A culture-independent molecular phylogenetic survey was carried out for the bacterial community in Obsidian Pool (OP), a Yellowstone National Park hot spring previously shown to contain remarkable archaeal diversity (S. M. Barns, R. E. Fundyga, M. W. Jeffries, and N. R. Page, Proc. Natl. Acad. Sci. USA 91:1609–1613, 1994). Small-subunit rRNA genes (rDNA) were amplified directly from OP sediment DNA by PCR with universally conserved orBacteria-specific rDNA primers and cloned. Unique rDNA types among >300 clones were identified by restriction fragment length polymorphism, and 122 representative rDNA sequences were determined. These were found to represent 54 distinct bacterial sequence types or clusters (≥98% identity) of sequences. A majority (70%) of the sequence types were affiliated with 14 previously recognized bacterial divisions (main phyla; kingdoms); 30% were unaffiliated with recognized bacterial divisions. The unaffiliated sequence types (represented by 38 sequences) nominally comprise 12 novel, division level lineages termed candidate divisions. Several OP sequences were nearly identical to those of cultivated chemolithotrophic thermophiles, including the hydrogen-oxidizing Calderobacterium and the sulfate reducers Thermodesulfovibrio andThermodesulfobacterium, or belonged to monophyletic assemblages recognized for a particular type of metabolism, such as the hydrogen-oxidizing Aquificales and the sulfate-reducing δ-Proteobacteria. The occurrence of such organisms is consistent with the chemical composition of OP (high in reduced iron and sulfur) and suggests a lithotrophic base for primary productivity in this hot spring, through hydrogen oxidation and sulfate reduction. Unexpectedly, no archaeal sequences were encountered in OP clone libraries made with universal primers. Hybridization analysis of amplified OP DNA with domain-specific probes confirmed that the analyzed community rDNA from OP sediment was predominantly bacterial. These results expand substantially our knowledge of the extent of bacterial diversity and call into question the commonly held notion that Archaea dominate hydrothermal environments. Finally, the currently known extent of division level bacterial phylogenetic diversity is collated and summarized.

Phylogenetic comparative analyses of RNase P RNA-encoding gene sequences from Chlorobium limicola, Chlorobium tepidum, Bacteroides thetaiotaomicron, and Flavobacterium yabuuchiae rerme the secondary structure model of the general (eu)bacterial RNase P RNA and show that a highly conserved feature of that RNA is not essential. Two helices, comprised of 2 base pairs each, are added to the secondary structure model and form part of a cruciform in the RNA. Novel sequence variations in the B. etaiotaomicron and F. yabuuchwae RNA indicate the likelihood that all secondary structure resulting from canonical base-pairing has been detected: there are no remaining unpaired, contiguous, canonical complementarities in the structure model common to all bacterial RNase P RNAs. A nomenclature for the elements of the completed secondary structure model is proposed. The Chlorobium RNase P RNAs lack a stem-oop structure that is otherwise universally present and highly conserved in structure in other (eu)bacterial RNase P RNAs. The Chlorobium RNAs are nevertheless catalytic, with kinetic properties similar to those of RNase P RNAs ofEscherichia coil and other Bacteria. Removal of this stem-oop structure from the E. colt RNA affects neither its affinity for nor its catalytic rate for cleavage of a precursor transfer RNA substrate. These results show that this structural element does not play a direct role in substrate binding or catalysis.RNase P is the site-specific endoribonuclease that removes 5' flanking sequences from precursors of transfer RNA (for reviews, see refs. 1-3). RNase P is a ribonucleoprotein; in Bacteria (formerly eubacteria), in which it is best studied, RNase P is composed of a single ca. 130-kDa RNA and a single ca. 14-kDa protein (see ref. 4 for review). The RNA component of bacterial RNase P is the catalytic moiety; at high ionic strength in vitro it is capable of efficient catalysis in the absence of protein (5). Understanding the mechanisms of substrate recognition and catalysis by RNase P RNA requires knowledge of the structure required for those functions. A model for the secondary structure § of bacterial RNase P RNA has been derived from phylogenetic comparisons (4, 6), which included sequences from representatives of 5 of the approximately 12 "Kingdoms" [sensu Woese (7)] of Bacteria. This secondary structure model was expected to be incomplete; additional sequence variation was required to provide evidence for either the presence or absence of additional structure. Sequences from organisms that are phylogenetically distant from previously examined ones are especially useful for inferring structure because they generally vary greatly from those known. Moreover, phylogenetically disparate sequences may allow the identification of elements in the RNA that are not universally present and, therefore, are presumably not essential for the function ofthe RNA. Naturally occurring gene sequences that lack specific structural elements also provide information useful in designing meaningful deletions or sub...

