For the last 25 years species delimitation in prokaryotes (Archaea and Bacteria) was to a large extent based on DNA-DNA hybridization (DDH), a tedious lab procedure designed in the early 1970s that served its purpose astonishingly well in the absence of deciphered genome sequences. With the rapid progress in genome sequencing time has come to directly use the now available and easy to generate genome sequences for delimitation of species. GBDP (Genome Blast Distance Phylogeny) infers genome-to-genome distances between pairs of entirely or partially sequenced genomes, a digital, highly reliable estimator for the relatedness of genomes. Its application as an in-silico replacement for DDH was recently introduced. The main challenge in the implementation of such an application is to produce digital DDH values that must mimic the wet-lab DDH values as close as possible to ensure consistency in the Prokaryotic species concept. Correlation and regression analyses were used to determine the best-performing methods and the most influential parameters. GBDP was further enriched with a set of new features such as confidence intervals for intergenomic distances obtained via resampling or via the statistical models for DDH prediction and an additional family of distance functions. As in previous analyses, GBDP obtained the highest agreement with wet-lab DDH among all tested methods, but improved models led to a further increase in the accuracy of DDH prediction. Confidence intervals yielded stable results when inferred from the statistical models, whereas those obtained via resampling showed marked differences between the underlying distance functions. Despite the high accuracy of GBDP-based DDH prediction, inferences from limited empirical data are always associated with a certain degree of uncertainty. It is thus crucial to enrich in-silico DDH replacements with confidence-interval estimation, enabling the user to statistically evaluate the outcomes. Such methodological advancements, easily accessible through the web service at http://ggdc.dsmz.de
 , are crucial steps towards a consistent and truly genome sequence-based classification of microorganisms.

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Genome sequence-based species delimitation with confidence intervals and improved distance functions

DNA-DNA hybridization (DDH) values have been used by bacterial taxonomists since the 1960s to determine relatedness between strains and are still the most important criterion in the delineation of bacterial species. Since the extent of hybridization between a pair of strains is ultimately governed by their respective genomic sequences, we examined the quantitative relationship between DDH values and genome sequence-derived parameters, such as the average nucleotide identity (ANI) of common genes and the percentage of conserved DNA. A total of 124 DDH values were determined for 28 strains for which genome sequences were available. The strains belong to six important and diverse groups of bacteria for which the intra-group 16S rRNA gene sequence identity was greater than 94 %. The results revealed a close relationship between DDH values and ANI and between DNA-DNA hybridization and the percentage of conserved DNA for each pair of strains. The recommended cut-off point of 70 % DDH for species delineation corresponded to 95 % ANI and 69 % conserved DNA. When the analysis was restricted to the protein-coding portion of the genome, 70 % DDH corresponded to 85 % conserved genes for a pair of strains. These results reveal extensive gene diversity within the current concept of "species". Examination of reciprocal values indicated that the level of experimental error associated with the DDH method is too high to reveal the subtle differences in genome size among the strains sampled. It is concluded that ANI can accurately replace DDH values for strains for which genome sequences are available.

DNA–DNA hybridization values and their relationship to whole-genome sequence similarities

Fluorometric hybridization in microdilution wells was developed to determine genetic relatedness among microorganisms. Total chromosomal deoxyribonucleic acid (DNA) for hybridization reactions was labeled with photoreactive biotin (photobiotin). The biotinylated DNA was hybridized with single-stranded unlabeled DNAs which had been immobilized on the surfaces of microdilution wells. After hybridization, biotinylated DNA was quantitatively detected with beta-D-galactosidase and a fluorogenic substrate, 4-methylumbelliferyl-beta-D-galactopyranoside. Homology values obtained with this fluorometric direct binding method were compared with values obtained with two membrane filter methods, one in which photobiotin labeling was used and one in which radioisotope labeling was used. The results showed that the fluorometric direct binding method in which microdilution wells are used could be an alternative to radioisotope and membrane filter hybridization methods.

Fluorometric Deoxyribonucleic Acid-Deoxyribonucleic Acid Hybridization in Microdilution Wells as an Alternative to Membrane Filter Hybridization in which Radioisotopes Are Used To Determine Genetic Relatedness among Bacterial Strains

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.

Acinetobacter baumannii has emerged as a highly troublesome pathogen for many institutions globally. As a consequence of its immense ability to acquire or upregulate antibiotic drug resistance determinants, it has justifiably been propelled to the forefront of scientific attention. Apart from its predilection for the seriously ill within intensive care units, A. baumannii has more recently caused a range of infectious syndromes in military personnel injured in the Iraq and Afghanistan conflicts. This review details the significant advances that have been made in our understanding of this remarkable organism over the last 10 years, including current taxonomy and species identification, issues with susceptibility testing, mechanisms of antibiotic resistance, global epidemiology, clinical impact of infection, host-pathogen interactions, and infection control and therapeutic considerations.

Acinetobacter baumannii: Emergence of a Successful Pathogen

A new method is proposed to measure relatedness amongst bacteria, based on renaturation rate determinations of DNA types and their mixture. The equations, relating the degree of binding with renaturation rates, are calculated for several cases. The optimal conditions for measurements are given. The method was checked in several ways. The advantages are summarized.

