High-performance liquid chromatography is a promising alternative for determining the G+C content of bacterial deoxyribonucleic acid (DNA). The method which we evaluated involves enzymatic degradation of the DNA to nucleosides by P1 nuclease and bovine intestinal mucosa alkaline phosphatase, separation of the nucleosides by high-performance liquid chromatography, and calculation of the G+C content from the apparent ratios of deoxyguanosine and thymidine. Because the nucleosides are released from the DNA at different rates, incomplete degradation produces large errors in the apparent G+C content. For partially purified DNA, salts are a major source of interference in degradation. However, when the salts are carefully removed, the preparation and degradation of DNA contribute little error to the determination of G+C content. This method also requires careful selection of the chromatographic conditions to ensure separation of the major nucleosides from the nucleosides of modified bases and precise control of the flow rates. Both of these conditions are achievable with standard equipment and C18 reversed-phase columns. Then the method is precise, and the relative standard deviations of replicate measurements are close to 0.1%. It is also rapid, and a single measurement requires about 15 min. It requires small amounts of sample, and the G+C content can be determined from DNA isolated from a single bacterial colony. It is not affected by contamination with ribonucleic acid. Because this method yields a direct measurement, it may also be more accurate than indirect methods, such as the buoyant density and thermal denaturation methods. In addition, for highly purified DNA, the extent of modification can be determined.

Precise Measurement of the G+C Content of Deoxyribonucleic Acid by High-Performance Liquid Chromatography

Peptidoglycan types of bacterial cell walls and their taxonomic implications.

Denitrification is a distinct means of energy conservation, making use of N oxides as terminal electron acceptors for cellular bioenergetics under anaerobic, microaerophilic, and occasionally aerobic conditions. The process is an essential branch of the global N cycle, reversing dinitrogen fixation, and is associated with chemolithotrophic, phototrophic, diazotrophic, or organotrophic metabolism but generally not with obligately anaerobic life. Discovered more than a century ago and believed to be exclusively a bacterial trait, denitrification has now been found in halophilic and hyperthermophilic archaea and in the mitochondria of fungi, raising evolutionarily intriguing vistas. Important advances in the biochemical characterization of denitrification and the underlying genetics have been achieved with Pseudomonas stutzeri, Pseudomonas aeruginosa, Paracoccus denitrificans, Ralstonia eutropha, and Rhodobacter sphaeroides. Pseudomonads represent one of the largest assemblies of the denitrifying bacteria within a single genus, favoring their use as model organisms. Around 50 genes are required within a single bacterium to encode the core structures of the denitrification apparatus. Much of the denitrification process of gram-negative bacteria has been found confined to the periplasm, whereas the topology and enzymology of the gram-positive bacteria are less well established. The activation and enzymatic transformation of N oxides is based on the redox chemistry of Fe, Cu, and Mo. Biochemical breakthroughs have included the X-ray structures of the two types of respiratory nitrite reductases and the isolation of the novel enzymes nitric oxide reductase and nitrous oxide reductase, as well as their structural characterization by indirect spectroscopic means. This revealed unexpected relationships among denitrification enzymes and respiratory oxygen reductases. Denitrification is intimately related to fundamental cellular processes that include primary and secondary transport, protein translocation, cytochrome c biogenesis, anaerobic gene regulation, metalloprotein assembly, and the biosynthesis of the cofactors molybdopterin and heme D1. An important class of regulators for the anaerobic expression of the denitrification apparatus are transcription factors of the greater FNR family. Nitrate and nitric oxide, in addition to being respiratory substrates, have been identified as signaling molecules for the induction of distinct N oxide-metabolizing enzymes.

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Cell biology and molecular basis of denitrification.

Publisher Summary Recent advances in the biochemistry of microorganisms have revealed that cell-component analysis can be effectively applied to bacterial systematics, providing the basis of chemotaxonomy. Analyses of cell components are coming to be essential tools not only for bacterial classification but also for identification. The sequence of chromosomal DNA holds the essential genetic information for an organism. Phylogenetic relationships are often examined using data on nucleotide sequences of ribosomal RNA. However, it is still impossible to determine the sequences of large polynucleotides for the number of organisms sufficient for taxonomic purposes. DNA (RNA) homologies, which have been applied to various kinds of bacteria, present useful information on bacterial systematics. Homology indices are values used in comparing microorganisms. DNA homology data contribute to the clarification, not only of the relatedness of two organisms, but also of the evaluation of phenotypic characteristics.