RNAPhylogenetic-comparative analysis of the eukaryal ribonuclease P ABSTRACTRibonuclease P (RNase P) is the ribonucleoprotein enzyme that cleaves 59-leader sequences from precursor-tRNAs. Bacterial and eukaryal RNase P RNAs differ fundamentally in that the former, but not the latter, are capable of catalyzing pre-tRNA maturation in vitro in the absence of proteins. An explanation of these functional differences will be assisted by a detailed comparison of bacterial and eukaryal RNase P RNA structures. However, the structures of eukaryal RNase P RNAs remain poorly characterized, compared to their bacterial and archaeal homologs. Hence, we have taken a phylogenetic-comparative approach to refine the secondary structures of eukaryal RNase P RNAs. To this end, 20 new RNase P RNA sequences have been determined from species of ascomycetous fungi representative of the genera Arxiozyma, Clavispora, Kluyveromyces, Pichia, Saccharomyces, Saccharomycopsis, Torulaspora, Wickerhamia, and Zygosaccharomyces. Phylogenetic-comparative analysis of these and other sequences refines previous eukaryal RNase P RNA secondary structure models. Patterns of sequence conservation and length variation refine the minimum-consensus model of the core eukaryal RNA structure. In comparison to bacterial RNase P RNAs, the eukaryal homologs lack RNA structural elements thought to be critical for both substrate binding and catalysis. Nonetheless, the eukaryal RNA retains the main features of the catalytic core of the bacterial RNase P. This indicates that the eukaryal RNA remains intrinsically a ribozyme.

The 16s rRNAs from nine rapidly growing Mycobacterium species were partially sequenced by using the dideoxynucleotide-terminated, primer extension method with cDNA generated by reverse transcriptase. The sequences were aligned with 47 16s rRNA or DNA sequences that represented 30 previously described and 5 undescribed species of the genus Mycobacterium, and a dendrogram was constructed by using equally weighted distance values. Our results confirmed the phylogenetic separation of the rapidly and slowly growing mycobacteria and showed that the majority of the slowly growing members of the genus represent the most recently evolved organisms. The 24 strains which represented 21 rapidly growing species constituted several sublines, which were defined by the following taxa: (i) Mycobacterium neoaumm and M. diernhoferi, (ii) M. gudium, (iii) the M. chubuense cluster, (iv) the M. fortuitum cluster, (v) M. kommossense, (vi) M. sphagni, (vii) M. fallax and M. chitae, (viii) M. aurum and M. vaccae, (ix) the M. fravescens cluster, and (x) M. chelonae subsp. abscessus. Our phylogenetic analysis confirmed the validity of the phenotypically defined species mentioned above, but our conclusions disagree with most of the conclusions about intrageneric relationships derived from numerical phenetic analyses.In the early 1980s, using 16s rRNA cataloging methods, workers determined that Mycobacterium phlei, one of the fast-growing Mycobacterium species, is a member of the phylogenetically defined order Actinomycetales (39). Together with other mycolic acid-containing organisms (i.e., members of the genera Rhodococcus and Nocardia), the members of the genus Mycobacterium form one of the several sublines of descent within the radiation of the actinomycetes. Later, the results of reverse transcriptase sequencing of 16s rRNAs from a larger number of strains confirmed the phylogenetic coherency of this subline, which was extended by the inclusion of the genera Corynebacterium (35), Gordona (36), and Tsukamurella (5). Within the genus Mycobacterium, species fall into one of two large groups which by and large correspond to the traditional groups containing the slowly growing and rapidly growing members of the genus (35).With the introduction of even faster