The quantitative measurement of DNA hybridization from renaturation rates.

A new method is described for the determination of the total molecular weight of haploid genome DNA. It is based on initial optical renaturation rate measurements of precisely known concentrations of fragmented DNA. The theoretical basis of the measurement is presented. The actual state of replication of the genome has little effect. The exact conditions and the effects of DNA concentration, renaturation time, size of DNA fragments, buffer concentration, optimal temperature and % (G + C) have been determined. Several types of bacterial DNA of known genome size were included as a control. As an application, the DNA genome size have been determined of 40 different bacteria. The method appears to offer advantages by its simplicity, rapidity and reproducibility.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1970.tb00831.x

The Determination of Molecular Weight of Bacterial Genome DNA from Renaturation Rates

The relationships of the yellow-pigmented hydrogen-oxidizing species Pseudomonas flava, Pseudomonas pseudoflava, Pseudomonas palleronii, Pseudomonas taeniospiralis, and “Pseudomonas carboxydoflava,” which are all members of the acidovorans ribosomal ribonucleic acid (rRNA) complex in rRNA superfamily III, were studied by using deoxyribonucleic acid (DNA):rRNA hybridization, immunotyping, numerical analysis of biochemical and auxanographic features, polyacrylamide gel electrophoresis of cellular proteins, numerical analysis of fatty acid patterns, and DNA:DNA hybridization. Our results show that these five yellow-pigmented hydrogen-oxidizing Pseudomonas species are more closely related to each other than to other taxa belonging to the acidovorans rRNA complex. We propose the transfer of these species to a new genus, Hydrogenophaga, with the following four species: Hydrogenophaga flava (formerly Pseudomonas flava), Hydrogenophaga pseudoflava (to accommodate both Pseudomonas pseudoflava and “Pseudomonas carboxydoflava”), Hydrogenophaga taeniospiralis (formerly Pseudomonas taeniospiralis), and Hydrogenophaga palleronii (formerly Pseudomonas palleronii). The type species is H. flava, with monotype strain DSM 619 (= LMG 2185 = CCUG 1658). Because H. flava grows slowly and unreliably, but is genotypically and protein electrophoretically very similar to H. pseudoflava, the latter species can be used as an alternative reference taxon for the new genus. The type strains of H. pseudoflava, H. taeniospiralis, and H. palleronii are strains GA3 (= LMG 5945 = CCUG 13799). DSM 2082 (= LMG 7170 = CCUG 15921), and Stanier 362t1 (= LMG 2366t1 = CCUG 20334), respectively.

Hydrogenophaga, a new genus of hydrogen-oxidizing bacteria that includes Hydrogenophaga flava comb. nov. (formerly Pseudomonas flava), Hydrogenophaga palleronii (formerly Pseudomonas palleronii), Hydrogenophaga pseudoflava (formerly Pseudomonas pseudoflava and Pseudomonas carboxydoflava), and Hydrogenophaga taeniospiralis (formerly Pseudomonas taeniospiralis)

Agrobacterium tumefaciens is the agent for crown gall disease of several plants, thus causing serious economic losses in many countries. Except for its phytopathogenic action, it does not display any characteristic physiological or morphological properties. Its final identification is established by experimentally induced tumours, a procedure which often demands too much time for quick intervention in an epidemic. A quick, easy and specific identification test would be very useful. We wish, therefore, to report that Agrobacterium tumefaciens and A. radiobacter produce 3-ketoglycosides from the corresponding disaccharides and bionic acids. We propose a test based on this property, which is very simple, can be carried out in any phytopathological laboratory with ordinary glassware and chemicals, gives a result after 1–2 days and is, so far as we can judge from our present experience, limited to members of the genus Agrobacterium.

A Biochemical Test for Crown Gall Bacteria

SUMMARY: Improved methods for the identification and grouping of bacteria poly-acrylamide gel electrophoresis of soluble proteins are described. Electrophoretic protein patterns were obtained in rigorously standardized conditions. The results were much more reproducible than any described previously. Some of the factors affecting reproducibility were: growth conditions, time and speed of centrifugation of extracts, and conditions of gel electrophoresis. Protein patterns were compared computing correlation coefficients from normalized densitometric tracings and clustering the strains the unweighted average pair group method. As model systems, both Agrobacterium and Zymomonas were used because of differences in the sharpness of the peaks. The method was applied to 42 Agrobacterium strains. The agreement with the results of clustering either phenotypic tests or DNA: DNA hybridization was excellent. Computerized comparisons of electrophoretic protein patterns can be a fast, easy and powerful tool for classification and identification of bacteria.

J. De Ley

Papers

The quantitative measurement of DNA hybridization from renaturation rates.

The Determination of Molecular Weight of Bacterial Genome DNA from Renaturation Rates

A Biochemical Test for Crown Gall Bacteria

Identification and grouping of bacteria by numerical analysis of their electrophoretic protein patterns.