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.

Phylogenetic analysis of ribosomal RNA sequences obtained from uncultivated organisms of a hot spring in Yellowstone National Park reveals several novel groups of Archaea, many of which diverged from the crenarchaeal line of descent prior to previously characterized members of that kingdom. Universal phylogenetic trees constructed with the addition of these sequences indicate monophyly of Archaea, with modest bootstrap support. The data also show a specific relationship between low-temperature marine Archaea and some hot spring Archaea. Two of the environmental sequences are enigmatic: depending upon the data set and analytical method used, these sequences branch deeply within the Crenarchaeota, below the bifurcation between Crenarchaeota and Euryarchaeota, or even as the sister group to Eukaryotes. If additional data confirm either of the latter two placements, then the organisms represented by these ribosomal RNA sequences would merit recognition as a new kingdom, provisionally named "Korarchaeota."

Ofthe three primary phylogenetic domains Archaea (archaebacteria), Bacteria (eubacteria), and Eucarya (eukaryotes) -Archaea is the least understood in terms of its diversity, physiologies, and ecological panorama. Although many species of Crenarchaeota {one of the two recognized archaeal kingdoms sensu Woese [Woese, C. R., Kandler, 0. & Wheelis, M. L. (1990) Proc. Nadl. Acad. Sci. USA 87, 4576-4579J} have been isolated, they constitute a relatively tight-knit cluster of lineages in phylogenetic analyses of rRNA sequences. It seemed possible that this limited diversity is merely apparent and reflects only a failure to culture organisms, not their absence. We report here phylogenetic characterization of many archaeal small subunit rRNA gene sequences obtained by polymerase chain reaction amplification of mixed population DNA extracted directly from sediment of a hot spring in Yellowstone National Park. This approach obviates the need for cultivation to identify organisms. The analyses document the existence not only of species belonging to well-characterized crenarchaeal genera or families but also ofcrenarchaeal species for which no close relatives have so far been found. The large number of distinct archaeal sequence types retrieved from this single hot spring was unexpected and demonstrates that Crenarchaeota is a much more diverse group than was previously suspected. The results have impact on our concepts of the phylogenetic organization of Archaea.Microbiologists have long understood the limitations of cultivation techniques in assessing the diversity of naturally occurring microbial communities. Commonly, only a small fraction of organisms observed microscopically can be cultivated using standard methods. Recently, sequence-based phylogenetic techniques have been used to alleviate the requirement for cultivation to identify microorganisms. Such studies have detected the presence of previously unknown organisms in each instance of their use (for review, see ref.1). These techniques sample microbial populations directly through isolating and sequencing specific genes from the environment. Phylogenetic comparative analysis of these sequences is then used to determine evolutionary relationships between members of the community and cultivated species. The results allow inference of some properties of otherwise unknown organisms in the environment, based on the properties of their studied relatives. In addition, the sequences can be used to design oligonucleotide probes for determination of morphotype and abundance of particular organisms and for assistance in cultivation efforts.We have employed molecular phylogenetic techniques to investigate the diversity of Archaea in a hot spring in Yellowstone National Park. Small-subunit rRNA genes were amplified by polymerase chain reaction (PCR) from DNA extracted directly from sediment, by using primers designed to amplify archaeal and eucaryal genes selectively. Amplification products were then cloned and the nucleotide sequences of the inserts were determined....

The 16S rRNAs from 29 cyanobacteria and the cyanelle of the phytoflagellate Cyanophora paradoxa were partially sequenced by a dideoxynucleotide-terminated, primer extension method. A least-squares distance matrix analysis was used to infer phylogenetic trees that include green chloroplasts (those of euglenoids, green algae, and higher plants only support the conclusion of previous workers that the cyanobacteria and green chloroplasts form a coherent phylogenetic group but also suggest that the chloroplast lineage, which includes the cyanelle of C. paradoxa, is not just a sister group to the free-living forms but rather is contained within the cyanobacterial radiation.The cyanobacteria are one of the most morphologically diverse and conspicuously successful procaryotic groups. It is generally believed that the cyanobacteria were the first major group of phototrophs to arise with a two-stage photosynthetic pathway capable of oxidizing water to produce molecular oxygen. Geochemical and fossil evidence indicates that in the Precambrian Era they caused the transition in the Earth's atmosphere from its primordial, anaerobic state to its current, aerobic condition (19,36,43). Moreover, molecular phylogenetic analysis of c-type cytochrome and rRNA sequences have established a relationship between cyanobacteria and the green (euglenoids, green algae, and higher plants) and red (rhodophyte) chloroplasts, thus supporting the procaryotic origins of chloroplasts.Because of their ubiquity, rRNA sequences are particularly useful for establishing evolutionary relationships among diverse organisms. Woese and colleagues, using partial (RNase Tl-generated oligonucleotide catalogs) and complete 16S rRNA sequences, have defined about 10 major divisions (phyla) of eubacteria (45). The cyanobacteria are one of these phyla. However, too few strains (eight) of cyanobacteria had been investigated to develop a comprehensive overview of the diversity of the group.We have used a method for directly sequencing 16S rRNA to explore the evolutionary relationships among 30 representatives of the diverse cyanobacterial groups, including the photosynthetic organelle of the phytoflagellate Cyanophora paradoxa. The results shed new light on the relative ages of * Corresponding author. t Present address:

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