Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics

Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols: a guide to methods and applications

We describe a new molecular approach to analyzing the genetic diversity of complex microbial populations. This technique is based on the separation of polymerase chain reaction-amplified fragments of genes coding for 16S rRNA, all the same length, by denaturing gradient gel electrophoresis (DGGE). DGGE analysis of different microbial communities demonstrated the presence of up to 10 distinguishable bands in the separation pattern, which were most likely derived from as many different species constituting these populations, and thereby generated a DGGE profile of the populations. We showed that it is possible to identify constituents which represent only 1% of the total population. With an oligonucleotide probe specific for the V3 region of 16S rRNA of sulfate-reducing bacteria, particular DNA fragments from some of the microbial populations could be identified by hybridization analysis. Analysis of the genomic DNA from a bacterial biofilm grown under aerobic conditions suggests that sulfate-reducing bacteria, despite their anaerobicity, were present in this environment. The results we obtained demonstrate that this technique will contribute to our understanding of the genetic diversity of uncharacterized microbial populations.

Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA

A set of oligonucleotide primers capable of initiating enzymatic amplification (polymerase chain reaction) on a phylogenetically and taxonomically wide range of bacteria is described along with methods for their use and examples. One pair of primers is capable of amplifying nearly full-length 16S ribosomal DNA (rDNA) from many bacterial genera; the additional primers are useful for various exceptional sequences. Methods for purification of amplified material, direct sequencing, cloning, sequencing, and transcription are outlined. An obligate intracellular parasite of bovine erythrocytes, Anaplasma marginale, is used as an example; its 16S rDNA was amplified, cloned, sequenced, and phylogenetically placed. Anaplasmas are related to the genera Rickettsia and Ehrlichia. In addition, 16S rDNAs from several species were readily amplified from material found in lyophilized ampoules from the American Type Culture Collection. By use of this method, the phylogenetic study of extremely fastidious or highly pathogenic bacterial species can be carried out without the need to culture them. In theory, any gene segment for which polymerase chain reaction primer design is possible can be derived from a readily obtainable lyophilized bacterial culture.

16S ribosomal DNA amplification for phylogenetic study.

A 16S rRNA gene database (http://greengenes.lbl.gov) addresses limitations of public repositories by providing chimera screening, standard alignment, and taxonomic classification using multiple published taxonomies. It was found that there is incongruent taxonomic nomenclature among curators even at the phylum level. Putative chimeras were identified in 3% of environmental sequences and in 0.2% of records derived from isolates. Environmental sequences were classified into 100 phylum-level lineages in the Archaea and Bacteria.

/pdf/greengenes-a-chimera-checked-16s-rrna-gene-database-and-4svk1mo01y.pdf

Greengenes, a Chimera-Checked 16S rRNA Gene Database and Workbench Compatible with ARB

Ribosomal RNA (rRNA) sequences were used to establish the phylogenetic affiliations of symbioses in which prokaryotes appear to confer sulfur-based chemoautotrophy on their invertebrate hosts. Two submarine hydrothermal vent animals, the vestimentiferan tube worm Riftia pachyptila and the clam Calyptogena magnifica, and a tidal-flat bivalve , Solemya velum, were inspected. 5S rRNA9s were extracted from symbiont-bearing tissues, separated into unique forms, and their nucleotide sequences determined and related to other 5S rRNA9s in a phylogenetic tree analysis. The prokaryotic symbionts are related to one another and affiliated with the same narrow phylogenetic grouping as Escherichia coli and Pseudomonas aeruginosa. The sequence comparisons suggest that Riftia is more closely related to the bivalves than their current taxonomic status would suggest .

https://science.sciencemag.org/content/sci/224/4647/409.full.pdf

Analysis of Hydrothermal Vent-Associated Symbionts by Ribosomal RNA Sequences

The microorganisms inhabiting a 91 degrees C hot spring in Yellowstone National Park were characterized by sequencing 5S rRNAs isolated from the mixed, natural microflora without cultivation. By comparisons of these sequences with reference sequences, the phylogenetic relationships of the hot spring organisms to better characterized ones were established. Quantitation of the total 5S-sized rRNAs revealed a complex microbial community of three dominant members, a predominant archaebacterium affiliated with the sulfur-metabolizing (dependent) branch of the archaebacteria, and two eubacteria distantly related to Thermus spp. The archaebacterial and the eubacterial 5S rRNAs each constituted about half the examined population.

Characterization of a Yellowstone hot spring microbial community by 5S rRNA sequences.

https://www.cell.com/cell/pdf/0092-8674(90)90592-3.pdf

The excision of intervening sequences from salmonella 23S ribosomal RNA

The inter- and intrageneric relationships of the genus Vibrio were investigated by performing a comparative analysis of the 16S rRNAs of 10 species, including four pathogenic representatives. The results of immunological and 5S rRNA studies were confirmed in that the genus is a neighboring taxon of the family Enterobacteriaceae. With regard to the intrageneric structure, Vibrio alginolyticus, Vibrio campbellii, Vibrio natriegens, Vibrio harveyi, Vibrio proteolyticus, Vibrio parahaemolyticus, and Vibrio vulnificus form the core of the genus, while Vibrio (Listonella) anguillarum, Vibrio diazotrophicus, and Vibrio hollisae are placed on the outskirts of the genus. Variable regions around positions 80, 180, and 450 could be used as target sites for genus- and species-specific oligonucleotide probes and polymerase chain reaction primers to be used in molecular identification.

Towards a phylogeny of the genus Vibrio based on 16S rRNA sequences.

Comparison of partial 16S rRNA sequences from representative Campylobacter species indicates that the Campylobacter species form a previously undescribed basic eubacterial group, which is related to the other major groups only by very deep branching. This analysis was extended to include the spiral bacterium associated with human gastritis, Campylobacter pylori (formerly Campylobacter pyloridis). The distance between C. pylori and the other Campylobacter species is sufficient to exclude the pyloric organism from the Campylobacter genus. The results indicate that C. pylori is more closely related to Wolinella succinogenes than it is to the other Campylobacter species inspected. Another close relative of the campylobacters was found to be Thiovulum, a sulfide-dependent marine bacterium.

David J. Lane

Papers

Analysis of Hydrothermal Vent-Associated Symbionts by Ribosomal RNA Sequences

Characterization of a Yellowstone hot spring microbial community by 5S rRNA sequences.

The excision of intervening sequences from salmonella 23S ribosomal RNA

Towards a phylogeny of the genus Vibrio based on 16S rRNA sequences.

Campylobacter pylori, the spiral bacterium associated with human gastritis, is not a true Campylobacter sp.