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

The application of molecular biological methods to study the diversity and ecology of microorganisms in natural environments has been practiced since the mid-1980s Since that time many new insights into the composition of uncultivated microbial communities have been gained Whole groups of organisms that are only known from molecular sequences are now believed to be quantitatively significant in many environments Molecular methods have also allowed characterization of many long-recognized but poorly understood organisms These organisms have eluded laboratory cultivation and, hence, have remained enigmatic This review provides an outline of the main methods used in molecular microbial ecology, and their limitations Some discoveries, made through the application of molecular biological methods, are highlighted, with reference to morphologically distinctive, uncultivated bacteria; an important biotechnological process (wastewater treatment); and symbiotic relationships between Bacteria, Archaea and Eukarya

Microbial Evolution, Diversity, and Ecology: A Decade of Ribosomal RNA Analysis of Uncultivated Microorganisms

The sequencing of 16S and 23S ribosomal RNA (rRNA) molecules is currently the gold standard for the classification of new microbial isolates. Comparative analyses of these sequences are for the first time in the history of microbiology facilitating the reconstruction of universal phylogenetic trees [38]. Among many other important findings the work of Carl Woese and his colleagues demonstrated that only certain (by far not all) phenotypic/physiological groups of micro-organisms are monophyletic (e.g., methanogenes, cyanobacteria, spirochetes). About 10 years ago it has been proposed to use an rRNA approach for studies in microbial ecology [21]. The microbial diversity should be analyzed in a cultivation-independent way by direct rRNA sequence retrieval, whereas nucleic acid probes complementary to rRNA or rRNA genes should be the tools to monitor population dynamics in the environmental samples. By their own nature rRNA-targeted probes track genotypes which are not necessarily linked to one phenotype. Microbial ecologists who want to apply this approach to investigate correlations between community structures and functions should be aware of this fact and design or apply rRNA-targeted probes accordingly.

In situ identification of micro-organisms by whole cell hybridization with rRNA-targeted nucleic acid probes

The presence of methanogenic bacteria was assessed in peat and soil cores taken from upland moors. The sampling area was largely covered by blanket bog peat together with small areas of red-brown limestone and peaty gley. A 30-cm-deep core of each soil type was taken, and DNA was extracted from 5-cm transverse sections. Purified DNA was subjected to PCR amplification with primers IAf and 1100Ar, which specifically amplify 1.1 kb of the archaeal 16S rRNA gene, and ME1 and ME2, which were designed to amplify a 0.75-kb region of the alpha-subunit gene for methyl coenzyme M reductase (MCR). Amplification with both primer pairs was obtained only with DNA extracted from the two deepest sections of the blanket bog peat core. This is consistent with the notion that anaerobiosis is required for activity and survival of the methanogen population. PCR products from both amplifications were cloned, and the resulting transformants were screened with specific oligonucleotide probes internal to the MCR or archaeal 16S rRNA PCR product. Plasmid DNA was extracted from probe-positive clones of both types and the insert was sequenced. The DNA sequences of 8 MCR clones were identical, as were those of 16 of the 17 16S rRNA clones. One clone showed marked variation from the remainder in specific regions of the sequence. From a comparison of these two different 16S rRNA sequences, an oligonucleotide was synthesized that was 100% homologous to a sequence region of the first 16 clones but had six mismatches with the variant. This probe was used to screen primary populations of PCR clones, and all of those that were probe negative were checked for the presence of inserts, which were then sequenced. By using this strategy, further novel methanogen 16S rRNA variants were identified and analyzed. The sequences recovered from the peat formed two clusters on the end of long branches within the methanogen radiation that are distinct from each other. These cannot be placed directly with sequences from any cultured taxa for which sequence information is available.

Isolation and identification of methanogen-specific DNA from blanket bog peat by PCR amplification and sequence analysis.

Most of the small ciliate protozoa, including Dasytricha ruminantium and Entodinium spp. living in the rumen of sheep, were found to have intracellular bacteria. These bacteria were not present in digestive vacuoles. They showed characteristic coenzyme F420 autofluorescence and they were detected with a rhodamine-labelled Archaea-specific oligonucleotide probe. The measured volume percent of autofluorescing bacteria (1%) was close to the total volume of intracellular bacteria estimated from TEM stereology. Thus it is likely that all of the bacteria living in the cytoplasm of these ciliates were endosymbiotic methanogens, using H2 evolved by the host ciliate to form methane. Intracellular methanogens appear to be much more numerous than those attached to the external cell surface of ciliates.

Some rumen ciliates have endosymbiotic methanogens

The polymerase chain reaction (PCR) was used to amplify small-subunit ribosomal DNA from the anaerobic ciliated protozoon Metopus palaeformis, and from its uncultured endosymbiotic bacteria. This was accomplished directly from total DNA extracted from protozoa without prior isolation or enrichment for symbiont cells. The double-stranded amplification products were precipitated and directly sequenced using the linear PCR reaction. Fluorescent oligonucleotide probes were designed and used in whole-cell hybridizations to provide direct visual evidence that the sequences originated from the host ciliate and from the endosymbiont. Phylogenetic analysis of the Metopus palaeformis sequence consistently placed it as a deep-branching lineage near the root of the ciliate tree. However, the present data were insufficient to resolve the detailed relationship between Blepharisma and Metopus and thus to determine if the heterotrichs are mono- or paraphyletic. Phylogenetic analysis of the symbiont partial sequence clearly demonstrated that it is an archaeobacterium and that it is closely related to, but distinct from, Methanobacterium formicicum.

The use of rRNA sequences and fluorescent probes to investigate the phylogenetic positions of the anaerobic ciliate Metopus palaeformis and its archaeobacterial endosymbiont.

Thomas Rh

Papers

The use of rRNA sequences and fluorescent probes to investigate the phylogenetic positions of the anaerobic ciliate Metopus palaeformis and its archaeobacterial endosymbiont.