Termite Gut Symbiotic Archaezoa Are Becoming Living Metabolic Fossils

ABSTRACT Over the course of several million years, the eukaryotic gut symbionts of lower termites have become adapted to a cellulolytic environment. Up to now it has been believed that they produce nutriments using their own cellulolytic enzymes for the benefit of their termite host. However, we have now isolated two endoglucanases with similar apparent molecular masses of approximately 36 kDa from the not yet culturable symbiotic Archaezoa living in the hindgut of the most primitive Australian termite, Mastotermes darwiniensis . The N-terminal sequences of these cellulases exhibited significant homology to cellulases of termite origin, which belong to glycosyl hydrolase family 9. The corre…

Occurrence of rhizobia in the gut of the higher termite Nasutitermes nigriceps

Wood-eating termites feed on a diet highly deficient in nitrogen. They must complement their diet with the aid of nitrogen-fixing bacteria. Nitrogen fixation in the gut has been demonstrated, but information about nitrogen-fixing bacteria in pure culture is scarce. From the higher termite Nasutitermes nigriceps the symbiotic bacterial strain M3A was isolated, which thrives in the hindgut contents. The Gram-negative strain exhibited similarities to the species of the genus Ensifer (including Sinorhizobium) on the basis of morphological and physiological/biochemical features. The 16S rRNA gene analysis showed the highest sequence similarity of the isolate M3A to Ensifer adhaerens (>99%; ATCC …

Spirochaeta coccoides sp. nov., a novel coccoid spirochete from the hindgut of the termite Neotermes castaneus.

ABSTRACT A novel spirochete strain, SPN1, was isolated from the hindgut contents of the termite Neotermes castaneus . The highest similarities (about 90%) of the strain SPN1 16S rRNA gene sequence are with spirochetes belonging to the genus Spirochaeta , and thus, the isolate could not be assigned to the so-called termite clusters of the treponemes or to a known species of the genus Spirochaeta . Therefore, it represents a novel species, which was named Spirochaeta coccoides . In contrast to all other known validly described spirochete species, strain SPN1 shows a coccoid morphology and is immotile. The isolated strain is obligately anaerobic and ferments different mono-, di-, and oligosacc…

Molecular Methods for Identification of Wine Microorganisms and Yeast Development

A prerequisite for the biochemical and physiological investigation of microorganisms is the isolation and management of pure cultures. Nevertheless, most of the environmental microorganisms are graded as “yet not cultivable” because the nutritional requirements are unknown or they could not be isolated due to the fact that fast-growing strains overgrow other microorganisms of a microbiota. In addition to plating techniques, isolation without cultivation and analysis of microbes could be performed by micromanipulation techniques or the application of optical tweezers followed by the utilization of PCR-based technologies.

Biology of Microorganisms on Grapes, in Must and in Wine

Diversity and Lignocellulolytic Activities of Cultured Microorganisms

Rapid isolation of single microbial cells from mixed natural and laboratory populations with the aid of a micromanipulator.

In order to facilitate the isolation of pure cultures from natural habitats we have developed a method for the isolation of single microbial cell clones from a mixed population, e.g. the flora of the termite gut, with the aid of a modern micromanipulator. The separated single prokaryotic or eukaryotic cells were grown after transfer in culture media or they were used for single cell PCR. The micromanipulator was also applied for the removal of nuclei from protozoa, of which the SSU rDNA was directly amplified.

Bacterial Ectosymbionts which Confer Motility: Mixotricha paradoxa from the Intestine of the Australian Termite Mastotermes darwiniensis

Use of a species-specific multiplex PCR for the identification of pediococci.

In this study, the 23S rRNA genes of nine different Pediococcus type strains were sequenced. By using a multiple sequence alignment with 23S rDNA sequences of related lactic acid bacteria two primer pairs were constructed, one for the general identification of the genus Pediococcus and one for the identification of the atypical species, P. dextrinicus. Furthermore, a primer set for a rapid multiplex PCR identification of the eight typical Pediococcus species was developed. With this technique, the species P. damnosus, P. parvulus, P. inopinatus, P. cellicola, P. pentosaceus, P. acidilactici, P. claussenii, and P. stilesii could be discriminated simultaneously in a single PCR. Experiments wi…

Isolation of Desulfovibrio intestinalis sp. nov. from the hindgut' of the lower termite Mastotermes darwiniensis.

