Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA fragments was used to explore the genetic diversity of hydrothermal vent microbial communities, specifically to determine the importance of sulfur-oxidizing bacteria therein. DGGE analysis of two different hydrothermal vent samples revealed one PCR band for one sample and three PCR bands for the other sample, which probably correspond to the dominant bacterial populations in these communities. Three of the four 16S rDNA fragments were sequenced. By comparison with 16S rRNA sequences of the Ribosomal Database Project, two of the DGGE-separated fragments were assigned to the genus Thiomicrospira. To identify these 'phylotypes' in more detail, a phylogenetic framework was created by determining the nearly complete 16S rRNA gene sequence (approx. 1500 nucleotides) from three described Thiomicrospira species, viz., Tms. crunogena, Tms. pelophila, Tms. denitrificans, and from a new isolate, Thiomicrospira sp. strain MA2-6. All Thiomicrospira species except Tms. denitrificans formed a monophyletic group within the gamma subdivision of the Proteobacteria. Tms. denitrificans was assigned as a member of the epsilon subdivision and was distantly affiliated with Thiovulum, another sulfur-oxidizing bacterium. Sequences of two dominant 16S rDNA fragments obtained by DGGE analysis fell into the gamma subdivision Thiomicrospira. The sequence of one fragment was in all comparable positions identical to the 16S rRNA sequence of Tms. crunogena. Identifying a dominant molecular isolate as Tms. crunogena indicates that this species is a dominant community member of hydrothermal vent sites. Another 'phylotype' represented a new Thiomicrospira species, phylogenetically in an intermediate position between Tms. crunogena and Tms. pelophila. The third 'phylotype' was identified as a Desulfovibrio, indicating that sulfate-reducing bacteria, as sources of sulfide, may complement sulfur- and sulfide-oxidizing bacteria ecologically in these sulfide-producing hydrothermal vents.

Phylogenetic relationships of Thiomicrospira species and their identification in deep-sea hydrothermal vent samples by denaturing gradient gel electrophoresis of 16S rDNA fragments

Medical microbiology is extremely reliant on the culture of bacteria from clinical specimens and their subsequent identification by biochemical and phenotypic characteristics for the diagnosis of disease. Following determination of the structure of DNA by Watson & Crick (1953), studies in bacteriology have seen a major shift from functional to molecular techniques for identifying bacteria (Towner & Cockayne, 1993). This review will deal with the 16s-23s spacer region of the rRNA operon (Fig. 1) and its use in the identification of micro-organisms at the species and strain (typing) levels.

New approaches to typing and identification of bacteria using the 16S-23S rDNA spacer region

Comparative 16S rRNA sequence analysis has demonstrated that the genusBacillus consists of at least five phyletic lines. rRNA group 3 bacilli of Ash, Farrow, Wallbanks and Collins (1991) comprisingBacillus polymyxa and close relatives is phylogenetically so removed fromBacillus subtilis, the type species of the genus and other aerobic, endospore-forming bacilli that they warrant reclassification in a new genusPaenibacillus. The genusPaenibacillus can be readily distinguished from otherBacillus groups using a battery of phenotypic characters and a highly specific gene probe based on 16S rRNA.

Molecular identification of rRNA group 3 bacilli (Ash, Farrow, Wallbanks and Collins) using a PCR probe test. Proposal for the creation of a new genus Paenibacillus

Comparative sequence analysis of small subunit rRNA is currently one of the most important methods for the elucidation of bacterial phylogeny as well as bacterial identification. Phylogenetic investigations targeting alternative phylogenetic markers such as large subunit rRNA, elongation factors, and ATPases have shown that 16S rRNA-based trees reflect the history of the corresponding organisms globally. However, in comparison with three to four billion years of evolution the phylogenetic information content of these markers is limited. Consequently, the limited resolution power of the marker molecules allows only a spot check of the evolutionary history of microorganisms. This is often indicated by locally different topologies of trees based on different markers, data sets or the application of different treeing approaches. Sequence peculiarities as well as methods and parameters for data analysis were studied with respect to their effects on the results of phylogenetic investigations. It is shown that only careful data analysis starting with a proper alignment, followed by the analysis of positional variability, rates and character of change, testing various data selections, applying alternative treeing methods and, finally, performing confidence tests, allows reasonable utilization of the limited phylogenetic information.