analytical methods (i.e., analysis of polymerase chain reaction-mediated DNA stretches [4,28]), the number of actinomycetes analyzed and consequently the knowledge about the natural relationships of these organisms have increased considerably during the last 3 years. From a phylogenetic point of view, the genus Mycobacterium is one of the most thoroughly investigated bacterial taxa. There are 55 or so validly described species in this genus (51), and 45 strains of 34 species have been subjected to either 16s rRNA analysis (31,35,40), 16s rDNA analysis (4, 28), or 23s rDNA analysis (21). The bifurcation within the genus Mycobactenurn has been confirmed, with the majority of the slowly growing strains forming a phylogenetically very shallow (i.e., recent) group. In addition to the phyloge...

The genus Hydrogenobacter consists of extremely thermophilic, obligately chemolithotrophic organisms that exhibit anaerobic anabolism but aerobic catabolism. Preliminary studies of the phylogenetic position of these organisms based on limited 16s ribosomal DNA sequence data suggested that they belong to one of the earliest branching orders of the Bacteria. In this study, the complete 16s ribosomal DNA sequences of two type strains, Hydrogenobacter thermophilus TK-6 and Calderobacterium hydrogenophilum 2-829, and another isolate, Hydrogenobacter sp. strain T3, were determined, and the phylogenetic positions of these organisms were examined. Our results revealed that the two type strains are members of a single genus, the genus Hydrogenobacter. Our results also verified the previous conclusion that the Aquifex-Hydrogenobacter complex belongs to a very early branching order, the "Aquificales." Within this order, the relationships among the various organisms are such that only a single family, the "AquiJcaceae," can be recognized at this time. Given the early branching point of the "Aqu$cales," the characteristics of these organisms support the view that the last common ancestor of existing life was thermophilic and suggest that this ancestor may have fixed carbon chemoautotrophically.It has been proposed that aerobic, thermophilic, hydrogenoxidizing, autotrophic bacteria played an important role in the primary productioln of organic matter on the early earth and that the ancestors of the Bacteria may have been thermophilic (4, 12). The increasing attention paid to thermophilic organisms as possible representatives of the earliest forms of life has resulted from the observation that the vast majority of the earliest branching organisms on 16s rRNA-based phylogenetic trees have this phenotype. The discovery of highly thermophilic, aerobic, hydrogen-oxidizing bacteria belonging to the genera Hydrogenobacter and Aquifex in geothermal hot springs was surprising, if one considers the hypothesis that free oxygen appeared only as a consequence of photosystem 11. It has been suggested that photosystem I1 developed much later in evolution, when the mean temperatures on the earth's surface were in the mesobiotic range (5). For this reason, the correct phylogenetic placement of these organisms deserves a thorough and detailed examination.Among the Bacteria, the Thermotoga lineage was previously thought to be the deepest phylogenetic branch and the most slowly evolving lineage (1,46,47). Interestingly, on the basis of its partial 16s rRNA sequence the more recently described highly t hermophilic hydrogen-oxidizing organism Aquifex pyrophilus, which was isolated from submarine hydrothermal vents, appeared to represent an even earlier branching event (12, 16). With an optimum growth temperature of 95"C, this organism is among the most thermophilic of the members of the Bacteria that have been described.On the basis of DNA-DNA hybridization data, the genus Aquifex was determined to be closely related to the genus Hydrogenobacter, ...