Manfred Berchtold

Aerobic and facultatively anaerobic cellulolytic bacteria from the gut of the termite Zootermopsis angusticollis

Aims: To demonstrate the occurrence of cellulolytic bacteria in the termite Zootermopsis angusticollis.

Differential enumeration and in situ localization of microorganisms in the hindgut of the lower termite Mastotermes darwiniensis by hybridization with rRNA-targeted probes

Abstract We examined the abundance and spatial distribution of major phylogenetic groups of the domain Bacteria in hindguts of the Australian lower termite Mastotermes darwiniensis by using in situ hybridization with group-specific, fluorescently labeled, rRNA-targeted oligonucleotide probes. Between 32.0 ± 7.2% and 52.3 ± 8.2% of the DAPI-stained cells in different hindgut fractions were detected with probe EUB338, specific for members of the domain Bacteria. About 85% of the prokaryotic cells were associated with the flagellates of the thin-walled anterior region (P3a) and the thick wall of the posterior region (P3b/P4) of the hindgut, as shown by DAPI staining. At most, half of the EUB338-detected cells hybridized with one of the other probes that targeted a smaller assemblage within the bacterial domain. In most fractions, cells were found in varying numbers with probe ALF1b, which targeted members of the α-Proteobacteria, whereas substantial amounts of sulfate-reducing bacteria, gram-positive bacteria with a high DNA G+C content and members of the Cytophaga-Flavobacterium cluster of the Cytophaga-Flavobacterium-Bacteroides (CFB) phylum could be detected only in the wall fraction of P3b/P4. This clearly indicates that the hindgut microhabitats differ in the composition of their microbial community. In situ hybridization of cryosections through the hindgut showed only low numbers of bacteria attached to the P3a wall. In contrast, the wall of P3b was densely colonized by rod- and coccus-shaped bacteria, which could be assigned to the Cytophaga-Flavobacterium cluster of the CFB phylum and to the group of gram-positive bacteria with a high DNA G+C content, respectively. Oxygen concentration profiles determined with microelectrodes revealed steep oxygen gradients both in P3a and P3b. Oxygen was consumed within 100 μm below the gut surface, and anoxic conditions prevailed in the central portions of both gut regions, indicating that oxygen consumption in the hindgut does not depend on the presence of a biofilm on the hindgut wall.

Psychrotolerant sulfate-reducing bacteria from an oxic freshwater sediment, description of Desulfovibrio cuneatus sp. nov. and Desulfovibrio litoralis sp. nov.

The most abundant culturable sulfate-reducing bacteria were isolated from the littoral sediment of the oligotrophic Lake Stechlin. The strains STL1 and STL4 were obtained from the oxic uppermost layer, while strain STL6 was isolated from the anoxic zone in 20 to 30 mm depth. The isolates showed a striking morphological feature in tapering off at one end of the cell. Physiological characteristics related them to the genus Desulfovibrio. They contained desulfoviridin. H2, formate, pyruvate, lactate, and fumarate were utilized with sulfate, sulfite, thiosulfate, or elemental sulfur as electron acceptors. All isolates were able to reduce oxygen and survived 120 h of aeration. However, aerobic growth was not observed. The isolates were psychrotolerant, and grew with rates of up to 0.29 d-1 at 4 degrees C. Analysis of the 16S rDNA confirmed that the strains belong to the genus Desulfovibrio. However, they were not closely related to any known member of this genus and formed a new cluster with at least two new species. Strain STL1 and STL4, exhibiting 99.7% sequence similarity in 16S rRNA, are proposed as the new species Desulfovibrio cuneatus sp. nov., while strain STL6 is assigned to the new species Desulfovibrio litoralis sp. nov.

Phylogenetic Analysis and In Situ Identification of Uncultivated Spirochetes from the Hindgut of the Termite Mastotermes darwiniensis

Summary Different small subunit ribosomal DNA (SSU rDNA) sequences of uncultivated spirochetes from the hindgut of the termite Mastotermes darwiniensis were determined and phylogenetically analysed. Starting with DNA isolated from hindgut contents we amplified SSU rDNA coding regions by PCR using bacteria- and spirochete-specific primers, respectively. Cloning and sequencing of the amplification products re-sulted in 11 different spirochetal SSU rDNA sequences with similarity values of 81.8% to 99.0% to each other. Phylogenetic trees showed that the obtained sequences are related to the genus Treponema with 84.0% to 91.1% similarity to Treponema spec. H1 and Spirochaeta stenostrepta as their nearest relatives among the cultivated spirochetes. Using fluorescence in situ hybridisation one of the SSU rDNA sequences could be assigned to a large spirochete. So at least one of the large hindgut spirochetes unique in termite hindguts and assigned to the genera Hollandina , Pillotina , Clevelandina or Diplocalyx phylogenetically belongs to the treponeme cluster of spirochetes.

