topologies are supported by alternative phylogenetic markers such as elongation factors and ATPase subunits. Besides their phylogenetic information content, the differently conserved primary structure regions of rRNAs provide target sites for specific hybridization probes which have been proven to be powerful tools for the identification of microbes on the basis of their phylogenetic relationships.

Bacteria↗