Bacterial phylogeny based on comparative sequence analysis.

Five hydrocarbon-oxidizing strains were isolated from formation waters of oilfields in Russia, Kazakhstan and China. These strains were moderately thermophilic, neutrophilic, motile, spore-forming rods, aerobic or facultatively anaerobic. The G+C content of their DNA ranged from 49.7 to 52.3 mol%. The major isoprenoid quinone was menaquinone-7; cellular fatty acid profiles consisted of significant amounts of iso-15:0, iso-16:0 and iso-17:0 fatty acids (61.7-86.8% of the total). Based on data from 16S rDNA analysis and DNA-DNA hybridization, the subsurface isolates could be divided into two groups, one of which consisted of strains UT and X and the other of which consisted of strains K, Sam and 34T. The new strains exhibited a close phylogenetic relationship to thermophilic bacilli of 'Group 5' of Ash et al. [Ash, C., Farrow, J. A. E., Wallbanks, S. & Collins, M. D. (1991). Lett Appl Microbiol 13, 202-206] and a set of corresponding signature positions of 16S rRNA. Comparative analysis of the 16S rDNA sequences and fatty acid compositions of the novel isolates and established species of thermophilic bacilli indicated that the subsurface strains represent two new species within a new genus, for which the names Geobacillus subterraneus gen. nov., sp. nov., and Geobacillus uzenensis sp. nov. are proposed. It is also proposed that Bacillus stearothermophilus, Bacillus thermoleovorans, Bacillus thermocatenulatus, Bacillus kaustophilus, Bacillus thermoglucosidasius and Bacillus thermodenitrificans be transferred to this new genus, with Geobacillus stearothermophilus (formerly Bacillus stearothermophilus) as the type species.

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Taxonomic study of aerobic thermophilic bacilli: descriptions of Geobacillus subterraneus gen. nov., sp. nov. and Geobacillus uzenensis sp. nov. from petroleum reservoirs and transfer of Bacillus stearothermophilus, Bacillus thermocatenulatus, Bacillus thermoleovorans, Bacillus kaustophilus, Bacillus thermodenitrificans to Geobacillus as the new combinations G. stearothermophilus, G. th.

Complete 23S Ribosomal RNA Sequences of Gram-positive Bacteria with a Low DNA G+C Content

SUMMARY: The phylogenetic incoherency of the genus Bacillus as presently described is demonstrated by analysis of both published and new data from comparative 16S rRNA cataloguing of nine Bacillus species and a number of related non-Bacillus taxa, i.e. Caryophanon latum, Filibacter limicola and Planococcus citreus. While the ellipsoidal-spore-forming bacilli, e.g. B. subtilis and allied species, formed a coherent cluster, the round-spore-forming bacilli showed a higher degree of relationship to the non-spore-forming organisms than these bacilli show among each other. Thus B. sphaericus clustered with C. latum, B. globisporus grouped with F. limicola, B. pasteurii with Sporosarcina ureae, and ‘B. aminovorans’ with P. citreus, respectively. These organisms formed two related subclusters which, in their phylogenetic depth, are comparable to that of the B. subtilis subline. With the exception of ‘B. aminovorans’ the 16S rRNA phylogeny was entirely consistent with the distribution of murein types. Even more distantly related to and grouping outside the main Bacillus cluster was B. stearothermophilus, which displayed a moderate relationship to Thermoactinomyces vulgaris. Taxonomic problems arising from the new insights into the intrageneric relationships of Bacillus are discussed.

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Michael Weizenegger

Papers

Complete 23S Ribosomal RNA Sequences of Gram-positive Bacteria with a Low DNA G+C Content

Comparative 16S rRNA oligonucleotide analyses and murein types of round-spore-forming bacilli and non-spore-forming relatives.

Comparative Sequence Analysis of 23S rRNA from Proteobacteria

The Phylogenetic Positions of Pelobacter acetylenicus and Pelobacter propionicus