A Gram-negative, anaerobic sulfate-reducing bacterium was isolated from hindgut contents of the lower termite Mastotermes darwiniensis Froggatt (strain KMS2). Strain KMS2 is motile by a single polar flagellum. The isolate possesses desulfoviridin and catalase activity. The G+C content of its DNA is in the range of 54.5-55.5 mol% (strain KMS2). It respires hydrogen and different low molecular weight organic compounds in the presence of sulfate, thiosulfate, and sulfite, and also oxygen. The isolated strain ferments pyruvate. Fastest growth with a doubling time of 12.5 h was obtained at 37°C and not at 28°C, the temperature at which the termites were grown. The isolate showed a 16S rDNA seque…

Species-specific identification of Dekkera/Brettanomyces yeasts by fluorescently labeled DNA probes targeting the 26S rRNA.

Sequencing of the complete 26S rRNA genes of all Dekkera/Brettanomyces species colonizing different beverages revealed the potential for a specific primer and probe design to support diagnostic PCR approaches and FISH. By analysis of the complete 26S rRNA genes of all five currently known Dekkera/Brettanomyces species (Dekkera bruxellensis, D. anomala, Brettanomyces custersianus, B. nanus and B. naardenensis), several regions with high nucleotide sequence variability yet distinct from the D1/D2 domains were identified. FISH species-specific probes targeting the 26S rRNA gene's most variable regions were designed. Accessibility of probe targets for hybridization was facilitated by the constr…

Lactic Acid Bacteria

A typical lactic acid bacterium grown under standard conditions is aerotolerant, acid tolerant, organotrophic, and a strictly fermentative rod or coccus, producing lactic acid as a major end product. It lacks cytochromes and is unable to synthesize porphyrins. Its features can vary under certain conditions. Catalase and cytochromes may be formed in the presence of hemes and lactic acid can be further metabolized, resulting in lower lactic acid concentrations. Cell division occurs in one plane, except pediococci. The cells are usually nonmotile. They have a requirement for complex growth factors such as vitamins and amino acids. An unequivocal definition of LAB is not possible (Axelsson, Lac…

Fast protocols for the 5S rDNA and ITS-2 based identification ofOenococcus oeni

To identify specific marker sequences for the rapid identification of Oenococcus oeni, we sequenced the 23S-5S internal transcribed spacer (ITS-2) region and the 5S rDNA of five different O. oeni strains and three phylogenetically related lactic acid bacteria (LAB). Comparative analysis revealed 100% identity among the ITS-2 region of the O. oeni strains and remarkable differences in length and sequence compared to related LAB. These results enabled us to develop a primer set for a rapid PCR-identification of O. oeni within three hours. Moreover, the comparison of the 5S rDNA sequences and the highly conserved secondary structure provided the template for the design of three fluorescence-la…

Multiplex PCR for species discrimination of Sclerotiniaceae by novel laccase introns

Common PCR-based targets for the identification of filamentous fungi and yeasts are the sequences of the internal transcribed spacer region (ITS1, 5.8S rDNA, ITS2). Within the Sclerotiniaceae the ITS-region is homogenous and the identification is almost impossible. Furthermore, the lack of IGS-data (intergenic spacer region) requires new specific marker genes for a rapid identification of phytopathogenic Sclerotiniaceae. We sequenced and analyzed new laccase2 (lcc2) genes from the phylogenetically related Sclerotinia sclerotiorum (Lib.) de Bary, Sclerotinia minor Jagger, and Monilinia fructigena Honey. Comparative analysis revealed remarkable differences in length and sequence compared to t…

Fast identification of wine related lactic acid bacteria by multiplex PCR

The microflora of must and wine consists of yeasts, acetic acid bacteria and lactic acid bacteria (LAB). The latter group plays an important role for wine quality. The malolactic fermentation carried out by LAB leads to deacidification and stabilisation of wines. Nevertheless, LAB are often associated with wine spoilage. They are mainly responsible for the formation of biogenic amines. Furthermore, some strains produce exopolysaccharide slimes, acetic acid, diacetyl and other off-flavours. In this context a better monitoring of the vinification process is crucial to improve wine quality. Moreover, a lot of biodiversity studies would also profit from a fast and reliable identification method…

Micromanipulation Techniques for the Isolation of Single Microorganisms

A prerequisite for the biochemical and physiological investigation of microorganisms is the isolation and management of pure cultures. The only absolute criterion of purity for a bacterial culture is that it has been derived from the progeny of a single cell. Failure to apply this criterion may lead to much effort in proving the purity of a culture. All strains upon which research is to be based should therefore be rigorously purified before starting to investigate the properties of individual organisms (Johnstone 1969). Ecologically oriented microbiologists are faced especially with the problem of how to obtain a pure culture of certain microbial strains from their densely populated natura…

Cellulose Digestion in the Termite Gut