4 Lipid and Cell-Wall Analysis in Bacterial Systematics

A method is described for the rapid isolation and purification of bacterial genomic DNA. A total of 215 bacterial strains representing species of Campylobacter, Corynebacterium, Escherichia, Legionella, Neisseria, Staphylococcus and Streptococcus, were lysed with guanidium thiocyanate. DNA was prepared using just three other reagents and one high-speed centrifugation step. The method, which was applicable to both Gram-positive and Gram-negative bacteria, eliminated endogenous nuclease activity and avoided the need for phenol, RNase and protease treatments. The DNA was of high purity, high molecular mass and double-stranded.

Rapid extraction of bacterial genomic DNA with guanidium thiocyanate

DNA base composition was determined by reversed-phase high-performance liquid chromatography (HPLC). DNA was hydrolysed into nucleosides with nuclease P1 and bacterial alkaline phosphatase. The mixture of nucleosides was applied to HPLC without any further purification. One determination by chromatography needed 2 μg of hydrolysed nucleosides and took only 8 min. The relative standard error of nucleoside analysis was less than 1%. The system described here gives a direct and precise method for determining DNA base composition.

Determination of DNA base composition by reversed-phase high-performance liquid chromatography

We determined the taxonomic status of six Bacillus species (Bacillus alginolyticus, Bacillus chondroitinus, Bacillus curdlanolyticus, Bacillus glucanolyticus, Bacillus kobensis, and Bacillus thiaminolyticus) by using the results of 16S rRNA gene sequence and cellular fatty acid composition analyses. Phylogenetic analysis clustered these species closely with the Paenibacillus species. Like the Paenibacillus species, the six Bacillus species contained anteiso-C15:0 fatty acid as a major cellular fatty acid. The use of a specific PCR primer designed for differentiating the genus Paenibacillus from other members of the Bacillaceae showed that the six Bacillus species had the same amplified 16S rRNA gene fragment as members of the genus Paenibacillus. Based on these observations and other taxonomic characteristics, the six Bacillus species were transferred to the genus Paenibacillus. In addition, we propose emendation of the genus Paenibacillus.

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Transfer of Bacillus Alginolyticus, Bacillus Chondroitinus, Bacillus Curdlanolyticus, Bacillus Glucanolyticus, Bacillus Kobensis, and Bacillus Thiaminolyticus to the Genus Paenibacillus and Emended Description of the Genus Paenibacillus

Research letterDetermination of DNA base composition by reversed-phase high-performance liquid chromatography

16S rRNA gene sequences of the type strains of 11 species belonging to the Bacillus brevis and Bacillus aneurinolyticus groups were determined. On the basis of the results of gene sequence analyses, these species were separated into two clusters. The B. brevis cluster included 10 species, namely, Bacillus brevis, Bacillus agri, Bacillus centrosporus, Bacillus choshinensis, Bacillus parabrevis, Bacillus reuszeri, Bacillus formosus, Bacillus borstelensis, Bacillus laterosporus, and Bacillus thermoruber. Bacillus aneurinolyticus and Bacillus migulanus belonged to the B. aneurinolyticus cluster. Moreover, the two clusters were phylogenetically distinct from other Bacillus, Amphibacillus, Sporolactobacillus, Paenibacillus, and Alicyclobacillus species. On the basis of our data, we propose reclassification of the B. brevis cluster as Brevibacillus gen. nov. and reclassification of the B. aneurinolyticus cluster as Aneurinibacillus gen. nov. By using 16S rRNA gene sequence alignments, two specific PCR amplification primers were designed for differentiating the two new genera from each other and from other aerobic, endospore-forming organisms.

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Kazuo Komagata

Papers

4 Lipid and Cell-Wall Analysis in Bacterial Systematics

Determination of DNA base composition by reversed-phase high-performance liquid chromatography

Transfer of Bacillus Alginolyticus, Bacillus Chondroitinus, Bacillus Curdlanolyticus, Bacillus Glucanolyticus, Bacillus Kobensis, and Bacillus Thiaminolyticus to the Genus Paenibacillus and Emended Description of the Genus Paenibacillus

Research letterDetermination of DNA base composition by reversed-phase high-performance liquid chromatography

Proposal for Two New Genera, Brevibacillus gen. nov. and Aneurinibacillus gen. nov.