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.

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

The phylogenetic interrelationships of members of the genus Listeria were investigated by using reverse transcriptase sequencing of 16S rRNA. The sequence data indicate that at the intrageneric level the genus Listeria consists of the following two closely related but distinct lines of descent: (i) the Listeria monocytogenes group of species (including Listeria innocua, Listeria ivanovii, Listeria seeligeri, and Listeria welshimeri) and (ii) the species Listeria grayi and Listeria murrayi. At the intergeneric level a specific phylogenetic relationship between the genera Listeria and Brochothrix was evident. The sequence data clearly demonstrated that the genus Listeria is phylogenetically remote from the genus Lactobacillus and should not be included in an extended family Lactobacillaceae.

Phylogenetic Analysis of the Genus Listeria Based on Reverse Transcriptase Sequencing of 16S rRNA

5S rRNA nucleotide sequences from Thiobacillus neapolitanus, Thiobacillus ferrooxidans, Thiobacillus thiooxidans, Thiobacillus intermedius, Thiobacillus perometabolis, Thiobacillus thioparus, Thiobacillus versutus, Thiobacillus novellus, Thiobacillus acidophilus, Thiomicrospira pelophila, Thiomicrospira sp. strain L-12, and Acidiphilium cryptum were determined. A phylogenetic tree, based upon comparison of these and other related 5S rRNA sequences, is presented. The results place the thiobacilli, Thiomicrospira spp., and Acidiphilium spp. in the "purple photosynthetic" bacterial grouping which also includes the enteric, vibrio, pseudomonad, and other familiar eubacterial groups in addition to the purple photosynthetic bacteria. The genus Thiobacillus is not an evolutionarily coherent grouping but rather spans the full breadth of the purple photosynthetic bacteria.

Phylogenetic analysis of the genera Thiobacillus and Thiomicrospira by 5S rRNA sequences.

Publisher Summary This chapter focuses on reverse transcriptase sequencing of ribosomal RNA for phylogenetic analysis. The cyanobacteria comprise a large group of structurally complex and ecologically important gram-negative prokaryotes whose taxonomic treatment has generated much interest and controversy over the years. By molecular taxonomic criteria (particularly 16 S rRNA oligonucleotide catalog analysis), the cyanobacteria, together with chloroplasts and prochlorophytes, are a coherent, eubacterial grouping that warrants phylum status. The sequencing protocol is a variation of the base-specific, dideoxynucleotide-terminated, chain-elongation method. It has been modified for the use of reverse transcriptase and ribosomal RNA templates. The method relies on the fact that certain regions of 16 S rRNA nucleotide sequence vary little or not at all among different organisms. A short oligodeoxynucleotide, complementary to such a universally conserved site, specifically anneals to its target site on the 16 S rRNA, even in the presence of other cellular RNAs. The RNA purification method is designed for use on pure cultures containing only one type of 16 S rRNA. Mixed cultures, however, can often be sufficiently purified by differential centrifugation or filtration, at the time of harvesting, for the method to work.

[12] Reverse transcriptase sequencing of ribosomal RNA for phylogenetic analysis

Water samples and suspended particulate matter were collected from three high temperature (156–319°C) hydrothermal vents in the Guaymas Basin during July 1985, and were analysed for a variety of chemical constituents and for the presence of viable microorganisms. Our results indicate that black smoker fluids (> 150°C) are devoid of recognizable bacteria and contain negligible concentrations of ATP ( -1 ) and low concentrations of particulate organic matter. In contrast, vent water samples collected in the hydrothermal plume at a distance of only 25 cm from the point of hot fluid discharge contain a diverse, metabolically active bacterial assemblage, high ATP concentrations (up to 372 ng 1 -1 ) and high concentrations of particulate organic carbon and nitrogen (up to 999 and 163 μg 1 -1 , respectively). The hottest waters (> 150°C) displayed a low, but measurable level of metabolic activity (incorporation of 3 H-adenine and 14 C-glutamate) at temperatures ranging from 25 to 80°C, 1 atm with maximal activity at 45°C. The maximum rate of incorporation, however, was only ∼ 1% of the activity measured in the samples collected in the hydrothermal plume. The strong metabolic preference for mesophilic growth temperatures (45°C) argues against a high temperature origin. Thermophilic sulfur-respiring bacteria were isolated from a variety of source materials including black smoker vent waters (112–319°C). All positive enrichments grew at 80°C, but none survived at temperatures in excess of 93°C. We conclude that these bacterial cells did not originate from the high temperature hydrothermal vent waters. Two different sample devices (reffered to as vent cap and smoker poker) were designed and employed during in situ collection-incubation experiments. Neither the vent cap attachment-colonization experiment nor the smoker poker collection device revealed the existence of bacterial cells in the black smoker fluids, despite evidence for a significant bacterial population in the hydrothermal plume. Our field and laboratory results indicate that thermophilic and mesophilic bacteria observed in hydrothermal vent plumes are not derived from the hot hydrothermal fluids but must originate in peripheral habitats.

A microbiological study of Guaymas Basin high temperature hydrothermal vents

Nucleic acid fragments capable of hybridizing to rRNA of a Salmonella species and not capable of hybridizing to rRNA of Escherichia coli.

David J. Lane

Papers

Phylogenetic Analysis of the Genus Listeria Based on Reverse Transcriptase Sequencing of 16S rRNA

Phylogenetic analysis of the genera Thiobacillus and Thiomicrospira by 5S rRNA sequences.

[12] Reverse transcriptase sequencing of ribosomal RNA for phylogenetic analysis

A microbiological study of Guaymas Basin high temperature hydrothermal vents

Detection of salmonella.