Phylogenetic Position of the Two Uncultivated Trichomonads Pentatrichomonoides scroa Kirby and Metadevescovina extranea Kirby from the Hindgut of the Termite Mastotermes darwiniensis Froggatt

Summary The phylogenetic position of the two uncultivated trichomonad flagellates Pentatrichomonoides scroa and Metadevescovina extranea symbiotic in the hindgut of the Australian termite Mastotermes darwiniensis was determined by analysis of small subunit ribosomal DNA (SSU rDNA) sequences. After enrichment of the two trichmonads from crude hindgut contents and isolation of DNA the SSU rDNA was amplified by the polymerase chain reaction (PCR). The amplification products were cloned and sequenced resulting in two different sequences. Excluding the amplification primers the sequences were 1526 and 1500 nucleotides in length, respectively. The two sequences were assigned to the corresponding flagellate cells by whole cell hybridization using specific oligonucleotide probes. The SSU rDNA of Pentatrichomonoides scroa showed 87.3% similarity to Metadevescovina extranea and 87.2% and 93.0% to the other so far examined trichomonads Tritrichomonas foetus and Trichonrinrs trypanoides, respectively. Metadevescovina extranea was 89.0% and 88.1% similar to Tritrichomonas foetus and Trichomitus trypanoides , respectively. The sequences were phylogenetically analysed using distance matrix and character state methods. Within the trichomonads Pentatrichomonoides scroa and Trichomitus trypanoides clustered together in all analyses and Tritrichomonas foetus was the first branching trichomonad. Depending on the data set used, the trichomonad cluster consisting of four SSU rDNA sequences was one of the three deepest branching eukaryotic lineages.

Culture and Phylogenetic Characterization of Trichomitus trypanoides Duboscq & Grassè 1924, n. comb.: a Trichomonad Flagellate Isolated from the Hindgut of the Termite Reticulitermes santonensis Feytaud

ABSTRACT. A trichomonad flagellate strain R1 was isolated from the hindgut contents of the termite Reticulitermes santonensis Feytaud. The flagellate was cultivated at 28° C in anaerobic medium containing yeast extract, minerals and vitamins. The isolate fed on living bacteria. It showed the typical morphological and ultrastructural features of the trichomonads. closely resembling Trichomitus trypanoides. In order to determine its phylogenetic position the small subunit ribosomal DNA (SSU rDNA) of the flagellate was amplified in vitro using the polymerase chain reaction (PCR), cloned in a plasmid vector and sequenced. Comparison of the obtained sequence with so far available SSU rRNA/rDNA sequences showed strongest similarity (89%) to the sequence of Tritrichomonas foetus. The phylogenetic analysis with parsimony and distance matrix methods placed Trichomitus trypanoides strain R1 near by the root of the phylogenetically so far analyzed eukaryotic organisms. This confirms that termites harbour hindgut symbionts, which originate from very early evolved eukaryotes.

The phylogenetic position of Dimastigella trypaniformis within the parasitic kinetoplastids.

The nuclear 16S-like rRNA coding regions of two strains of the kinetoplastid flagellateDimastigella trypaniformis Sandon (strain Ulm and strain Glasgow) were sequenced and phylogenetically analyzed. Strain Ulm was isolated from the hindgut contents of the Australian termiteMastotermes darwiniensis Frogatt, whereas strain Glasgow originates from a soil sample in Scotland. After preparation of genomic DNA the 16S-like rRNA coding regions were amplified using polymerase chain reaction (PCR) technology. The amplification products were cloned in a plasmid vector and sequenced according to standard methods. The sequence of the 16S-like rRNA coding region of strain Ulm differs less than 2% from the sequence of strain glasgow, indicating that the two strains are most probably members of one species. Phylogenetic analysis of the sequence data positionedD. trypaniformis Sandon as a deep branching lineage near the root of the kinetoplastid group of flagellates.

16S-like rDNA sequence and phylogenetic position of the diplomonad Spironucleus muris (Lavier 1936)

Summary The sequence of the small subunit (SSU) rDNA gene of the facultative pathogenic flagellate Spironucleus muris has been determined. This flagellate belongs to the class Diplomonadida. We will set out its phylogenetic position within the eukaryotic tree according to its morphological characteristics. Cells of Spironucleus muris were enriched from the digestive tract of immunodeficient mice and the DNA was extracted. After sequencing the SSU rDNA insert (1206 bp), we carried out a comparative sequence analysis with accessible SSU rRNA sequences from related eukaryotes. Escherichia coli and Methanospirilium hungatei were used as outgroups. Spironucleus muris shows strongest structural similarity (73.2%) to the parasitic flagellate Hexamita inflata (class Diplomonadida), which originated very early in the eukaryotic tree.